Detection of Second Primary Lymphoma in Late Diffuse Large B-cell Lymphoma Recurrences.

Approximately one-third of patients with diffuse large B-cell lymphoma (DLBCL) relapse and often require salvage chemotherapy followed by autologous stem cell transplantation. In most cases, the clonal relationship between the first diagnosis and subsequent relapse is not assessed, thereby potentially missing the identification of second primary lymphoma. In this study, the clonal relationship of 59 paired DLBCL diagnoses and recurrences was established by next-generation sequencing-based detection of immunoglobulin gene rearrangements. Among 50 patients with interpretable results, 43 patients (86%) developed clonally related relapsed disease. This was observed in 100% of early recurrences (<2 years), 80% of the recurrences with an interval between 2 and 5 years, and 73% of late recurrences (≥5 years). On the other hand, 7 (14%) out of 50 patients displayed different dominant clonotypes in primary DLBCL and clinical recurrences, confirming the occurrence of second primary DLBCL; 37% of DLBCL recurrences that occurred ≥4 years after diagnosis were shown to be second primary lymphomas. The clonally unrelated cases were Epstein-Barr virus positive in 43% of the cases, whereas this was only 5% in the relapsed DLBCL cases. In conclusion, next-generation sequencing-based clonality testing in late recurrences should be considered in routine diagnostics to distinguish relapse from second primary lymphoma, as this latter group of patients with DLBCL may benefit from less-intensive treatment strategies.

Reclassification of diffuse large B cell lymphoma to large B cell lymphoma with IRF4 rearrangement in an adult population.

AIMS: Large B cell lymphoma with IRF4 rearrangement (LBCL-IRF4) is a new entity in the 2017 revised World Health Organisation (WHO) classification that was initially mainly reported in children. After identification of a 79-year-old patient, we assessed how often IRF4 rearrangements can be detected in adult diffuse large B cell lymphomas (DLBCLs) which have to be reclassified to LBCL-IRF4 based on fluorescence in-situ hybridisation (FISH) for IRF4. METHODS AND RESULTS: With FISH, we studied the presence of IRF4 rearrangements in 238 lymphomas that were diagnosed as DLBCL according to the previous WHO classification of 2008. CONCLUSIONS: In addition to the index patient, an IRF4 rearrangement was detected in another five of 237 patients (2%). The immunohistochemical profile of these five IRF4 rearranged lymphomas was consistent with previous reports of LBCL-IRF4. One case was recognised to represent transformation of follicular lymphoma rather than de-novo LBCL-IRF4. BCL6 rearrangements were found in two cases of LBCL-IRF4; BCL2 and MYC rearrangements were excluded. Patients presented with limited stage disease with involvement of the head and neck in three patients, and involvement of the lung and thyroid in two others. This study shows that, although rare, LBCL-IRF4 should also be considered in older patients and at localisations other than the head and neck region.

Persistent symptoms of fatigue, neuropathy and role-functioning impairment among indolent non-Hodgkin lymphoma survivors: A longitudinal PROFILES registry study.

Little is known about the long-term health-related quality of life (HRQoL) and persistence of symptoms among patients with indolent non-Hodgkin lymphoma (iNHL). This large population-based longitudinal study therefore investigated the long-term HRQoL and persistence of symptoms and identified associated sociodemographic, clinical and psychological factors. Patients diagnosed between 1999 and 2014 and four or more months after diagnosis were invited to participate in a longitudinal survey. Sociodemographic and clinical data were obtained from the Netherlands Cancer Registry. The EORTC QLQ-C30 and CLL-16 were completed by 669 patients (74% response rate). Patients completed on average four questionnaires. Primary treatment was active surveillance (52%), systemic therapy (31%) or radiotherapy (13%). Respectively, 36% reported persistent fatigue, 33% persistent neuropathy and 25% persistent role-functioning impairment. This was 2-3 times higher than in the age- and sex-matched normative population. Up to 10 years after diagnosis, scores remained relatively stable without clinically relevant changes. Comorbidities, psychological distress, shorter time since diagnosis, systemic therapy, younger age, education level and having no partner were associated with worse outcomes (all ps < 0.05). Up to a third of patients with iNHL experience long-term persistent symptoms which do not improve over time. Early recognition of symptoms will help in providing tailored supportive care for those in need.

