PDGFRß promotes oncogenic progression via STAT3/STAT5 hyperactivation in anaplastic large cell lymphoma.

BACKGROUND: Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin T cell lymphoma commonly driven by NPM-ALK. AP-1 transcription factors, cJUN and JUNb, act as downstream effectors of NPM-ALK and transcriptionally regulate PDGFRß. Blocking PDGFRß kinase activity with imatinib effectively reduces tumor burden and prolongs survival, although the downstream molecular mechanisms remain elusive. METHODS AND RESULTS: In a transgenic mouse model that mimics PDGFRß-driven human ALCL in vivo, we identify PDGFRß as a driver of aggressive tumor growth. Mechanistically, PDGFRß induces the pro-survival factor Bcl-xL and the growth-enhancing cytokine IL-10 via STAT5 activation. CRISPR/Cas9 deletion of both STAT5 gene products, STAT5A and STAT5B, results in the significant impairment of cell viability compared to deletion of STAT5A, STAT5B or STAT3 alone. Moreover, combined blockade of STAT3/5 activity with a selective SH2 domain inhibitor, AC-4-130, effectively obstructs tumor development in vivo. CONCLUSIONS: We therefore propose PDGFRß as a novel biomarker and introduce PDGFRß-STAT3/5 signaling as an important axis in aggressive ALCL. Furthermore, we suggest that inhibition of PDGFRß or STAT3/5 improve existing therapies for both previously untreated and relapsed/refractory ALK+ ALCL patients.

SPARC-positive macrophages are the superior prognostic factor in the microenvironment of diffuse large B-cell lymphoma and independent of MYC rearrangement and double-/triple-hit status.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with respect to outcome. Features of the tumor microenvironment (TME) are associated with prognosis when assessed by gene expression profiling. However, it is uncertain whether assessment of the microenvironment can add prognostic information to the most relevant and clinically well-established molecular subgroups when analyzed by immunohistochemistry (IHC). PATIENTS AND METHODS: We carried out a histopathologic analysis of biomarkers related to TME in a very large cohort (n = 455) of DLBCL treated in prospective trials and correlated with clinicopathologic and molecular data, including chromosomal rearrangements and gene expression profiles for cell-of-origin and TME. RESULTS: The content of PD1+, FoxP3+ and CD8+, as well as vessel density, was not associated with outcome. However, we found a low content of CD68+ macrophages to be associated with inferior progression-free survival (PFS) and overall survival (OS; P = 0.023 and 0.040, respectively) at both univariable and multivariable analyses, adjusted for the factors of the International Prognostic Index (IPI), MYC break and BCL2/MYC and BCL6/MYC double-hit status. The subgroup of PDL1+ macrophages was not associated with survival. Instead, secreted protein acidic and cysteine rich (SPARC)-positive macrophages were identified as the subtype of macrophages most associated with survival. SPARC-positive macrophages and stromal cells directly correlated with favorable PFS and OS (both, P[log rank] <0.001, P[trend] < 0.001). The association of SPARC with prognosis was independent of the factors of the IPI, MYC double-/triple-hit status, Bcl2/c-myc double expression, cell-of-origin subtype and a recently published gene expression signature [lymphoma-associated macrophage interaction signature (LAMIS)]. CONCLUSIONS: SPARC expression in the TME detected by a single IHC staining with fair-to-good interobserver reproducibility is a powerful prognostic parameter. Thus SPARC expression is a strong candidate for risk assessment in DLBCL in daily practice.

First-line treatment with R-CHOP or rituximab-bendamustine in patients with follicular lymphoma grade 3A-results of a retrospective analysis.

