Panel Sequencing of Primary Cutaneous B-Cell Lymphoma.

BACKGROUND: Primary cutaneous follicular B-cell lymphoma (PCFBCL) represents an indolent subtype of Non-Hodgkin's lymphomas, being clinically characterized by slowly growing tumors of the skin and common cutaneous relapses, while only exhibiting a low propensity for systemic dissemination or fatal outcome. Up to now, only few studies have investigated underlying molecular alterations of PCFBCL with respect to somatic mutations. OBJECTIVES: Our aim was to gain deeper insight into the pathogenesis of PCFBCL and to delineate discriminatory molecular features of this lymphoma subtype. METHODS: We performed hybridization-based panel sequencing of 40 lymphoma-associated genes of 10 cases of well-characterized PCFBCL. In addition, we included two further ambiguous cases of atypical B-cell-rich lymphoid infiltrate/B-cell lymphoma of the skin for which definite subtype attribution had not been possible by routine investigations. RESULTS: In 10 out of 12 analyzed cases, we identified genetic alterations within 15 of the selected 40 target genes. The most frequently detected alterations in PCFBCL affected the TNFRSF14, CREBBP, STAT6 and TP53 genes. Our analysis unrevealed novel mutations of the BCL2 gene in PCFBCL. All patients exhibited an indolent clinical course. Both the included arbitrary cases of atypical B-cell-rich cutaneous infiltrates showed somatic mutations within the FAS gene. As these mutations have previously been designated as subtype-specific recurrent alterations in primary cutaneous marginal zone lymphoma (PCMZL), we finally favored the diagnosis of PCMZL in these two cases based on these molecular findings. CONCLUSIONS: To conclude, our molecular data support that PCFBCL shows distinct somatic mutations which may aid to differentiate PCFBCL from pseudo-lymphoma as well as from other indolent and aggressive cutaneous B-cell lymphomas. While the detected genetic alterations of PCFBCL did not turn out to harbor any prognostic value in our cohort, our molecular data may add adjunctive discriminatory features for diagnostic purposes on a molecular level.

Clinical, histopathological and prognostic features of primary cutaneous acral CD8+ T-cell lymphoma and other dermal CD8+ cutaneous lymphoproliferations: results of an EORTC Cutaneous Lymphoma Group workshop.

BACKGROUND: The differential diagnosis of atypical dermal nonepidermotropic CD8+ lymphocytic infiltrates includes a heterogeneous spectrum of lymphoproliferations with overlapping histological and phenotypic features, but divergent clinical manifestations and prognoses. As these neoplasms are rare, more data on their clinicopathological presentation and course are needed. OBJECTIVES: To assess the clinical, histological and immunophenotypic features; outcomes of; and differences between dermal CD8+ lymphoproliferations. METHODS: Retrospective analysis of a series of 46 patients and biopsies by the international EORTC Cutaneous Lymphoma Group. RESULTS: The dermal CD8+ lymphoproliferations (n = 46) could be assigned to one of three groups: (i) cutaneous acral CD8+ T-cell lymphoma (n = 31), characterized mostly by a solitary nodule arising at acral sites, a monotonous dermal infiltrate of small-to-medium-sized CD8+ lymphocytes with a characteristic dot-like pattern of CD68, a low proliferation rate and an excellent prognosis; (ii) primary cutaneous CD8+ peripheral T-cell lymphoma, unspecified/NOS (n = 11), presenting with one or multiple rapidly evolving tumours, mostly medium-sized pleomorphic CD8+ tumour cells with expression of several cytotoxic markers, and high proliferative activity; and (iii) cutaneous CD8+ lymphoproliferations (n = 4), associated with congenital immunodeficiency syndromes in two patients with persisting localized or disseminated violaceous to brownish plaques on the extremities, a histiocyte-rich infiltrate of mostly small CD8+ lymphocytes with subtle atypia and a protracted course; and papular CD8+ eruptions in two patients with acquired immunosuppression. CONCLUSIONS: A constellation of distinct clinical, histopathological and phenotypic features allows discrimination and assignment of dermal CD8+ infiltrates into distinct disease entities. Primary cutaneous acral CD8+ lymphoma, assigned a provisional category in current lymphoma classifications, is a distinct and reproducible entity. A correct diagnosis is essential to avoid unnecessarily aggressive treatment for indolent CD8+ lymphoproliferations and to identify cases with underlying immuno-deficiency or potential for dismal outcome.