Rituximab in patients with primary CNS lymphoma (HOVON 105/ALLG NHL 24): a randomised, open-label, phase 3 intergroup study.

BACKGROUND: The prognosis for primary CNS lymphoma has improved with the use of high-dose methotrexate-based chemotherapy, but patient outcomes remain poor. Rituximab, a chimeric monoclonal antibody that targets the CD20 cell surface protein, has substantial activity in systemic CD20-positive diffuse large B-cell lymphoma, but its efficacy in primary CNS lymphoma is unknown and low penetration of the large rituximab molecule through the blood-brain barrier could limit its effect. We aimed to investigate the addition of rituximab to a high-dose methotrexate-based chemotherapy regimen in patients with newly diagnosed primary CNS lymphoma. METHODS: This intergroup, multicentre, open-label, randomised phase 3 study was done at 23 hospitals in the Netherlands, Australia, and New Zealand. Non-immunocompromised patients aged 18-70 years with newly diagnosed primary CNS lymphoma were randomly assigned (1:1) to receive methotrexate-based chemotherapy with or without intravenous rituximab. We used a web-based randomisation system with stratification by centre, age, and Eastern Cooperative Oncology Group-WHO performance status, and a minimisation procedure. All group assignment was open label and neither investigators nor patients were masked to allocation. All patients were treated with two 28-day cycles of induction chemotherapy, consisting of intravenous methotrexate 3 g per m2 on days 1 and 15, intravenous carmustine 100 mg per m2 on day 4, intravenous teniposide 100 mg per m2 on days 2 and 3, and oral prednisone 60 mg per m2 on days 1-5, with (R-MBVP) or without (MBVP) intravenous rituximab 375 mg per m2 on days 0, 7, 14, and 21 in cycle one and days 0 and 14 in cycle two. Patients with response at the end of induction subsequently received high-dose cytarabine and, in patients aged 60 years or younger, low-dose whole-brain radiotherapy. The primary endpoint was event-free survival, with events defined as not reaching complete response or complete response unconfirmed at the end of treatment, or progression or death after response; analysis was adjusted for age and performance score. Patients were analysed on a modified intention-to-treat basis. This trial is registered with the Nederlands Trial Register, number NTR2427, and the Australian New Zealand Clinical Trials Registry, number ACTRN12610000908033. The trial was closed on May 27, 2016, after achieving complete accrual, and follow-up is ongoing. FINDINGS: Between Aug 3, 2010, and May 27, 2016, we recruited 200 patients (109 men and 91 women; median age was 61 years [IQR 55-67]). We randomly assigned 100 patients to MBVP and 99 patients to R-MBVP. One patient was randomly assigned to the R-MBVP group but found to be ineligible because of an incorrect diagnosis and was excluded from all analyses. After a median follow-up of 32·9 months (IQR 23·9-51·5), 98 patients had had an event (51 in the MBVP group and 47 in the R-MBVP group), of whom 79 had died (41 in the MBVP group and 38 in the R-MBVP group). Event-free survival at 1 year was 49% (95% CI 39-58) in the MBVP group (no rituximab) and 52% (42-61) in the R-MBVP group (with rituximab; hazard ratio 1·00, 95% CI 0·70-1·43, p=0·99). Grade 3 or 4 adverse events occurred in 58 (58%) patients in the MBVP group and 63 (64%) patients in the R-MBVP group, with infections (24 [24%] patients receiving MBVP vs 21 [21%] patients receiving R-MBVP), haematological toxicity (15 [15%] vs 12 [12%]), and nervous system disorders (ten [10%] vs 15 [15%]) being the most common. Life-threatening or fatal serious adverse events occurred in 12 (12%) patients in the MBVP group and ten (10%) patients in the R-MBVP group, and five (5%) patients in the MBVP group and three (3%) in the R-MBVP group died from treatment-related causes. INTERPRETATION: We found no clear benefit of addition of rituximab to methotrexate, carmustine, teniposide, and prednisone chemotherapy in primary CNS lymphoma. Therefore, the results of this study do not support the use of rituximab as a component of standard treatment in primary CNS lymphoma. FUNDING: Roche, the Dutch Cancer Society, and Stichting STOPhersentumoren.

