Application of CD54 in diagnosing bone marrow involvement by using flow cytometry in patients with diffuse large B-cell lymphoma.

BACKGROUND: Flow cytometry plays a key role in detecting bone marrow (BM) involvement in patients with diffuse large B-cell lymphoma (DLBCL). To improve its detection sensitivity, we need to explore novel markers. In this study, we detected the expression CD54 on lymphoma cells in BM specimens from DLBCL patients and clarified its diagnostic significance in BM involvement by DLBCL. METHODS: We collected BM specimens from 76 patients with DLBCL (germinal center B-cell (GCB) = 25, non-GCB = 51) and 10 control patients without lymphoma. We detected and compared the expression of CD54 on lymphoma cells and normal mature B cells by using 10-color panels. RESULTS: Normal plasma cells expressed a higher level of CD54 as compared with hematogones (p < 0.05) and normal mature B cells (p < 0.05). Among 76 patients, 23 of them (GCB = 12, non-GCB = 11) had BM involvement. Lymphoma B cells from 12 cases (GBC = 4, non-GCB = 8) expressed a higher level of CD54 compared to normal mature B cells (p < 0.05). Additionally, lymphoma cells of the non-GCB subtype frequently expressed a higher level of CD54 in comparison to the GCB subtype (p < 0.05). And the high expression of CD54 was not related to plasmacytoid differentiation. CONCLUSION: Aberrant expression of CD54 on lymphoma cells is frequently seen in patients' BM specimens involved by DLBCL, especially in the non-GCB subtype. CD54 could be used as a new marker to gate on lymphoma cells and improve the detection sensitivity of BM involvement in patients with DLBCL.

Insertion of atypical glycans into the tumor antigen-binding site identifies DLBCLs with distinct origin and behavior.

Glycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma-associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell-like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell-like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN-expressing M2-polarized macrophages.

c-MYC and p53 expression highlight starry-sky pattern as a favourable prognostic feature in R-CHOP-treated diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a clinically heterogeneous entity, in which the first-line treatment currently consists of an immuno-chemotherapy regimen (R-CHOP). However, around 30% of patients will not respond or will relapse. Overexpression of c-MYC or p53 is frequently found in DLBCL, but an association with prognosis remains controversial, as for other biomarkers previously linked with DLBCL aggressivity (CD5, CD23, or BCL2). The aim of this study was to explore the expression of these biomarkers and their correlation with outcome, clinical, or pathological features in a DLBCL cohort. Immunohistochemical (c-MYC, p53, BCL2, CD5, and CD23), morphological ('starry-sky' pattern [SSP]), targeted gene panel sequencing by next-generation sequencing (NGS), and fluorescence in situ hybridisation analyses were performed on tissue microarray blocks for a retrospective cohort of 94 R-CHOP-treated de novo DLBCL. In univariate analyses, p53 overexpression (p53high ) was associated with unfavourable outcome (p = 0.04) and with c-MYC overexpression (p = 0.01), whereas c-MYC overexpression was linked with an SSP (p = 0.004), but only tended towards an inferior prognosis (p = 0.06). Presence of a starry-sky morphology was found to be correlated with better survival in p53high DLBCL (p = 0.03) and/or c-MYC-positive DLBCL (p = 0.002). Furthermore, NGS data revealed that these three variables were associated with somatic mutations (PIM1, TNFRSF14, FOXO1, and B2M) involved in B-cell proliferation, survival, metabolism, and immune signalling. Taken together, these results show that the SSP pattern seems to be a protective factor in high-risk DLBCL subgroups and highlight cell death as a built-in failsafe mechanism to control tumour growth.

The Positivity of Phosphorylated STAT3 Is a Novel Marker for Favorable Prognosis in Germinal Center B-Cell Type of Diffuse Large B-Cell Lymphoma.

