Motive and opportunity: MYC rearrangements in high-grade B-cell lymphoma with MYC and BCL2 rearrangements (an LLMPP study).

ABSTRACT: Rearrangements that place the oncogenes MYC, BCL2, or BCL6 adjacent to superenhancers are common in mature B-cell lymphomas. Lymphomas with diffuse large B-cell lymphoma (DLBCL) or high-grade morphology with both MYC and BCL2 rearrangements are classified as high-grade B-cell lymphoma with MYC and BCL2 rearrangements ("double hit"; HGBCL-DH-BCL2) and are associated with aggressive disease and poor outcomes. Although it is established that MYC rearrangements involving immunoglobulin (IG) loci are associated with inferior outcomes relative to those involving other non-IG superenhancers, the frequency of and mechanisms driving IG vs non-IG MYC rearrangements have not been elucidated. Here, we used custom targeted capture and/or whole-genome sequencing to characterize oncogene rearrangements across 883 mature B-cell lymphomas including Burkitt lymphoma, follicular lymphoma, DLBCL, and HGBCL-DH-BCL2 tumors. We demonstrate that, although BCL2 rearrangement topology is consistent across entities, HGBCL-DH-BCL2 have distinct MYC rearrangement architecture relative to tumors with single MYC rearrangements or with both MYC and BCL6 rearrangements (HGBCL-DH-BCL6), including both a higher frequency of non-IG rearrangements and different architecture of MYC::IGH rearrangements. The distinct MYC rearrangement patterns in HGBCL-DH-BCL2 occur on the background of high levels of somatic hypermutation across MYC partner loci in HGBCL-DH-BCL2, creating more opportunity to form these rearrangements. Furthermore, because 1 IGH allele is already disrupted by the existing BCL2 rearrangement, the MYC rearrangement architecture in HGBCL-DH-BCL2 likely reflects selective pressure to preserve both BCL2 and B-cell receptor expression. These data provide new mechanistic explanations for the distinct patterns of MYC rearrangements observed across different lymphoma entities.

Evaluation of clinical parameters and biomarkers in older, untreated mantle cell lymphoma patients receiving bendamustine-rituximab.

Mantle cell lymphoma (MCL) is clinically and biologically heterogeneous. While various prognostic features have been proposed, none currently impact therapy selection, particularly in older patients, for whom treatment is primarily dictated by age and comorbidities. Herein, we undertook a comprehensive comparison of clinicopathological features in a cohort of patients 60 years and older, uniformly treated with bendamustine and rituximab, with a median survival of >8 years. The strongest prognostic indicators in this cohort were a high-risk call by a simplified MCL international prognostic index (s-MIPI) (HR: 3.32, 95% CI: 1.65-6.68 compared to low risk), a high-risk call by MCL35 (HR: 10.34, 95% CI: 2.37-45.20 compared to low risk) and blastoid cytology (HR: 4.21, 95% CR: 1.92-9.22 compared to classic). Patients called high risk by both the s-MIPI and MCL35 had the most dismal prognosis (HR: 11.58, 95% CI: 4.10-32.72), while those with high risk by either had a moderate but clinically relevant prognosis (HR: 2.95, 95% CI: 1.49-5.82). A robust assay to assess proliferation, such as MCL35, along with stringent guidelines for cytological evaluation of MCL, in combination with MIPI, may be a strong path to risk-stratify older MCL patients in future clinical trials.

Characterization and clinical impact of the tumor microenvironment in post-transplant aggressive B-cell lymphomas.

Post-transplant lymphoproliferative disorders (PTLD) are iatrogenic immune deficiency-associated lymphoid/plasmacytic proliferations developing due to immunosuppression in solid organ or hematopoietic stem cell allograft patients. PTLD are characterized by abnormal proliferation of lymphoid cells and have a heterogeneous clinical behavior. We profiled expression of >700 tumor microenvironment (TME)-related genes in 75 post-transplant aggressive B-cell lymphomas (PTABCL). Epstein-Barr virus (EBV)-positive PT-ABCL clustered together and were enriched for type I interferon pathway and antiviral-response genes. Additionally, a cytotoxicity gene signature associated with EBV-positivity and favorable overall survival (OS) (hazard ratio =0.61; P=0.019). In silico immunophenotyping revealed two subgroups with distinct immune cell compositions. The inflamed subgroup with higher proportions of immune cells had better outcome compared to noninflamed subgroup (median OS >200.0 vs. 15.2 months; P=0.006). In multivariable analysis with EBV status, International Prognostic Index, and rituximab-containing treatment, inflamed TME remained as an independent predictor for favorable outcome. We also compared TME between post-transplant and immunocompetent host diffuse large B-cell lymphomas (n=75) and discovered that the proportions of T cells were lower in PT-diffuse large B-cell lymphomas. In conclusion, we provide a comprehensive phenotypic characterization of PT-ABCL, highlighting the importance of immune cell composition of TME in determining the clinical behavior and prognosis of PT-ABCL.