Recurrent mutations in genes involved in nuclear factor-κB signalling in nodal marginal zone lymphoma-diagnostic and therapeutic implications.

AIMS: To investigate the spectrum of mutations in 20 genes involved in B-cell receptor and/or Toll-like receptor signalling resulting in activation of nuclear factor-κB (NF-κB) in 20 nodal marginal zone lymphomas (NMZLs), 20 follicular lymphomas (FLs), and 11 cases of B-cell lymphoma, unclassifiable (BCL-u). METHODS AND RESULTS: Nodal marginal zone lymphomas were diagnosed according to strict criteria, including the expression of at least one putative marginal zone marker (MNDA and/or IRTA1). Cases that showed features of NMZL but did not fulfil all criteria were included as BCL-u. All FLs were required to have a BCL2 rearrangement. Mutations were found in: nine NMZLs, with recurrent mutations in TNFAIP3 and CD79B; 12 FLs, with recurrent mutations in TNFRSF14, TNFAIP3, and CARD11; and five cases of BCL-u, with recurrent mutations in TNFRSF14. TNFRSF14 mutations were present in FL and BCL-u, but not in any of the NMZLs. In the BCL-u group, TNFRSF14 mutations clustered with a FL immunophenotype. CONCLUSIONS: These results suggest that TNFRSF14 mutations point towards a diagnosis of FL, and can be used in the sometimes difficult distinction between NMZL and FL, but to apply this in diagnostics would require confirmation in an independent cohort. In addition, the presence or absence of specific mutations in pathways converging on NF-κB could be important for decisions regarding targeted treatment.

Prognostic relevance of CD163 and CD8 combined with EZH2 and gain of chromosome 18 in follicular lymphoma: a study by the Lunenburg Lymphoma Biomarker Consortium.

In follicular lymphoma, studies addressing the prognostic value of microenvironment-related immunohistochemical markers and tumor cell-related genetic markers have yielded conflicting results, precluding implementation in practice. Therefore, the Lunenburg Lymphoma Biomarker Consortium performed a validation study evaluating published markers. To maximize sensitivity, an end of spectrum design was applied for 122 uniformly immunochemotherapy-treated follicular lymphoma patients retrieved from international trials and registries. The criteria were: early failure, progression or lymphoma-related death <2 years versus long remission, response duration of >5 years. Immunohistochemical staining for T cells and macrophages was performed on tissue microarrays from initial biopsies and scored with a validated computer-assisted protocol. Shallow whole-genome and deep targeted sequencing was performed on the same samples. The 96/122 cases with complete molecular and immunohistochemical data were included in the analysis. EZH2 wild-type (P=0.006), gain of chromosome 18 (P=0.002), low percentages of CD8+ cells (P=0.011) and CD163+ areas (P=0.038) were associated with early failure. No significant differences in other markers were observed, thereby refuting previous claims of their prognostic significance. Using an optimized study design, this Lunenburg Lymphoma Biomarker Consortium study substantiates wild-type EZH2 status, gain of chromosome 18, low percentages of CD8+ cells and CD163+ area as predictors of early failure to immunochemotherapy in follicular lymphoma treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP [-like]), while refuting the prognostic impact of various other markers.

'Trained immunity': consequences for lymphoid malignancies.

