Julia Steinhilber

MYD88 L265P and CXCR4 mutations in lymphoplasmacytic lymphoma identify cases with high disease activity.

Recurrent mutations in MYD88 have been identified in >90% of lymphoplasmacytic lymphoma (LPL). Recently, WHIM (warts, hypogammaglobulinaemia, infections, myelokathexis) syndrome‐like mutations in CXCR4 have been described in 28% of LPL cases, and seem to impact clinical presentation and response to therapy. We investigated the presence of the MYD88 L265P mutation in 90 decalcified, formalin‐fixed, paraffin‐embedded (FFPE) bone marrow (BM) biopsies, including 51 cases of LPL, 14 cases of B‐cell chronic lymphocytic leukaemia (CLL), 13 cases of marginal zone lymphoma (MZL) and 12 normal controls. In addition, the C‐terminal domain of CXCR4 was sequenced in LPL cases. MYD88 L265P was found in 49/51 (96%) LPL cases and in 1/13 (7·6%) MZL (splenic type), whereas all CLL samples remained negative. The two MYD88 wild type LPL cases were associated with cold agglutinin disease. Mutations in CXCR4 were detected in 17/47 (36·2%) LPL cases, which showed a higher extent of BM infiltration and lower leucocyte counts (P = 0·02), haemoglobin (P = 0·05) and platelet counts (P = 0·01). In conclusion the detection of MYD88 L265P mutation in FFPE samples is reliable and useful for subtyping small B‐cell lymphomas in BM biopsies. In addition, the presence of CXCR4 mutations identifies a subgroup of LPL patients with higher disease activity.

Development of monocytosis in patients with primary myelofibrosis indicates an accelerated phase of the disease.

Primary myelofibrosis is a type of chronic myeloproliferative neoplasm characterized by progressive bone marrow failure with worsening cytopenia and in a subset of patients, progression to acute leukemia. Published data in patients with myelodysplastic syndromes have shown that the development of monocytosis in the course of myelodysplastic syndromes is associated with a poor prognosis. A similar occurrence has been only sporadically reported in patients with primary myelofibrosis. Over a period of four years we identified 10 out of 237 cases of primary myelofibrosis who developed persistent absolute monocytosis (>1 × 109/l) during the course of disease (5 men and 5 women; median age/range: 68 years/52–82). Monocytosis developed at a median interval of 42 months from diagnosis (range: 1–180) and persisted for a median period of 23 months (range: 2–57). Five patients died after developing monocytosis (range: 20–188 months) and two experienced worsening disease and became transfusion dependent. Monocytosis was associated with increased white blood cells, decreased hemoglobin, decreased platelet count, and the presence of circulating blasts. In three cases, bone marrow biopsies after the onset of monocytosis showed marked myelomonocytic proliferation with morphological shifting from a typical primary myelofibrosis marrow appearance to aspects compatible with an overt ‘secondary’ chronic myelomonocytic leukemia. Before the development of monocytosis, 5 of 10 patients carried the JAK2V617F mutation; five patients showed karyotypic alterations. No change in JAK2 mutational status or cytogenetic evolution were associated with the development of monocytosis. Four of nine patients analyzed showed KRAS mutation in codon 12 or 13 with low allele burden. This is the first study correlating monocytosis developing in primary myelofibrosis patients with bone marrow morphology, laboratory data, molecular analysis and clinical follow-up. Development of monocytosis in patients with established primary myelofibrosis is associated with rapid disease progression and these patients should be considered as a high-risk group associated with short survival.

Identification of C/EBPβ Target Genes in ALK+ Anaplastic Large Cell Lymphoma (ALCL) by Gene Expression Profiling and Chromatin Immunoprecipitation

C/EBPβ (CCAAT enhancer binding protein) is a transcription factor that plays a crucial role in survival and transformation of ALK+ anaplastic large cell lymphoma (ALCL). The aim of this study was to identify the downstream targets of C/EBPβ responsible for ALK-mediated oncogenesis. C/EBPβ was knocked down in ALK+ ALCL cell lines with a C/EBPβ-shRNA, followed by gene expression profiling (GEP). GEP analysis revealed a reproducible signature of genes that were significantly regulated by C/EBPβ. Classification into biological categories revealed overrepresentation of genes involved in the immune response, apoptosis and cell proliferation. Transcriptional regulation by C/EBPβ was found in 6 of 11 (BCL2A1, G0S2, TRIB1, S100A9, DDX21 and DDIT4) genes investigated by chromatin immunoprecipitation. We demonstrated that BCL2A1, G0S2 and DDX21 play a crucial role in survival and proliferation of ALK+ ALCL cells. DDX21, a gene involved in rRNA biogenesis, was found differentially overexpressed in primary ALK+ ALCL cases. All three candidate genes were validated in primary ALCL cases by either immunohistochemistry or RT-qPCR. In conclusion, we identified and validated several key C/EBPβ-regulated genes with major impact on survival and cell growth in ALK+ ALCL, supporting the central role of C/EBPβ in ALK-mediated oncogenesis.