Early-phase clinical trial eligibility and response evaluation criteria for refractory, relapsed, or progressive neuroblastoma: A consensus statement from the National Cancer Institute Clinical Trials Planning Meeting.

BACKGROUND: International standardized criteria for eligibility, evaluable disease sites, and disease response assessment in patients with refractory, progressive, or relapsed high-risk neuroblastoma enrolled in early-phase clinical trials are lacking. METHODS: A National Cancer Institute-sponsored Clinical Trials Planning Meeting was convened to develop an international consensus to refine the tumor site eligibility criteria and evaluation of disease response for early-phase clinical trials in children with high-risk neuroblastoma. RESULTS: Standardized data collection of patient and disease characteristics (including specified genomic data), eligibility criteria, a definition of evaluable disease, and response evaluations for primary and metastatic sites of disease were developed. Eligibility included two distinct patient groups: progressive disease and refractory disease. The refractory disease group was subdivided into responding persistent disease and stable persistent disease to better capture the clinical heterogeneity of refractory neuroblastoma. Requirements for defining disease evaluable for a response assessment were provided; they included requirements for biopsy to confirm viable neuroblastoma and/or ganglioneuroblastoma in those patients with soft tissue or bone disease not avid for iodine-123 meta-iodobenzylguanidine. Standardized evaluations for response components and time intervals for response evaluations were established. CONCLUSIONS: The use of international consensus eligibility, evaluability, and response criteria for early-phase clinical studies will facilitate the collection of comparable data across international trials and promote more rapid identification of effective treatment regimens for high-risk neuroblastoma.

Immune cell constitution in the tumor microenvironment predicts the outcome in diffuse large B-cell lymphoma.

The tumor microenvironment (TME) and limited immune surveillance play important roles in lymphoma pathogenesis. Here we aimed to characterize immunological profiles of diffuse large B-cell lymphoma (DLBCL) and predict the outcome in response to immunochemotherapy. We profiled the expression of 730 immune-related genes in tumor tissues of 81 patients with DLBCL utilizing the Nanostring platform, and used multiplex immunohistochemistry to characterize T-cell phenotypes, including cytotoxic T cells (CD8, Granzyme B, OX40, Ki67), T-cell immune checkpoint (CD3, CD4, CD8, PD1, TIM3, LAG3), as well as regulatory T-cells and Th1 effector cells (CD3, CD4, FOXP3, TBET) in 188 patients. We observed a high degree of heterogeneity at the transcriptome level. Correlation matrix analysis identified gene expression signatures with highly correlating genes, the main cluster containing genes for cytolytic factors, immune checkpoint molecules, T cells and macrophages, together named a TME immune cell signature. Immunophenotyping of the distinct cell subsets revealed that a high proportion of immune checkpoint positive T cells translated to unfavorable survival. Together, our results demonstrate that the immunological profile of DLBCL TME is heterogeneous and clinically meaningful. This highlights the potential impact of T-cell immune checkpoint in regulating survival and resistance to immunochemotherapy. (Registered at clinicaltrials.gov identifiers: NCT01502982 and NCT01325194.)

Prognostic implications of the microenvironment for follicular lymphoma under immunomodulation therapy.