In hematological malignancies complex interactions exist between the immune system, microorganisms and malignant cells. On one hand, microorganisms can induce cancer, as illustrated by specific infection-induced lymphoproliferative diseases such as Helicobacter pylori-associated gastric mucosa-associated lymphoid tissue lymphoma. On the other hand, malignant cells create an immunosuppressive environment for their own benefit, but this also results in an increased risk of infections. Disrupted innate immunity contributes to the neoplastic transformation of blood cells by several mechanisms, including the uncontrolled clearance of microbial and autoantigens resulting in chronic immune stimulation and proliferation, chronic inflammation, and defective immune surveillance and anti-cancer immunity. Restoring dysfunction or enhancing responsiveness of the innate immune system might therefore represent a new angle for the prevention and treatment of hematological malignancies, in particular lymphoid malignancies and associated infections. Recently, it has been shown that cells of the innate immune system, such as monocytes/macrophages and natural killer cells, harbor features of immunological memory and display enhanced functionality long-term after stimulation with certain microorganisms and vaccines. These functional changes rely on epigenetic reprogramming and have been termed 'trained immunity'. In this review the concept of 'trained immunity' is discussed in the setting of lymphoid malignancies. Amelioration of infectious complications and hematological disease progression can be envisioned to result from the induction of trained immunity, but future studies are required to prove this exciting new hypothesis.

Survival, neurocognitive function, and health-related quality of life outcomes after rituximab-methotrexate, BCNU, teniposide, and prednisolone for primary CNS lymphoma: Final results of the HOVON 105/ALLG NHL 24 study.

BACKGROUND: Studies on the efficacy of rituximab in primary CNS lymphoma (PCNSL) reported conflicting results. Our international randomized phase 3 study showed that the addition of rituximab to high-dose methotrexate, BCNU, teniposide, and prednisolone (MBVP) in PCNSL was not efficacious in the short term. Here we present long-term results after a median follow-up of 82.3 months. METHODS: One hundred and ninety-nine eligible newly diagnosed, nonimmunocompromised patients with PCNSL aged 18-70 years with WHO performance status 0-3 was randomized between treatment with MBVP chemotherapy with or without rituximab, followed by high-dose cytarabine consolidation in responding patients, and reduced-dose WBRT in patients aged ≤ 60 years. Event-free survival was the primary endpoint. Overall survival rate, neurocognitive functioning (NCF), and health-related quality of life (HRQoL) were additionally assessed, with the IPCG test battery, EORTC QLQ-C30 and QLQ-BN20 questionnaires, respectively. RESULTS: For event-free survival, the hazard ratio was 0.85, 95% CI 0.61-1.18, P = .33. Overall survival rate at 5 years for MBVP and R-MBVP was 49% (39-59) and 53% (43-63) respectively. In total, 64 patients died in the MBVP arm and 55 in the R-MBVP arm, of which 69% were due to PCNSL. At the group level, all domains of NCF and HRQoL improved to a clinically relevant extent after treatment initiation, and remained stable thereafter up to 60 months of follow-up, except for motor speed which deteriorated between 24 and 60 months. Although fatigue improved initially, high levels persisted in the long term. CONCLUSIONS: Long-term follow-up confirms the lack of added value of rituximab in addition to MBVP and HD-cytarabine for PCNSL.

Genomic profiling of post-transplant lymphoproliferative disorders using cell-free DNA.

Diagnosing post-transplant lymphoproliferative disorder (PTLD) is challenging and often requires invasive procedures. Analyses of cell-free DNA (cfDNA) isolated from plasma is minimally invasive and highly effective for genomic profiling of tumors. We studied the feasibility of using cfDNA to profile PTLD and explore its potential to serve as a screening tool. We included seventeen patients with monomorphic PTLD after solid organ transplantation in this multi-center observational cohort study. We used low-coverage whole genome sequencing (lcWGS) to detect copy number variations (CNVs) and targeted next-generation sequencing (NGS) to identify Epstein-Barr virus (EBV) DNA load and somatic single nucleotide variants (SNVs) in cfDNA from plasma. Seven out of seventeen (41%) patients had EBV-positive tumors, and 13/17 (76%) had stage IV disease. Nine out of seventeen (56%) patients showed CNVs in cfDNA, with more CNVs in EBV-negative cases. Recurrent gains were detected for 3q, 11q, and 18q. Recurrent losses were observed at 6q. The fraction of EBV reads in cfDNA from EBV-positive patients was 3-log higher compared to controls and EBV-negative patients. 289 SNVs were identified, with a median of 19 per sample. SNV burden correlated significantly with lactate dehydrogenase levels. Similar SNV burdens were observed in EBV-negative and EBV-positive PTLD. The most commonly mutated genes were TP53 and KMT2D (41%), followed by SPEN, TET2 (35%), and ARID1A, IGLL5, and PIM1 (29%), indicating DNA damage response, epigenetic regulation, and B-cell signaling/NFkB pathways as drivers of PTLD. Overall, CNVs were more prevalent in EBV-negative lymphoma, while no difference was observed in the number of SNVs. Our data indicated the potential of analyzing cfDNA as a tool for PTLD screening and response monitoring.

