[Malignant lymphomas - quo vadis? - What developments await us in diagnostics and therapy?] / Maligne Lymphome ­ Quo vadis?

The diagnosis and treatment of malignant lymphoma is rapidly advancing, offering hope but also highlighting inherent limitations. Technological breakthroughs in sequencing technologies enable more precise subtyping and risk stratification. For example, in diffuse large B-cell lymphoma (DLBCL), exome sequencing revealed molecular subtypes. Understanding these subtypes sheds light on lymphomagenesis and prognosis, and may provide targets for tailored therapies. Additionally, tumor-derived cell-free DNA (ctDNA) detected in blood plasma allows for genotyping, risk stratification, and measurement of minimal residual disease (MRD). Current studies often examine drug effectiveness through "all-comer" approaches or in transcriptionally defined subtypes. Molecular agnostic studies increasingly focus on clinically defined high-risk patients (e.g., using the IPI) to better demonstrate the statistical significance of therapy effects. Improved patient selection can enhance the cost-effectiveness of modern, often expensive, therapies.

Distinct Hodgkin lymphoma subtypes defined by noninvasive genomic profiling.

The scarcity of malignant Hodgkin and Reed-Sternberg cells hampers tissue-based comprehensive genomic profiling of classic Hodgkin lymphoma (cHL). By contrast, liquid biopsies show promise for molecular profiling of cHL due to relatively high circulating tumour DNA (ctDNA) levels1-4. Here we show that the plasma representation of mutations exceeds the bulk tumour representation in most cases, making cHL particularly amenable to noninvasive profiling. Leveraging single-cell transcriptional profiles of cHL tumours, we demonstrate Hodgkin and Reed-Sternberg ctDNA shedding to be shaped by DNASE1L3, whose increased tumour microenvironment-derived expression drives high ctDNA concentrations. Using this insight, we comprehensively profile 366 patients, revealing two distinct cHL genomic subtypes with characteristic clinical and prognostic correlates, as well as distinct transcriptional and immunological profiles. Furthermore, we identify a novel class of truncating IL4R mutations that are dependent on IL-13 signalling and therapeutically targetable with IL-4Rα-blocking antibodies. Finally, using PhasED-seq5, we demonstrate the clinical value of pretreatment and on-treatment ctDNA levels for longitudinally refining cHL risk prediction and for detection of radiographically occult minimal residual disease. Collectively, these results support the utility of noninvasive strategies for genotyping and dynamic monitoring of cHL, as well as capturing molecularly distinct subtypes with diagnostic, prognostic and therapeutic potential.

Improved early outcome prediction by MRI-based 3D tumor volume assessment in patients with CNS lymphomas.

BACKGROUND: Central nervous system lymphomas (CNSL) display remarkable clinical heterogeneity, yet accurate prediction of outcomes remains challenging. The IPCG criteria are widely used in routine practice for the assessment of treatment response. However, the value of the IPCG criteria for ultimate outcome prediction is largely unclear, mainly due to the uncertainty in delineating complete from partial responses during and after treatment. METHODS: We explored various MRI features including semi-automated 3D tumor volume measurements at different disease milestones and their association with survival in 93 CNSL patients undergoing curative-intent treatment. RESULTS: At diagnosis, patients with more than 3 lymphoma lesions, periventricular involvement, and high 3D tumor volumes showed significantly unfavorable PFS and OS. At first interim MRI during treatment, the IPCG criteria failed to discriminate outcomes in responding patients. Therefore, we randomized these patients into training and validation cohorts to investigate whether 3D tumor volumetry could improve outcome prediction. We identified a 3D tumor volume reduction of ≥97% as the optimal threshold for risk stratification (=3D early response, 3D_ER). Applied to the validation cohort, patients achieving 3D_ER had significantly superior outcomes. In multivariate analyses, 3D_ER was independently prognostic of PFS and OS. Finally, we leveraged prognostic information from 3D MRI features and circulating biomarkers to build a composite metric that further improved outcome prediction in CNSL. CONCLUSIONS: We developed semi-automated 3D tumor volume measurements as strong and independent early predictors of clinical outcomes in CNSL patients. These radiologic features could help improve risk stratification and help guide future treatment approaches.

