Genetic Characterization of Primary Mediastinal B-Cell Lymphoma: Pathogenesis and Patient Outcomes.

PURPOSE: Primary mediastinal large B-cell lymphoma (PMBCL) is a rare aggressive lymphoma predominantly affecting young female patients. Large-scale genomic investigations and genetic markers for risk stratification are lacking. PATIENTS AND METHODS: To elucidate the full spectrum of genomic alterations, samples from 340 patients with previously untreated PMBCL were investigated by whole-genome (n = 20), whole-exome (n = 78), and targeted (n = 308) sequencing. Statistically significant prognostic variables were identified using a multivariable Cox regression model and confirmed by L1/L2 regularized regressions. RESULTS: Whole-genome sequencing revealed a commonly disrupted p53 pathway with nonredundant somatic structural variations (SVs) in TP53-related genes (TP63, TP73, and WWOX) and identified novel SVs facilitating immune evasion (DOCK8 and CD83). Integration of mutation and copy-number data expanded the repertoire of known PMBCL alterations (eg, ARID1A, P2RY8, and PLXNC1) with a previously unrecognized role for epigenetic/chromatin modifiers. Multivariable analysis identified six genetic lesions with significant prognostic impact. CD58 mutations (31%) showed the strongest association with worse PFS (hazard ratio [HR], 2.52 [95% CI, 1.50 to 4.21]; P < .001) and overall survival (HR, 2.33 [95% CI, 1.14 to 4.76]; P = .02). IPI high-risk patients with mutated CD58 demonstrated a particularly poor prognosis, with 5-year PFS and OS rates of 41% and 58%, respectively. The adverse prognostic significance of the CD58 mutation status was predominantly observed in patients treated with nonintensified regimens, indicating that dose intensification may, to some extent, mitigate the impact of this high-risk marker. By contrast, DUSP2-mutated patients (24%) displayed durable responses (PFS: HR, 0.2 [95% CI, 0.07 to 0.55]; P = .002) and prolonged OS (HR, 0.11 [95% CI, 0.01 to 0.78]; P = .028). Upon CHOP-like treatment, these patients had very favorable outcome, with 5-year PFS and OS rates of 93% and 98%, respectively. CONCLUSION: This large-scale genomic characterization of PMBCL identified novel treatment targets and genetic lesions for refined risk stratification. DUSP2 and CD58 mutation analyses may guide treatment decisions between rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone and dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab.

Novel insights into the pathogenesis of follicular lymphoma by molecular profiling of localized and systemic disease forms.

Knowledge on the pathogenesis of FL is mainly based on data derived from advanced/systemic stages of FL (sFL) and only small cohorts of localized FL (lFL) have been characterized intensively so far. Comprehensive analysis with profiling of somatic copy number alterations (SCNA) and whole exome sequencing (WES) was performed in 147 lFL and 122 sFL. Putative targets were analyzed for gene and protein expression. Overall, lFL and sFL, as well as BCL2 translocation-positive (BCL2+) and -negative (BCL2-) FL showed overlapping features in SCNA and mutational profiles. Significant differences between lFL and sFL, however, were detected for SCNA frequencies, e.g., in 18q-gains (14% lFL vs. 36% sFL; p = 0.0003). Although rare in lFL, gains in 18q21 were associated with inferior progression-free survival (PFS). The mutational landscape of lFL and sFL included typical genetic lesions. However, ARID1A mutations were significantly more often detected in sFL (29%) compared to lFL (6%, p = 0.0001). In BCL2 + FL mutations in KMT2D, BCL2, ABL2, IGLL5 and ARID1A were enriched, while STAT6 mutations more frequently occurred in BCL2- FL. Although the landscape of lFL and sFL showed overlapping features, molecular profiling revealed novel insights and identified gains in 18q21 as prognostic marker in lFL.

Frequent ZNF217 mutations lead to transcriptional deregulation of interferon signal transduction via altered chromatin accessibility in B cell lymphoma.