Next-Generation Sequencing Identifies Deregulation of MicroRNAs Involved in Both Innate and Adaptive Immune Response in ALK+ ALCL

Anaplastic large cell lymphoma (ALCL) is divided into two systemic diseases according to the expression of the anaplastic lymphoma kinase (ALK). We investigated the differential expression of miRNAs between ALK+ ALCL, ALK- ALCL cells and normal T-cells using next generation sequencing (NGS). In addition, a C/EBPβ-dependent miRNA profile was generated. The data were validated in primary ALCL cases. NGS identified 106 miRNAs significantly differentially expressed between ALK+ and ALK- ALCL and 228 between ALK+ ALCL and normal T-cells. We identified a signature of 56 miRNAs distinguishing ALK+ ALCL, ALK- ALCL and T-cells. The top candidates significant differentially expressed between ALK+ and ALK- ALCL included 5 upregulated miRNAs: miR-340, miR-203, miR-135b, miR-182, miR-183; and 7 downregulated: miR-196b, miR-155, miR-146a, miR-424, miR-503, miR-424*, miR-542-3p. The miR-17-92 cluster was also upregulated in ALK+ cells. Additionally, we identified a signature of 3 miRNAs significantly regulated by the transcription factor C/EBPβ, which is specifically overexpressed in ALK+ ALCL, including the miR-181 family. Of interest, miR-181a, which regulates T-cell differentiation and modulates TCR signalling strength, was significantly downregulated in ALK+ ALCL cases. In summary, our data reveal a miRNA signature linking ALK+ ALCL to a deregulated immune response and may reflect the abnormal TCR antigen expression known in ALK+ ALCL.

Expression of miRNA-26b-5p and its target TRPS1 is associated with radiation exposure in post-Chernobyl breast cancer

Ionizing radiation is a well‐recognized risk factor for the development of breast cancer. However, it is unknown whether radiation‐specific molecular oncogenic mechanisms exist. We investigated post‐Chernobyl breast cancers from radiation‐exposed female clean‐up workers and nonexposed controls for molecular changes. Radiation‐associated alterations identified in the discovery cohort (n = 38) were subsequently validated in a second cohort (n = 39). Increased expression of hsa‐miR‐26b‐5p was associated with radiation exposure in both of the cohorts. Moreover, downregulation of the TRPS1 protein, which is a transcriptional target of hsa‐miR‐26b‐5p, was associated with radiation exposure. As TRPS1 overexpression is common in sporadic breast cancer, its observed downregulation in radiation‐associated breast cancer warrants clarification of the specific functional role of TRPS1 in the radiation context. For this purpose, the impact of TRPS1 on the transcriptome was characterized in two radiation‐transformed breast cell culture models after siRNA‐knockdown. Deregulated genes upon TRPS1 knockdown were associated with DNA‐repair, cell cycle, mitosis, cell migration, angiogenesis and EMT pathways. Furthermore, we identified the interaction partners of TRPS1 from the transcriptomic correlation networks derived from gene expression data on radiation‐transformed breast cell culture models and sporadic breast cancer tissues provided by the TCGA database. The genes correlating with TRPS1 in the radiation‐transformed breast cell lines were primarily linked to DNA damage response and chromosome segregation, while the transcriptional interaction partners in the sporadic breast cancers were mostly associated with apoptosis. Thus, upregulation of hsa‐miR‐26b‐5p and downregulation of TRPS1 in radiation‐associated breast cancer tissue samples suggests these molecules representing radiation markers in breast cancer.