Follicular lymphoma (FL) constitutes a significant proportion of lymphomas and shows frequent relapses. Beyond conventional chemotherapy, new therapeutic approaches have emerged, focussing on the interplay between lymphoma cells and the microenvironment. Here we report the immunophenotypic investigation of the microenvironment of a clinically well-characterized prospective cohort (study SAKK35/10, NCT01307605) of 154 treatment-naïve FL patients in need of therapy, who have been treated with rituximab only or a combination of rituximab and the immunomodulatory drug lenalidomide/Revlimid® A high ratio of CD4- to CD8-positive T cells (P = 0·009) and increased amounts of PD1+ tumour-infiltrating T cells (P = 0·007) were associated with inferior progression-free survival in the whole cohort. Interestingly, the prognostic impact of PD1+ T cells and the CD4/CD8 ratio lost its significance in the subgroup treated with R2 . In the latter group, high amounts of GATA3+ T helper (Th2) equivalents were associated with better progression-free survival (P < 0·001). We identified tumour microenvironmental features that allow prognostic stratification with respect to immuno- and combined immuno- and immunomodulatory therapy. Our analysis indicates that lenalidomide may compensate the adverse prognostic implication of higher amounts of CD4+ and, particularly, PD1+ T cells and that it has favourable effects mainly in cases with higher amounts of Th2 equivalents. [Correction added on 11 February 2020, after online publication: The NCT-trial number was previously incorrect and has been updated in this version].

Molecularly matched therapy in the context of sensitivity, resistance, and safety; patient outcomes in end-stage cancer - the MetAction study.

Background: In precision cancer medicine, the challenge is to prioritize DNA driver events, account for resistance markers, and procure sufficient information for treatment that maintains patient safety. The MetAction project, exploring how tumor molecular vulnerabilities predict therapy response, first established the required workflow for DNA sequencing and data interpretation (2014-2015). Here, we employed it to identify molecularly matched therapy and recorded outcome in end-stage cancer (2016-2019).Material and methods: Metastatic tissue from 26 patients (16 colorectal cancer cases) was sequenced by the Oncomine assay. The study tumor boards interpreted called variants with respect to sensitivity or resistance to matched therapy and recommended single-agent or combination treatment if considered tolerable. The primary endpoint was the rate of progression-free survival 1.3-fold longer than for the most recent systemic therapy. The objective response rate and overall survival were secondary endpoints.Results: Both common and rare actionable alterations were identified. Thirteen patients were found eligible for therapy following review of tumor sensitivity and resistance variants and patient tolerability. The interventions were inhibitors of ALK/ROS1-, BRAF-, EGFR-, FGFR-, mTOR-, PARP-, or PD-1-mediated signaling for 2-3 cases each. Among 10 patients who received treatment until radiologic evaluation, 6 (46% of the eligible cases) met the primary endpoint. Four colorectal cancer patients (15% of the total study cohort) had objective response. The only serious adverse event was a transient colitis, which appeared in 1 of the 2 patients given PD-1 inhibitor with complete response. Apart from those two, overall survival was similar for patients who did and did not receive study treatment.Conclusions: The systematic MetAction approach may point forward to a refined framework for how to interpret the complexity of sensitivity versus resistance and patient safety that resides in tumor sequence data, for the possibly improved outcome of precision cancer medicine in future studies. ClinicalTrials.gov, identifier: NCT02142036.

Distinct patterns of B-cell receptor signaling in non-Hodgkin lymphomas identified by single-cell profiling.

Kinases downstream of B-cell antigen receptor (BCR) represent attractive targets for therapy in non-Hodgkin lymphoma (NHL). As clinical responses vary, improved knowledge regarding activation and regulation of BCR signaling in individual patients is needed. Here, using phosphospecific flow cytometry to obtain malignant B-cell signaling profiles from 95 patients representing 4 types of NHL revealed a striking contrast between chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) tumors. Lymphoma cells from diffuse large B-cell lymphoma patients had high basal phosphorylation levels of most measured signaling nodes, whereas follicular lymphoma cells represented the opposite pattern with no or very low basal levels. MCL showed large interpatient variability in basal levels, and elevated levels for the phosphorylated forms of AKT, extracellular signal-regulated kinase, p38, STAT1, and STAT5 were associated with poor outcome. CLL tumors had elevated basal levels for the phosphorylated forms of BCR-signaling nodes (Src family tyrosine kinase, spleen tyrosine kinase [SYK], phospholipase Cγ), but had low α-BCR-induced signaling. This contrasted MCL tumors, where α-BCR-induced signaling was variable, but significantly potentiated as compared with the other types. Overexpression of CD79B, combined with a gating strategy whereby signaling output was directly quantified per cell as a function of CD79B levels, confirmed a direct relationship between surface CD79B, immunoglobulin M (IgM), and IgM-induced signaling levels. Furthermore, α-BCR-induced signaling strength was variable across patient samples and correlated with BCR subunit CD79B expression, but was inversely correlated with susceptibility to Bruton tyrosine kinase (BTK) and SYK inhibitors in MCL. These individual differences in BCR levels and signaling might relate to differences in therapy responses to BCR-pathway inhibitors.