Recent exome-wide studies discovered frequent somatic mutations in the epigenetic modifier ZNF217 in primary mediastinal B cell lymphoma (PMBCL) and related disorders. As functional consequences of ZNF217 alterations remain unknown, we comprehensively evaluated their impact in PMBCL. Targeted sequencing identified genetic lesions affecting ZNF217 in 33% of 157 PMBCL patients. Subsequent gene expression profiling (n = 120) revealed changes in cytokine and interferon signal transduction in ZNF217-aberrant PMBCL cases. In vitro, knockout of ZNF217 led to changes in chromatin accessibility interfering with binding motifs for crucial lymphoma-associated transcription factors. This led to disturbed expression of interferon-responsive and inflammation-associated genes, altered cell behavior, and aberrant differentiation. Mass spectrometry demonstrates that ZNF217 acts within a histone modifier complex containing LSD1, CoREST and HDAC and interferes with H3K4 methylation and H3K27 acetylation. Concluding, our data suggest non-catalytic activity of ZNF217, which directs histone modifier complex function and controls B cell differentiation-associated patterns of chromatin structure.

Large B-Cell Lymphomas in the 5th Edition of the WHO-Classification of Haematolymphoid Neoplasms-Updated Classification and New Concepts.

The family/class of the large B-cell lymphomas (LBCL) in the 5th edition of the World Health Organization (WHO) classification of haematolymphoid tumors (WHO-HAEM5) features only a few major changes as compared to the 4th edition. In most entities, there are only subtle changes, many of them only representing some minor modifications in diagnostic terms. Major changes have been made in the diffuse large B-cell lymphomas (DLBCL)/high-grade B-cell lymphomas (HGBL) associated with MYC and BCL2 and/or BCL6 rearrangements. This category now consists of MYC and BCL2 rearranged cases exclusively, while the MYC/BCL6 double hit lymphomas now constitute genetic subtypes of DLBCL, not otherwise specified (NOS) or of HGBL, NOS. Other major changes are the conceptual merger of lymphomas arising in immune-privileged sites and the description of LBCL arising in the setting of immune dysregulation/deficiency. In addition, novel findings concerning underlying biological mechanisms in the pathogenesis of the different entities are provided.

Follicular Lymphoma in the 5th Edition of the WHO-Classification of Haematolymphoid Neoplasms-Updated Classification and New Biological Data.

The conceptual description of Follicular lymphoma (FL) in the 5th edition of the World Health Organization (WHO) classification of haematolymphoid tumors (WHO-HAEM5) has undergone significant revision. The vast majority of FL (85%) with a follicular growth pattern are composed of centrocytes and centroblasts, harbor the t(14;18)(q32;q21) translocation and are now termed classic FL (cFL). They are set apart from three related subtypes, FL with predominantly follicular growth pattern, FL with unusual cytological features (uFL) and follicular large B-cell lymphoma (FLBCL). In contrast to the revised 4th edition of the WHO classification of haematolymphoid tumors (WHO-HAEM4R), grading of cFL is no longer mandatory. FL with a predominantly diffuse growth pattern had been previously recognized in WHO-HAEM4R. It frequently occurs as a large tumor in the inguinal region and is associated with CD23 expression. An absence of the IGH::BCL2 fusion and frequent STAT6 mutations along with 1p36 deletion or TNFRSF14 mutation is typical. The newly introduced subtype of uFL includes two subsets that significantly diverge from cFL: one with "blastoid" and one with "large centrocyte" variant cytological features. uFL more frequently displays variant immunophenotypic and genotypic features. FLBCL is largely identical to WHO-HAEM4R FL grade 3B and renaming was done for reasons of consistency throughout the classification. In-situ follicular B-cell neoplasm, pediatric-type FL, duodenal-type FL and primary cutaneous follicle center lymphoma are categorized as discrete entities. In addition, novel findings concerning underlying biological mechanisms in the pathogenesis of early and systemic follicular lymphoma will be presented.