On the basis of immunohistochemistry, diffuse large B-cell lymphoma (DLBCL) is categorized as a germinal center B-cell (GCB) or non-GCB subtype. Recent integrated genomic analyses have highlighted the importance of the JAK-STAT3 pathway in the molecular pathogenesis of DLBCL. However, its relevance to clinical outcomes remains controversial. Therefore, we evaluated the extent of the nuclear expression of phosphorylated STAT3 (pSTAT3), a surrogate marker of signal transducer and activator of transcription 3 (STAT3) activation, by immunohistochemistry. We also analyzed the potential relationship between pSTAT3 positivity (defined as ≥40% positive neoplastic cells) and clinicopathologic characteristics in 294 patients with DLBCL. pSTAT3 was detected in 122 patients (42%), with a higher rate in the non-GCB subtype than in the GCB subtype (57% vs. 28%, P<0.001). Factors potentially activating STAT3, MYD88L265P, and Epstein-Barr virus-encoded small RNA were identified in the pSTAT3-positive non-GCB subtype, whereas the pSTAT3-positive GCB subtype often showed STAT3 mutations and lacked EZH2 mutations and the rearrangements of BCL2 and MYC. Multivariate analyses revealed that the pSTAT3-positive GCB subtype showed a favorable prognosis (HR: 0.17; 95% confidence interval, 0.04-0.7; P=0.014). These findings suggest that pSTAT3 positivity may have a unique impact on the clinicopathologic characteristics of DLBCL, making it a promising novel marker for the favorable prognosis of patients with the GCB subtype.

Lack of expression of LMO2 clone SP51 identifies MYC rearrangements in aggressive large B-cell lymphomas.

MYC rearrangements (MYC-R) confer unfavorable prognosis to large B-cell lymphomas (LBCL). Because of the low incidence of such genetic alteration, surrogates to screen MYC-R may be useful in daily practice. Previous studies suggested that clone 1A9-1 of LMO2 loss may be a good predictor for the presence of MYC-R in LBCL. The present study examines the utility of LMO2 clone SP51. For this purpose, we have analyzed 20 Burkitt lymphomas and 325 LBCL. Among them, 245 cases were studied prospectively using whole tissue sections, and 100 retrospectively by tissue microarrays. The cohort of CD10-positive prospective cases achieved the best results. Lack of LMO2 SP51 expression predicted the presence of MYC-R with high specificity, accuracy, positive and negative predictive value (PPV/NPV), and positive and negative likelihood ratios (PLR/NLR). Compared with MYC protein expression, LMO2 SP51 obtained significantly higher specificity, accuracy, PPV, and PLR (94%, 91%, 85%, and 14.33 vs 73%, 77%, 56%, and 3.26, respectively), and similar NPV and NLR (92% and 0.22 vs 95% and 0.12). Compared with LMO2 clone 1A9-1, the sensitivity of LMO2 SP51 was lower (79% vs 89%). We conclude that LMO2 SP51 may be a useful marker to screen MYC-R in CD10-positive LBCL.

Artificial intelligence to detect MYC translocation in slides of diffuse large B-cell lymphoma.

In patients with suspected lymphoma, the tissue biopsy provides lymphoma confirmation, classification, and prognostic factors, including genetic changes. We developed a deep learning algorithm to detect MYC rearrangement in scanned histological slides of diffuse large B-cell lymphoma. The H&E-stained slides of 287 cases from 11 hospitals were used for training and evaluation. The overall sensitivity to detect MYC rearrangement was 0.93 and the specificity 0.52, showing that prediction of MYC translocation based on morphology alone was possible in 93% of MYC-rearranged cases. This would allow a simple and fast prescreening, saving approximately 34% of genetic tests with the current algorithm.

Fibrin-associate diffuse large B-Cell lymphoma arising in a left atrial myxoma: A case report and literature review✰,✰✰.

We report a 60-year-old male with fibrin-associated diffuse large B-cell lymphoma (fa-DLBCL) in left atrial myxoma. Echocardiography showed a mass (63 mm × 33 mm) in the left atrium. Histological inspection indicated fa-DLBCL on the surface of atrial myxoma incidentally, together with extensive fibrinous like exudation on myxoma surface. Malignant cells were localized in solid sheets and nests at the peripheral area of the fibrinous exudation which were positive for B-lineage markers (CD20+, CD79a+, PAX-5+) and in situ hybridization of EBV-encoded RNA (EBER). PCR amplification showed clonal rearrangement of immunoglobulin heavy chain (IgH) genes. The patient was still alive with no recurrence in the 35-month follow-up after surgery. We also did a detailed clinicopathological analysis and literature review, which indicated that fa-DLBCL was a heterogeneous entity.