Clonal Relationship and Mutation Analysis in Lymphoplasmacytic Lymphoma/Waldenström Macroglobulinemia Associated With Diffuse Large B-cell Lymphoma.

Patients with lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) occasionally develop diffuse large B-cell lymphoma (DLBCL). This mostly results from LPL/WM transformation, although clonally unrelated DLBCL can also arise. LPL/WM is characterized by activating MYD88L265P (>95%) and CXCR4 mutations (~30%), but the genetic drivers of transformation remain to be identified. Here, in thirteen LPL/WM patients who developed DLBCL, the clonal relationship of LPL and DLBCL together with mutations contributing to transformation were investigated. In 2 LPL/WM patients (15%), high-throughput sequencing of immunoglobulin gene rearrangements showed evidence of >1 clonal B-cell population in LPL tissue biopsies. In the majority of LPL/WM patients, DLBCL presentations were clonally related to the dominant clone in LPL, providing evidence of transformation. However, in 3 patients (23%), DLBCL was clonally unrelated to the major malignant B-cell clone in LPL, of which 2 patients developed de novo DLBCL. In this study cohort, LPL displayed MYD88L265P mutation in 8 out of eleven patients analyzed (73%), while CXCR4 mutations were observed in 6 cases (55%). MYD88WT LPL biopsies present in 3 patients (27%) were characterized by CD79B and TNFAIP3 mutations. Upon transformation, DLBCL acquired novel mutations targeting BTG1, BTG2, CD79B, CARD11, TP53, and PIM1. Together, we demonstrate variable clonal B-cell dynamics in LPL/WM patients developing DLBCL, and the occurrence of clonally unrelated DLBCL in about one-quarter of LPL/WM patients. Moreover, we identified commonly mutated genes upon DLBCL transformation, which together with preserved mutations already present in LPL characterize the mutational landscape of DLBCL occurrences in LPL/WM patients.

R-CODOX-M/R-IVAC versus DA-EPOCH-R in patients with newly diagnosed Burkitt lymphoma (HOVON/SAKK): final results of a multicentre, phase 3, open-label, randomised trial.