Outcome prediction by interim positron emission tomography and IgM monoclonal gammopathy in diffuse large B-cell lymphoma.

In diffuse large B-cell lymphoma (DLBCL), a positive interim positron emission tomography (PET) scan predicts treatment failure, but the proportion of high-risk patients thus identified is small. To improve prediction, we combined the interim PET result with the presence or absence of an associated IgM gammopathy. Of 108 DLBCL patients participating in a prospective trial, nine (8%) were interim PET positive and 19 (18%) had an IgM gammopathy. The monoclonal protein was not associated with distinguishing genetic features, and its light chain restriction was not always concordant with the light chain restriction of the lymphoma. The information provided by interim PET and IgM gammopathy was combined to dichotomize the population into sizeable high-risk (1-2 adverse factors) and low-risk groups (no adverse factor) with widely different outcomes (population size, 25% vs. 75%; 3-year risk of progression, 51% vs. 10%; 3-year overall survival, 64% vs. 95%). Multivariable analyses including established risk factors revealed the interim PET result and the IgM gammopathy status to be the only factors significantly associated with outcome. Information about interim PET response and IgM gammopathy may be useful in studies testing risk-adapted treatment strategies.

Concurrent pembrolizumab with AVD for untreated classic Hodgkin lymphoma.

Concurrent administration of pembrolizumab with chemotherapy in untreated classic Hodgkin lymphoma (CHL) has not been studied previously. To investigate this combination, we conducted a single-arm study of concurrent pembrolizumab with AVD (doxorubicin, vinblastine, and dacarbazine; APVD) for untreated CHL. We enrolled 30 patients and met the primary safety end point with no observed significant treatment delays in the first 2 cycles. Twelve patients experienced grade 3 or 4 nonhematologic adverse events (AEs), most commonly febrile neutropenia and infection/sepsis. Grade 3 or 4 immune-related AEs, including alanine aminotransferase elevation and aspartate aminotransferase elevation were observed in 3 patients. One patient experienced an episode of grade 2 colitis and arthritis. Six patients missed at least 1 dose of pembrolizumab because of AEs, primarily grade 2 or higher transaminitis. Among 29 response-evaluable patients, the best overall response rate was 100% and the complete response rate was 90%. With a median follow-up of 2.1 years, the 2-year progression-free survival (PFS) and overall survival were 97% and 100%, respectively. To date, no patient who has withheld or discontinued pembrolizumab because of toxicity has progressed. Clearance of circulating tumor DNA (ctDNA) was associated with superior PFS when measured after cycle 2 and at the end of treatment (EOT). None of the 4 patients with persistent uptake by fluorodeoxyglucose positron emission tomography (PET) at EOT yet negative ctDNA have relapsed to date. Concurrent APVD shows promising safety and efficacy but may yield spurious PET findings in some patients. This trial was registered at www.clinicaltrials.gov as #NCT03331341.

Circulating Tumor DNA Profiling for Detection, Risk Stratification, and Classification of Brain Lymphomas.

PURPOSE: Clinical outcomes of patients with CNS lymphomas (CNSLs) are remarkably heterogeneous, yet identification of patients at high risk for treatment failure is challenging. Furthermore, CNSL diagnosis often remains unconfirmed because of contraindications for invasive stereotactic biopsies. Therefore, improved biomarkers are needed to better stratify patients into risk groups, predict treatment response, and noninvasively identify CNSL. PATIENTS AND METHODS: We explored the value of circulating tumor DNA (ctDNA) for early outcome prediction, measurable residual disease monitoring, and surgery-free CNSL identification by applying ultrasensitive targeted next-generation sequencing to a total of 306 tumor, plasma, and CSF specimens from 136 patients with brain cancers, including 92 patients with CNSL. RESULTS: Before therapy, ctDNA was detectable in 78% of plasma and 100% of CSF samples. Patients with positive ctDNA in pretreatment plasma had significantly shorter progression-free survival (PFS, P < .0001, log-rank test) and overall survival (OS, P = .0001, log-rank test). In multivariate analyses including established clinical and radiographic risk factors, pretreatment plasma ctDNA concentrations were independently prognostic of clinical outcomes (PFS HR, 1.4; 95% CI, 1.0 to 1.9; P = .03; OS HR, 1.6; 95% CI, 1.1 to 2.2; P = .006). Moreover, measurable residual disease detection by plasma ctDNA monitoring during treatment identified patients with particularly poor prognosis following curative-intent immunochemotherapy (PFS, P = .0002; OS, P = .004, log-rank test). Finally, we developed a proof-of-principle machine learning approach for biopsy-free CNSL identification from ctDNA, showing sensitivities of 59% (CSF) and 25% (plasma) with high positive predictive value. CONCLUSION: We demonstrate robust and ultrasensitive detection of ctDNA at various disease milestones in CNSL. Our findings highlight the role of ctDNA as a noninvasive biomarker and its potential value for personalized risk stratification and treatment guidance in patients with CNSL.[Media: see text].