Oncogenic Mutations and Gene Fusions in CD30-Positive Lymphoproliferations and Clonally Related Mycosis Fungoides Occurring in the Same Patients.

The emergence of a common progenitor cell has been postulated for the association of CD30-positive lymphoproliferative disease (LPD) and mycosis fungoides (MF) within the same patient. Up to now, no comprehensive analysis has yet addressed the genetic profiles of such concurrent lymphoma subtypes. We aimed to delineate the molecular alterations of clonally related CD30-positive LPD and MF occurring in the same two patients. We analyzed the molecular profile of 16 samples of two patients suffering both from CD30-positive LPD and MF being obtained over a time course of at least 5 years. To detect oncogenic mutations, we applied targeted sequencing technologies with a hybrid capture-based DNA library preparation approach, and for the identification of fusion transcripts, an anchored multiplex PCR enrichment kit was used. In all samples of CD30-positive LPD and MF, oncogenic fusions afflicting the Jak/signal transducer and activator of transcription signaling pathway were present, namely NPM1‒TYK2 in patient 1 and ILF3‒JAK2 in patient 2. Additional signal transducer and activator of transcription 5A gene STAT5A mutations exclusively occurred in lesions of CD30-positive LPD in one patient. CD30-positive LPD and MF may share genetic events when occurring within the same patients. Constitutive activation of the Jak/signal transducer and activator of transcription signaling pathway may play a central role in the molecular pathogenesis of both entities.

Targeted Deep Sequencing of Mycosis Fungoides Reveals Intracellular Signaling Pathways Associated with Aggressiveness and Large Cell Transformation.

INTRODUCTION: Large-cell transformation (LCT) of mycosis fungoides (MF) has been associated with a higher risk of relapse and progression and, consequently, restricted prognosis. Its molecular pathogenesis has not been elucidated yet. MATERIALS AND METHODS: In order to address molecular mechanisms of LCT, we performed hybrid capture panel-based sequencing of skin biopsies from 10 patients suffering from MF with LCT versus 17 patients without LCT including follow-up biopsies during clinical course, respectively (51 samples in total). The analyzed patients were attributed to three different groups based on the presence of LCT and clinical behavior. RESULTS: While indolent MF cases without LCT did not show pathogenic driver mutations, a high rate of oncogenic alterations was detected in patients with LCT and aggressive clinical courses. Various genes of different oncogenic signaling pathways, including the MAPK and JAK-STAT signaling pathways, as well as epigenetic modifiers were affected. A high inter-individual and distinctive intra-individual mutation diversity was observed. Oncogenic RAS mutations were exclusively detected in patients with LCT. CONCLUSION: Our data demonstrate that LCT transition of MF is associated with increased frequency of somatic mutations in cancer-associated genes. In particular, the activation of RAS signaling-together with epigenetic dysregulation-may crucially contribute to the molecular pathogenesis of the LCT phenotype, thus conveying its adverse clinical behavior.

Clonal expansion of large granular lymphocytes in patients with spondyloarthritis and psoriatic arthritis treated with TNFα inhibitors.

To determine the prevalence of clonal T-large granular lymphocyte (T-LGL) cells in patients with spondyloarthritis (SpA) and psoriatic arthritis (PsA) and to define possible risk factors for this condition. We present a cross-sectional analysis with retrospective and prospective aspects. 115 SpA patients, 48 PsA patients and 51 controls were recruited between December 28, 2017 and January 23, 2019. Flow cytometry (FACS) was performed to screen for aberrant T-LGL cells. Molecular analysis was then employed to confirm the diagnosis in patients with suggestive FACS findings. Patients with clonal T-LGL populations were followed prospectively by FACS analysis. Electronic patient files were retrospectively analyzed to determine risk factors. Median age was 49 years for SpA, 55.5 years for PsA, and 54 years for controls. Median disease duration of SpA and PsA was 15 years and 11 years, respectively. 79.8% of patients had received biologics at some point, 75.5% had ever received tumor necrosis factor (TNF) inhibitors. 59.5% were treated with TNF inhibitors at the time of study inclusion. We identified clonal T-LGL expansions in 13 individuals equaling a prevalence of 6% (13/214). T-LGL patients were taking TNF inhibitors more frequently at the time of study inclusion (p = 0.022) and were more likely to have ever been treated with TNF inhibition (p = 0.046). Clonal T-LGL expansions can be detected in patients with SpA, PsA and also in healthy controls. Confirming earlier results, exposure to TNFα-blocking agents appears to increase the risk of developing clonal expansions of T-LGL cells.