Clinical significance of 'double-hit' and 'double-expression' lymphomas.

BACKGROUND: 'Double-hit' lymphoma (DHL) and 'double-expression' lymphoma (DEL) involving gene rearrangement and protein expression of MYC and BCL2/BCL6 have recently become the most commonly used terms to describe the poor prognostic types of diffuse large B-cell lymphoma (DLBCL). However, the clinical and pathological spectra of these rare entities have not been well defined. The aim of this study was to determine the frequency of DHL and DEL in DLBCL and their prognostic impacts in the era of cyclophosphamide, doxorubicin, vincristine and prednisone plus rituximab therapy. METHODS: The data and tissues from 98 patients diagnosed with DLBCL were used in this study. Formalin-fixed and paraffin-embedded tissues were constructed for immunohistochemistry (IHC) and interphase fluorescene in situ hybridisation (FISH) analysis for MYC, BCL6 and BCL2 rearrangements. RESULTS: There were 14 cases (14.29%, 14/98) and 34 cases (34.70%, 34/98) of lymphomas with the DHL and DEL of MYC and BCL2/BCL6, respectively. DLBCL patients with MYC/BCL2 rearrangements more frequently had bone marrow involvement (p=0.002), and their tumours were commonly of the germinal centre B cell (GCB) subtype. Patients with MYC+/BCL2 +coexpression were more often assessed using the International Prognostic Index (IPI), (Performance Status) PS score and bone marrow involvement. Patients with MYC+/BCL6 +coexpression were associated with their IPI values, Eastern Cooperative Oncology Group scores and occurrence of B symptoms. Their lymphomas were more often of the non-GCB subtype (p=0.010). Multivariate analysis showed that IPI, bone marrow involvement, rearrangements of BCL2, BCL6 and MYC, expression concurrent with rearrangements and coexpression were all significantly associated with overall survival and progression-free survival, with the exception of MYC+/BCL6 +coexpression. CONCLUSIONS: MYC/BCL2 DHL, MYC/BCL6 DHL and MYC/BCL2 DEL are an aggressive B cell lymphoma and patients have a poor prognosis. IPI and bone marrow involvement were independent prognostic factors for DHL and DEL. BCL2, BCL6 and MYC rearrangements translocation, expression concurrent rearrangements and coexpression were were independent prognostic factors for survival. POTENTIAL IMPLICATIONS: The present study analysed the genomic alterations and protein expression levels of MYC, BCL2 and BCL6 using FISH and IHC in Chinese patients with DLBCL.We assessed the frequency, clinicopathological features and the prognostic impacts of DHL and DEL in a cohort of de novo DLBCL patients and evaluated the role of each genetic translocation separately and in combination in order to provide reliable conclusions and practical recommendations for diagnostic workups and prognostic predictions.

CD20-negative primary middle ear diffuse large B-cell lymphoma coexpressing MYC and BCL-2 secondary to acute lymphoblastic leukemia: A case report.

RATIONALE: Second diffuse large B-cell lymphoma (DLBCL) after treatment of acute lymphoblastic leukemia (ALL) is uncommon. To our knowledge, primary middle ear DLBCL which presents CD20-negative and coexpression of MYC and BCL-2 has not been reported yet. PATIENT CONCERNS: A 20-year-old Chinese man complained fever and weakness for 2 months. Subsequently bone marrow morphology and flow cytometry immunophenotype suggested ALL. Administrated with 9 cycles of multiagent combined chemotherapy, he felt right ear progressive hearing loss, otalgia, aural fullness. Otoendoscopic examination revealed a pitchy mass obstructing the right external auditory canal. Then the mass resection was performed for biopsy and immunohistochemistry examination. DIAGNOSIS: The mass was diagnosed as DLBCL which was negative for CD20 and double expression of MYC and BCL-2. INTERVENTIONS: Chemotherapy. OUTCOMES: The patient eventually gave up and died of severe infection. LESSONS: Although intensive chemotherapy has markedly improved the survival of ALL, more and more secondary cancers have been reported. In addition, primary middle ear lymphoma is much rare; hence, it is easy to be misdiagnosed. Furthermore, DLBCL with negative CD20 and double expression of MYC and BCL-2 is aggressive, which is characterized by chemotherapy resistance and inferior survival rates. We discuss this case aiming at raising awareness of tumors secondary to ALL and exploring the appropriate treatment options for the rare DLBCL.