Determinants of resistance to engineered T cell therapies targeting CD19 in large B cell lymphomas.

Most relapsed/refractory large B cell lymphoma (r/rLBCL) patients receiving anti-CD19 chimeric antigen receptor (CAR19) T cells relapse. To characterize determinants of resistance, we profiled over 700 longitudinal specimens from two independent cohorts (n = 65 and n = 73) of r/rLBCL patients treated with axicabtagene ciloleucel. A method for simultaneous profiling of circulating tumor DNA (ctDNA), cell-free CAR19 (cfCAR19) retroviral fragments, and cell-free T cell receptor rearrangements (cfTCR) enabled integration of tumor and both engineered and non-engineered T cell effector-mediated factors for assessing treatment failure and predicting outcomes. Alterations in multiple classes of genes are associated with resistance, including B cell identity (PAX5 and IRF8), immune checkpoints (CD274), and those affecting the microenvironment (TMEM30A). Somatic tumor alterations affect CAR19 therapy at multiple levels, including CAR19 T cell expansion, persistence, and tumor microenvironment. Further, CAR19 T cells play a reciprocal role in shaping tumor genotype and phenotype. We envision these findings will facilitate improved chimeric antigen receptor (CAR) T cells and personalized therapeutic approaches.

A phase 1/2 study of lenalidomide and obinutuzumab with CHOP for newly diagnosed DLBCL.

Diffuse large B-cell lymphoma (DLBCL) can be cured with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP); however, one-third of patients experience refractory or relapsed disease. Studies comparing R-CHOP with modified regimens replacing R with obinutuzumab (O) or adding lenalidomide (L) did not result in improved outcomes; however, L and O together may enhance natural killer-cell mediated antibody-dependent cellular toxicity when paired with CHOP. Here, we report on a phase 1b/2 study of 53 patients with newly diagnosed DLBCL who received 6 cycles of LO-CHOP. The end of treatment overall and complete response rates of the 50 evaluable patients were 98% and 90%, respectively. After a median follow-up of 4.5 years, the 4-year progression free and overall survival rates were 87.4% and 91.3%, respectively. Grade 3 to 4 adverse events were experienced by 70% of patients, including neutropenia (38%), thrombocytopenia (17%), fatigue (13%), and neutropenic fever (13%). Of the 33 patients profiled with circulating tumor DNA (ctDNA) sequencing, 31 (94%) had detectable pretreatment ctDNA with cancer personalized profiling by deep sequencing, 24 (73%) were classifiable by the LymphGen classifier, and 15/20 (75%) and 12/17 (71%) patients achieved early and major molecular responses after 1 and 2 cycles, respectively. Using phased variant enrichment and detection sequencing, 16/18 evaluable patients (89%) showed no detectable ctDNA after at least 5 cycles of LO-CHOP. LO-CHOP demonstrates high efficacy and tolerability in newly diagnosed DLBCL, leading to a high rate of undetectable minimal residual disease by ctDNA. This trial has been registered at www.clinicaltrials.gov as NCT02529852.

Molecular Monitoring of Lymphomas.

