Heike Horn

MYC status in concert with BCL2 and BCL6 expression predicts outcome in diffuse large B-cell lymphoma

MYC rearrangements occur in 5% to 10% of diffuse large B-cell lymphomas (DLBCL) and confer an increased risk to cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisone (CHOP) and rituximab (R)-CHOP treated patients. We investigated the prognostic relevance of MYC-, BCL2- and BCL6-rearrangements and protein expression in a prospective randomized trial. Paraffin-embedded tumor samples from 442 de novo DLBCL treated within the RICOVER study of the German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL) were investigated using immunohistochemistry and fluorescence in situ hybridization (FISH) to detect protein expression and breaks of MYC, BCL2, and BCL6. Rearrangements of MYC, BCL2, and BCL6 were detected in 8.8%, 13.5%, and 28.7%, respectively. Protein overexpression of MYC (>40%) was encountered in 31.8% of tumors; 79.6% and 82.8% of tumors expressed BCL2 and BCL6, respectively. MYC translocations, MYChigh, BCL2high, and BCL6low protein expressions were associated with inferior survival. In multivariate Cox regression modeling, protein expression patterns of MYC, BCL2 and BCL6, and MYC rearrangements were predictive of outcome and provided prognostic information independent of the International Prognostic Index (IPI) for overall survival and event-free survival. A combined immunohistochemical or FISH/immunohistochemical score predicts outcome in DLBCL patients independent of the IPI and identifies a subset of 15% of patients with dismal prognosis in the high-risk IPI group following treatment with R-CHOP. Registered at http://www.cancer.gov/clinicaltrials: RICOVER trial of the DSHNHL is NCT 00052936.

Immunoblastic morphology but not the immunohistochemical GCB/nonGCB classifier predicts outcome in diffuse large B-cell lymphoma in the RICOVER-60 trial of the DSHNHL

The survival of diffuse large B-cell lymphoma patients varies considerably, reflecting the molecular diversity of tumors. In view of the controversy whether cytologic features, immunohistochemical markers or gene expression signatures may capture this molecular diversity, we investigated which features provide prognostic information in a prospective trial in the R-CHOP treatment era. Within the cohort of DLBCLs patients treated in the RICOVER-60 trial of the German High-Grade Lymphoma Study Group (DSHNHL), we tested the prognostic impact of IB morphology in 949 patients. The expression of immunohistochemical markers CD5, CD10, BCL2, BCL6, human leukocyte antigen (HLA)-DR, interferon regulatory factor-4/multiple myeloma-1 (IRF4/MUM1), and Ki-67 was assessed in 506 patients. Expression of the immunohistochemical markers tested was of modest, if any, prognostic relevance. Moreover, the Hans algorithm using the expression patterns of CD10, BCL6, and interferon regulatory factor-4/multiple myeloma-1 failed to show prognostic significance in the entire cohort as well as in patient subgroups. IB morphology, however, emerged as a robust, significantly adverse prognostic factor in multivariate analysis, and its diagnosis showed a good reproducibility among expert hematopathologists. We conclude, therefore, that IB morphology in DLBCL is likely to capture some of the adverse molecular alterations that are currently not detectable in a routine diagnostic setting, and that its recognition has significant prognostic power.

Integration of gene mutations in risk prognostication for patients receiving first-line immunochemotherapy for follicular lymphoma: a retrospective analysis of a prospective clinical trial and validation in a population-based registry