Nuclear factor erythroid 2-related factors 1 and 2 are able to define the worst prognosis group among high-risk diffuse large B cell lymphomas treated with R-CHOEP.

AIMS: Oxidative stress markers and antioxidant enzymes have previously been shown to have prognostic value and associate with adverse outcome in patients with diffuse large B cell lymphoma (DLBCL). Nuclear factor erythroid 2-related factor 1 (Nrf1) and factor 2 (Nrf2) are among the principal inducers of antioxidant enzyme production. Kelch ECH associating protein 1 (Keap1) is a negative regulator of Nrf2, and BTB (BR-C, ttk and bab) domain and CNC homolog 1 (Bach1) represses the function of both factors. Their significance in DLBCL prognosis is unknown. METHODS: Diagnostic biopsy samples of 76 patients with high-risk DLBCL were retrospectively stained with immunohistochemistry for Nrf1, Nrf2, Keap1 and Bach1, and correlated with clinical data and outcome. RESULTS: Nuclear Nrf2 and nuclear Bach1 expression were associated with adverse clinical features (anaemia, advanced stage, high IPI, high risk of neutropaenic infections), whereas cytoplasmic Nrf1 and Nrf2 were associated with favourable clinical presentation (normal haemoglobin level, no B symptoms, limited stage). None of the evaluated factors could predict survival alone. However, when two of the following parameters were combined: high nuclear score of Nrf2, low nuclear score of Nrf1, high cytoplasmic score of Nrf1 and low cytoplasmic score of Keap1 were associated with significantly worse overall survival. CONCLUSIONS: Nrf1 and Nrf2 are relevant in disease presentation and overall survival in high-risk DLBCL. Low nuclear expression of Nrf1, high cytoplasmic expression of Nrf1, high nuclear expression of Nrf2 and low cytoplasmic expression of Keap1 are associated with adverse outcome in this patient group.

Complete Remission and Long-term Survival of a Patient with a Diffuse Large B-cell Lymphoma Under Viscum album Extracts After Resistance to R-CHOP: A Case Report.

BACKGROUND: A nodular lymphocyte predominant Hodgkin's lymphoma (NLPHL) is a lymphoproliferative neoplasm with a fair prognosis, but the possibility of a malignant transformation into a diffuse large B-cell lymphoma (DLBCL) is high. DLBCL progresses aggressively. Introduction of rituximab into therapy had led to improved outcomes. The use of Viscum album extracts (VAE) in cancer is established, but their application in lymphoma are rare. CASE PRESENTATION: A 65-year-old patient was diagnosed with DLBCL stage IIa with splenomegaly, transformed from a NLPHL, after a 30-year history of repeatedly enlarged inguinal lymph nodes. The patient initially rejected chemotherapy. After his tumor pain increased, he accepted the consecutive therapies bendamustine plus vincristine plus prednisolone, trofosfamide, and rituximab plus cyclophosphamide plus hydroxydaunorubicin plus vincristine plus prednisone (R-CHOP), inducing only a slight regression of the splenic lesions. VAE was additionally applied to R-CHOP. Five months after termination of chemotherapy - under continued VAE therapy in increasing dosage- regression of paraaortal lesions was found. The patient fully recovered under continuous VAE application and is in ongoing complete remission and in a good state of health 17 years after the initial diagnosis. CONCLUSION: As complete remission of lymphoproliferative disorders after VAE treatment has been previously reported, further investigations of VAE in lymphoma seem highly worthwhile.

Simultaneous occurrence of splenic diffuse large B cell lymphoma and gastrointestinal stromal tumor in the stomach: a case report.

BACKGROUND: Although the primary malignant spleen tumor is relatively rare, lymphoma is the most common splenic malignancy. It can have quite different clinical manifestations that usually lead to relatively poor outcomes, and thus early and accurate diagnosis are of utmost importance. CASE PRESENTATION: The present study reports a case of a 67-year-old female with high fever, abnormal spleen (diagnosed by PET/CT) and no obvious lymph node enlargement. After being subjected to splenectomy, the patient was diagnosed with splenic diffuse large B cell lymphoma coexisting with gastrointestinal stromal tumor in the stomach. CONCLUSIONS: To our knowledge, splenic lymphoma accompanied by gastrointestinal stromal tumor in the stomach is rarely reported. This case report discusses the diagnosis and case management of a patient referring to the existing literature.