Molecular monitoring of tumor-derived alterations has an established role in the surveillance of leukemias, and emerging nucleic acid sequencing technologies are likely to similarly transform the clinical management of lymphomas. Lymphomas are well suited for molecular surveillance due to relatively high cell-free DNA and circulating tumor DNA concentrations, high somatic mutational burden, and the existence of stereotyped variants enabling focused interrogation of recurrently altered regions. Here, we review the clinical scenarios and key technologies applicable for the molecular monitoring of lymphomas, summarizing current evidence in the literature regarding molecular subtyping and classification, evaluation of treatment response, the surveillance of active cellular therapies, and emerging clinical trial strategies.

Inferring gene expression from cell-free DNA fragmentation profiles.

Profiling of circulating tumor DNA (ctDNA) in the bloodstream shows promise for noninvasive cancer detection. Chromatin fragmentation features have previously been explored to infer gene expression profiles from cell-free DNA (cfDNA), but current fragmentomic methods require high concentrations of tumor-derived DNA and provide limited resolution. Here we describe promoter fragmentation entropy as an epigenomic cfDNA feature that predicts RNA expression levels at individual genes. We developed 'epigenetic expression inference from cell-free DNA-sequencing' (EPIC-seq), a method that uses targeted sequencing of promoters of genes of interest. Profiling 329 blood samples from 201 patients with cancer and 87 healthy adults, we demonstrate classification of subtypes of lung carcinoma and diffuse large B cell lymphoma. Applying EPIC-seq to serial blood samples from patients treated with PD-(L)1 immune-checkpoint inhibitors, we show that gene expression profiles inferred by EPIC-seq are correlated with clinical response. Our results indicate that EPIC-seq could enable noninvasive, high-throughput tissue-of-origin characterization with diagnostic, prognostic and therapeutic potential.

The molecular ontogeny of follicular lymphoma: gene mutations succeeding the BCL2 translocation define common precursor cells.

Relapsed follicular lymphoma (FL) can arise from common progenitor cells (CPCs). Conceptually, CPC-defining mutations are somatic alterations shared by the initial and relapsed tumours, mostly B-cell leukaemia/lymphoma 2 (BCL2)/immunoglobulin heavy locus (IGH) translocations and other recurrent gene mutations. Through complementary approaches for highly sensitive mutation detection, we do not find CPC-defining mutations in highly purified BCL2/IGH-negative haematopoietic progenitor cells in clinical remission samples from three patients with relapsed FL. Instead, we find cells harbouring the same BCL2/IGH translocation but lacking CREB binding protein (CREBBP), lysine methyltransferase 2D (KMT2D) and other recurrent gene mutations. Thus, (i) the BCL2/IGH translocation can precede CPC-defining mutations in human FL, and (ii) BCL2/IGH-translocated cells can persist in clinical remission.

Enhanced detection of minimal residual disease by targeted sequencing of phased variants in circulating tumor DNA.

Circulating tumor-derived DNA (ctDNA) is an emerging biomarker for many cancers, but the limited sensitivity of current detection methods reduces its utility for diagnosing minimal residual disease. Here we describe phased variant enrichment and detection sequencing (PhasED-seq), a method that uses multiple somatic mutations in individual DNA fragments to improve the sensitivity of ctDNA detection. Leveraging whole-genome sequences from 2,538 tumors, we identify phased variants and their associations with mutational signatures. We show that even without molecular barcodes, the limits of detection of PhasED-seq outperform prior methods, including duplex barcoding, allowing ctDNA detection in the ppm range in participant samples. We profiled 678 specimens from 213 participants with B cell lymphomas, including serial cell-free DNA samples before and during therapy for diffuse large B cell lymphoma. In participants with undetectable ctDNA after two cycles of therapy using a next-generation sequencing-based approach termed cancer personalized profiling by deep sequencing, an additional 25% have ctDNA detectable by PhasED-seq and have worse outcomes. Finally, we demonstrate the application of PhasED-seq to solid tumors.

Short Diagnosis-to-Treatment Interval Is Associated With Higher Circulating Tumor DNA Levels in Diffuse Large B-Cell Lymphoma.