EMMPRIN (CD147) is induced by C|[sol]|EBP|[beta]| and is differentially expressed in ALK|[plus]| and ALK|[minus]| anaplastic large-cell lymphoma

Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is characterized by expression of oncogenic ALK fusion proteins due to the translocation t(2;5)(p23;q35) or variants. Although genotypically a T-cell lymphoma, ALK+ ALCL cells frequently show loss of T-cell-specific surface antigens and expression of monocytic markers. C/EBPβ, a transcription factor constitutively overexpressed in ALK+ ALCL cells, has been shown to play an important role in the activation and differentiation of macrophages and is furthermore capable of transdifferentiating B-cell and T-cell progenitors to macrophages in vitro. To analyze the role of C/EBPβ for the unusual phenotype of ALK+ ALCL cells, C/EBPβ was knocked down by RNA interference in two ALK+ ALCL cell lines, and surface antigen expression profiles of these cell lines were generated using a Human Cell Surface Marker Screening Panel (BD Biosciences). Interesting candidate antigens were further analyzed by immunohistochemistry in primary ALCL ALK+ and ALK- cases. Antigen expression profiling revealed marked changes in the expression of the activation markers CD25, CD30, CD98, CD147, and CD227 after C/EBPβ knockdown. Immunohistochemical analysis confirmed a strong, membranous CD147 (EMMPRIN) expression in ALK+ ALCL cases. In contrast, ALK- ALCL cases showed a weaker CD147 expression. CD274 or PD-L1, an immune inhibitory receptor ligand, was downregulated after C/EBPβ knockdown. PD-L1 also showed stronger expression in ALK+ ALCL compared with ALK- ALCL, suggesting an additional role of C/EBPβ in ALK+ ALCL in generating an immunosuppressive environment. Finally, no expression changes of T-cell or monocytic markers were detected. In conclusion, surface antigen expression profiling demonstrates that C/EBPβ plays a critical role in the activation state of ALK+ ALCL cells and reveals CD147 and PD-L1 as important downstream targets. The multiple roles of CD147 in migration, adhesion, and invasion, as well as T-cell activation and proliferation suggest its involvement in the pathogenesis of ALCL.

EMMPRIN (CD147) is induced by C/EBPβ and is differentially expressed in ALK+ and ALK− anaplastic large-cell lymphoma

Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is characterized by expression of oncogenic ALK fusion proteins due to the translocation t(2;5)(p23;q35) or variants. Although genotypically a T-cell lymphoma, ALK+ ALCL cells frequently show loss of T-cell-specific surface antigens and expression of monocytic markers. C/EBPβ, a transcription factor constitutively overexpressed in ALK+ ALCL cells, has been shown to play an important role in the activation and differentiation of macrophages and is furthermore capable of transdifferentiating B-cell and T-cell progenitors to macrophages in vitro. To analyze the role of C/EBPβ for the unusual phenotype of ALK+ ALCL cells, C/EBPβ was knocked down by RNA interference in two ALK+ ALCL cell lines, and surface antigen expression profiles of these cell lines were generated using a Human Cell Surface Marker Screening Panel (BD Biosciences). Interesting candidate antigens were further analyzed by immunohistochemistry in primary ALCL ALK+ and ALK- cases. Antigen expression profiling revealed marked changes in the expression of the activation markers CD25, CD30, CD98, CD147, and CD227 after C/EBPβ knockdown. Immunohistochemical analysis confirmed a strong, membranous CD147 (EMMPRIN) expression in ALK+ ALCL cases. In contrast, ALK- ALCL cases showed a weaker CD147 expression. CD274 or PD-L1, an immune inhibitory receptor ligand, was downregulated after C/EBPβ knockdown. PD-L1 also showed stronger expression in ALK+ ALCL compared with ALK- ALCL, suggesting an additional role of C/EBPβ in ALK+ ALCL in generating an immunosuppressive environment. Finally, no expression changes of T-cell or monocytic markers were detected. In conclusion, surface antigen expression profiling demonstrates that C/EBPβ plays a critical role in the activation state of ALK+ ALCL cells and reveals CD147 and PD-L1 as important downstream targets. The multiple roles of CD147 in migration, adhesion, and invasion, as well as T-cell activation and proliferation suggest its involvement in the pathogenesis of ALCL.

The Pathological Spectrum of Systemic Anaplastic Large Cell Lymphoma (ALCL)

Anaplastic large cell lymphoma (ALCL) represents a group of malignant T-cell lymphoproliferations that share morphological and immunophenotypical features, namely strong CD30 expression and variable loss of T-cell markers, but differ in clinical presentation and prognosis. The recognition of anaplastic lymphoma kinase (ALK) fusion proteins as a result of chromosomal translocations or inversions was the starting point for the distinction of different subgroups of ALCL. According to their distinct clinical settings and molecular findings, the 2016 revised World Health Organization (WHO) classification recognizes four different entities: systemic ALK-positive ALCL (ALK+ ALCL), systemic ALK-negative ALCL (ALK− ALCL), primary cutaneous ALCL (pC-ALCL), and breast implant-associated ALCL (BI-ALCL), the latter included as a provisional entity. ALK is rearranged in approximately 80% of systemic ALCL cases with one of its partner genes, most commonly NPM1, and is associated with favorable prognosis, whereas systemic ALK− ALCL shows heterogeneous clinical, phenotypical, and genetic features, underlining the different oncogenesis between these two entities. Recognition of the pathological spectrum of ALCL is crucial to understand its pathogenesis and its boundaries with other entities. In this review, we will focus on the morphological, immunophenotypical, and molecular features of systemic ALK+ and ALK− ALCL. In addition, BI-ALCL will be discussed.

