Non-germinal Center Diffuse Large B-cell Research Articles

Final Results of a Phase I/II Trial of Carfilzomib + R-CHOP for Frontline Treatment of Patients with Non-Germinal Center Diffuse Large B-Cell Lymphoma (DLBCL)

INTRODUCTION: Recent data from the REMoDL-B trial show that addition of the proteasome inhibitor bortezomib to R-CHOP improves progression free survival (PFS) of patients (Pts) with activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL) compared to R-CHOP, but peripheral neuropathy limited dose intensity. Relative to bortezomib, carfilzomib (K) displays comparable clinical activity in plasma cell neoplasms but does not cause neurotoxicity. K has preferential clinical activity in relapsed non-germinal center (non-GC) DLBCL treated with chemotherapy (Torka, et al. Blood Adv. 2023). To explore the safety and efficacy of K in combination with R-CHOP (KR-CHOP), we performed a phase I trial to identify the recommended phase 2 dose (RP2D) in all DLBCL Pts followed by a phase II extension exclusively in non-GC DLBCL. METHODS: Between 2015-2020, Pts with untreated de novo or transformed DLBCL, adequate organ function and ECOG PS ≤ 3 were enrolled at 3 US academic medical centers. As previously reported [Hill, et al, Blood (2018), 132 (Supl.1):1692], during phase I dose escalation, the RP2D of K was 20 mg/m 2 on days (D) 1 & 2 of cycle 1 and 56 mg/m 2 on D1 & 2 of cycles 2-6. Rituximab was given on D2 and CHOP was given on D3. All Pts received pegfilgrastim and zoster prophylaxis. To assess the cardiac safety of combining K with anthracycline, we compared echocardiogram data at baseline and conclusion of therapy from study Pts and control NHL Pts treated with CHOP. To gauge for differential clinical activity of KR-CHOP vs R-CHOP, we compared PFS and overall survival (OS) rates for study Pts with non-GC DLBCL vs. Pts with non-GC DLBCL receiving standard of-care (SOC) R-CHOP at the Cleveland Clinic during the same period. The outcomes were also compared between propensity score (PS)-matched pairs, to account for differences in baseline demographics and clinical risk. RESULTS: 48 Pts were enrolled, and 47 treated; 18 in dose levels < RP2D and 29 at RP2D.Baseline demographics and treatment outcomes are shown in the Table. All-grade (G) hematologic adverse events (AEs) were: anemia, 35% (22% ≤ G2, 13% G3); thrombocytopenia, 24% (15% ≤ G2, 2% G3, 7% G4); febrile neutropenia, 9% (all G3/G4). Non-hematologic AEs included: nausea, 49% (47% ≤ G2, 2% G3); vomiting, 23% (21% ≤ G2, 2% G3), peripheral sensory neuropathy, 27% (all ≤ G2); dyspnea, 24% (all ≤G2). There was one case of G3 congestive heart failure. Echocardiograms from 42 study Pts who completed 6 cycles of KR-CHOP showed no statistically significant difference in change in ejection fraction from baseline compared to 39 control Pts [95% vs. 94%, respectively (P = 0.59)]. The overall response rate was 89% (70% CR, 19% PR). At a median follow-up of 31 months, 3-year Kaplan-Meier estimates of PFS and OS for the study population were 79% and 87%, respectively. There were 8 deaths in the study: 4 due to lymphoma; 2 due to infection; 2 due to other causes. Comparing outcomes of the 36 study Pts with non-GC DLBCL to 172 control Pts receiving R-CHOP as SOC, more control Pts had stage IV disease (54% vs 34%, p=0.03) and older age (median 67 vs 66 years old, p=0.03), but less frequently >1 extranodal site of disease (23% vs. 42%, P = 0.02). PS matching using logistic regression identified 25 case-control pairs of Pts with non-GC DLBCL according to gender, age, and other International Prognostic Index (IPI) elements (see Table). In analysis of PS-matched pairs of Pts with non-GC DLBCL, Pts treated with KR-CHOP had better 3-year PFS and OS relative to Pts treated with R-CHOP as SOC [96% vs. 50% (p=0.002) (see Figure) and 67% vs. 96% (p = 0.02), respectively]. In Cox univariate analysis, treatment with R-CHOP vs. KR-CHOP was associated with an increased risk of PFS and OS with hazard ratios (HR) of 6.15 [95% confidence interval (CI) 1.71 - 22.05, P = 0.002] and 3.19 [(95% CI, 1.01 -10.11), P = 0.02], respectively. CONCLUSION: KR-CHOP can be delivered safely, without excess cardiac toxicity or peripheral neuropathy. The superior PFS and OS rates for Pts with non-GC DLBCL vs. controls receiving R-CHOP are encouraging but should be interpreted with caution due to inherent selection bias in clinical trial populations. These data suggest that, like bortezomib, carfilzomib (K) may have preferential clinical activity in ABC DLBCL. Future randomized trials are justified using advanced molecular profiling to further investigate which Pts with DLBCL may benefit from addition of a proteasome inhibitor as part of frontline treatment.