BACKGROUND Follicular lymphoma is a clinically and genetically heterogeneous disease, but the prognostic value of somatic mutations has not been systematically assessed. We aimed to improve risk stratification of patients receiving first-line immunochemotherapy by integrating gene mutations into a prognostic model. METHODS We did DNA deep sequencing to retrospectively analyse the mutation status of 74 genes in 151 follicular lymphoma biopsy specimens that were obtained from patients within 1 year before beginning immunochemotherapy consisting of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). These patients were recruited between May 4, 2000, and Oct 20, 2010, as part of a phase 3 trial (GLSG2000). Eligible patients had symptomatic, advanced stage follicular lymphoma and were previously untreated. The primary endpoints were failure-free survival (defined as less than a partial remission at the end of induction, relapse, progression, or death) and overall survival calculated from date of treatment initiation. Median follow-up was 7·7 years (IQR 5·5-9·3). Mutations and clinical factors were incorporated into a risk model for failure-free survival using multivariable L1-penalised Cox regression. We validated the risk model in an independent population-based cohort of 107 patients with symptomatic follicular lymphoma considered ineligible for curative irradiation. Pretreatment biopsies were taken between Feb 24, 2004, and Nov 24, 2009, within 1 year before beginning first-line immunochemotherapy consisting of rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP). Median follow-up was 6·7 years (IQR 5·7-7·6). FINDINGS We established a clinicogenetic risk model (termed m7-FLIPI) that included the mutation status of seven genes (EZH2, ARID1A, MEF2B, EP300, FOXO1, CREBBP, and CARD11), the Follicular Lymphoma International Prognostic Index (FLIPI), and Eastern Cooperative Oncology Group (ECOG) performance status. In the training cohort, m7-FLIPI defined a high-risk group (28%, 43/151) with 5-year failure-free survival of 38·29% (95% CI 25·31-57·95) versus 77·21% (95% CI 69·21-86·14) for the low-risk group (hazard ratio [HR] 4·14, 95% CI 2·47-6·93; p<0·0001; bootstrap-corrected HR 2·02), and outperformed a prognostic model of only gene mutations (HR 3·76, 95% CI 2·10-6·74; p<0·0001; bootstrap-corrected HR 1·57). The positive predictive value and negative predictive value for 5-year failure-free survival were 64% and 78%, respectively, with a C-index of 0·80 (95% CI 0·71-0·89). In the validation cohort, m7-FLIPI again defined a high-risk group (22%, 24/107) with 5-year failure-free survival of 25·00% (95% CI 12·50-49·99) versus 68·24% (58·84-79·15) in the low-risk group (HR 3·58, 95% CI 2·00-6·42; p<0.0001). The positive predictive value for 5-year failure-free survival was 72% and 68% for negative predictive value, with a C-index of 0·79 (95% CI 0·69-0·89). In the validation cohort, risk stratification by m7-FLIPI outperformed FLIPI alone (HR 2·18, 95% CI 1·21-3·92), and FLIPI combined with ECOG performance status (HR 2·03, 95% CI 1·12-3·67). INTERPRETATION Integration of the mutational status of seven genes with clinical risk factors improves prognostication for patients with follicular lymphoma receiving first-line immunochemotherapy and is a promising approach to identify the subset at highest risk of treatment failure. FUNDING Deutsche Krebshilfe, Terry Fox Research Institute.

Follicular lymphoma grade 3b is a distinct neoplasm according to cytogenetic and immunohistochemical profiles

Background According to the current World Health Organization Classification of Lymphoid Tumours, follicular lymphoma is subclassified into three grades according to the number of centroblasts. Follicular lymphoma grade 3 can be further divided into types A and B. Almost all available genetic data on grade 3B follicular lymphomas have been generated from tumors with an additional diffuse large B-cell lymphoma component. The purely follicular type of follicular lymphoma grade 3B is a rare neoplasm. Design and Methods We performed a detailed immunohistochemical (CD10, IRF4/MUM1, BCL2, Ig light chains) and genetic (translocations of BCL2, BCL6, MYC, IRF4) characterization of the largest series of purely follicular cases of grade 3B follicular lymphoma available to date, comprising 23 tumor samples. We also included 25 typical grade 1 or 2 follicular lymphomas, 9 follicular lymphomas with large centrocytes and/or high proliferation indices (FL/LCC), 12 cases of follicular lymphoma grade 3A, 16 cases of diffuse large B-cell lymphoma/follicular lymphoma grade 3B and 15 follicular lymphomas in which a straightforward distinction between grades 3A and 3B was not possible. Results Translocations affecting BCL2 and BCL6 genes are rare in grade 3B follicular lymphomas (2/23, 9% and 4/23, 17%) when compared with grade 1 or 2 follicular lymphomas (22/25, 88%, P<0.001 and 0/25, P<0.05), FL/LCC (7/9, 78%, P<0.001 and 2/9, 22%), grade 3A follicular lymphomas (7/12, 58%, P<0.01 and 2/12, 17%), unclassified grade 3 follicular lymphomas (6/15, 40% and 2/15, 13%) and diffuse large B-cell lymphoma/follicular lymphoma grade 3B (2/16, 13% and 8/16, 50%, P<0.05). MYC translocations were detected occasionally in FL/LCC, follicular lymphoma grade 3B, and diffuse large B-cell lymphoma/follicular lymphoma grade 3B (13%–22%), but not in grade 1, 2 or 3A follicular lymphomas (P<0.05 when compared with follicular lymphoma grade 3B). Both follicular lymphoma grade 3B and diffuse large B-cell lymphoma/follicular lymphoma grade 3B were enriched in samples with a CD10−IRF4/MUM1+ immunophenotype (8/19, 42% and 7/16, 44%), with the vast majority of them lacking BCL2 translocations. In contrast, 42/46 grade 1 or 2 follicular lymphomas, FL/LCC and grade 3A follicular lymphomas were CD10+ (91%) while 0/46 expressed IRF4/MUM1. None of the tumor samples tested with increased IRF4/MUM1-expression harbored a translocation of the IRF4 gene locus. Conclusions Our results show that grade 3B follicular lymphomas form a distinct category of follicular lymphomas with infrequent BCL2 and BCL6 translocations, while grades 1, 2 and 3A follicular lymphomas and FL/LCC display homogeneous features with frequent BCL2 translocations and a CD10+IRF4/MUM1− immunophenotype.