Based on centroblast frequency, follicular lymphoma (FL) is subdivided into grades 1-2, 3A, and 3B. Grade FL3A frequently coexists with FL1-2 (FL1-2-3A). Based on clinical trials, FL1-2 is treated with rituximab (R) or obinutuzumab plus bendamustine (B) or CHOP, while FL3B is treated with R-CHOP. In contrast, there are little data guiding therapy in FL3A. We present a retrospective, multicenter analysis of 95 FL3A or FL1-2-3A and 203 FL1-2 patients treated with R-CHOP or R-B first-line. R-CHOP facilitated a higher response rate (95% versus 76%) and longer overall survival (OS) (3-year OS 89% versus 73%, P = 0.008) in FL3A or FL1-2-3A, whereas the difference in progression-free survival (PFS) did not reach statistical significance. While transformation rates into aggressive lymphoma were similar between both groups, there were more additional malignancies after R-B compared with R-CHOP (6 versus 2 cases). In FL1-2, R-B achieved a higher 3-year PFS (79% versus 47%, P < 0.01), while there was no significant difference regarding OS or transformation. With the limitations of a retrospective analysis, these results suggest a benefit for R-CHOP over R-B in FL3A or FL1-2-3A. Confirmatory data from prospective clinical trials are needed.

[Hematological neoplasias as differential diagnoses of soft tissue tumors]. / Hämatologische Neoplasien als Differenzialdiagnosen von Weichgewebstumoren.

BACKGROUND: Several hematological malignancies might morphologically present as differential diagnoses of sarcomas. OBJECTIVES: To illustrate entities of hematological malignancies that might cause difficulties in differentiation from sarcomas and to introduce immunohistochemical and molecular tests that facilitate the diagnosis. MATERIAL AND METHODS: Selective literature research ( http://www.ncbi.nlm.nih.gov ) was combined with the clinico-pathological experience of the authors. RESULTS: In particular, hematologic malignancies with small blue round cell cytology, as well as lymphomas with anaplastic or spindle cell morphology, may mimic sarcomas. Identification of the correct diagnosis is usually possible by applying immunohistochemical and molecular analyses. Lymphomas without expression of CD45 and hematological neoplasias with expression of markers characteristic of sarcomas may cause difficulties in differential diagnosis. CONCLUSION: Hematological malignancies should be kept in mind as differential diagnoses of sarcomas and should be excluded by immunohistochemical and molecular analyses according to morphology and the clinical picture.

[Aggressive B­cell lymphomas : Recommendations from the German Panel of Reference Pathologists in the Competence Network on Malignant Lymphomas on diagnostic procedures according to the current WHO classification, update 2017]. / Aggressive B­Zell-Lymphome : Empfehlungen des Deutschen Panels der Referenzpathologen im Kompetenznetz Maligne Lymphome e. V. zum diagnostischen Vorgehen nach der aktuellen WHO-Klassifikation, Update 2017.

The update of the 4th edition of the WHO classification for hematopoietic neoplasms introduces changes in the field of mature aggressive B­cell lymphomas that are relevant to diagnostic pathologists. In daily practice, the question arises of which analysis should be performed when diagnosing the most common lymphoma entity, diffuse large B­cell lymphoma. We discuss the importance of the cell of origin, the analysis of MYC translocations, and the delineation of the new WHO entities of high-grade B­cell lymphomas.

[Revised version of the 4th edition of the WHO classification of malignant lymphomas : What is new?] / Revidierte Fassung der 4. Ausgabe der WHO-Klassifikation maligner Lymphome : Was ist neu?

After 8 years, the WHO has now published the updated version of the 4th edition of the classification of hematopoietic and lymphoid tumors. This update provides a conceptual rewrite of existing entities as well as some new provisional entities and categories, particularly among the aggressive B­cell lymphomas. Important new diagnostic categories include the high-grade B­cell lymphomas, the large B­cell lymphoma with IRF4 rearrangement, and the Burkitt-like lymphoma with 11q aberrations. Of particular importance, new concepts concerning the taxonomy and classification of early lymphoid lesions or precursor lesions are included, such as the in situ follicular neoplasia or the in situ mantle cell neoplasia. In addition, the concept of indolent lymphoproliferations, such as breast-implant-associated anaplastic large cell lymphoma and the indolent T­cell lymphoproliferative disorder of the gastrointestinal tract, has been strengthened. Finally, diagnostic criteria for existing lymphoma entities have been refined.