Joint Analyses of Serum Proteins and Circulating Tumor DNA Improves Risk Assessment and Subtype Stratification in Large B-Cell Lymphomas

Introduction Inadequate dismantling of clinical and biological heterogeneity is an obstacle for improving outcome in large B-cell lymphoma (LBCL). Circulating tumor DNA (ctDNA)-based stratification holds great potential in resolving these challenges. However, the assessment is limited to genetic content, and excludes host response from the estimates. Previously, we have shown that an inflammatory signature identified from the pretreatment serum proteins translates to poor survival, and multiple serum proteins correlate with LBCL genetic subtypes. To further improve risk stratification and molecular characterization of LBCL from the liquid biopsy, we co-analyzed serum protein and ctDNA levels in LBCL patients treated in two Nordic Lymphoma Group (NLG) trials. Materials and methods Our discovery cohort consisted of 122 patients less than 65 years of age with clinically high-risk (age adjusted IPI ≥ 2) LBCL, who were treated in NLG-LBC-06 trial with immunochemotherapy. Plasma and serum samples were collected prior to treatment. Cell free DNA was extracted from plasma and sequenced with in-house adapted duplex sequencing platform using custom targeted panel of 347 lymphoma driver genes and immunoglobulin loci. An inflammation score was composed as log transformed mean of the most pertinent inflammatory serum proteins detected in the inflammatory signature (IL10, IL2RA, CXCL9, IL18, GZMB, PD-L1 and IFNG), and their levels were measured using antibody-based Luminex assay (pg/ml, R&D Systems). In addition, CCL17 and TNFRSF13B levels were analyzed to uncover their associations with genetic subtypes. An independent cohort of 91 patients treated in our previous NLG-LBC-05 trial was used for validation. Results Combined analysis of serum proteins and ctDNA identified a group of patients (n=12, 10%) with high serum inflammation scores and ctDNA burden (≥ 3.75 hGE/ml), which translated to poor survival compared to the patients with low concentrations of either inflammatory proteins or ctDNA, or both (progression free survival, PFS, hazard ratio 7.33, 95%-CI 2.59-20.79, P < 0.001, Figure 1A). This finding was confirmed in the independent validation cohort. In both cohorts, the combined analysis showed higher specificity in survival prediction as compared to the ctDNA-based assessment alone, indicating that inflammatory serum markers alongside with ctDNA can identify those patients who, despite of having high ctDNA burden, have excellent survival. Beyond inflammation, the levels of serum TNFRSF13B and CCL17 correlated with mutations associating with non-germinal center diffuse large B-cell lymphoma (non-GCB DLBCL)/MCD subtype and primary mediastinal B-cell lymphoma/grey-zone lymphoma (PMBCL/GZL), respectively. For example, serum TNFRSF13B levels correlated positively with mutations in MCD genes, such as CD79B, SETD1B, PIM2 and PRDM1, and negatively with mutations in EZB genes EZH2 and CREBBP in both cohorts (P < 0.05, Figure 1B). Supporting our earlier findings, high CCL17 levels revealed a group of patients diagnosed with DLBCL not otherwise specified or high-grade lymphoma but with a mutational landscape resembling PMBCL/GZL, including mutations in TNFAIP3, ITPKB, B2M, BTG1 and STAT6 (P < 0.05). Conclusion Our findings demonstrate that co-analysis of inflammatory serum markers along with the ctDNA improves ctDNA-based survival prediction. Moreover, analysis of serum proteins refines molecular classification. Altogether, joint analysis of serum proteins and ctDNA provides new tools to dismantle LBCL heterogeneity.

