Interim PET-guided treatment for early-stage NLPHL: a subgroup analysis of the randomized GHSG HD16 and HD17 studies.

The optimal first-line treatment for nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) diagnosed in early stages is largely undefined. We, therefore, analyzed 100 NLPHL patients treated in the randomized HD16 (early-stage favorable; n = 85) and HD17 (early-stage unfavorable; n = 15) studies. These studies investigated the omission of consolidation radiotherapy (RT) in patients with a negative interim positron emission tomography (iPET) (ie, Deauville score <3) after chemotherapy (HD16: 2× doxorubicin, bleomycin, vinblastine, and dacarbazine [ABVD]; HD17: 2× escalated bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone [BEACOPP] plus 2× ABVD). Patients with NLPHL treated in the HD16 and HD17 studies had 5-year progression-free survival (PFS) rates of 90.3% and 92.9%, respectively. Thus, the 5-year PFS did not differ significantly from that of patients with classical Hodgkin lymphoma treated within the same studies (HD16: P = .88; HD17: P = .50). Patients with early-stage favorable NLPHL who had a negative iPET after 2× ABVD and did not undergo consolidation RT tended to have a worse 5-year PFS than patients with a negative iPET who received consolidation RT (83% vs 100%; P = .05). There were 10 cases of NLPHL recurrence. However, no NLPHL patient died during follow-up. Hence, the 5-year overall survival rate was 100%. Taken together, contemporary Hodgkin lymphoma-directed treatment approaches result in excellent outcomes for patients with newly diagnosed early-stage NLPHL and, thus, represent valid treatment options. In early-stage favorable NLPHL, consolidation RT appears necessary after 2× ABVD to achieve the optimal disease control irrespective of the iPET result.

Intrathecal IgM synthesis as a diagnostic marker in patients with suspected CNS lymphoma.

For CNS lymphomas (CNSL), there is a high need for minimally invasive and easily obtainable diagnostic markers. Intrathecal IgM synthesis can easily be determined in routine CSF diagnostics. The aim of this study was to systematically investigate the diagnostic potential of intrathecal IgM synthesis in primary and secondary CNSL (PCNSL and SCNSL). In this retrospective study, patients with a biopsy-proven diagnosis of PCNSL or SCNSL were compared with patients with other neurological diseases in whom CNSL was initially the primary radiological differential diagnosis based on MRI. Sensitivity and specificity of intrathecal IgM synthesis were calculated using receiver operating characteristic curves. Seventy patients with CNSL were included (49 PCNSL and 21 SCNSL) and compared to 70 control patients. The sensitivity and specificity for the diagnosis of CNSL were 49% and 87%, respectively, for the entire patient population and 66% and 91% after selection for cases with tumor access to the CSF system and isolated intrathecal IgM synthesis. In cases with MRI-based radiological suspicion of CNSL, intrathecal IgM synthesis has good specificity but limited sensitivity. Because of its low-threshold availability, analysis of intrathecal IgM synthesis has the potential to lead to higher diagnostic accuracy, especially in resource-limited settings, and deserves further study.

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.

Fluorescence confocal microscopy can accelerate diagnosis of cervical lymphadenopathy.

Fluorescence confocal microscopy (FCM) is an optical technique that uses laser light sources of different wavelengths to generate real-time images of fresh, unfixed tissue specimens. Unlike conventional histological evaluation methods, FCM is able to assess fresh tissue samples without the associated cryo artifacts typically observed after frozen sectioning. The purpose of this study was to evaluate the utility of FCM imaging in the differential diagnosis of cervical lymphadenopathy. Twenty-two cervical lymph node specimens from patients with lymphadenopathy of unknown origin were imaged by FCM. Two pathologists independently evaluated the scans for suspicion of malignancy and preliminary diagnosis. Malignancy was reliably excluded or confirmed by both pathologists with a sensitivity of 90.9% for pathologist 1 and 100% for pathologist 2. The specificity was 100% for both pathologists. For the preliminary diagnosis, almost perfect agreement with the final diagnosis was observed for both pathologists (κ= 0.94 for pathologist 1 and κ= 1.00 for pathologist 2). This is the first study to investigate lymph node specimens with different diagnoses, including lymphoma, using FCM. Our results indicate that differential diagnosis of lymph node specimens is feasible in FCM images, thus encouraging further exploration of FCM imaging in lymph node specimens to accelerate diagnosis and open the possibility of digitizing diagnosis on fresh, unfixed tissue.