PURPOSE: Patients with Diffuse Large B-cell Lymphoma (DLBCL) in need of immediate therapy are largely under-represented in clinical trials. The diagnosis-to-treatment interval (DTI) has recently been described as a metric to quantify such patient selection bias, with short DTI being associated with adverse risk factors and inferior outcomes. Here, we characterized the relationships between DTI, circulating tumor DNA (ctDNA), conventional risk factors, and clinical outcomes, with the goal of defining objective disease metrics contributing to selection bias. PATIENTS AND METHODS: We evaluated pretreatment ctDNA levels in 267 patients with DLBCL treated across multiple centers in Europe and the United States using Cancer Personalized Profiling by Deep Sequencing. Pretreatment ctDNA levels were correlated with DTI, total metabolic tumor volumes (TMTVs), the International Prognostic Index (IPI), and outcome. RESULTS: Short DTI was associated with advanced-stage disease (P < .001) and higher IPI (P < .001). We also found an inverse correlation between DTI and TMTV (RS = -0.37; P < .001). Similarly, pretreatment ctDNA levels were significantly associated with stage, IPI, and TMTV (all P < .001), demonstrating that both DTI and ctDNA reflect disease burden. Notably, patients with shorter DTI had higher pretreatment ctDNA levels (P < .001). Pretreatment ctDNA levels predicted short DTI independent of the IPI (P < .001). Although each risk factor was significantly associated with event-free survival in univariable analysis, ctDNA level was prognostic of event-free survival independent of DTI and IPI in multivariable Cox regression (ctDNA: hazard ratio, 1.5; 95% CI [1.2 to 2.0]; IPI: 1.1 [0.9 to 1.3]; -DTI: 1.1 [1.0 to 1.2]). CONCLUSION: Short DTI largely reflects baseline tumor burden, which can be objectively measured using pretreatment ctDNA levels. Pretreatment ctDNA levels therefore have utility for quantifying and guarding against selection biases in prospective DLBCL clinical trials.

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.

Cathepsin S Alterations Induce a Tumor-Promoting Immune Microenvironment in Follicular Lymphoma.

Tumor cells orchestrate their microenvironment. Here, we provide biochemical, structural, functional, and clinical evidence that Cathepsin S (CTSS) alterations induce a tumor-promoting immune microenvironment in follicular lymphoma (FL). We found CTSS mutations at Y132 in 6% of FL (19/305). Another 13% (37/286) had CTSS amplification, which was associated with higher CTSS expression. CTSS Y132 mutations lead to accelerated autocatalytic conversion from an enzymatically inactive profrom to active CTSS and increased substrate cleavage, including CD74, which regulates major histocompatibility complex class II (MHC class II)-restricted antigen presentation. Lymphoma cells with hyperactive CTSS more efficiently activated antigen-specific CD4+ T cells in vitro. Tumors with hyperactive CTSS showed increased CD4+ T cell infiltration and proinflammatory cytokine perturbation in a mouse model and in human FLs. In mice, this CTSS-induced immune microenvironment promoted tumor growth. Clinically, patients with CTSS-hyperactive FL had better treatment outcomes with standard immunochemotherapies, indicating that these immunosuppressive regimens target both the lymphoma cells and the tumor-promoting immune microenvironment.

Selective Inhibition of HDAC3 Targets Synthetic Vulnerabilities and Activates Immune Surveillance in Lymphoma.

CREBBP mutations are highly recurrent in B-cell lymphomas and either inactivate its histone acetyltransferase (HAT) domain or truncate the protein. Herein, we show that these two classes of mutations yield different degrees of disruption of the epigenome, with HAT mutations being more severe and associated with inferior clinical outcome. Genes perturbed by CREBBP mutation are direct targets of the BCL6-HDAC3 onco-repressor complex. Accordingly, we show that HDAC3-selective inhibitors reverse CREBBP-mutant aberrant epigenetic programming, resulting in: (i) growth inhibition of lymphoma cells through induction of BCL6 target genes such as CDKN1A and (ii) restoration of immune surveillance due to induction of BCL6-repressed IFN pathway and antigen-presenting genes. By reactivating these genes, exposure to HDAC3 inhibitors restored the ability of tumor-infiltrating lymphocytes to kill DLBCL cells in an MHC class I and II-dependent manner, and synergized with PD-L1 blockade in a syngeneic model in vivo. Hence, HDAC3 inhibition represents a novel mechanism-based immune epigenetic therapy for CREBBP-mutant lymphomas. SIGNIFICANCE: We have leveraged the molecular characterization of different types of CREBBP mutations to define a rational approach for targeting these mutations through selective inhibition of HDAC3. This represents an attractive therapeutic avenue for targeting synthetic vulnerabilities in CREBBP-mutant cells in tandem with promoting antitumor immunity.This article is highlighted in the In This Issue feature, p. 327.

