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Formalin-fixed, paraffin-embedded (FFPE) specimens are an underutilized resource in medical research, particularly in the setting of transcriptome sequencing, as RNA from these samples is often degraded. We took advantage of an exome capture-based RNA-sequencing protocol to explore global gene expression in paired fresh-frozen (FF) and FFPE samples from 16 diffuse large B-cell lymphoma (DLBCL) patients. While FFPE samples generated fewer mapped reads compared to their FF counterparts, these reads captured the same library complexity and had a similar number of genes expressed on average. Furthermore, gene expression demonstrated a high correlation when comparing housekeeping genes only or across the entire transcriptome (r = 0.99 for both comparisons). Differences in gene expression were primarily seen in lowly expressed genes and genes with small or large coding sequences. Using cell-of-origin classifiers and clinically relevant gene expression signatures for DLBCL, FF, and FFPE samples from the same biopsy paired nearly perfectly in clustering analysis. This was further confirmed in a validation cohort of 50 FFPE DLBCL samples. In summary, we found the biological differences between tumors to be far greater than artifacts created as a result of degraded RNA. We conclude that exome capture transcriptome sequencing data from archival samples can confidently be used for cell-of-origin classification of DLBCL samples.

Background Programmed cell death 1 (PD-1) and its ligands PD-L1 and PD-L2, as well as Indoleamine 2,3-deoxygenase (IDO1) can be expressed both by tumor and microenvironmental cells and are crucial for tumor immune escape. We aimed to evaluate the role of PD-1, its ligands and IDO1 in a cohort of patients with primary diffuse large B-cell lymphoma of the CNS (PCNSL). Material and methods Tissue microarrays (TMAs) were constructed in 45 PCNSL cases. RNA extraction from whole tissue sections and RNA sequencing were successfully performed in 33 cases. Immunohistochemical stainings for PD-1, PD-L1/paired box protein 5 (PAX-5), PD-L2/PAX-5 and IDO1, and Epstein-Barr virus encoding RNA (EBER) in situ hybridization were analyzed. Results High proportions of PD-L1 and PD-L2 positive tumor cells were observed in 11% and 9% of cases, respectively. High proportions of PD-L1 and PD-L2 positive leukocytes were observed in 55% and 51% of cases, respectively. RNA sequencing revealed that gene expression of IDO1 was high in patients with high proportion of PD-L1 positive leukocytes (p = .01). Protein expression of IDO1 in leukocytes was detected in 14/45 cases, in 79% of these cases a high proportion of PD-L1 positive leukocytes was observed. Gene expression of IDO1 was high in EBER-positive cases (p = .0009) and protein expression of IDO1 was detected in five of six EBER-positive cases. Conclusion Our study shows a significant association between gene and protein expression of IDO1 and protein expression of PD-L1 in the tumor microenvironment of PCNSL, possibly of importance for prediction of response to immunotherapies. Title in Thesis: PD-L1 and IDO1 are important immunosuppressive molecules in primary diffuse large B-cell lymphoma of the CNS

OBJECTIVE: The prognostic value of site of nodal involvement in diffuse large B-cell lymphomas (DLBCL) is mainly unknown. We aimed to determine the prognostic significance of nodal abdominal involvement in relation to tumour cell markers and clinical characteristics of 249 DLBCL patients in a retrospective single-centre study. METHODS: Contrast-enhanced computed tomography (CT) of the abdomen and thorax revealed pathologically enlarged abdominal lymph nodes in 156 patients, while in 93 patients there were no pathologically enlarged lymph nodes in the abdomen. In 81 cases, the diagnosis of DLBCL was verified by histopathological biopsy obtained from abdominal lymph node. RESULTS: Patients with abdominal nodal disease had inferior lymphoma-specific survival (P = .04) and presented with higher age-adjusted IPI (P

The tumor cells in diffuse large B‐cell lymphomas (DLBCL) are considered to originate from germinal center derived B‐cells (GCB) or activated B‐cells (ABC). Gene expression profiling (GEP) is preferably used to determine the cell of origin (COO). However, GEP is not widely applied in clinical practice and consequently, several algorithms based on immunohistochemistry (IHC) have been developed. Our aim was to evaluate the concordance of COO assignment between the Lymph2Cx GEP assay and the IHC‐based Hans algorithm, to decide which model is the best survival predictor. Both GEP and IHC were performed in 359 homogenously treated Swedish and Danish DLBCL patients, in a retrospective multicenter cohort. The overall concordance between GEP and IHC algorithm was 72%; GEP classified 85% of cases assigned as GCB by IHC, as GCB, while 58% classified as non‐GCB by IHC, were categorized as ABC by GEP. There were significant survival differences (overall survival and progression‐free survival) if cases were classified by GEP, whereas if cases were categorized by IHC only progression‐free survival differed significantly. Importantly, patients assigned as non‐GCB/ABC both by IHC and GEP had the worst prognosis, which was also significant in multivariate analyses. Double expression of MYC and BCL2 was more common in ABC cases and was associated with a dismal outcome. In conclusion, to determine COO both by IHC and GEP is the strongest outcome predictor to identify DLBCL patients with the worst outcome.