Round-robin test for the cell-of-origin classification of diffuse large B-cell lymphoma-a feasibility study using full slide staining.

Diffuse large B-cell lymphoma (DLBCL) is subdivided by gene expression analysis (GEP) into two molecular subtypes named germinal center B-cell-like (GCB) and activated B-cell-like (ABC) after their putative cell-of-origin (COO). Determination of the COO is considered mandatory in any new-diagnosed DLBCL, not otherwise specified according to the updated WHO classification. Despite the fact that pathologists are free to choose the method for COO classification, immunohistochemical (IHC) assays are most widely used. However, to the best of our knowledge, no round-robin test to evaluate the interlaboratory variability has been published so far. Eight hematopathology laboratories participated in an interlaboratory test for COO classification of 10 DLBCL tumors using the IHC classifier comprising the expression of CD10, BCL6, and MUM1 (so-called Hans classifier). The results were compared with GEP for COO signature and, in a subset, with results obtained by image analysis. In 7/10 cases (70%), at least seven laboratories assigned a given case to the same COO subtype (one center assessed one sample as not analyzable), which was in agreement with the COO subtype determined by GEP. The results in 3/10 cases (30%) revealed discrepancies between centers and/or between IHC and GEP subtype. Whereas the CD10 staining results were highly reproducible, staining for MUM1 was inconsistent in 50% and for BCL6 in 40% of cases. Image analysis of 16 slides stained for BCL6 (N = 8) and MUM1 (N = 8) of the two cases with the highest disagreement in COO classification were in line with the score of the pathologists in 14/16 stainings analyzed (87.5%). This study describes the first round-robin test for COO subtyping in DLBCL using IHC and demonstrates that COO classification using the Hans classifier yields consistent results among experienced hematopathologists, even when variable staining protocols are used. Data from this small feasibility study need to be validated in larger cohorts.

CD30 expression and its correlation with MYC and BCL2 in de novo diffuse large B-cell lymphoma.

AIM: CD30+ diffuse large B-cell lymphoma (DLBCL) has emerged as a new immunophenotypic variant of de novo DLBCLs. However, the prevalence of CD30 positivity is variable according to different studies, and the prognostic significance of CD30 is also controversial. This study aimed to investigate the positive expression rate and prognostic impact of CD30 in de novo DLBCLs and try to find the correlated influences. METHODS: A total of 241 patients with de novo DLBCL in east China from 2008 to 2015 were included to investigate the prevalence, clinicopathological features and outcomes of CD30+ de novo DLBCLs. Immunohistochemical evaluation for CD10, CD30, BCL2, BCL6, MUM1/IRF4, MYC and Ki67, and fluorescence in situ hybridisation for MYC and BCL2 gene alterations were performed. RESULTS: Using a >0% threshold, CD30 expression was detected in approximately 10% patient with de novo DLBCL. These predominately presented with centroblastic or anaplastic morphological patterns, less frequently showing immunoblastic morphology or 'starry sky' pattern, mutually exclusive with MYC gene rearrangement, and negatively associated with BCL2 protein expression. CD30 expression was associated with a favourable prognosis of patients' outcomes. However, the multivariate analysis revealed that it was not an independent prognostic factor in de novo DLBCLs. The impact of CD30 might be influenced by the international prognostic index and the expression of MYC and BCL2 proteins. CONCLUSION: CD30+ DLBCL may be a subset of de novo DLBCLs with characteristic clinicopathological features, but the prognostic role of CD30 is limited.

Intralymphatic Spread is a Rare Finding Associated With Poor Prognosis in Diffuse Large B-Cell Lymphoma With Extranodal Involvements.