Primary thyroid lymphoma (PTL): a rare case report and review of the literature

Primary thyroid lymphoma (PTL) is a rare malignancy of the thyroid, accounting for 5% of all thyroid malignancies and approximately 2% of extranodal lymphomas. PTL occurs mainly in elderly females and diffuse large B-cell lymphoma (DLBCL) is most common. Here we report a case of non-germinal center diffuse large B-cell lymphoma (non-GC DLBCL) occurring in a 62-year-old female who was admitted to the hospital because of multiple palpable left neck masses. The patient then underwent thyroid ultrasound, detecting multiple nodules in the two lobes and isthmus of the thyroid gland, in which the largest nodule was in the left lobe with TIRADS 5 measuring 47x33 mm, and many cervical lymph nodes. Microscopically, thyroid tissue showed proliferation of tumor cells with large nuclei, coarse chromatin, narrow rims of cytoplasm, and scattered mitoses. There was 01 destructured lymph node, proliferating cells with characteristics similar to those in the thyroid gland; the remaining lymph nodes were chronically inflamed. Immunohistochemically, the tumor cells were positive for CD20, CD79a, Ki67 (40%), and negative for CD10, BCL6, MUM1, CD5, BCL2, c-MYC, CD15, CD30, EBV. Primary thyroid lymphomas are rare with the prognosis of patients depending on the histological classification of the tumor and the stage of the disease. They need to be evaluated and treated individually because there is no unified treatment due to their rarity.

Zanubrutinib monotherapy for relapsed or refractory non-germinal center diffuse large B-cell lymphoma

AbstractThe non-germinal center B-cell like (non-GCB) subtype of diffuse large B-cell lymphoma (DLBCL) has poor clinical outcomes. Bruton tyrosine kinase (BTK) inhibitors have established therapeutic activity in B-cell malignancies, with modest activity in DLBCL. Zanubrutinib, a potent and selective BTK inhibitor, was evaluated in patients with relapsed or refractory (R/R) non-GCB DLBCL. The BGB-3111-207 study (NCT03145064) was a multicenter single-arm phase 2 study. Patients received twice-daily oral zanubrutinib, 160 mg, until disease progression or unacceptable toxicity. The primary end point was the overall response rate (ORR). Secondary end points included progression-free survival (PFS) and duration of response (DOR). Overall survival (OS) was an exploratory end point. Forty-one patients were enrolled in China after having progressed or not responded to prior therapy. At data cutoff, 4 patients continued treatment with 37 discontinuations. The median follow-up was 6.8 months, the ORR was 29.3%, and the complete response rate was 17.1%. Median DOR, PFS, and OS were 4.5, 2.8, and 8.4 months, respectively. Adverse events (AEs) leading to treatment discontinuation were reported in 4 patients, and grade ≥ 3 AEs were reported in 48.8% of patients. Major hemorrhage, atrial fibrillation, and/or flutter were not observed. Zanubrutinib demonstrated modest antitumor activity in non-GCB DLBCL, like other BTK inhibitors, as well as a safety profile consistent with previous studies. Through retrospective biomarker testing, potential antitumor activity was observed in patients with both CD79B and MYD88 mutations, who have inferior outcomes to immunochemotherapy. Future studies of zanubrutinib in R/R non-GCB DLBCL will focus on developing mechanism-based treatment combinations and biomarker-driven patient selection.

Zanubrutinib monotherapy for patients with relapsed or refractory non-germinal center diffuse large B-cell lymphoma: Results from a phase II, single-arm, multicenter, study.