Heterogeneous MYCN amplification in neuroblastoma: a SIOP Europe Neuroblastoma Study.

BACKGROUND: In neuroblastoma (NB), the most powerful prognostic marker, the MYCN amplification (MNA), occasionally shows intratumoural heterogeneity (ITH), i.e. coexistence of MYCN-amplified and non-MYCN-amplified tumour cell clones, called heterogeneous MNA (hetMNA). Prognostication and therapy allocation are still unsolved issues. METHODS: The SIOPEN Biology group analysed 99 hetMNA NBs focussing on the prognostic significance of MYCN ITH. RESULTS: Patients <18 months (18 m) showed a better outcome in all stages as compared to older patients (5-year OS in localised stages: <18 m: 0.95 ± 0.04, >18 m: 0.67 ± 0.14, p = 0.011; metastatic: <18 m: 0.76 ± 0.15, >18 m: 0.28 ± 0.09, p = 0.084). The genomic 'background', but not MNA clone sizes, correlated significantly with relapse frequency and OS. No relapses occurred in cases of only numerical chromosomal aberrations. Infiltrated bone marrows and relapse tumour cells mostly displayed no MNA. However, one stage 4s tumour with segmental chromosomal aberrations showed a homogeneous MNA in the relapse. CONCLUSIONS: This study provides a rationale for the necessary distinction between heterogeneous and homogeneous MNA. HetMNA tumours have to be evaluated individually, taking age, stage and, most importantly, genomic background into account to avoid unnecessary upgrading of risk/overtreatment, especially in infants, as well as in order to identify tumours prone to developing homogeneous MNA.

The MCL35 gene expression proliferation assay predicts high-risk MCL patients in a Norwegian cohort of younger patients given intensive first line therapy.

Patients with mantle cell lymphoma (MCL) generally have a dismal prognosis. Intensified induction treatment with rituximab and high dose cytarabine (R_HDAC), and consolidation with high-dose therapy with autologous stem cell support has resulted in 10-year overall survival (OS) higher than 60%. However, the clinical course varies. Diagnostic tools capable of stratifying patients include the MCL International Prognostic Index (MIPI), gene expression-based proliferation signature, Ki-67 proliferation index or tumour cell morphology. Here, we tested the performance of a newly developed Nanostring-based RNA expression-based proliferation assay (MCL35) on formalin-fixed paraffin-embedded tumour tissue from younger patients recruited in or treated according to Nordic MCL protocols compared to the prognosticators listed above. Seventy-four patients were included and the assay performed well in all cases except four, which had inadequate RNA quality. The patients were evenly distributed in the MCL35 low-, intermediate- and high-risk categories. MCL35 low- and intermediate- risk groups had overlapping progression-free survival (PFS), while patients in the high-risk category had significantly inferior PFS. Combining MCL35 with MIPI or the MIPI-C (MIPI with the addition of binary Ki67 score +/-30%) showed a better discrimination than either assessment alone. In conclusion, the MCL35 assay alone or combined with MIPI or MIPI-C scores can identify patients who still have a dismal outcome despite intensified treatment.

Efficacy of the Nordic and the MSKCC chemotherapy protocols on the overall and progression-free survival in intracranial PCNSL.

BACKGROUND: To compare the Nordic and the Memorial Sloan-Kettering Cancer Center (MSKCC) chemotherapy protocols for Overall Survival (OS) and Progression-Free Survival (PFS) for intracranial primary CNS lymphoma (PCNSL). METHODS: A prospective database at Oslo University Hospital of PCNSL was reviewed over a 12-year period (2003-2014). RESULTS: Overall, 79 patients with PCNSL were identified, of whom 57 received chemotherapy. MSKCC with Rituximab (RTX) was used in 18 patients (32%) who had median OS of 46.3 months [9.8-131.9] and median PFS of 34.6 months [6.4-131.9]. The Nordic protocol was used in 14 patients (25%) who had median OS of 30.9 months [2.7-106.3] and PFS of 14.3 months [0.0-106.3]. The MSKCC was used without RTX in 25 patients (44%) who had OS of 15.2 months [0.7-136.5] and PFS of 12.0 months [0.0-117.0]. MSKCC with RTX had a significantly longer median OS (p < 0.05) compared to the other regimens in univariate analysis. In multivariate analysis, the only prognostic factor for OS and PFS of significance was deep brain involvement (p < 0.005). CONCLUSIONS: In univariate analysis, the MSKCC with RTX achieved significantly longer median OS compared to the Nordic protocol. However, in multivariate analysis, the only prognostic factor for survival of statistical significance was deep brain involvement.