BACKGROUND: Patients with newly diagnosed high-risk Burkitt lymphoma are treated with high-intensity immune-chemotherapy regimens such as R-CODOX-M/R-IVAC or with lower-intensity regimens such as DA-EPOCH-R. The aim of this study was to make a formal comparison between these regimens. METHODS: This multicentre, phase 3, open-label, randomised study was done in 26 clinical centres in the Netherlands, Belgium, and Switzerland. Eligible patients were aged 18-75 years with newly diagnosed high-risk Burkitt lymphoma without CNS involvement. Patients were randomly assigned to two cycles of R-CODOX-M/R-IVAC (R-CODOX-M: rituximab 375 mg/m2 on day 1 and 9, cyclophosphamide 800 mg/m2 on day 1, cyclophosphamide 200 mg/m2 on days 2-5, vincristine 1·5 mg/m2 on days 1 and 8, doxorubicin 40 mg/m2 on day 1, and methotrexate 3000 mg/m2 on day 10; R-IVAC: rituximab 375 mg/m2 on days 3 and 7, iphosphamide 1500 mg/m2 on days 1-5, etoposide 60 mg/m2 on days 1-5, and cytarabin 2000 mg/m2 on day 1 and 2) or six cycles of DA-EPOCH-R (dose-adjusted etoposide 50-124 mg/m2 on days 1-4, prednisolone 120 mg/m2 on days 1-5, vincristine 0·4 mg/m2 on days 1-4, dose-adjusted cyclophosphamide 480-1866 mg/m2 on day 5, dose-adjusted doxorubicin 10-24·8 mg/m2 on days 1-4, rituximab 375 mg/m2 on days 1 and 5). Patients older than 65 years received a dose modified R-CODOX-M/R-IVAC. All drugs were intravenous except for prednisolone, which was oral. Patients also received four intrathecal CNS administrations with cytarabin (70 mg) and four with methotrexate (15 mg). Patients were stratified by centre, leukemic disease, and HIV-positivity. The primary endpoint was progression-fee survival. All analyses were done by modified intention-to-treat, excluding randomly assigned patients who were subsequently found to have CNS involvement or diagnosis other than Burkitt lymphoma at study entry. This study is registered with the European Clinical Trial Register, EudraCT2013-004394-27. FINDINGS: Due to a slow accrual, the study was closed prematurely on Nov 15, 2021. Between Aug 4, 2014, and Sept 17, 2021, 89 patients were enrolled and randomly assigned to receive R-CODOX-M/R-IVAC (n=46) or DA-EPOCH-R (n=43). Five patients were excluded after random assignment (three in the R-CODOX-M/R-IVAC group [one diagnosis other than Burkitt lymphoma at study entry according to local pathology and two CNS involvement] and two in the DA-EPOCH-R group [one diagnosis other than Burkitt lymphoma at study entry according to local pathology and one CNS involvement]. 84 remaining patients were included in the modified intention-to-treat analysis. 73 (87%) of 84 patients were male, 76 (90%) presented with stage III or IV disease, and nine (11%) had HIV-positive Burkitt lymphoma. Median patient age was 52 years (IQR 37-64). With a median follow-up of 28·5 months (IQR 13·2-43·7), 2-year progression-free survival was 76% (95% CI 60-86%) in the R-CODOX-M/R-IVAC group and 70% (54-82%) in the DA-EPOCH-R group (hazard ratio 1·42, 95% CI 0·63-3·18; p=0·40). There were two deaths in the R-CODOX-M/R-IVAC group (one infection [treatment related] and one due to disease progression [not treatment related]) and one death in the DA-EPOCH-R group (COVID-19 infection [treatment related]). In the R-CODOX-M/R-IVAC group, four patients went off-protocol because of toxic effects, versus none in the DA-EPOCH-R group. Patients treated with R-CODOX-M/R-IVAC had more infectious adverse events (24 [56%] of 43 patients had at least one grade 3-5 infection vs 14 [34%] of 41 patients in the DA-EPOCH-R group). INTERPRETATION: The trial stopped early, but the available data suggest that while DA-EPOCH-R did not result in superior progression-free survival compared with R-CODOX-M/R-IVAC, it was associated with fewer toxic effects and need for supportive care. DA-EPOCH-R appears to be an additional valid therapeutic option for patients with high-risk Burkitt lymphoma without CNS involvement. FUNDING: The Dutch Cancer Society and the Schumacher-Kramer Foundation.

A significant proportion of classic Hodgkin lymphoma recurrences represents clonally unrelated second primary lymphoma.

Despite high cure rates in classic Hodgkin lymphoma (cHL), relapses are observed. Whether relapsed cHL represents second primary lymphoma or an underlying T-cell lymphoma (TCL) mimicking cHL is underinvestigated. To analyze the nature of cHL recurrences, in-depth clonality testing of immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements was performed in paired cHL diagnoses and recurrences among 60 patients, supported by targeted mutation analysis of lymphoma-associated genes. Clonal Ig rearrangements were detected by next-generation sequencing (NGS) in 69 of 120 (58%) diagnoses and recurrence samples. The clonal relationship could be established in 34 cases, identifying clonally related relapsed cHL in 24 of 34 patients (71%). Clonally unrelated cHL was observed in 10 of 34 patients (29%) as determined by IG-NGS clonality assessment and confirmed by the identification of predominantly mutually exclusive gene mutations in the paired cHL samples. In recurrences of >2 years, ∼60% of patients with cHL for whom the clonal relationship could be established showed a second primary cHL. Clonal TCR gene rearrangements were identified in 14 of 125 samples (11%), and TCL-associated gene mutations were detected in 7 of 14 samples. Retrospective pathology review with integration of the molecular findings were consistent with an underlying TCL in 5 patients aged >50 years. This study shows that cHL recurrences, especially after 2 years, sometimes represent a new primary cHL or TCL mimicking cHL, as uncovered by NGS-based Ig/TCR clonality testing and gene mutation analysis. Given the significant therapeutic consequences, molecular testing of a presumed relapse in cHL is crucial for subsequent appropriate treatment strategies adapted to the specific lymphoma presentation.