e20051 Background: The non-germinal center (non-GCB) subtype of diffuse large B-cell lymphoma (DLBCL) is associated with poor clinical outcomes. Inhibitors of Bruton’s tyrosine kinase (BTK) have established therapeutic activity in mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenstrom macroglobulinemia and have shown modest activity in DLBCL. Zanubrutinib, a potent and selective BTK inhibitor, was evaluated in a phase 2 study in patients with relapsed or refractory (R/R) non-GCB DLBCL. Methods: The BGB-3111-207 study (NCT03145064) is a multicenter, single arm, phase 2 study in patients with R/R non-GCB DLBCL who were ineligible for intensive chemotherapy and bone marrow transplantation. Subjects received zanubrutinib 160 mg by mouth twice daily until disease progression or unacceptable toxicity. The primary endpoint was the overall response rate (ORR) using the Lugano criteria. Secondary endpoints included progression free survival (PFS) and duration of response (DOR). Overall survival (OS) was the exploratory endpoint. Results: Forty-one patients were enrolled at 11 sites in China between June 2017 and May 2018. Patients were 61% male, with a median age of 62 years. The median number of prior therapies was 2. International Prognostic Index risk was high-intermediate or high for 48.8% of patients, and 56.1% of patients had extranodal disease at study entry. As of the data cutoff date of May 24, 2019, four patients were continuing treatment and 37 patients had discontinued. With a median follow-up time of 6.8 months, the ORR was 29.3% and the complete response rate was 17.1%. The median DOR was 4.5 months, the median PFS was 2.8 months, and the median OS was 8.4 months. Adverse events (AEs) leading to treatment discontinuation were reported in four patients. Grade 3 or higher AEs were reported in 48.8% of patients. Major hemorrhage, atrial fibrillation and/or flutter, second primary malignancy, and tumor lysis syndrome were not observed. Conclusions: In this study zanubrutinib demonstrated modest antitumor activity in non-GCB DLBCL, similar to other BTK inhibitors. The safety profile was tolerable and consistent with previous reports of zanubrutinib treatment. Future studies of zanubrutinib in R/R non-GCB DLBCL will focus on the development of mechanism-based treatment combinations and biomarker-driven patient selection. Clinical trial information: NCT03145064 .

Activity and complexes of mTOR in diffuse large B-cell lymphomas—a tissue microarray study

Diffuse large B-cell lymphoma is a heterogeneous group of diseases with different responses to therapy. Targeting mTOR (mammalian target of rapamycin) offers a new approach to improve the treatment. mTOR inhibitors are being developed and are in clinical use in mantle cell lymphoma therapy and clinical trials are ongoing in other high-grade lymphomas as well. However, there is limited data about mTOR activity and the expression of its different complexes in diffuse large B-cell lymphomas. Tissue microarray blocks were constructed from paraffin-embedded biopsy specimens. More than 700 immunohistochemical stainings (mTOR signaling-related proteins and phosphoproteins, markers for lymphoma classification) were evaluated from 68 diffuse large B-cell lymphoma biopsies from conventionally treated and followed patients. Approximately 30% of cases were characterized as germinal center-derived diffuse large B-cell lymphomas, which showed virtually no mTOR activity, as determined by phospho-ribosomal S6 expression, the most sensitive marker of mTOR activity. In about 80% of non-germinal center-derived diffuse large B-cell lymphoma cases, positivity of mTOR-related phosphoproteins was observed, denoting mTOR activity. Moreover, Rictor (a characteristic protein of the mTOR complex2) was overexpressed in 43% of all diffuse large B-cell lymphomas and in 63% of mTOR-active non-germinal center diffuse large B-cell lymphoma samples. Rictor overexpression with mTOR activity indicated significantly worse survival for patients than mTOR inactivity or mTOR activity with low Rictor expression. These results suggest that mTOR activity is characteristic in most non-germinal center-derived diffuse large B-cell lymphomas with potentially variable mTOR-inhibitor sensitivity. Taken together, mTOR inhibitors may be useful in addition to regular therapy in diffuse large B-cell lymphomas, however, patient and inhibitor selection criteria must be carefully considered.