Is deep brain involvement in intracranial primary central nervous system lymphoma of importance for penetration of chemotherapeutic agents?

PURPOSE: The purposes of this study are to study the impact of deep brain involvement on overall survival (OS) and progression-free survival (PFS) in intracranial primary CNS lymphoma (PCNSL), and to explore possible mechanisms for this impact using advanced MRI techniques. METHODS: Seventy-nine patients with histologically verified PCNSL were identified from a prospective clinical database of patients treated at Oslo University Hospital between 2003 and 2014. Patients were treated per standard chemotherapeutic regimens (N = 57) or no chemotherapy (N = 22). Anatomical MRIs were available in all patients to assess tumor load and location based on contrast agent enhancement visible on T1-weighted images. Diffusion MRIs were available in 33 (42%) patients and perfusion MRI in 13 (16%) patients that received active treatment. RESULTS: Across all patients, OS and PFS were 16.4 and 9.8 months, respectively. In multivariate analysis, MRI-based deep brain involvement (80%) was the only negative significant factor of OS (OR = 14.2; P < 0.005). While a reduced Karnofsky performance status was associated with deep brain involvement (P < 0.05), neither chemotherapy regimen, neurologic status, nor patient age were independent significant factors for OS or PFS in this setting. Compared to other tumors and healthy tissue levels, MRI perfusion showed more pathologic hemodynamic flow signatures in tumors with deep brain involvement. CONCLUSION: In intracranial PCNSL, the only significant prognostic factor for OS and PFS in multivariate analysis was age and deep brain involvement. While contingent on a small study sample, we hypothesize this may in part be explained by regional differences in vascular supply and delivery from a dysfunctional perfusion signature.

The role of surgery in intracranial PCNSL.

This aimed to evaluate the effect of surgery for overall survival (OS) and progression-free survival (PFS) in intracranial primary CNS lymphoma (PCNSL) of all patients diagnosed at a single center. A prospective database at Oslo University Hospital of PCNSL was reviewed over a 12-year period (2003-2014). Seventy-nine patients with intracranial PCNSL were identified. Deep brain involvement was shown in 63 patients. Thirty-two patients underwent craniotomy with resection, while all other patients had a biopsy. Fifty-seven patients were given chemotherapy: 18 were treated with the MSKCC (Memorial Sloan-Kettering Cancer Center) with rituximab, 21 with the MSKCC without rituximab, and 14 within a Nordic prospective phase II protocol. Forty-four patients achieved complete response (CR) and had OS of 46.3 months. Patients who underwent resection had a median OS of 28.6 versus 11.7 months for those who had a biopsy performed. Resection showed an insignificant prolongation of OS. Multivariate analysis confirmed statistical significance of deep brain involvement only (p < 0.005). Neither chemotherapy regimen, Karnofsky Performance Status (KPS), type of surgery, nor patient age was significant factors for OS or PFS. Resective surgery played no role in significantly improving either OS or PFS and therefore it is not recommended as treatment for PCNSL.

Recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma on behalf of the International Neuroblastoma Response Criteria Bone Marrow Working Group.