Checkpoint inhibition enhances cell contacts between CD4+ T cells and Hodgkin-Reed-Sternberg cells of classic Hodgkin lymphoma.

Although checkpoint molecules like CTLA-4 and PD1 have been described several years ago, checkpoint inhibitors such as Nivolumab (an anti-PD-1 antibody) have only recently been used to treat classic Hodgkin lymphoma (cHL). Several studies have shown convincing therapeutic effects of Nivolumab in cHL. However, the mechanism of action of Nivolumab in cHL is not fully understood. The aim of this study was to monitor changes in cell motility and cell contacts after administration of Nivolumab to an in vitro model of cHL as well as to native hyperplastic lymphoid tissue and native human tissue from cHL. In both tissue and in vitro, CD4+, CD8+, CD30+ and CD20+ cell velocities were unchanged after Nivolumab incubation. In contrast, in primary cHL tissue, the duration of cell contacts between CD4+ T cells and HRS cells was significantly increased after 5 h Nivolumab treatment, and the number of contacts with HRS cells was also slightly increased for CD4+ T cells (not significant), suggesting that CD4+ T cells in particular contribute to the cytotoxicity observed as a result of Nivolumab therapy. There was no change in the duration of cell contacts in the hyperplastic lymphoid tissue after Nivolumab incubation. In conclusion, we show here for the first time by imaging of native lymphoma tissue an enhanced interaction of CD4+ T cells and HRS cells in cHL after Nivolumab administration.

Immune phenotypes and checkpoint molecule expression of clonally expanded lymph node-infiltrating T cells in classical Hodgkin lymphoma.

Lymph node-infiltrating T cells have been of particular interest in classical Hodgkin lymphoma (cHL). High rates of complete therapeutic responses to antibody-mediated immune checkpoint blockade, even in relapsed/refractory patients, suggest the existence of a T cell-dominated, antigen-experienced, functionally inhibited and lymphoma-directed immune microenvironment. We asked whether clonally expanded T cells (1) were detectable in cHL lymph nodes, (2) showed characteristic immune phenotypes, and (3) were inhibited by immune checkpoint molecule expression. We applied high-dimensional FACS index sorting and single cell T cell receptor αß sequencing to lymph node-infiltrating T cells from 10 treatment-naïve patients. T cells were predominantly CD4+ and showed memory differentiation. Expression of classical immune checkpoint molecules (CTLA-4, PD-1, TIM-3) was generally low (< 12.0% of T cells) and not different between CD4+ and CD8+ T cells. Degrees of clonal T cell expansion varied between patients (range: 1-18 expanded clones per patient) and was almost exclusively restricted to CD8+ T cells. Clonally expanded T cells showed non-naïve phenotypes and low checkpoint molecule expression similar to non-expanded T cells. Our data suggest that the therapeutic effects of immune checkpoint blockade require mechanisms in addition to dis-inhibition of pre-existing lymphoma-directed T cell responses. Future studies on immune checkpoint blockade-associated effects will identify molecular T cell targets, address dynamic aspects of cell compositions over time, and extend their focus beyond lymph node-infiltrating T cells.

LRPAP1 autoantibodies in mantle cell lymphoma are associated with superior outcome.

Low-density lipoprotein (LDL) receptor-related protein-associated protein 1 (LRPAP1) had been identified by B-cell receptor (BCR) expression cloning and subsequent protein array screening as a frequent and proliferation-inducing autoantigen of mantle cell lymphoma (MCL). Of interest, high-titered and light chain-restricted LRPAP1 autoantibodies were detected in 8 of 28 patients with MCL. In the present study, LRPAP1 autoantibodies in sera of patients treated within the Younger and Elderly trials of the European MCL Network were analyzed regarding frequency, association with disease characteristics, and prognostic impact. LRPAP1 autoantibodies were detected in 41 (13%) of 312 evaluable patients with MCL. These LRPAP1 autoantibodies belonged predominantly to the immunoglobulin G (IgG) class and were clonally light chain restricted (27 with κ light chains, 14 patients with λ light chains). Titers ranged between 1:400 and 1:3200. The presence of LRPAP1 autoantibodies was not significantly associated with any baseline clinical characteristic, however, it was associated with a superior 5-year probability for failure-free survival (FFS) of 70% (95% confidence interval [CI], 57% to 87%) vs 51% (95% CI, 44% to 58%), P = .0052; and for overall survival (OS) of 93% (95% CI, 85% to 100%) vs 68% (95% CI, 62% to 74%), P = .0142. LRPAP1-seropositive patients had a Mantle Cell Lymphoma International Prognostic Index-adjusted hazard ratio for FFS of 0.48 (95% CI 0.27-0.83, P = .0083) and for OS of 0.47 (95% CI 0.24-0.94, P = .032). LRPAP1 autoantibodies were frequently detected in a large cohort of MCL patients treated within prospective multicenter clinical trials. Our results suggest better outcomes for LRPAP1-autoantibody seropositive patients.