Thyroid carcinoma-associated genetic mutations also occur in thyroid lymphomas

Molecular testing for mutations activating the mitogen-associated protein kinase signaling pathway is being used to help diagnose thyroid carcinomas. However, the prevalence of these mutations in thyroid lymphomas has not been reported. Therefore, we studied the prevalence of BRAF, NRAS, HRAS, and KRAS mutations in 33 thyroid lymphomas and correlated the mutational status with the clinical, pathological, cytogenetic, and immunophenotypic findings. Eleven cases were also tested for PAX8/PPARγ translocations. The lymphomas included 25 diffuse large B-cell lymphomas, 6 extranodal marginal-zone lymphomas of mucosa-associated lymphoid tissue type, and 2 follicular lymphomas. Seventeen diffuse large B-cell lymphomas were germinal center type, six non-germinal center type, and two unclassifiable (Hans algorithm). None of the cases had an associated thyroid carcinoma. Mutations of the BRAF gene were identified in six (24%) diffuse large B-cell lymphomas (D594G in three germinal center diffuse large B-cell lymphomas, K601N in two germinal center diffuse large B-cell lymphomas, and V600E in one non-germinal center diffuse large B-cell lymphoma) and of the NRAS gene in two (8%) non-germinal center diffuse large B-cell lymphomas (Q61K and Q61H). BRAF and NRAS mutations were not found in any extranodal marginal-zone lymphomas of mucosa-associated lymphoid tissue type or follicular lymphomas. HRAS and KRAS mutations were not identified in any of the cases, nor were PAX8/PPARγ translocations found. Thus, interpretation of finding a BRAF or NRAS mutation in the thyroid, particularly in preoperative thyroid aspirates, must take into account the differential diagnosis of a lymphoma. In addition to the diagnostic importance, our data also demonstrate that alteration in the mitogen-associated protein kinase pathway may have a role in the pathogenesis of some large B-cell lymphomas of the thyroid with potential therapeutic implications.

The novel anti-MEK small molecule AZD6244 induces BIM-dependent and AKT-independent apoptosis in diffuse large B-cell lymphoma

AbstractThe RAS/RAF/MEK/ERK signaling pathway has been largely unexplored as a potential therapeutic target in lymphoma. The novel 2nd generation anti-MEK small molecule, AZD6244, down-regulated its direct downstream target, phospho-ERK (pERK) in germinal center and nongerminal center diffuse large B-cell lymphoma (DLBCL) cell lines and primary cells. Similar decreased pERK levels were noted despite constitutive activation (CA) of MEK. Consequently, several lymphoma-related ERK substrates were down-regulated by AZD6244 including MCT-1, c-Myc, Bcl-2, Mcl-1, and CDK1/2. AZD6244 induced time- and dose-dependent antiproliferation and apoptosis in all DLBCL cell lines and fresh/primary cells (IC50 100nM-300nM). Furthermore, AZD6244 resulted in significantly less tumor compared with control in an in vivo DLBCL SCID xenograft model. Cell death was associated with cleaved PARP, caspases-8, -9, and -3, and apoptosis was caspase-dependent. In addition, there was stabilization of FoxO3a, activation of BIM and PUMA, and a significant decrease in c-Myc transcripts. Moreover, siRNA knockdown of BIM abrogated AZD6244-related apoptosis, while shRNA knockdown of ERK minimally sensitized cells. Finally, manipulation of AKT with transfection of OCI-LY3 cells with CA-AKT or through chemical inhibition (LY294002) had minimal effect on AZD6244-induced cell death. Altogether, these findings show that the novel anti-MEK agent, AZD6244, induced apoptosis in DLBCL and that cell death was BIM-dependent.

BCL-2 Expression at the Time of Diagnosis Affects the Clinical Outcome of Patients with Germinal Center and Non-Germinal Center Diffuse Large B-Cell Lymphoma (DLBCL) Treated with Chemo-Immunotherpy