Molecular profiling of EBV associated diffuse large B-cell lymphoma.

Epstein-Barr virus (EBV) associated diffuse large B-cell lymphoma (DLBCL) represents a rare aggressive B-cell lymphoma subtype characterized by an adverse clinical outcome. EBV infection of lymphoma cells has been associated with different lymphoma subtypes while the precise role of EBV in lymphomagenesis and specific molecular characteristics of these lymphomas remain elusive. To further unravel the biology of EBV associated DLBCL, we present a comprehensive molecular analysis of overall 60 primary EBV positive (EBV+) DLBCLs using targeted sequencing of cancer candidate genes (CCGs) and genome-wide determination of recurrent somatic copy number alterations (SCNAs) in 46 cases, respectively. Applying the LymphGen classifier 2.0, we found that less than 20% of primary EBV + DLBCLs correspond to one of the established molecular DLBCL subtypes underscoring the unique biology of this entity. We have identified recurrent mutations activating the oncogenic JAK-STAT and NOTCH pathways as well as frequent amplifications of 9p24.1 contributing to immune escape by PD-L1 overexpression. Our findings enable further functional preclinical and clinical studies exploring the therapeutic potential of targeting these aberrations in patients with EBV + DLBCL to improve outcome.

Molecular Cytogenetic Profiling Reveals Similarities and Differences Between Localized Nodal and Systemic Follicular Lymphomas.

Recently, we have developed novel highly promising gene expression (GE) classifiers discriminating localized nodal (LFL) from systemic follicular lymphoma (SFL) with prognostic impact. However, few data are available in LFL especially concerning hotspot genetic alterations that are associated with the pathogenesis and prognosis of SFL. A total of 144 LFL and 527 SFL, enrolled in prospective clinical trials of the German Low Grade Lymphoma Study Group, were analyzed by fluorescence in situ hybridization to detect deletions in chromosomes 1p, 6q, and 17p as well as BCL2 translocations to determine their impact on clinical outcome of LFL patients. The frequency of chromosomal deletions in 1p and 17p was comparable between LFL and SFL, while 6q deletions and BCL2 translocations more frequently occurred in SFL. A higher proportion of 1p deletions was seen in BCL2-translocation-positive LFL, compared with BCL2-translocation-negative LFL. Deletions in chromosomes 1p, 6q, and 17p predicted clinical outcome of patients with SFL in the entire cohort, while only deletions in chromosome 1p retained its negative prognostic impact in R-CHOP-treated SFL. In contrast, no deletions in one of the investigated genetic loci predicted clinical outcome in LFL. Likewise, the presence or absence of BCL2 translocations had no prognostic impact in LFL. Despite representing a genetic portfolio closely resembling SFL, LFL showed some differences in deletion frequencies. BCL2 translocation and 6q deletion frequency differs between LFL and SFL and might contribute to distinct genetic profiles in LFL and SFL.

PARP14 is a novel target in STAT6 mutant follicular lymphoma.

The variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of tumor cells and complex interactions within the tumor microenvironment (TME). IL-4 producing follicular helper T cells (TFH) are critical components of the FL TME. Binding of IL-4 to IL-4R on FL cells activates JAK/STAT signaling. We identified STAT6 mutations (STAT6MUT) in 13% of FL (N = 33/258), all clustered within the DNA binding domain. Gene expression data and immunohistochemistry showed upregulation of IL-4/STAT6 target genes in STAT6MUT FL, including CCL17, CCL22, and FCER2 (CD23). Functionally, STAT6MUT was gain-of-function by serial replating phenotype in pre-B CFU assays. Expression of STAT6MUT enhanced IL-4 induced FCER2/CD23, CCL17 and CCL22 expression and was associated with nuclear accumulation of pSTAT6. RNA sequencing identified PARP14 -a transcriptional switch and co-activator of STAT6- among the top differentially upregulated genes in IL-4 stimulated STAT6MUT lymphoma cells and in STAT6MUT primary FL cells. Quantitative chromatin immunoprecipitation (qChIP) demonstrated binding of STAT6MUT but not STAT6WT to the PARP14 promotor. Reporter assays showed increased IL-4 induced transactivation activity of STAT6MUT at the PARP14 promotor, suggesting a self-reinforcing regulatory circuit. Knock-down of PARP14 or PARP-inhibition abrogated the STAT6MUT gain-of-function phenotype. Thus, our results identify PARP14 as a novel therapeutic target in STAT6MUT FL.