The age of the bone marrow microenvironment influences B-cell acute lymphoblastic leukemia progression via CXCR5-CXCL13.

B-cell acute lymphoblastic leukemia (B-ALL) occurs most commonly in children, whereas chronic myeloid leukemia is more frequent in adults. The myeloid bias of hematopoiesis in elderly individuals has been considered causative, but the age of the bone marrow microenvironment (BMM) may be contributory. Using various murine models of B-ALL in young vs old mice, we recapitulated B-ALL preponderance in children vs adults. We showed differential effects of young vs old BM macrophages on B-ALL cell function. Molecular profiling using RNA- and ATAC-sequencing revealed pronounced differences in young vs old BMM-derived macrophages and enrichment for gene sets associated with inflammation. In concordance with the role of C-X-C motif chemokine (CXCL) 13 for disease-associated B-cell chemoattraction, we found CXCL13 to be highly expressed in young macrophages on a translational compared with a transcriptional level. Inhibition of CXCL13 in BM macrophages impaired leukemia cell migration and decreased the proliferation of cocultured B-ALL cells, whereas recombinant CXCL13 increased pAKT and B-ALL cell expansion. Pretreatment of B-ALL-initiating cells with CXCL13 accelerated B-ALL progression. Deficiency of Cxcr5, the receptor for CXCL13, on B-ALL-initiating cells prolonged murine survival, whereas high expression of CXCR5 in pediatric B-ALL may predict central nervous system relapse. CXCL13 staining was increased in bone sections from pediatric compared with adult patients with B-ALL. Taken together, our study shows that the age of the BMM and, in particular, BM macrophages influence the leukemia phenotype. The CXCR5-CXCL13 axis may act as prognostic marker and an attractive novel target for the treatment of B-ALL.

Massive parallel sequencing unveils homologous recombination deficiency in follicular dendritic cell sarcoma.

Standardized treatment options are lacking for patients with unresectable or multifocal follicular dendritic cell sarcoma (FDCS) and disease-related mortality is as high as 20%. Applying whole genome sequencing (WGS) in one case and whole exome sequencing (WES) in additional twelve, this study adds information on the molecular landscape of FDCS, expanding knowledge on pathobiological mechanisms and identifying novel markers of potential theragnostic significance. Massive parallel sequencing showed high frequency of mutations on oncosuppressor genes, particularly in RB1, CARS and BRCA2 and unveiled alterations on homologous recombination DNA damage repair related genes in 70% (9/13) of cases. This indicates that patients with high stage FDCS may be eligible for poly ADP ribose polymerase inhibition protocols. Low tumor mutational burden was confirmed in this study despite common PDL1 expression in FDCS arguing on the efficacy of immune checkpoint inhibitors. CDKN2A deletion, detected by WGS and confirmed by FISH in 41% of cases (9/22) indicates that impairment of cell cycle regulation may sustain oncogenesis in FDCS. Absence of mutations in the RAS/RAF/MAPK pathway and lack of clonal hematopoiesis related mutations in FDCS sanction its differences from dendritic cell-derived neoplasms of haematopoietic derivation. WGS and WES in FDCS provides additional information on the molecular landscape of this rare tumor, proposing novel candidate genes for innovative therapeutical approaches to improve survival of patients with multifocal disease.