AbstractAbstract 3130DLBCL has been recognized as a heterogeneous disease varying in molecular biology and clinical outcome. The use of genetic expression profiling has led to the sub-classification of DLBCL into germinal center B-cell like (GCB) and non-germinal center B cell like (non-GCB) based on the cell of origin of the neoplastic B-cell. Immunohistochemistry (IHC) algorithms had been developed and validated to identify GCB or non-GCB DLBCL. Deregulation of Bcl-2 family member of proteins plays an important role in the development, progression, and prognosis of various subtypes of B-cell neoplasms, including DLBCL. Bcl-2 protein expression is a previously known negative prognostic indicator of clinical outcome in DLBCL treated with antracycline-containing combination chemotherapy (e.g. CHOP) in the past. In the post-rituximab (R) era (e.g. use of upfront R-CHOP), the negative prognostic value of Bcl-2 protein expression needs to be reevaluated to ensure its validity. To study the prognostic value of Bcl-2 in patients with either GCB or non-GCB DLBCL, we retrospectively analyzed differences in progression-free survival (PFS) and overall survival (OS) between Bcl-2+ and Bcl-2- de novo DLBCL (GCB or non-GCB subtypes). Using the RPCI tumor registry and pharmacy database, we identified 201 DLBCL patients treated with equivalent doses of rituximab and anthracycline-based therapy (i.e. R=CHOP or R+DA-EPOCH) at RPCI between 1997 and 2007. Demographic, clinical and pathological characteristics were obtained for each patient. Patients were classified into GCB or non-GCB DLBCL according to the Han's algorithm based on the expression of CD10, Bcl-6 and MUM-1. Bcl-2 was determined by IHC and was available for 101 patients. Using the Han's algorithm, fifty-three patients (26.4%) were classified as GCB, 54 patients (26.9%) non-GCB, and 94 patients (46.8%) could not be classified due to inadequate data/sample. Bcl-2 expression was detected in 67% and 73% of the GCB- and non-GCB DLBCL subtypes respectively. Demographics and clinical characteristics were equally distributed between GCB- and non-GCB DLBCL. Patients received either R+CHOP (90%) or R+DA-EPOCH (10%). The complete response (CR) rate of the entire cohort was 82.6% and no differences were observed between GCB- or non-GCB DLBCL (78.4% vs. 75.5%, P=0.73) or by Bcl-2 expression (+:72.5% vs. -:81.3%, P=0.35]. After a median follow up period of 74 months, significant differences were observed between Bcl-2 positive or negative and GCB or non-GCB DLBCL. GCB-DLBCL had a longer 5-yr PFS and 5-yr OS than non-GCB DLBCL (58.5% vs. 37%, P=0.026; 81.1% vs. 53.7% and, P=0.002; respectively). By itself, Bcl2 over-expression, had a negative impact in PFS (P=0.002) and OS (P=0.001) in R+CHOP/R+DA-EPOCH treated de novo DLBCL. The combined prognostic value of the Han's algorithm and Bcl-2 expression was also evaluated. Bcl-2 expression in the context of both GBC and non-GCB subtypes remains an unfavorable prognostic indicator for overall survival, with a more pronounced influence in the GCB-DLBCL phenotype (See table). Our data supports the predictive value of the Han's algorithm and Bcl-2 expression in DLBCL patients undergoing front-line chemo-immunotherapy. Bcl-2 expression is associated with a poor prognosis in GCB and non-GCB DLBCL. It is possible that intrinsic biological pathways involved in lymphomagenesis and/or “resistance” of these subtypes of DLBCL may play a role in their responsiveness to rituximab-based therapies and could be influenced by the net balance between pro- and anti-apoptotic proteins. Attempts to further delineate the biological heterogeneity of DLBCL may help identify subgroups of patients at high risk of resistance to chemo-immunotherapy and lead to the development of new therapeutic strategies. In conclusion, our data analysis confirms that the DLBCL immunophenotypes based on cell of origin and Bcl-2 status continues to have predictive significance on clinical outcomes in DLBCL in the rituximab era. Disclosures:No relevant conflicts of interest to declare.

Specific expression of miR-17-5p and miR-127 in testicular and central nervous system diffuse large B-cell lymphoma

Recent studies have shown that certain non-coding short RNAs, called miRNAs, play an important role in diffuse large B-cell lymphomas. Patients with diffuse large B-cell lymphoma have great diversity in both clinical characteristics, site of presentation and outcome. The aim of our study is to validate the differential expression in germinal center and non-germinal center diffuse large B-cell lymphoma,s and to study to the extent to which the primary site of differentiation is associated with the miRNA expression profile. We studied 50 cases of de novo diffuse large B-cell lymphoma for the expression of 15 miRNAs (miR-15a, miR-15b, miR-16, miR-17-3p, miR-17-5p, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-92, miR-127, miR-155, miR-181a and miR-221). Apart from 19 nodal cases without extranodal dissemination (stages I and II), we selected two groups with unambiguous stages I and II extranodal presentation; 9 cases of primary central nervous system, 11 cases of primary testicular and 11 cases of other primary extranodal diffuse large B-cell lymphomas. All cases were analyzed with qRT-PCR. In situ hybridization for the most differentially expressed miRNAs was performed to show miRNA expression in tumor cells, but not in background cells. MiR-21 and miR-19b showed the highest expression levels. No significant differences were seen between germinal center and non-germinal center diffuse large B-cell lymphomas in either the total or the nodal group for any of the 15 miRNAs. Two miRNAs showed significant differences in expression levels for diffuse large B-cell lymphoma subgroups according to the site of presentation. MiR-17-5p showed a significant higher expression level in the central nervous system compared with testicular and nodal diffuse large B-cell lymphomas (P<0.05). MiR-127 levels were significantly higher in testicular than in central nervous system and in nodal diffuse large B-cell lymphomas (P<0.05). We conclude that the location of diffuse large B-cell lymphoma is an important factor in determining the differential expression of miRNAs.