Novel Approaches in Molecular Characterization of Classical Hodgkin Lymphoma.

Classical Hodgkin lymphoma (cHL) represents a B-cell lymphoproliferative disease characterized by clonal immunoglobulin gene rearrangements and recurrent genomic aberrations in the Hodgkin Reed-Sternberg cells in a reactive inflammatory background. Several methods are available for the molecular analysis of cHL on both tissue and cell-free DNA isolated from blood, which can provide detailed information regarding the clonal composition and genetic alterations that drive lymphoma pathogenesis. Clonality testing involving the detection of immunoglobulin and T cell receptor gene rearrangements, together with mutation analysis, represent valuable tools for cHL diagnostics, especially for patients with an atypical histological or clinical presentation reminiscent of a reactive lesion or another lymphoma subtype. In addition, clonality assessment may establish the clonal relationship of composite or subsequent lymphoma presentations within one patient. During the last few decades, more insight has been obtained on the molecular mechanisms that drive cHL development, including recurrently affected signaling pathways (e.g., NF-κB and JAK/STAT) and immune evasion. We provide an overview of the different approaches to characterize the molecular composition of cHL, and the implementation of these next-generation sequencing-based techniques in research and diagnostic settings.

Biological and Clinical Implications of Gene-Expression Profiling in Diffuse Large B-Cell Lymphoma: A Proposal for a Targeted BLYM-777 Consortium Panel as Part of a Multilayered Analytical Approach.

Gene-expression profiling (GEP) is used to study the molecular biology of lymphomas. Here, advancing insights from GEP studies in diffuse large B-cell lymphoma (DLBCL) lymphomagenesis are discussed. GEP studies elucidated subtypes based on cell-of-origin principles and profoundly changed the biological understanding of DLBCL with clinical relevance. Studies integrating GEP and next-generation DNA sequencing defined different molecular subtypes of DLBCL entities originating at specific anatomical localizations. With the emergence of high-throughput technologies, the tumor microenvironment (TME) has been recognized as a critical component in DLBCL pathogenesis. TME studies have characterized so-called "lymphoma microenvironments" and "ecotypes". Despite gained insights, unexplained chemo-refractoriness in DLBCL remains. To further elucidate the complex biology of DLBCL, we propose a novel targeted GEP consortium panel, called BLYM-777. This knowledge-based biology-driven panel includes probes for 777 genes, covering many aspects regarding B-cell lymphomagenesis (f.e., MYC signature, TME, immune surveillance and resistance to CAR T-cell therapy). Regarding lymphomagenesis, upcoming DLBCL studies need to incorporate genomic and transcriptomic approaches with proteomic methods and correlate these multi-omics data with patient characteristics of well-defined and homogeneous cohorts. This multilayered methodology potentially enhances diagnostic classification of DLBCL subtypes, prognostication, and the development of novel targeted therapeutic strategies.

Genomic and microenvironmental landscape of stage I follicular lymphoma, compared with stage III/IV.