Only strongly enhanced residual FDG uptake in early response PET (Deauville 5 or qPET ≥ 2) is prognostic in pediatric Hodgkin lymphoma: Results of the GPOH-HD2002 trial.

PURPOSE: In 2014, we published the qPET method to quantify fluorodeoxyglucose positron emission tomography (FDG-PET) responses. Analysis of the distribution of the quantified signals suggested that a clearly abnormal FDG-PET response corresponds to a visual Deauville score (vDS) of 5 and high qPET values ≥ 2. Evaluation in long-term outcome data is still pending. Therefore, we analyzed progression-free survival (PFS) by early FDG-PET response in a subset of the GPOH-HD2002 trial for pediatric Hodgkin lymphoma (PHL). PATIENTS/METHODS: Pairwise FDG-PET scans for initial staging and early response assessment after two cycles of chemotherapy were available in 93 PHL patients. vDS and qPET measurement were performed and related to PFS. RESULTS: Patients with a qPET value ≥ 2.0 or vDS of 5 had 5-year PFS rates of 44%, respectively 50%. Those with qPET values < 2.0 or vDS 1 to 4 had 5-year PFS rates of 90%, respectively 80%. The positive predictive value of FDG-PET response assessment increased from 18% (9%; 33%) using a qPET threshold of 0.95 (vDS ≤ 3) to 30% (13%; 54%) for a qPET threshold of 1.3 (vDS ≤ 4) and to 56% (23%; 85%) when the qPET threshold was ≥ 2.0 (vDS 5). The negative predictive values remained stable at ≥92% (CI: 82%; 98%). CONCLUSION: Only strongly enhanced residual FDG uptake in early response PET (vDS 5 or qPET ≥ 2, respectively) seems to be markedly prognostic in PHL when treatment according to the GPOH-HD-2002 protocol is given.

Differences between BCL2-break positive and negative follicular lymphoma unraveled by whole-exome sequencing.

Depending on disease stage follicular lymphoma (FL) lack the t(14;18) in ~15-~50% of cases. Nevertheless, most of these cases express BCL2. To elucidate mechanisms triggering BCL2 expression and promoting pathogenesis in t(14;18)-negative FL, exonic single-nucleotide variant (SNV) profiles of 28 t(14;18)-positive and 13 t(14;18)-negative FL were analyzed, followed by the integration of copy-number changes, copy-neutral LOH and published gene-expression data as well as the assessment of immunoglobulin N-glycosylation sites. Typical FL mutations also affected t(14;18)-negative FL. Curated gene set/pathway annotation of genes mutated in either t(14;18)-positive or t(14;18)-negative FL revealed a strong enrichment of same or similar gene sets but also a more prominent or exclusive enrichment of immune response and N-glycosylation signatures in t(14;18)-negative FL. Mutated genes showed high BCL2 association in both subgroups. Among the genes mutated in t(14;18)-negative FL 555 were affected by copy-number alterations and/or copy-neutral LOH and 96 were differently expressed between t(14;18)-positive and t(14;18)-negative FL (P<0.01). N-glycosylation sites were detected considerably less frequently in t(14;18)-negative FL. These results suggest a diverse portfolio of genetic alterations that may induce or regulate BCL2 expression or promote pathogenesis of t(14;18)-negative FL as well as a less specific but increased crosstalk with the microenvironment that may compensate for the lack of N-glycosylation.

Molecular signatures that can be transferred across different omics platforms.