Intralymphatic spread is common in solid cancers, but has been rarely studied in lymphomas. Review of 635 extranodal specimens from 475 diffuse large B-cell lymphoma (DLBCL) patients revealed intralymphatic spread in 10 surgical resection specimens from 10 patients including 9 de novo DLBCLs and 1 Richter transformation. The prevalence in de novo DLBCL with extranodal involvements was 1.65%. The most common involved site of intralymphatic spread was the gastrointestinal tract, followed by the female genital tract and breasts. Lymphatic vessels, lined by D2-40-positive endothelial cells, were expanded by lymphoma cells, reminiscent of intravascular lymphoma or tumor emboli. None of the involved lymphatic vessels were located in the mucosa. Patients with intralymphatic spread had a trend of lower overall response rate and a trend of higher progressive disease than those without intralymphatic spread. Compared with patients without intralymphatic spread, those patients with intralymphatic spread had a shorter median overall survival (14.3 vs. 96.2 mo; P=0.004) and a shorter median progression-free survival (11.2 vs. 64.2 mo; P=0.01), respectively. Multivariate analyses showed that intralymphatic spread was an independent poor prognostic factor for overall survival (hazard ratio, 3.029; 95% confidence interval, 1.315-6.978; P=0.009), irrespective of the National Comprehensive Cancer Network-International Prognostic Index, B symptoms, and serum albumin levels. Among patients who underwent surgical resection, intralymphatic spread was still an independent prognostic factor. In conclusion, our study demonstrated extranodal intralymphatic spread in DLBCL. Inspiringly, this rare morphologic finding may serve as a new negative prognostic indicator in DLBCL with extranodal involvements.

Clinical Impact of the Cell-of-Origin Classification and the MYC/ BCL2 Dual Expresser Status in Diffuse Large B-Cell Lymphoma Treated Within Prospective Clinical Trials of the German High-Grade Non-Hodgkin's Lymphoma Study Group.

Purpose To explore the prognostic impact and interdependence of the cell-of-origin (COO) classification, dual expression (DE) of MYC and BCL2 proteins, and MYC, BCL2, and BCL6 translocations in two prospectively randomized clinical trials of patients with diffuse large B-cell lymphoma (DLBCL). Patients and Methods Overall, 452 formalin-fixed paraffin-embedded samples from two prospective, randomized DLBCL trials (RICOVER-60, prospective, randomized study for patients > 60 years, all IPI groups; and R-MegaCHOEP, prospective, randomized study for patients ≤ 60 years with age-adjusted IPI 2,3) of the German High-Grade Non-Hodgkin Lymphoma Study Group were analyzed with the Lymph2Cx assay for COO classification, with immunohistochemistry for MYC and BCL2, and with fluorescent in situ hybridization for MYC, BCL2, and BCL6 rearrangements. Results COO classification was successful in 414 of 452 samples. No significant differences with respect to COO (activated B-cell [ABC]-like DLBCL v germinal center B-cell [GCB]-like DLBCL) were observed in event-free survival, progression-free survival, and overall survival in patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in the RICOVER-60 trial. Also, no differences with respect to COO were observed in multivariable analyses adjusted for International Prognostic Index factors in event-free survival (hazard ratio [HR] of ABC-like disease v GCB-like disease, 1.0; 95% CI, 0.6 to 1.6; P = .93), progression-free survival (HR, 1.1; 95% CI, 0.6 to 1.8; P = .82), and overall survival (HR, 1.0; 95% CI, 0.6 to 1.8; P = .96). Similar results were observed in the R-MegaCHOEP trial. In patients treated with R-CHOP, DE status was associated with significantly inferior survival compared with nonDE within the GCB, but not within the ABC subgroup. DE status was associated with significantly inferior outcome compared with patients with ABC-like DLBCL without DE (5-year PFS rate, 39% [95% CI,19% to 59%] v 68% [95% CI, 52% to 85%]; P = .03) and compared with patients with GCB-like DLBCL without DE. When data from patients with nonDE were analyzed separately, the outcome of patients in the ABC subgroup was inferior to that of patients in the GCB subgroup (5-year PFS rate, 68% [95% CI, 52% to 85%] v 85% [95% CI, 74% to 96%]; P = .04). Conclusion COO profiling in two prospective randomized DLBCL trials failed to identify prognostic subgroups, whereas dual expression of MYC and BCL2 was predictive of poor survival. Evaluation of prognostic or predictive biomarkers in the management of DLBCL, such as the COO, within prospective clinical trials will be important in the future.