The different epidemiologic subtypes of Burkitt lymphoma share a homogenous micro RNA profile distinct from diffuse large B-cell lymphoma

Sporadic Burkitt lymphoma (sBL) can be delineated from diffuse large B-cell lymphoma (DLBCL) by a very homogeneous mRNA expression signature. However, it remained unclear whether all three BL variants—sBL, endemic BL (eBL) and human immunodeficiency virus-associated BL (HIV-BL)—represent a uniform biological entity despite their differences in geographical occurrence, association with immunodeficiency and/or incidence of Epstein–Barr virus (EBV) infection. To address this issue, we generated micro RNA (miRNA) profiles from 18 eBL, 31 sBL and 15 HIV-BL cases. In addition, we analyzed the miRNA expression of 86 DLBCL to determine whether miRNA profiles recapitulate the molecular differences between BL and DLBCL evidenced by mRNA profiling. A signature of 38 miRNAs containing MYC regulated and nuclear factor-kB pathway-associated miRNAs was obtained that differentiated BL from DLBCL. The miRNA profiles of sBL and eBL displayed only six differentially expressed miRNAs, whereas HIV and EBV infection had no impact on the miRNA profile of BL. In conclusion, miRNA profiling confirms that BL and DLBCL represent distinct lymphoma categories and demonstrates that the three BL variants are representatives of the same biological entity with only marginal miRNA expression differences between eBL and sBL.

Clinicogenetic risk models predict early progression of follicular lymphoma after first-line immunochemotherapy

Follicular lymphoma (FL) is a clinically and molecularly heterogeneous disease. Posttreatment surrogate end points, such as progression of disease within 24 months (POD24) are promising predictors for overall survival (OS) but are of limited clinical value, primarily because they cannot guide up-front treatment decisions. We used the clinical and molecular data from 2 independent cohorts of symptomatic patients in need of first-line immunochemotherapy (151 patients from a German Low-Grade Lymphoma Study Group [GLSG] trial and 107 patients from a population-based registry of the British Columbia Cancer Agency [BCCA]) to validate the predictive utility of POD24, and to evaluate the ability of pretreatment risk models to predict early treatment failure. POD24 occurred in 17% and 23% of evaluable GLSG and BCCA patients, with 5-year OS rates of 41% (vs 91% for those without POD24, P < .0001) and 26% (vs 86%, P < .0001), respectively. The m7-FL International Prognostic Index (m7-FLIPI), a prospective clinicogenetic risk model for failure-free survival, had the highest accuracy to predict POD24 (76% and 77%, respectively) with an odds ratio of 5.82 in GLSG (P = .00031) and 4.76 in BCCA patients (P = .0052). A clinicogenetic risk model specifically designed to predict POD24, the POD24-PI, had the highest sensitivity to predict POD24, but at the expense of a lower specificity. In conclusion, the m7-FLIPI prospectively identifies the smallest subgroup of patients (28% and 22%, respectively) at highest risk of early failure of first-line immunochemotherapy and death, including patients not fulfilling the POD24 criteria, and should be evaluated in prospective trials of precision medicine approaches in FL.