EBER in situ hybridization in subcutaneous aluminum granulomas/lymphoid hyperplasia: A diagnostic clue to differentiate injection-associated lymphoid hyperplasia from other forms of pseudolymphomas and cutaneous lymphomas.

BACKGROUND: Subcutaneous vaccination or desensitization may induce persistent nodules at the injection sites. Without the knowledge of prior injection, histopathological work-up may be challenging. OBJECTIVE: Aim of this study was to contribute to the histopathological work-up of unclear subcutaneous nodules, especially their differentiation from cutaneous lymphoma. METHODS: We retrospectively reviewed clinical data and histopathological slides of four patients with subcutaneous nodules, which were suspected to suffer from cutaneous T- or B-cell lymphoma. Sections of these cases and 12 negative controls were stained with hematoxylin and eosin and a standardized immunohistochemical panel of B- and T-cell markers including EBER in situ hybridization as well as electron microscopy. RESULTS: In all cases, large histiocytes with granular cytoplasm compatible with intracellular aluminum hydroxide were present. EBER in situ hybridization revealed positive staining of these granular histiocytes while staining was absent in negative controls. LIMITATIONS: Post hoc completion of medical history revealed that vaccination or specific immunotherapy had been applied before at the biopsy site in only three out of four patients; one patient was lost to follow-up. CONCLUSION: EBER in situ hybridization is an adjunctive tool to differentiate aluminum-induced granuloma/lymphoid hyperplasia from other forms of pseudolymphoma and cutaneous B- or T-cell lymphomas.

The transcription factor NRF2 enhances melanoma malignancy by blocking differentiation and inducing COX2 expression.

The transcription factor NRF2 is the major mediator of oxidative stress responses and is closely connected to therapy resistance in tumors harboring activating mutations in the NRF2 pathway. In melanoma, such mutations are rare, and it is unclear to what extent melanomas rely on NRF2. Here we show that NRF2 suppresses the activity of the melanocyte lineage marker MITF in melanoma, thereby reducing the expression of pigmentation markers. Intriguingly, we furthermore identified NRF2 as key regulator of immune-modulating genes, linking oxidative stress with the induction of cyclooxygenase 2 (COX2) in an ATF4-dependent manner. COX2 is critical for the secretion of prostaglandin E2 and was strongly induced by H2O2 or TNFα only in presence of NRF2. Induction of MITF and depletion of COX2 and PGE2 were also observed in NRF2-deleted melanoma cells in vivo. Furthermore, genes corresponding to the innate immune response such as RSAD2 and IFIH1 were strongly elevated in absence of NRF2 and coincided with immune evasion parameters in human melanoma datasets. Even in vitro, NRF2 activation or prostaglandin E2 supplementation blunted the induction of the innate immune response in melanoma cells. Transcriptome analyses from lung adenocarcinomas indicate that the observed link between NRF2 and the innate immune response is not restricted to melanoma.

Recurrent Oncogenic JAK and STAT Alterations in Cutaneous CD30-Positive Lymphoproliferative Disorders.

The group of cutaneous CD30-positive lymphoproliferative disorders (LPD) comprises two different entities, namely lymphomatoid papulosis (LyP) and cutaneous anaplastic large T-cell lymphoma (cALCL). LyP constitutes a benign lymphoproliferation with spontaneously regressing papules, whereas cALCL presents with solitary or multiple skin tumors with a low propensity to disseminate. To elucidate the hitherto largely unknown molecular pathogenesis of these entities, we performed comprehensive next-generation sequencing in a well-characterized cohort of 12 patients. Considering the low tumor cell content of LyP, we applied targeted sequencing technologies with a hybrid capture-based DNA library preparation approach and for the identification of fusion transcripts an anchored multiplex PCR enrichment kit. As the major finding, we detected, in 50% of LPD, genetic events that implied a constitutively activated Janus kinase-signal transducer and activator of transcription signaling (JAK-STAT) pathway in these entities. The identified molecular aberrations comprised either pathogenic STAT mutations or oncogenic fusion transcripts comprising effector domains of JAK. With respect to LyP, we report to our knowledge such previously unreported genetic aberrations in this specific entity. The detection of these convergent aberrations within the JAK-STAT signaling pathway deciphers common potential driving mechanisms of lymphomagenesis within LPD being shared between LyP and cALCL. Moreover, the presence of these oncogenic alterations paves the way to develop novel personalized treatment strategies.