Epstein-Barr virus status of sporadic Burkitt lymphoma is associated with patient age and mutational features.

Sporadic Burkitt lymphoma (BL) is the most frequent tumour of children and adolescents but a rare subtype of lymphomas in adults. To date most molecular data have been obtained from lymphomas arising in the young. Recently, Epstein-Barr virus (EBV) positive and negative BL in young patients was shown to differ in molecular features. In the present study, we present a large age-overarching cohort of sporadic BL (n = 162) analysed by immunohistochemistry, translocations of MYC proto-oncogene, basic helix-loop-helix transcription factor (MYC), B-cell leukaemia/lymphoma 2 (BCL2) and B-cell leukaemia/lymphoma 6 (BCL6) and by targeted sequencing. We illustrate an age-associated inter-tumoral molecular heterogeneity in this disease. Mutations affecting inhibitor of DNA binding 3, HLH protein (ID3), transcription factor 3 (TCF3) and cyclin D3 (CCND3), which are highly recurrent in paediatric BL, and expression of sex determining region Y-box transcription factor 11 (SOX11) declined with patient age at diagnosis (P = 0·0204 and P = 0·0197 respectively). In contrast, EBV was more frequently detected in adult patients (P = 0·0262). Irrespective of age, EBV-positive sporadic BL showed significantly less frequent mutations in ID3/TCF3/CCND3 (P = 0·0088) but more often mutations of G protein subunit alpha 13 (GNA13; P = 0·0368) and forkhead box O1 (FOXO1; P = 0·0044) compared to EBV-negative tumours. Our findings suggest that among sporadic BL an EBV-positive subgroup of lymphomas increases with patient age that shows distinct pathogenic features reminiscent of EBV-positive endemic BL.

Molecular and functional profiling identifies therapeutically targetable vulnerabilities in plasmablastic lymphoma.

Plasmablastic lymphoma (PBL) represents a rare and aggressive lymphoma subtype frequently associated with immunosuppression. Clinically, patients with PBL are characterized by poor outcome. The current understanding of the molecular pathogenesis is limited. A hallmark of PBL represents its plasmacytic differentiation with loss of B-cell markers and, in 60% of cases, its association with Epstein-Barr virus (EBV). Roughly 50% of PBLs harbor a MYC translocation. Here, we provide a comprehensive integrated genomic analysis using whole exome sequencing (WES) and genome-wide copy number determination in a large cohort of 96 primary PBL samples. We identify alterations activating the RAS-RAF, JAK-STAT, and NOTCH pathways as well as frequent high-level amplifications in MCL1 and IRF4. The functional impact of these alterations is assessed using an unbiased shRNA screen in a PBL model. These analyses identify the IRF4 and JAK-STAT pathways as promising molecular targets to improve outcome of PBL patients.

Genetic mechanisms of HLA-I loss and immune escape in diffuse large B cell lymphoma.