B-cell receptor reactivity against Rothia mucilaginosa in nodular lymphocyte-predominant Hodgkin lymphoma.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a Hodgkin lymphoma expressing functional B-cell receptors (BCR). Recently, we described a dual stimulation model of IgD+ lymphocyte-predominant cells by Moraxella catarrhalis antigen RpoC and its superantigen MID/hag, associated with extralong CDR3 and HLA-DRB1*04 or HLADRB1* 07 haplotype. The aim of the present study was to extend the antigen screening to further bacteria and viruses. The fragment antibody-binding (Fab) regions of seven new and 15 previously reported cases were analyzed. The reactivity of non-Moraxella spp.-reactive Fab regions against lysates of Rothia mucilaginosa was observed in 5/22 (22.7%) cases. Galactofuranosyl transferase (Gltf) and 2,3-butanediol dehydrogenase (Bdh) of R. mucilaginosa were identified by comparative silver- and immuno-staining in two-dimensional gels, with subsequent mass spectrometry and validation by western blots and enzyme-linked immunosorbent assay. Both R. mucilaginosa Gltf and Bdh induced BCR pathway activation and proliferation in vitro. Apoptosis was induced by recombinant Gltf/ETA'-immunotoxin conjugates in DEV cells expressing recombinant R. mucilaginosa-reactive BCR. Reactivity against M. catarrhalis RpoC was confirmed in 3/7 newly expressed BCR (total 10/22 reactive to Moraxella spp.), resulting in 15/22 (68.2%) cases with BCR reactivity against defined bacterial antigens. These findings strengthen the hypothesis of bacterial trigger contributing to subsets of NLPHL.

T-cell-derived Hodgkin lymphoma has motility characteristics intermediate between Hodgkin and anaplastic large cell lymphoma.

Classic Hodgkin lymphoma (cHL) is usually characterized by a low tumour cell content, derived from crippled germinal centre B cells. Rare cases have been described in which the tumour cells show clonal T-cell receptor rearrangements. From a clinicopathological perspective, it is unclear if these cases should be classified as cHL or anaplastic large T-cell lymphoma (ALCL). Since we recently observed differences in the motility of ALCL and cHL tumour cells, here, we aimed to obtain a better understanding of T-cell-derived cHL by investigating their global proteomic profiles and their motility. In a proteomics analysis, when only motility-associated proteins were regarded, T-cell-derived cHL cell lines showed the highest similarity to ALK- ALCL cell lines. In contrast, T-cell-derived cHL cell lines presented a very low overall motility, similar to that observed in conventional cHL. Whereas all ALCL cell lines, as well as T-cell-derived cHL, predominantly presented an amoeboid migration pattern with uropod at the rear, conventional cHL never presented with uropods. The migration of ALCL cell lines was strongly impaired upon application of different inhibitors. This effect was less pronounced in cHL cell lines and almost invisible in T-cell-derived cHL. In summary, our cell line-derived data suggest that based on proteomics and migration behaviour, T-cell-derived cHL is a neoplasm that shares features with both cHL and ALCL and is not an ALCL with low tumour cell content. Complementary clinical studies on this lymphoma are warranted.

Tumour cell characteristics and microenvironment composition correspond to clinical presentation in newly diagnosed nodular lymphocyte-predominant Hodgkin lymphoma.

Different studies have characterized the microenvironment and its prognostic impact in classic Hodgkin lymphoma whereas such analyses are pending for nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). We thus investigated characteristics of tumour cells and microenvironment in NLPHL and evaluated possible correlations with the clinical presentation. Lymph node samples from 152 NLPHL patients who had first-line treatment within the randomized German Hodgkin Study Group HD16-HD18 trials were available and analysed with regard to IgD status and nuclear size of the tumour cells as well as presence of PD1-positive follicular T helper cells and CD163-positive macrophages in the microenvironment. While large tumour cell nuclei and high numbers of PD1-positive follicular T helper cells in the microenvironment were more common in patients presenting with early/intermediate stages than in patients with advanced-stage disease (p < 0.0001, unpaired t-test; p = 0.0022, Mann-Whitney test), no differences between risk groups were observed in terms of the IgD status of the tumour cells and the content of CD163-positive macrophages in the microenvironment. PD1-positive follicular T helper cells were present in both cases with typical and variant growth patterns and rosetting around the tumour cells was observed in 96% of patients, indicating an important role of PD1-positive follicular T helper cells in NLPHL.

Histopathological growth patterns in patients with advanced nodular lymphocyte-predominant Hodgkin lymphoma treated within the randomized HD18 study: a report from the German Hodgkin Study Group.

We retrospectively investigated histopathological growth patterns in individuals with advanced nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) treated within the randomized HD18 study. In all, 35/60 patients (58%) presented with atypical growth patterns. Patients with atypical growth patterns more often had stage IV disease (P = 0·0354) and splenic involvement (P = 0·0048) than patients with typical growth patterns; a positive positron emission tomography after two cycles of chemotherapy (PET-2) tended to be more common (P = 0·1078). Five-year progression-free survival [hazard ratio (HR) = 0·86; 95% confidence interval (CI) = 0·49-1·47] and overall survival (HR = 0·85; 95% CI = 0·49-1·51) did not differ between the groups after study treatment with PET-2-guided escalated BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisone). Thus, advanced NLPHL is often associated with atypical growth patterns but their prognostic impact is compensated by PET-2-guided escalated BEACOPP.