We recently reported a truncating deletion in the NFKBIE gene, which encodes IκBε, a negative feedback regulator of NF-κB, in clinically aggressive chronic lymphocytic leukemia (CLL). Because preliminary data indicate enrichment of NFKBIE aberrations in other lymphoid malignancies, we screened a large patient cohort (n = 1460) diagnosed with different lymphoid neoplasms. While NFKBIE deletions were infrequent in follicular lymphoma, splenic marginal zone lymphoma, and T-cell acute lymphoblastic leukemia (

To determine the prognostic significance of co-expression of MYC, BCL-2 and BCL-6 proteins in combination with other biomarkers and clinical characteristics within a population-based cohort of diffuse large B-cell lymphoma (DLBCL) patients uniformly treated with R-CHOP.The immunohistochemical (IHC) expression of CD10, BCL-2, BCL-6, MUM1, MYC, CD5, CD30, Ki-67 and p53 was evaluated in a retrospective, population-based study comprising 188 DLBCL patients treated with R-CHOP and diagnosed in Sweden between 2002 and 2012.Patients had a median age at diagnosis of 64 years (26-85 years) with a male:female ratio of 1.4:1. Approximately half (52%) of the patients presented with an International Prognostic Index (IPI) age adjusted (IPIaa) ≥ 2. Median follow-up time was 51 months (range 0.4-158) and the five-year lymphoma-specific survival (LSS) was 76%, five-year overall survival (OS) was 65% and five-year progression-free survival (PFS) was 61%. A high Ki-67 value was found in 59% of patients, while p53 overexpression was detected in 12% of patients and MYC, BCL-2 and BCL-6 expression were detected in 42%, 55% and 74% of patients, respectively. IPIaa ≥2 (p = 0.002), Ki-67 ≥ 70% (p = 0.04) and p53 overexpression ≥50% (p = 0.02) were associated with inferior LSS and OS. Co-expression of both MYC (40%) and BCL-2 (70%) proteins was detected in 27% of patients and correlated with a significantly inferior LSS (p = 0.0002), OS (p = 0.009) and PFS (p = 0.03). In addition, triple expression of MYC, BCL-2 and BCL-6, also correlated with a significantly inferior LSS (p = 0.02).Concurrent expression of MYC and BCL-2 proteins, as detected by IHC, was strongly associated with an inferior survival in DLBCL patients treated with R-CHOP. Other markers affecting survival were triple expression of MYC, BCL-2 and BCL-6, IPIaa, high Ki-67 and p53 overexpression.

Core needle biopsies for the diagnosis of diffuse large B-cell lymphoma - a great concern for research

Deregulated NF-κB signaling is a hallmark of most, if not all, lymphoid malignancies, and recurrent gene mutations in both the canonical and non-canonical NF-κB pathway are known to lead to NF-κB activation. However, the full compendium of NF-κB gene mutations in lymphoid malignancies remains to be elucidated. Recently, we reported a 4-bp truncating mutation in the NFKBIE gene, which encodes IκBε, a negative regulator of NF-κB, in patients with chronic lymphocytic leukemia (CLL). The NFKBIE deletion was enriched in clinically aggressive CLL patients (7-8%) and associated with a worse clinical outcome. At the functional level, NFKBIE-deleted CLL showed reduced IκBε levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65, compared to wildtype patients. Preliminary data has indicated an increased frequency of NFKBIE aberrations in other lymphoid malignancies as well. To explore this further, we screened for NFKBIE deletions in a large cohort of patients diagnosed with a range of different lymphoid neoplasms. Overall, NFKBIE deletions were identified in 76 of 1414 patients (5.4%). While NFKBIE deletions were relatively infrequent in patients diagnosed with follicular lymphoma (3/225, 1.3%), splenic marginal zone lymphoma (3/175, 1.7%), and T-cell acute lymphoblastic leukemia (1/94, 1.1%), moderate frequencies were observed among diffuse large B-cell lymphoma (18/521, 3.5%), mantle cell lymphoma (8/189, 4.2%), and primary CNS lymphoma (1/34, 2.9%) patients. In contrast, a remarkably high frequency of NFKBIE deletions (41/176 cases, 23%) was observed among primary mediastinal B-cell lymphoma (PMBL) patients. Noteworthy, the prevalence of NFKBIE-deleted PMBL cases was similar in the different contributing centers. All PMBL patients in the present series received a CHOP based treatment regime; in ~75% of cases rituximab was added and ~25% were treated with dose intensified schemes. For the latter, the vast majority of patients received CHOEP, while individual cases were treated with MegaCHOEP, DA-EPOCH or ACVBP. Regarding clinicobiological associations, there were no significant differences between NFKBIE-deleted and wildtype PMBL patients with respect to age, sex, Ann Arbor stage, IPI risk-groups, extranodal or bone marrow involvement, bulky disease, and LDH elevation. However, NFKBIE-deleted patients were more likely to be refractory to primary chemotherapy (31% vs. 3%, P=.001) and had a shorter overall survival compared to wildtype patients (5-year overall survival: 63% vs 84%, P=.013). In multivariate analysis (including age, gender, Ann Arbor stage, IPI, and NFKBIE mutation status), NFKBIE mutation status (95% CI: 1.23-10.61; HR: 3.61; P=0.020) remained an independent factor for poor prognosis. In summary, we document NFKBIE deletions as a common genetic event across B-cell malignancies, albeit at varying frequencies. The high frequency of NFKBIE deletions in PMBL alludes to the critical role of this aberration in the pathophysiology of the disease. NFKBIE deletions were associated witha worse clinical outcome, hence potentially representing a novel poor-prognostic marker in PMBL. *Contributed equally as first authors. **Contributed equally as senior authors. Disclosures Stamatopoulos: Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.