Fifty percent of diffuse large B cell lymphoma (DLBCL) cases lack cell-surface expression of the class I major histocompatibility complex (MHC-I), thus escaping recognition by cytotoxic T cells. Here we show that, across B cell lymphomas, loss of MHC-I, but not MHC-II, is preferentially restricted to DLBCL. To identify the involved mechanisms, we performed whole exome and targeted HLA deep-sequencing in 74 DLBCL samples, and found somatic inactivation of B2M and the HLA-I loci in 80% (34 of 42) of MHC-INEG tumors. Furthermore, 70% (22 of 32) of MHC-IPOS DLBCLs harbored monoallelic HLA-I genetic alterations (MHC-IPOS/mono), indicating allele-specific inactivation. MHC-INEG and MHC-IPOS/mono cases harbored significantly higher mutational burden and inferred neoantigen load, suggesting potential coselection of HLA-I loss and sustained neoantigen production. Notably, the analysis of >500,000 individuals across different cancer types revealed common germline HLA-I homozygosity, preferentially in DLBCL. In mice, germinal-center B cells lacking HLA-I expression did not progress to lymphoma and were counterselected in the context of oncogene-driven lymphomagenesis, suggesting that additional events are needed to license immune evasion. These results suggest a multistep process of HLA-I loss in DLBCL development including both germline and somatic events, and have direct implications for the pathogenesis and immunotherapeutic targeting of this disease.

Rituximab plus high-dose chemotherapy (MegaCHOEP) or conventional chemotherapy (CHOEP-14) in young, high-risk patients with aggressive B-cell lymphoma: 10-year follow-up of a randomised, open-label, phase 3 trial.

BACKGROUND: R-MegaCHOEP was the first phase 3 study comparing high-dose chemotherapy plus rituximab followed by autologous haematopoietic stem-cell transplantation (HSCT) with conventional chemotherapy plus rituximab in first-line therapy for patients aged 60 years or younger with high-risk aggressive B-cell lymphoma. Little is known about the long-term outcomes of these patients. We aimed to evaluate the long-term efficacy and safety of conventional chemotherapy versus high-dose chemotherapy after 10 years of follow-up in the R-MegaCHOEP trial. METHODS: In this open-label, randomised, phase 3 trial done across 61 centres in Germany, patients aged 18-60 years with newly diagnosed, high-risk (age-adjusted International Prognostic Index [IPI] 2 or 3) aggressive B-cell lymphoma were randomly assigned (1:1, using Pocock minimisation) to eight cycles of conventional chemotherapy (cyclosphosphamide, doxorubicin, vincristine, etoposide, and prednisolone) plus rituximab (R-CHOEP-14) or four cycles of high-dose chemotherapy plus rituximab followed by autologous HSCT (R-MegaCHOEP). The trial was unmasked. Patients were stratified by age-adjusted IPI factors, presence of bulky disease (tumour mass ≥7·5 cm diameter), and treatment centre. The primary endpoint was event-free survival, analysed here 10 years after randomisation. 10-year overall survival, progression-free survival, conditional survival, relapse patterns, secondary malignancies, and molecular characteristics were also analysed. All analyses were done on the intention-to-treat population. This trial is registered with ClinicalTrials.gov, NCT00129090. FINDINGS: Between March 3, 2003, and April 7, 2009, 275 patients were randomly assigned to R-CHOEP-14 (n=136) or R-MegaCHOEP (n=139). 130 patients in the R-CHOEP-14 group and 132 patients in the R-MegaCHOEP group were included in the intention-to-treat population. After a median follow-up of 9·3 years (IQR 5·1-11·1), 10-year event-free survival was 51% (95% CI 42-61) in the R-MegaCHOEP group and 57% (47-67) in the R-CHOEP-14 group (adjusted hazard ratio [HR] 1·3 [95% CI 0·9-1·8], p=0·23). 10-year progression-free survival was 59% (50-68) in the R-MegaCHOEP group and 60% (51-70) in the R-CHOEP-14 group (adjusted HR 1·1 [0·7-1·7], p=0·64). 10-year overall survival was 66% (57-76) in the R-MegaCHOEP group and 72% (63-81) in the R-CHOEP-14 group (adjusted HR 1·3 [0·8-2·1], p=0·26). Relapse occurred in 30 (16% [95% CI 11-22]) of 190 patients who had complete remission or unconfirmed complete remission; 17 (17%) of 100 patients in the R-CHOEP-14 group and 13 (14%) of 90 patients in the R-MegaCHOEP group. Seven (23%) of 30 patients had low-grade histology at relapse and had better outcomes compared with patients who relapsed with aggressive histologies. Lymphoma affected the CNS in 18 (28%) of 64 patients with treatment failure. 22 secondary malignancies were reported in the intention-to-treat population; in 12 (9%) of 127 patients in the R-CHOEP-14 group and ten (8%) of 126 patients in the R-MegaCHOEP group. INTERPRETATION: Event-free survival and overall survival were similar between groups after 10 years of follow-up; outcomes were not improved in the R-MegaCHOEP group by high-dose chemotherapy and autologous HSCT. Patients who relapsed with aggressive histology showed a high incidence of CNS involvement and poor prognosis. For these patients, novel therapies are greatly warranted. FUNDING: Deutsche Krebshilfe (German Cancer Aid).