MOTIVATION: Molecular signatures for treatment recommendations are well researched. Still it is challenging to apply them to data generated by different protocols or technical platforms. RESULTS: We analyzed paired data for the same tumors (Burkitt lymphoma, diffuse large B-cell lymphoma) and features that had been generated by different experimental protocols and analytical platforms including the nanoString nCounter and Affymetrix Gene Chip transcriptomics as well as the SWATH and SRM proteomics platforms. A statistical model that assumes independent sample and feature effects accounted for 69-94% of technical variability. We analyzed how variability is propagated through linear signatures possibly affecting predictions and treatment recommendations. Linear signatures with feature weights adding to zero were substantially more robust than unbalanced signatures. They yielded consistent predictions across data from different platforms, both for transcriptomics and proteomics data. Similarly stable were their predictions across data from fresh frozen and matching formalin-fixed paraffin-embedded human tumor tissue. AVAILABILITY AND IMPLEMENTATION: The R-package 'zeroSum' can be downloaded at https://github.com/rehbergT/zeroSum . Complete data and R codes necessary to reproduce all our results can be received from the authors upon request. CONTACT: rainer.spang@ur.de.

[Recovery of paraffin blocks and central archiving : Experiences of the Kiel lymph node registry and the German study group for Hodgkin lymphoma]. / Rückforderung von Paraffinblöcken und zentrale Archivierung : Erfahrungen des Lymphknotenregisters Kiel und der Deutschen Studiengruppe für Hodgkin Lymphome.

BACKGROUND: Central collection of tissue blocks for pathological and translational research is particularly important in rare diseases. Transfer of tissue blocks from primary to central pathology is of crucial importance. OBJECTIVES: We aimed to answer the following questions: Has the transfer of tissue blocks sent for consultation or within clinical trials changed over the last 20 years? What are the reasons for reclaiming tissue blocks by the primary pathology and what actions would convince primary pathologists to leave the blocks in the reference pathology? MATERIAL AND METHODS: The first 100 biopsies of each year between 1995 and 2015 (n = 2100), as well as all tissue transfers within therapeutic studies (n = 1405, German Hodgkin Study Group, GHSG) between 1998 and 2015, were analyzed separately for block reclaims using the Department of Pathology database. A questionnaire evaluated the reasons for block reclaiming by the peripheral pathologists. RESULTS: There is a significant increase in block reclaims during the period analyzed among submissions for consultation as well as in clinical trials (linear regression, p = 0.0195 and p = 0.0107). The percentage of block reclaims does not differ between consultations and cases submitted upon request within clinical trials (p = 0.2404, t-test). A survey among pathologies that reclaim the block showed that their willingness to leave the block at the reference center would increase if the compatibility with accreditation guidelines (39.3%), a positive statement from professional associations (25%), or a formal confirmation of availability (53.6%) is provided. DISCUSSION: In particular, to improve research on rare diseases, it is desirable to point out the compatibility of central archiving in a designated center with accreditation guidelines.

WNT5A: a motility-promoting factor in Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) has a typical clinical manifestation, with dissemination involving functionally neighboring lymph nodes. The factors involved in the spread of lymphoma cells are poorly understood. Here we show that cHL cell lines migrate with higher rates compared with non-Hodgkin lymphoma cell lines. cHL cell migration, invasion and adhesion depend on autocrine WNT signaling as revealed by the inhibition of WNT secretion with the porcupine inhibitors Wnt-C59/IWP-2, but did not affect cell proliferation. While application of recombinant WNT5A or WNT5A overexpression stimulates HL cell migration, neither WNT10A, WNT10B nor WNT16 did so. Time-lapse studies revealed an amoeboid type of cell migration modulated by WNT5A. Reduced migration distances and velocity of cHL cells, as well as altered movement patterns, were observed using porcupine inhibitor or WNT5A antagonist. Knockdown of Frizzled5 and Dishevelled3 disrupted the WNT5A-mediated RHOA activation and cell migration. Overexpression of DVL3-K435M or inhibition of ROCK (Rho-associated protein kinase) by Y-27632/H1152P disrupted cHL cell migration. In addition to these mechanistic insights into the role of WNT5A in vitro, global gene expression data revealed an increased WNT5A expression in primary HL cells in comparison with normal B-cell subsets and other lymphomas. Furthermore, the activity of both porcupine and WNT5A in cHL cells had an impact on lymphoma development in the chick chorionallantoic membrane assay. Massive bleeding of these lymphomas was significantly reduced after inhibition of WNT secretion by Wnt-C59. Therefore, a model is proposed where WNT signaling has an important role in regulating tumor-promoting processes.