BACKGROUND: The current study was conducted to expedite international standardized reporting of bone marrow disease in children with neuroblastoma and to improve equivalence of care. METHODS: A multidisciplinary International Neuroblastoma Response Criteria Bone Marrow Working Group was convened by the US National Cancer Institute in January 2012 with representation from Europe, North America, and Australia. Practical transferable recommendations to standardize the reporting of bone marrow disease were developed. RESULTS: To the authors' knowledge, the current study is the first to comprehensively present consensus criteria for the collection, analysis, and reporting of the percentage area of bone marrow parenchyma occupied by tumor cells in trephine-biopsies. The quantitative analysis of neuroblastoma content in bone marrow aspirates by immunocytology and reverse transcriptase-quantitative polymerase chain reaction are revised. The inclusion of paired-like homeobox 2b (PHOX2B) for immunohistochemistry and reverse transcriptase-quantitative polymerase chain reaction is recommended. Recommendations for recording bone marrow response are provided. The authors endorse the quantitative assessment of neuroblastoma cell content in bilateral core needle biopsies-trephines and aspirates in all children with neuroblastoma, with the exception of infants, in whom the evaluation of aspirates alone is advised. It is interesting to note that 5% disease is accepted as an internationally achievable level for disease assessment. CONCLUSIONS: The quantitative assessment of neuroblastoma cells is recommended to provide data from which evidence-based numerical criteria for the reporting of bone marrow response can be realized. This is particularly important in the minimal disease setting and when neuroblastoma detection in bone marrow is intermittent, where clinical impact has yet to be validated. The wide adoption of these harmonized criteria will enhance the ability to compare outcomes from different trials and facilitate collaborative trial design. Cancer 2017;123:1095-1105. © 2016 American Cancer Society.

Folliculotropic Mycosis Fungoides with Skewed T-cell Receptor CDR3 Motif: Suggestive of Lipid-antigen Selection?

Folliculotropic mycosis fungoides (FMF), a variant of mycosis fungoides (MF) with distinct clinical features, is characterized by infiltration of malignant T cells in hair follicles. This raises the hypothesis that antigens in the hair follicle may contribute to the pathogenesis of FMF. T-cell receptor ß gene (TRB) sequences as well as dendritic cell subsets in patients with FMF (n = 21) and control patients with MF (n = 20) were studied to explore this hypothesis. A recurrent usage of the TRB junctional genes TRBJ2-1 and TRBJ2-7 was found in patients with FMF compared with those with MF. These genes contribute to an amino acid motif in the complementarity-determining region 3 (CDR3) of the T-cell receptor. This motif was previously found in T cells stimulated by lipids bound to CD1 on antigen-presenting cells. Additional immunohistochemical analysis revealed abundant CD1c- and CD1a- expressing dendritic cells in FMF. The combined findings support a role for lipid-antigen stimulation in FMF.

The tumour microenvironment influences survival and time to transformation in follicular lymphoma in the rituximab era.

The tumour microenvironment influences outcome in patients with follicular lymphoma (FL), but its impact on transformation is less studied. We investigated the prognostic significance of the tumour microenvironment on transformation and survival in FL patients treated in the rituximab era. We examined diagnostic and transformed biopsies from 52 FL patients using antibodies against CD3, CD4, CD8, CD21 (CR2), CD57 (B3GAT1), CD68, FOXP3, TIA1, PD-1 (PDCD1), PD-L1 (CD274) and PAX5. Results were compared with a second cohort of 40 FL patients without signs of transformation during a minimum of five years observation time. Cell numbers and localization were semi-quantitatively assessed. Better developed CD21+  follicular dendritic cell (FDC) meshworks at diagnosis was a negative prognostic factor for overall survival (OS), progression-free survival (PFS) and time to transformation (TTT) in patients with subsequently transformed FL. Remnants of FDC meshworks at transformation were associated with shorter OS and PFS from transformation. High degrees of intrafollicular CD68+ and PD-L1+  macrophage infiltration, high total area scores and an extrafollicular/diffuse pattern of FOXP3+  T cells and high intrafollicular scores of CD4+  T cells at diagnosis were associated with shorter TTT. Scores of several T-cell subset markers from the combined patient cohorts were predictive for transformation, especially CD4 and CD57.

Primary cold agglutinin-associated lymphoproliferative disease: a B-cell lymphoma of the bone marrow distinct from lymphoplasmacytic lymphoma.