The "Burkitt-like" immunophenotype and genotype is rarely encountered in diffuse large B cell lymphoma and high-grade B cell lymphoma, NOS.

Burkitt lymphoma (BL) is a B cell lymphoma composed of monomorphic medium-sized blastic cells with basophilic cytoplasm and a high proliferation index. BL has a characteristic immunophenotype of CD10 and BCL6 positive and BCL2 negative and harbours MYC gene rearrangements (MYCR) in >90% of the cases. Owing to its highly aggressive nature, intensified chemotherapy regimens are usually administered, requiring an exact diagnosis. Since the diagnosis usually warrants an integration of morphologic, immunophenotypic and genetic findings and because there is a morphologic overlap with the new WHO category of high-grade B cell lymphoma, not otherwise specified (HGBL, NOS) and some cases of diffuse large B cell lymphoma (DLBCL), we wanted to test the distinctiveness of the CD10+, BCL6+, BCL2- and MYCR positive immunopheno-genotype in a large cohort of >1000 DLBCL and HGBL. Only 9/982 DLBCL classified by an expert panel of haematopathologists (0.9%) displayed a single MYCR and were CD10+, BCL6+ and BCL2-. In a similar fashion, only one out of 32 HGBL, NOS (3%) displayed the "Burkitt-like" genetic/immunophenotypic constitution. The samples of non-BL showing the BL-typic immunopheno-genotype, interestingly, harboured higher copy number variations (CNV) by OncoScan analysis (mean 7.3 CNVs/sample; range: 2-13 vs. 2.4; range 0-6) and were also distinct from pleomorphic BL cases regarding their mutational spectrum by NGS analysis. This implies that the characteristic immunophenotype of BL, in concert with a single MYCR, is uncommon in these aggressive lymphomas, and that this constellation favours BL.

Identification of a miRNA based model to detect prognostic subgroups in patients with aggressive B-cell lymphoma.

In order to differentiate prognostic subgroups of patients with aggressive B-cell lymphoma, we analyzed the expression of 800 miRNAs with the NanoString nCounter human miRNA assay on a cohort of 228 FFPE samples of patients enrolled in the RICOVER-60 and MegaCHOEP trials. We identified significant miRNA signatures for overall survival (OS) and progression-free survival (PFS) by LASSO-penalized linear Cox-regression. High expression levels of miR-130a-3p and miR-423-5p indicate a better prognosis, whereas high levels of miR-374b-5p, miR-590-5p, miR-186-5p, and miR-106b-5p increase patients' risk levels for OS. Regarding PFS high expression of miR-365a-5p in addition to the other two miRNAs improves the prognosis and high levels of miR374a-5p, miR-106b-5p, and miR-590-5p, connects with increased risk and poor prognosis. We identified miRNA signatures to subdivide patients into two different risk groups. These prognostic models may be used in risk stratification in future clinical trials and help making personalized therapy decisions.

A Diagnostic Approach to the Identification of Burkitt-like Lymphoma With 11q Aberration in Aggressive B-Cell Lymphomas.