Inactivation of the putative ubiquitin-E3 ligase PDLIM2 in classical Hodgkin and anaplastic large cell lymphoma.

Apart from its unique histopathological appearance with rare tumor cells embedded in an inflammatory background of bystander cells, classical Hodgkin lymphoma (cHL) is characterized by an unusual activation of a broad range of signaling pathways involved in cellular activation. This includes constitutive high-level activity of nuclear factor-κB (NF-κB), Janus kinase/signal transducer and activator of transcription (JAK/STAT), activator protein-1 (AP-1) and interferon regulatory factor (IRF) transcription factors (TFs) that are physiologically only transiently activated. Here, we demonstrate that inactivation of the putative ubiquitin E3-ligase PDLIM2 contributes to this TF activation. PDLIM2 expression is lost at the mRNA and protein levels in the majority of cHL cell lines and Hodgkin and Reed-Sternberg (HRS) cells of nearly all cHL primary samples. This loss is associated with PDLIM2 genomic alterations, promoter methylation and altered splicing. Reconstitution of PDLIM2 in HRS cell lines inhibits proliferation, blocks NF-κB transcriptional activity and contributes to cHL-specific gene expression. In non-Hodgkin B-cell lines, small interfering RNA-mediated PDLIM2 knockdown results in superactivation of TFs NF-κB and AP-1 following phorbol 12-myristate 13-acetate (PMA) stimulation. Furthermore, expression of PDLIM2 is lost in anaplastic large cell lymphoma (ALCL) that shares key biological aspects with cHL. We conclude that inactivation of PDLIM2 is a recurrent finding in cHL and ALCL, promotes activation of inflammatory signaling pathways and thereby contributes to their pathogenesis.

Microenvironmental interactions between endothelial and lymphoma cells: a role for the canonical WNT pathway in Hodgkin lymphoma.

The interaction between vascular endothelial cells (ECs) and cancer cells is of vital importance to understand tumor dissemination. A paradigmatic cancer to study cell-cell interactions is classical Hodgkin Lymphoma (cHL) owing to its complex microenvironment. The role of the interplay between cHL and ECs remains poorly understood. Here we identify canonical WNT pathway activity as important for the mutual interactions between cHL cells and ECs. We demonstrate that local canonical WNT signaling activates cHL cell chemotaxis toward ECs, adhesion to EC layers and cell invasion using not only the Wnt-inhibitor Dickkopf, tankyrases and casein kinase 1 inhibitors but also knockdown of the lymphocyte enhancer binding-factor 1 (LEF-1) and ß-catenin in cHL cells. Furthermore, LEF-1- and ß-catenin-regulated cHL secretome promoted EC migration, sprouting and vascular tube formation involving vascular endothelial growth factor A (VEGF-A). Importantly, high VEGFA expression is associated with a worse overall survival of cHL patients. These findings strongly support the concept that WNTs might function as a regulator of lymphoma dissemination by affecting cHL cell chemotaxis and promoting EC behavior and thus angiogenesis through paracrine interactions.

Clinical, pathological and genetic features of follicular lymphoma grade 3A: a joint analysis of the German low-grade and high-grade lymphoma study groups GLSG and DSHNHL.