Different biological risk factors in young poor-prognosis and elderly patients with diffuse large B-cell lymphoma

Prognostically relevant risk factors in patients with diffuse large B-cell lymphoma (DLBCL) have predominantly been evaluated in elderly populations. We tested whether previously described risk factors are also valid in younger, poor-prognosis DLBCL patients. Paraffin-embedded samples from 112 patients with de novo DLBCL, enrolled in the R-MegaCHOEP trial of the German High Grade Non-Hodgkin Lymphoma Study Group (DSHNHL) were investigated using immunohistochemistry (MYC, FOXP1, LMO2, GCET1, CD5, CD10, BCL2, BCL6, IRF4/MUM1) and fluorescence in situ hybridization (MYC, BCL2, BCL6). MYC, BCL2 and BCL6 breaks occurred in 14, 21 and 31%, respectively. In the majority of cases, MYC was simultaneously rearranged with BCL2 and/or BCL6. The adverse impact of MYC rearrangements was confirmed, but the sole presence of BCL2 breaks emerged as a novel prognostic marker associated with inferior overall survival (OS) (P=0.002). Combined overexpression of MYC and BCL2 showed only limited association with inferior OS. All immunohistochemical cell of origin classifiers applied failed to predict survival time. DLBCL tumors with significant proportion of immunoblastic and/or immunoblastic-plasmacytoid cells had inferior OS, independently from from BCL2 break. Younger, poor-prognosis DLBCL patients, therefore, display different biological risk factors compared with an elderly population, with BCL2 translocations emerging as a powerful negative prognostic marker.

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.

Recurrent loss of heterozygosity in 1p36 associated with TNFRSF14 mutations in IRF4 translocation negative pediatric follicular lymphomas

Pediatric follicular lymphoma is a rare disease that differs genetically and clinically from its adult counterpart. With the exception of pediatric follicular lymphoma with IRF4-translocation, the genetic events associated with these lymphomas have not yet been defined. We applied array-comparative genomic hybridization and molecular inversion probe assay analyses to formalin-fixed paraffin-embedded tissues from 18 patients aged 18 years and under with IRF4 translocation negative follicular lymphoma. All evaluable cases lacked t(14;18). Only 6 of 16 evaluable cases displayed chromosomal imbalances with gains or amplifications of 6pter-p24.3 (including IRF4) and deletion and copy number neutral-loss of heterozygosity in 1p36 (including TNFRSF14) being most frequent. Sequencing of TNFRSF14 located in the minimal region of loss in 1p36.32 showed nine mutations in 7 cases from our series. Two subsets of pediatric follicular lymphoma were delineated according to the presence of molecular alterations, one with genomic aberrations associated with higher grade and/or diffuse large B-cell lymphoma component and more widespread disease, and another one lacking genetic alterations associated with more limited disease.

Diffuse large B-cell lymphomas of immunoblastic type are a major reservoir for MYC-IGH translocations.

The immunoblastic variant of diffuse large B-cell lymphoma (IB-DLBCL) has recently been recognized as an aggressive lymphoma type with inferior prognosis as compared with other DLBCL variants. At the same time, the presence of MYC rearrangements in DLBCL has been shown to indicate shorter survival in R-CHOP-treated patients. In this study, we investigated the occurrence of MYC gene rearrangements in IB-DLBCL versus non-IB-DLBCL in a large series. Using fluorescence in situ hybridization with an MYC break-apart and MYC-IGH fusion probe, we found that 13/39 evaluable IB-DLBCLs (33%) harbor translocations involving MYC, in contrast with only 5/68 (7%) in the non-IB-DLBCL group (P<0.01). The immunoglobulin heavy chain gene (IGH) was the translocation partner in all rearrangements (100%) involving MYC in IB-DLBCL, which is in contrast to what has been reported for DLBCL in the literature (50% to 70%). Moreover, MYC rearrangements occurred as the sole translocation in the majority of cases (77%), whereas across all DLBCLs the majority of MYC-rearranged cases carry additional rearrangements of either BCL2 and/or BCL6 genes (between 58% and 83% of cases). Finally, MYC-rearranged IB-DLBCLs were CD10 positive in 62% (8/13), whereas this was an uncommon feature in MYC germline IB-DLBCLs (15%). In conclusion, IB-DLBCLs are genetically characterized by frequent MYC-IGH translocations that often occur without additional BCL2 and/or BCL6 translocations. The activation of MYC, therefore, may be an important pathogenetic feature in IB-DLBCL.