3D analyses reveal T cells with activated nuclear features in T-cell/histiocyte-rich large B-cell lymphoma.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) can show variable histological growth patterns and present remarkable overlap with T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL). Previous studies suggest that NLPHL histological variants represent progression forms of NLPHL and THRLBCL transformation in aggressive disease. Since molecular studies of both lymphomas are limited due to the low number of tumor cells, the present study aimed to learn if a better understanding of these lymphomas is possible via detailed measurements of nuclear and cell size features in 2D and 3D sections. Whereas no significant differences were visible in 2D analyses, a slightly increased nuclear volume and a significantly enlarged cell size were noted in 3D measurements of the tumor cells of THRLBCL in comparison to typical NLPHL cases. Interestingly, not only was the size of the tumor cells increased in THRLBCL but also the nuclear volume of concomitant T cells in the reactive infiltrate when compared with typical NLPHL. Particularly CD8+ T cells had frequent contacts to tumor cells of THRLBCL. However, the nuclear volume of B cells was comparable in all cases. These results clearly demonstrate that 3D tissue analyses are superior to conventional 2D analyses of histological sections. Furthermore, the results point to a strong activation of T cells in THRLBCL, representing a cytotoxic response against the tumor cells with unclear effectiveness, resulting in enhanced swelling of the tumor cell bodies and limiting proliferative potential. Further molecular studies combining 3D tissue analyses and molecular data will help to gain profound insight into these ill-defined cellular processes.

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

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

Landscape of T Follicular Helper Cell Dynamics in Human Germinal Centers.

T follicular helper (Tfh) cells play a very important role in mounting a humoral response. Studies conducted in mouse models have revealed with good kinetic and spatial resolution the dynamics of these cells in germinal centers (GC) and their cross-talk with B cells upon an immune response. However, whether a similar migratory behavior is performed by human Tfh cells is unclear, as technology to track them in situ has been lacking. In this study, we combined traditional immunohistochemistry and real-time fluorescent imaging approaches on fresh human adenoid slices to provide static and dynamic information on Tfh cells. Our data indicate that GC light zones are composed of two distinct areas in terms of Tfh cell distribution and migration. In the outer GC light zones, Tfh cells migrate actively and with a high ability to form dynamic clusters showing intense and rapid reorganization. In these outer regions, Tfh cells demonstrate multiple interactions between each other. Conversely, in central regions of GC light zones, Tfh cells are much more static, forming long-lasting conjugates. These findings reveal for the first time, to our knowledge, the dynamic behavior whereby Tfh cells migrate in human GC and highlight the heterogeneity of GC for Tfh cell motility.

Genetic drivers of oncogenic pathways in molecular subgroups of peripheral T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) is a group of complex clinicopathological entities, often associated with an aggressive clinical course. Angioimmunoblastic T-cell lymphoma (AITL) and PTCL-not otherwise specified (PTCL-NOS) are the 2 most frequent categories, accounting for >50% of PTCLs. Gene expression profiling (GEP) defined molecular signatures for AITL and delineated biological and prognostic subgroups within PTCL-NOS (PTCL-GATA3 and PTCL-TBX21). Genomic copy number (CN) analysis and targeted sequencing of these molecular subgroups revealed unique CN abnormalities (CNAs) and oncogenic pathways, indicating distinct oncogenic evolution. PTCL-GATA3 exhibited greater genomic complexity that was characterized by frequent loss or mutation of tumor suppressor genes targeting the CDKN2A /B-TP53 axis and PTEN-PI3K pathways. Co-occurring gains/amplifications of STAT3 and MYC occurred in PTCL-GATA3. Several CNAs, in particular loss of CDKN2A, exhibited prognostic significance in PTCL-NOS as a single entity and in the PTCL-GATA3 subgroup. The PTCL-TBX21 subgroup had fewer CNAs, primarily targeting cytotoxic effector genes, and was enriched in mutations of genes regulating DNA methylation. CNAs affecting metabolic processes regulating RNA/protein degradation and T-cell receptor signaling were common in both subgroups. AITL showed lower genomic complexity compared with other PTCL entities, with frequent co-occurring gains of chromosome 5 (chr5) and chr21 that were significantly associated with IDH2 R172 mutation. CN losses were enriched in genes regulating PI3K-AKT-mTOR signaling in cases without IDH2 mutation. Overall, we demonstrated that novel GEP-defined PTCL subgroups likely evolve by distinct genetic pathways and provided biological rationale for therapies that may be investigated in future clinical trials.