Primary chronic cold agglutinin disease is a rare hemolytic disease mediated by monoclonal IGHV4-34-encoded cold agglutinins with a predominant specificity for the blood group antigen I. Bone marrow from 54 patients was studied to type the underlying lymphoproliferative disorder better. Bone marrow biopsies showed circumscribed intra-parenchymatous nodules with small monotonous monoclonal B cells in 40/54 patients (median infiltration: 10% of marrow cells) with a CD20(+), IgMs(+), IgDs(+), CD27(+), CD5(-/+), CD11c(-), CD23(-), CD38(-) immunophenotype. Neither plasmacytoid cytological features nor expression of plasma cell differentiation-associated transcription factors MUM1, XBP1 and BLIMP1 were noted in these B cells. However, a limited number of mature monoclonal IgM(+), IgD(-) plasma cells were present outside the lymphoid nodules and were diffusely scattered throughout the marrow. Of interest, the MYD88 L265P mutation, typical of lymphoplasmacytic lymphoma, was not detected (17/17 cases). Somatically mutated monoclonal IGHV4-34 gene rearrangement was demonstrated in eight patients with frozen samples (mean sequence homology 95.4%). However, mutations of BCL6 intron 1 were not demonstrated, except in one patient, suggesting that the lymphoma cells had not matured in the germinal center. In conclusion, cold agglutinin-associated lymphoproliferative disease displays homogeneous histological and immunophenotypic features. The absence of plasmacytoid cells, the presence of plasma cells predominantly outside the nodular lymphoid infiltrates, IGHV4-34 restriction and absence of MYD88 L265P mutation strongly suggest that cold agglutinin-associated lymphoproliferative disease is a distinct entity that is different from lymphoplasmacytic lymphoma.

Correction: Whole exome sequencing of high-risk neuroblastoma identifies novel non-synonymous variants.

[This corrects the article DOI: 10.1371/journal.pone.0273280.].

Inflammatory and subtype-dependent serum protein signatures predict survival beyond the ctDNA in aggressive B cell lymphomas.

BACKGROUND: Biological heterogeneity of large B cell lymphomas (LBCLs) is poorly captured by current prognostic tools, hampering optimal treatment decisions. METHODS: We dissected the levels of 1,463 serum proteins in a uniformly treated trial cohort of 109 patients with high-risk primary LBCL (ClinicalTrials.gov: NCT01325194) and correlated the profiles with molecular data from tumor tissue and circulating tumor DNA (ctDNA) together with clinical data. FINDINGS: We discovered clinically and biologically relevant associations beyond established clinical estimates and ctDNA. We identified an inflamed serum protein profile, which reflected host response to lymphoma, associated with inflamed and exhausted tumor microenvironment features and high ctDNA burden, and translated to poor outcome. We composed an inflammation score based on the identified inflammatory proteins and used the score to predict survival in an independent LBCL trial cohort (ClinicalTrials.gov: NCT03293173). Furthermore, joint analyses with ctDNA uncovered multiple serum proteins that correlate with tumor burden. We found that SERPINA9, TACI, and TARC complement minimally invasive subtype profiling and that TACI and TARC can be used to evaluate treatment response in a subtype-dependent manner in the liquid biopsy. CONCLUSIONS: Altogether, we discovered distinct serum protein landscapes that dissect the heterogeneity of LBCLs and provide agile, minimally invasive tools for precision oncology. FUNDING: This research was funded by grants from the Research Council of Finland, Finnish Cancer Organizations, Sigrid Juselius Foundation, University of Helsinki, iCAN Digital Precision Cancer Medicine Flagship, Orion Research Foundation sr, and Helsinki University Hospital.

Multi-omics profiling of longitudinal samples reveals early genomic changes in follicular lymphoma.

Follicular lymphoma (FL) is the most common indolent type of B-cell non-Hodgkin lymphoma. Advances in treatment have improved overall survival, but early relapse or transformation to aggressive disease is associated with inferior outcome. To identify early genetic events and track tumor clonal evolution, we performed multi-omics analysis of 94 longitudinal biopsies from 44 FL patients; 22 with transformation (tFL) and 22 with relapse without transformation (nFL). Deep whole-exome sequencing confirmed recurrent mutations in genes encoding epigenetic regulators (CREBBP, KMT2D, EZH2, EP300), with similar mutational landscape in nFL and tFL patients. Calculation of genomic distances between longitudinal samples revealed complex evolutionary patterns in both subgroups. CREBBP and KMT2D mutations were identified as genetic events that occur early in the disease course, and cases with CREBBP KAT domain mutations had low risk of transformation. Gains in chromosomes 12 and 18 (TCF4), and loss in 6q were identified as early and stable copy number alterations. Identification of such early and stable genetic events may provide opportunities for early disease detection and disease monitoring. Integrative analysis revealed that tumors with EZH2 mutations exhibited reduced gene expression of numerous histone genes, including histone linker genes. This might contribute to the epigenetic dysregulation in FL.