Impact of age on clinical risk scores in follicular lymphoma.

The Follicular Lymphoma (FL) International Prognostic Index (FLIPI) and FLIPI-2 are well-described clinical risk models. Age >60 years at diagnosis is a risk factor in both scores. Recently, we showed that older age is not associated with higher risk of disease progression or inferior treatment efficacy. Instead, shorter survival of older patients results mainly from an increased risk of nonrelapse deaths. This questions the value of age as a meaningful component of scores intended to predict disease-specific survival. The newly proposed PRIMA-prognostic index (PRIMA-PI) only includes ß2-microglobulin levels and bone marrow infiltration as risk factors. Here, we independently validate the PRIMA-PI in a clinical trial cohort of 475 patients with advanced FL who uniformly received cyclophosphamide, doxorubicin hydrochloride, vincristine sulfate, prednisone, and rituximab (R-CHOP) as frontline therapy. The PRIMA-PI separated 3 similar sized risk cohorts with 5-year progression-free survival (PFS) rates of 74%, 59%, and 39%, respectively (P < .0001). Furthermore, we compare the PRIMA-PI with the FLIPI and FLIPI-2. We demonstrate that the PRIMA-PI has the highest specificity to identify high-risk patients (80% for 5-year PFS) because of its superior risk stratification in patients >60 years (73% vs 33% [FLIPI] and 47% [FLIPI-2] for 5-year PFS). Thus, the PRIMA-PI is a promising clinical tool to stringently identify patients at highest risk of poor outcome after frontline R-CHOP for advanced FL, and is particularly useful in patients with older age. Further validation in non-R-CHOP treated cohorts is needed.

Duodenal-type and nodal follicular lymphomas differ by their immune microenvironment rather than their mutation profiles.

Duodenal-type follicular lymphoma (DTFL) is a rare and highly indolent follicular lymphoma (FL) variant. It is morphologically and immunophenotypically indistinguishable from typical FL, characterized by restricted involvement of intestinal mucosa, and lacks extraintestinal manifestations. The molecular determinants of this distinct clinical behavior are largely unknown. Thirty-eight diagnostic biopsies from patients with DTFL were evaluated. The 10-year overall survival rate was 100% in clinically evaluable patients (n = 19). We compared the targeted mutation profile of DTFL (n = 31), limited-stage typical FL (LSTFL; n = 17), and advanced-stage typical FL (ASTFL; n = 241). The mutation frequencies of recurrently mutated genes, including CREBBP, TNFRSF14/HVEM, and EZH2 were not significantly different. However, KMT2D was less commonly mutated in DTFL (52%) and LSTFL (24%) as compared with ASTFL (79%). In ASTFL, 41% of KMT2D-mutated cases harbored multiple mutations in KMT2D, as compared with only 12% in LSTFL (P = .019) and 0% in DTFL (P < .0001). Whole exome and targeted sequencing of DTFL revealed high mutation frequencies of EEF1A1 (35%) and HVCN1 (22%). We compared the immune microenvironment gene expression signatures of DTFL (n = 8) and LSTFL (n = 7). DTFL clearly separated from LSTFL by unsupervised, hierarchical clustering of 147 chemokines and cytokines and was enriched for a chronic inflammation signature. In conclusion, the mutational landscape of DTFL is highly related to typical FL. The lower frequency of multiple mutations in KMT2D in DTFL and LSTFL indicates an increasing selection pressure for complete KMT2D loss in ASTFL pathogenesis. The highly dissimilar immune microenvironment of DTFL suggests a central role in the biology of this disease.