Although the genomic and immune microenvironmental landscape of follicular lymphoma (FL) has been extensively investigated, little is known about the potential biological differences between stage I and stage III/IV disease. Using next-generation sequencing and immunohistochemistry, 82 FL nodal stage I cases were analyzed and compared with 139 FL stage III/IV nodal cases. Many similarities in mutations, chromosomal copy number aberrations, and microenvironmental cell populations were detected. However, there were also significant differences in microenvironmental and genomic features. CD8+ T cells (P = .02) and STAT6 mutations (false discovery rate [FDR] <0.001) were more frequent in stage I FL. In contrast, programmed cell death protein 1-positive T cells, CD68+/CD163+ macrophages (P < .001), BCL2 translocation (BCL2trl+) (P < .0001), and KMT2D (FDR = 0.003) and CREBBP (FDR = 0.04) mutations were found more frequently in stage III/IV FL. Using clustering, we identified 3 clusters within stage I, and 2 clusters within stage III/IV. The BLC2trl+ stage I cluster was comparable to the BCL2trl+ cluster in stage III/IV. The two BCL2trl- stage I clusters were unique for stage I. One was enriched for CREBBP (95%) and STAT6 (64%) mutations, without BLC6 translocation (BCL6trl), whereas the BCL2trl- stage III/IV cluster contained BCL6trl (64%) with fewer CREBBP (45%) and STAT6 (9%) mutations. The other BCL2trl- stage I cluster was relatively heterogeneous with more copy number aberrations and linker histone mutations. This exploratory study shows that stage I FL is genetically heterogeneous with different underlying oncogenic pathways. Stage I FL BCL2trl- is likely STAT6 driven, whereas BCL2trl- stage III/IV appears to be more BCL6trl driven.

Extent of radiological response does not reflect survival in primary central nervous system lymphoma.

BACKGROUND: In primary central nervous system lymphoma (PCNSL), small enhancing lesions can persist after treatment. It is unknown whether a difference in response category (complete response [CR], complete response unconfirmed [CRu], or partial response [PR]) reflects survival. We aimed to determine the value of a central radiology review on response assessment and whether the extent of response influenced progression-free and/or overall survival. METHODS: All patients in the HOVON 105/ALLG NHL 24 study with at least a baseline MRI and one MRI made for response evaluation available for central review were included. Tumor measurements were done by 2 independent central reviewers, disagreements were adjudicated by a third reviewer. Crude agreement and interobserver agreement (Cohen's kappa) were calculated. Differences in progression-free and overall survival between different categories of response at the end-of-protocol-treatment were assessed by the log-rank test in a landmark survival-analysis. RESULTS: Agreement between the central reviewers was 61.7% and between local and central response assessment was 63.0%. Cohen's kappa's, which corrects for expected agreement, were 0.44 and 0.46 (moderate), respectively. Progression agreement or not was 93.3% (kappa 0.87) between local and central response assessment. There were no significant differences in progression-free and overall survival between patients with CR, CRu, or PR at the end-of-protocol-treatment, according to both local and central response assessment. CONCLUSIONS: Reliability of response assessment (CR/CRu/PR) is moderate even by central radiology review and these response categories do not reliably predict survival. Therefore, primary outcome in PCNSL studies should be survival rather than CR or CR/CRu-rate.

Molecular Genetics of Relapsed Diffuse Large B-Cell Lymphoma: Insight into Mechanisms of Therapy Resistance.

The majority of patients with diffuse large B-cell lymphoma (DLBCL) can be treated successfully with a combination of chemotherapy and the monoclonal anti-CD20 antibody rituximab. Nonetheless, approximately one-third of the patients with DLBCL still experience relapse or refractory (R/R) disease after first-line immunochemotherapy. Whole-exome sequencing on large cohorts of primary DLBCL has revealed the mutational landscape of DLBCL, which has provided a framework to define novel prognostic subtypes in DLBCL. Several studies have investigated the genetic alterations specifically associated with R/R DLBCL, thereby uncovering molecular pathways linked to therapy resistance. Here, we summarize the current state of knowledge regarding the genetic alterations that are enriched in R/R DLBCL, and the corresponding pathways affected by these gene mutations. Furthermore, we elaborate on their potential role in mediating therapy resistance, also in connection with findings in other B-cell malignancies, and discuss alternative treatment options. Hence, this review provides a comprehensive overview on the gene lesions and molecular mechanisms underlying R/R DLBCL, which are considered valuable parameters to guide treatment.