Rare cases of aggressive B-cell lymphomas with a morphology similar to Burkitt lymphoma (BL) present with the BL-typical immunophenotype, but lacked MYC translocation (MYC-negative Burkitt-like lymphoma: mnBLL). A proportion of those with an imbalance pattern in chromosome 11q has been designated Burkitt-like lymphoma with 11q aberration in the recent update of the World Health Organization (WHO) classification. Because of the problems in the identification of Burkitt-like lymphoma with 11q aberration, our goal was to retrospectively analyze their frequency in a cohort of "candidate" aggressive lymphomas (cohort 1, n=35) such as mnBLL (n=16), diffuse large B-cell lymphoma with similarities to Burkitt lymphoma (DLBCL-BL; n=3), high-grade B-cell lymphomas, not otherwise specified (NOS) (n=16), as well as in a cohort of MYC-negative diffuse large B-cell lymphoma NOS (cohort 2, n=62). In total, 17/33 cohort 1 cases (52%) harbored the typical 11q aberration pattern, predominantly those that had been classified as mnBLL (12/16, 75%), but also as DLBCL-BL (2/3, 67%) and high-grade B-cell lymphomas, NOS (3/14; 21%). The specimens with this typical 11q aberration pattern were usually negative for the BCL2 protein. Of interest and as a new finding, samples harboring the 11q aberration pattern were often characterized by strikingly coarse apoptotic debris within starry sky macrophages facilitating their recognition. In contrast, only 1 of 62 garden variety DLBCL, NOS was positive for the 11q aberration pattern. In 2 DLBCL-BL, a dual MYC translocation/11q aberration pattern was detected. As a diagnostic algorithm, we, therefore, propose analysis of 11q status in MYC-negative high-grade lymphomas with features of BL, especially showing BCL2 negativity and a conspicuous coarse apoptotic debris in starry sky macrophages.

Cytokeratin expression in plasmablastic lymphoma - a possible diagnostic pitfall in the routine work-up of tumours.

AIMS: Plasmablastic lymphoma (PBL) is a rare aggressive B-cell lymphoma that frequently arises at extranodal sites in the setting of immunosuppression. The diagnosis of PBL is complex, owing to a frequent solid or cohesive growth pattern, and an often unusual immunophenotype. Several case reports have described cytokeratin (CK) expression in PBL, introducing a diagnostic pitfall. The aim of this study was to determine the frequency of CK expression in PBL in the largest series available to date. METHODS AND RESULTS: By using immunohistochemistry in a cohort of 72 PBLs, we identified CK8/18 positivity in 11 of 72 cases (15%) and AE1/3 positivity in six of 65 cases (9%), clearly contrasting with a control series of non-PBL aggressive B-cell lymphomas (one of 96 diffuse large B-cell lymphomas), as well as with data in the literature describing only occasional CK expression in haematological neoplasms. CONCLUSIONS: Our data indicate CK expression in a substantial number (15%) of PBLs. In view of the particular morphological features of PBL and its frequent negativity for the common leukocyte antigen and B-cell markers, this feature represents a pitfall in the routine diagnostic work-up of PBL, and requires more extensive immunohistochemical and molecular characterisation of cases entering the differential diagnosis.

Evaluating upfront high-dose consolidation after R-CHOP for follicular lymphoma by clinical and genetic risk models.