Correlation of high numbers of intratumoral FOXP3+ regulatory T cells with improved survival in germinal center-like diffuse large B-cell lymphoma, follicular lymphoma and classical Hodgkin's lymphoma

The tumor microenvironment is important for the behavior of cancer. We assessed the distribution and biological significance of FOXP3(+) regulatory T-cells (Treg) in lymphomas. The absolute number of intratumoral FOXP3(+) cells was immunohistochemically studied on lymphoma tissue microarrays from 1019 cases of different types of lymphomas and correlated to phenotypic and clinical parameters in uni- and multivariate models. Receiver operating characteristic -curves were used to determine prognostic cut-off values of FOXP3(+) cell density. Of the 1019 cases, 926 (91%) were evaluable. FOXP3(+) cell density varied between the lymphoma entities, and was highest in follicular lymphoma. An increased number of tumor-infiltrating FOXP3(+) cells over the receiver operating characteristic-determined cut-offs positively influenced both disease-specific and failure-free survival in follicular lymphoma (p=0.053) and disease-specific survival in germinal center-like diffuse large B-cell lymphoma (p=0.051) and overall and failure-free survival in classical Hodgkin's lymphoma (p=0.004), but had a negative prognostic effect in non-germinal center diffuse large B-cell lymphoma (p=0.059). In a Cox regression model, considering stage and age, the amount of FOXP3(+) cells was of independent prognostic significance for failure-free survival in classical Hodgkin's lymphoma and of borderline significance for overall survival in classical Hodgkin's lymphoma and disease-specific survival in germinal center-like and non-germinal center diffuse large B-cell lymphoma. FOXP3(+) cells represent important lymphoma/host microenvironment modulators. Assessment of FOXP3(+) cell density can contribute to the prediction of outcome in diffuse large B-cell lymphoma, fallicular lymphoma and classical Hodgkin's lymphoma.

High throughput tissue microarray analysis of FHIT expression in diffuse large cell B-cell lymphoma from Saudi Arabia

Recent studies have suggested a potential prognostic role of alterations of the fragile histidine triad (FHIT) gene in diffuse large B-cell lymphoma. To evaluate possible mechanisms of FHIT inactivation and to further clarify its potential prognostic relevance, we analyzed a set of 114 diffuse large B-cell lymphoma with clinical follow-up information. Tissue microarrays were analyzed by immunohistochemistry for protein expression, and corresponding DNA samples were analyzed for FHIT promotor hypermethlyation. Reduced or absent FHIT expression was found in 75 of 114 diffuse large B-cell lymphoma (66%), but was unrelated to clinical tumor stage or patient prognosis. FHIT promotor hypermethylation was observed in 29 of 93 (23%) interpretable diffuse large B-cell lymphoma. Hypermethylation was not significantly correlated to protein expression loss, which could be explained by competing mechanisms for FHIT inactivation in a substantial fraction of non FHIT hypermethylated diffuse large B-cell lymphoma. Hypermethylation was significantly associated with poor prognosis of diffuse large B-cell lymphoma patients and predominantly seen in nongerminal center diffuse large B-cell lymphoma (27%), but less frequent (13%) in germinal center diffuse large B-cell lymphoma. In summary, these data suggest that promotor hypermethylation is responsible for reduced FHIT expression in a substantial subset of diffuse large B-cell lymphoma, which is primarily composed of nongerminal center subtype with poor patient prognosis.