Evolutionary clonal trajectories in nodular lymphocyte-predominant Hodgkin lymphoma with high risk of transformation.

The B-cell architecture of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is complex since it is composed of malignant lymphocyte-predominant cells along with a rich B-cell bystander environment. To gain insight into molecular determinants of disease transformation, we studied B-cell evolutionary trajectories in lymphoma tissue from diagnosis to relapse or transformation to non- Hodgkin lymphoma by next-generation sequencing of immunoglobulin heavy chains. Patients with NLPHL that later transformed were older and showed IgD negativity, absence of the characteristic IGHV3/IGHD3/IGHJ6 lymphocyte-predominant rearrangement and high repertoire clonality. We constructed phylogenetic trees within the compartment of the malignant clone to investigate clonal evolution. In all relapsing cases, the lymphocyte-predominant rearrangement was identical at diagnosis and relapse. NLPHL cases with transformation showed more complex trajectories with strong intraclonal diversification. The dominant founder clone in transformations showed clonal evolution if derived from the same cell of origin, or arose from a different cell of origin. Together, our data point to a significant role of antigenic drive in the transformation of NLHPL and identify high B-cell repertoire clonality with dominant intraclonal lymphocyte-predominant cell diversification as a hallmark of transformation. Sequencing of initial paraffin-embedded tissue may therefore be applied diagnostically to identify NLPHL cases with high risk of transformation.

Identification of the atypically modified autoantigen Ars2 as the target of B-cell receptors from activated B-cell-type diffuse large B-cell lymphoma.

It has been suggested that B-cell receptor (BCRs) stimulation by specific antigens plays a pathogenic role in diffuse large B-cell lymphoma (DLBCL). Here, it was the aim to screen for specific reactivities of DLBCL-BCRs in the spectrum of autoantigens and antigens of infectious origin. Arsenite resistance protein 2 (Ars2) was identified as the BCR target of 3/5 ABC-type DLBCL cell lines and 2/11 primary DLBCL cases. Compared to controls, Ars2 was hypo-phosphorylated exclusively in cases and cell lines with Ars2-specific BCRs. In a validation cohort, hypo-phosphorylated Ars2 was found in 8/31 ABC-type, but only 1/20 germinal center B cell (GBC)-like type DLBCL. Incubation with Ars2 induced BCR-pathway activation and increased proliferation, while an Ars2/ETA-toxin conjugate induced killing of cell lines with Ars2-reactive BCRs. Ars2 appears to play a role in a subgroup of ABC-type DLBCLs. Moreover, transformed DLBCL lines with Ars2-reactive BCRs still show growth advantage after incubation with Ars2. These results provide knowledge about the pathogenic role of a specific antigen stimulating the BCR pathway in DLCBL.

Bioinformatics analysis of whole slide images reveals significant neighborhood preferences of tumor cells in Hodgkin lymphoma.

In pathology, tissue images are evaluated using a light microscope, relying on the expertise and experience of pathologists. There is a great need for computational methods to quantify and standardize histological observations. Computational quantification methods become more and more essential to evaluate tissue images. In particular, the distribution of tumor cells and their microenvironment are of special interest. Here, we systematically investigated tumor cell properties and their spatial neighborhood relations by a new application of statistical analysis to whole slide images of Hodgkin lymphoma, a tumor arising in lymph nodes, and inflammation of lymph nodes called lymphadenitis. We considered properties of more than 400, 000 immunohistochemically stained, CD30-positive cells in 35 whole slide images of tissue sections from subtypes of the classical Hodgkin lymphoma, nodular sclerosis and mixed cellularity, as well as from lymphadenitis. We found that cells of specific morphology exhibited significantly favored and unfavored spatial neighborhood relations of cells in dependence of their morphology. This information is important to evaluate differences between Hodgkin lymph nodes infiltrated by tumor cells (Hodgkin lymphoma) and inflamed lymph nodes, concerning the neighborhood relations of cells and the sizes of cells. The quantification of neighborhood relations revealed new insights of relations of CD30-positive cells in different diagnosis cases. The approach is general and can easily be applied to whole slide image analysis of other tumor types.