BACKGROUND: Histologically, follicular lymphoma (FL) grades 1, 2 and 3A are composed of two distinct cell types, centroblasts and centrocytes. FL grade 3B is composed only of centroblasts and has been shown to differ in immunophenotype and genetics from FL that contain centrocytes. We aimed to understand the pathogenetic and clinical relation between FL grade 3A to FL grade 1/2 on the one hand and FL grade 3B on the other hand. PATIENTS AND METHODS: Trial patients with long-term follow-up and diagnosis of FL grade 3 were selected and samples underwent a second central pathological review using a multiple-observer approach to assess grading. RESULTS: Interobserver variability for diagnosing FL grade 3 was high. FL grade 3A frequently harbored areas of FL grade 1/2 within the same tissue specimen. FL grade 3B rarely coexisted with grade 1/2 or 3A, suggesting divergent pathogenesis. There was no statistically significant difference in outcome between 47 cases of FL grade 3A and 14 cases of grade 3B. Compared with grade 1/2 FL, both groups showed longer progression-free survival without late events, especially after immunochemotherapy; this outcome difference was retained after adjustment for clinical prognostic factors. The subgroup of FL grade 3A with an additional FL grade 1/2 component or a translocation t(14;18) showed a poorer outcome. In contrast, the FL grade 3A lacking t(14;18) and of localized stage resembled the pediatric type of FL and showed a very good outcome. FL3 with MYC breaks showed a poor outcome. CONCLUSIONS: The results suggest that first-line immunochemotherapy might allow long-lasting remissions in a subgroup of FL grade 3A similar to diffuse large B-cell lymphoma. Within FL3A, prognostic subgroups can be identified by analyzing for coexisting FL1/2 and MYC breaks.

[Classification of malignant lymphomas. Current situation]. / Klassifikation maligner Lymphome. Aktueller Stand.

BACKGROUND: The World Health Organization (WHO) is planning an update of the WHO classification of malignant lymphomas. OBJECTIVE: To present new findings concerning the diagnostics and subclassification of malignant lymphomas. MATERIAL AND METHODS: A selective literature search ( http://www.ncbi.nlm.nih.gov ) was carried out and combined with the practical experiences of the authors in clinicopathological diagnostics. RESULTS: In recent years an increasing number of early lesions of malignant lymphomas have been described but the potential malignancy of these lesions is at least for some entities still uncertain. Newly defined entities have been described within the group of T-cell lymphomas and prognostic subgroups have been identified in the heterogeneous group of diffuse large B-cell lymphomas. Detection of mutations facilitates the differential diagnostics of morphologically similar diseases and can be an important component of the diagnostics. CONCLUSION: Recent scientific insights are being included more and more into the diagnostics of lymphomas. The update of the WHO classification is a consequence of these developments.

Similar clinical features in follicular lymphomas with and without breaks in the BCL2 locus.

Approximately 15% of follicular lymphomas (FLs) lack breaks in the BCL2 locus. The aim of this study was to better define molecular and clinical features of BCL2-breakpoint/t(14;18)-negative FLs. We studied the presence of BCL2, BCL6 and MYC breaks by fluorescence in situ hybridization and the expression of BCL2, MUM1, CD10, P53 and Ki67 in large clinical trial cohorts of 540 advanced-stage FL cases and 116 early-stage disease FL patients treated with chemotherapy regimens and radiation, respectively. A total of 86% and 53% of advanced- and early-stage FLs were BCL2-breakpoint-positive, respectively. BCL2 was expressed in almost all FLs with BCL2 break and also in 86% and 69% of BCL2-breakpoint-negative advanced- and early-stage FLs, respectively. CD10 expression was significantly reduced in BCL2-breakpoint-negative FLs of all stages and MUM1 and Ki67 expression were significantly increased in BCL2-break-negative early-stage FLs. Patient characteristics did not differ between FLs with and without BCL2 breaks and neither did survival times in advanced-stage FLs. These results suggest that the molecular profile differs to some extent between FLs with and without BCL2 breaks and support the notion that FLs with and without BCL2 breaks belong to the same lymphoma entity.