Diagnosis of Richter transformation in chronic lymphocytic leukemia: histology tips the scales

Development of diffuse large B-cell lymphoma in chronic lymphocytic leukemia, so-called Richter transformation (RT), occurs in 2–5% of patients and is associated with poor outcome. The clinical features of RT are fairly non-specific and unable to discriminate transformation from other mimics. In case of clinically suspected RT, a CT/MRT is recommended, and FDG-PET/CT may help to select the site of biopsy. Radiological features suggestive of RT have been defined, but there are only limited data about their predictive value, and histological confirmation is still considered the gold standard for RT diagnosis. We retrospectively analyzed 34 patients with clinically suspected RT and available radiological and histological data. A histopathological diagnosis of RT with concordant clinical and radiological findings was obtained in 13 patients. In 18 patients, CT did not show features of transformation, concordant with lack of RT in the biopsy. Of interest, a distinct lymphoma other than DLBCL was identified in two of these cases. A false-positive radiological diagnosis of RT was rendered in two patients, including a case of Herpes simplex virus lymphadenitis. In conclusion, our findings confirm the central role of tissue biopsy in the diagnostic work up in case of clinically suspected RT.

Langerhans cell histiocytosis associated with classical Hodgkin lymphoma contains BRAF V600E mutation

Clinical History A 13-year-old boy presented with generalized lymphadenopathy, fatigue and weight loss for five months. An excisional biopsy of enlarged left supraclavicular lymph node showed extensive nodal architectural effacement by a nodular proliferation containing scattered large atypical mononucleated, binucleated, and multinucleated cells with prominent eosinophilic nucleoli in the background of eosinophils, small lymphocytes, and plasma cells. The large atypical cells were positive for PAX-5 (weak), CD30 and CD15 (partial), and negative for CD3, CD20, CD45RB, and ALK-1 by immunohistochemical stains (Fig. 1a–c). In addition, there were multiple distinct sheets of large cells with nuclear grooves and abundant eosinophilic cytoplasm, which were positive for CD43, CD1a, S-100, and langerin, confirming them to be Langerhans cells (Fig. 1d–f). To investigate if there is BRAF V600E, c.1799T>A mutation, we performed the next-generation sequencing (NGS). Briefly, DNA was extracted from the microdissected Langerhans cells, and the NGS analysis was independently performed two times with the Ion Torrent PGM as described in the manufac ture r ’s pro tocol (Fus ion Method, Li fe Technologies, https://tools.thermofisher.com/content/sfs/ m a nu a l s / 4 4 6 8 3 26 _ I o nAmp l i c o nL i b r a r y P r e p _ FusionMethod_UG.pdf). The BRAF mutation p.V600E, c.1799T>A was detected in 2% in both runs and the reads on target have been in the first run 35053 reads and in the second run 31496 reads (Fig. 1g). There were no MAP2K1 hotspot mutations. Since the DNA extraction could not be repeated due to exhaustion of material, we employed allele-specific PCR followed by melting curve analysis and direct Sanger sequencing as previously described [1]. BRAF V600E (c.1799T>A) mutation was confirmed by melting curve analysis of the amplification products (Fig. 1h) and Sanger sequencing (Fig. 1i). A diagnosis of nodular sclerosis classical Hodgkin lymphoma (CHL) with foci of Langerhans cell histiocytosis (LCH) was rendered. The patient was treated as stage IIIA CHL with chemotherapy and focal radiation therapy. He remained disease free five years from his last treatment. It has been controversial if LCH associated with lymphoma represents a true neoplasm, highlighted by a recent finding that there were no BRAF or MAP2K1 mutations in LCH [2–5]. Our case may represent the first demonstration of BRAF V600E mutation in a typical case of LCH associated with lymphoma, supporting that at least some of the LCHs associated with lymphoma are true neoplasm. The twice-confirmed low allele frequency for the BRAF V600E mutation despite microdissection could point to an early clonal event in the background of an initially reactive Langerhans cell proliferation. * Shanxiang Zhang sz5@iupui.edu