High-dose therapy and autologous stem cell transplantation (HDT/ASCT) is an effective salvage treatment for eligible patients with follicular lymphoma (FL) and early progression of disease (POD). Since the introduction of rituximab, HDT/ASCT is no longer recommended in first remission. We here explored whether consolidative HDT/ASCT improved survival in defined subgroups of previously untreated patients. We report survival analyses of 431 patients who received frontline rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for advanced FL, and were randomized to receive consolidative HDT/ASCT. We performed targeted genotyping of 157 diagnostic biopsies, and calculated genotype-based risk scores. HDT/ASCT improved failure-free survival (FFS; hazard ratio [HR], 0.8, P = .07; as-treated: HR, 0.7, P = .04), but not overall survival (OS; HR, 1.3, P = .27; as-treated: HR, 1.4, P = .13). High-risk cohorts identified by FL International Prognostic Index (FLIPI), and the clinicogenetic risk models m7-FLIPI and POD within 24 months-prognostic index (POD24-PI) comprised 27%, 18%, and 22% of patients. HDT/ASCT did not significantly prolong FFS in high-risk patients as defined by FLIPI (HR, 0.9; P = .56), m7-FLIPI (HR, 0.9; P = .91), and POD24-PI (HR, 0.8; P = .60). Similarly, OS was not significantly improved. Finally, we used a machine-learning approach to predict benefit from HDT/ASCT by genotypes. Patients predicted to benefit from HDT/ASCT had longer FFS with HDT/ASCT (HR, 0.4; P = .03), but OS did not reach statistical significance. Thus, consolidative HDT/ASCT after frontline R-CHOP did not improve OS in unselected FL patients and subgroups selected by genotype-based risk models.

A novel lymphoma-associated macrophage interaction signature (LAMIS) provides robust risk prognostication in diffuse large B-cell lymphoma clinical trial cohorts of the DSHNHL.

Diffuse large B-cell lymphoma (DLBCL) is a disease with heterogeneous outcome. Stromal signatures have been correlated to survival in DLBCL. Their use, however, is hampered by the lack of assays for formalin-fixed paraffin-embedded material (FFPE). We constructed a lymphoma-associated macrophage interaction signature (LAMIS) interrogating features of the microenvironment using a NanoString assay applicable to FFPE. The clinical impact of the signature could be validated in a cohort of 466 patients enrolled in prospective clinical trials of the German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL). Patients with high expression of the signature (LAMIShigh) had shorter EFS, PFS, and OS. Multivariate analyses revealed independence from IPI factors in EFS (HR 1.7, 95% CI 1.2-2.4, p-value = 0.001), PFS (HR 1.8, 95% CI 1.2-2.5, p-value = 0.001) and OS (HR 1.8, 95% CI 1.3-2.7, p-value = 0.001). Multivariate analyses adjusted for the IPI factors showed the signature to be independent from COO, MYC rearrangements and double expresser status (DE). LAMIShigh and simultaneous DE status characterized a patient subgroup with dismal prognosis and early relapse. Our data underline the importance of the microenvironment in prognosis. Combined analysis of stromal features, the IPI and DE may provide a new rationale for targeted therapy.

Localized- and advanced-stage follicular lymphomas differ in their gene expression profiles.

The genetic background of follicular lymphomas (FLs) diagnosed in advanced clinical stages III/IV, and which are frequently characterized by t(14;18), has been substantially unraveled. Molecular features, as exemplified in the clinicogenetic risk model m7FLIPI, are important tools in risk stratification. In contrast, little information is available concerning localized-stage FL (clinical stages I/II), which accounts for ∼20% of newly diagnosed FL in which the detection rate of t(14;18) is only ∼50%. To investigate the genetic background of localized-stage FL, patient cohorts with advanced-stage FL or localized-stage FL, uniformly treated within phase 3 trials of the German Low-Grade Lymphoma Study Group, were comparatively analyzed. Targeted gene expression (GE) profiling of 184 genes using nCounter technology was performed in 110 localized-stage and 556 advanced-stage FL patients. By penalized Cox regression, a prognostic GE signature could not be identified in patients with advanced-stage FL, consistent with results from global tests and univariate regression. In contrast, it was possible to define robust GE signatures discriminating localized-stage and advanced-stage FL (area under the curve, 0.98) by penalized logistic regression. Of note, 3% of samples harboring an "advanced-stage signature" in the localized-stage cohort exhibited inferior failure-free survival (hazard ratio [HR], 7.1; P = .0003). Likewise, in the advanced-stage cohort, 7% of samples with a "localized-stage signature" had prolonged failure-free survival (HR, 2.3; P = .017) and overall survival (HR, 3.4; P = .072). These data support the concept of a biological difference between localized-stage and advanced-stage FL that might contribute to the superior outcome of localized FL.