T-cell pseudolymphoma in recurrent herpes simplex virus infection.

BACKGROUND: In cases of herpes virus infection without typical histologic (and clinical) signs it is difficult to achieve the correct diagnosis by histology alone. Some of those cases are prone to be misdiagnosed as cutaneous lymphoma. METHODS: This retrospective study included five patients with herpes simplex virus (HSV)-associated pseudolymphoma. We investigated clinical, histomorphologic and immunophenotypic features of all patients. RESULTS: All biopsy specimens presented a superficial and deep perivascular lymphohistiocytic infiltrate with epidermotropism, atypia and admixed plasma cells to varying degrees. Four of five samples showed lining-up of lymphocytes in the junctional zone with predominance of CD8+ lymphocytes, in contrast to the dermal part (inverse CD8:CD4 ratio). Papillary edema was found in four of five cases. Clinically, patchy erythema located on the buttocks and adjacent areas was typical, sometimes with erosions and crusts. Medical history of recurrent blisters, pain or itching was additionally helpful. CONCLUSION: We point out subtle but consistent histomorphologic criteria, which were helpful to diagnose HSV-associated pseudolymphoma in context with the clinical presentation.

Primary cutaneous diffuse large B-cell lymphoma, NOS and leg type: Clinical, morphologic and prognostic differences.

BACKGROUND AND OBJECTIVES: Primary cutaneous diffuse large B-cell lymphoma, NOS (PCLBCL/NOS) is a rare PCLBCL. Only few data are available for this tumor. The aim of this study was to identify clinical and/or immunohistochemical markers (in addition to Bcl-2) that characterize PCLBCL/NOS, assist in differentiating it from PCLBCL, leg type (PCLBCL/LT) and help to assess the clinical course/prognosis. PATIENTS AND METHODS: Bcl-2- PCLBCL/NOS) cases (n = 14 were compared with Bcl-2+ PCLBCL/LT cases (n = 29). RESULTS: PCLBCL/NOS patients were younger, predominantly male and had better survival rates than patients with PCLBCL/LT. Patients with PCLBCL/NOS presented more often with larger plaques limited to one or two contiguous body regions, whereas PCLBCL/LT cases often presented with disseminated lesions. Neoplastic cells had a higher proliferation rate (Ki67) in PCLBCL/LT patients. The tumor microenvironment of PCLBCL/NOS had a more prominent CD3+ infiltrate. Overall survival data for the whole cohort (n = 37) revealed that female gender and Bcl-2 expression correlated with a worse survival rate. Bcl-6 expression and centroblastic subtype correlated with better outcomes. None of the other markers studied (e.g. GCB/non-GCB subtype) correlated with survival rate. CONCLUSIONS: PCLBCL/NOS and PCLBCL/LT differ in their clinical behavior and outcomes. Bcl-2 still seems to be the best marker for discriminating between these two subgroups. Bcl-2, female gender and Bcl-6 represent prognostic markers for PCLBCL.

Genomic Profiling Identifies Outcome-Relevant Mechanisms of Innate and Acquired Resistance to Third-Generation Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Therapy in Lung Cancer.

PURPOSE: Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are effective in acquired resistance (AR) to early-generation EGFR TKIs in EGFR-mutant lung cancer. However, efficacy is marked by interindividual heterogeneity. We present the molecular profiles of pretreatment and post-treatment samples from patients treated with third-generation EGFR TKIs and their impact on treatment outcomes. METHODS: Using the databases of two lung cancer networks and two lung cancer centers, we molecularly characterized 124 patients with EGFR p.T790M-positive AR to early-generation EGFR TKIs. In 56 patients, correlative analyses of third-generation EGFR TKI treatment outcomes and molecular characteristics were feasible. In addition, matched post-treatment biopsy samples were collected for 29 patients with progression to third-generation EGFR TKIs. RESULTS: Co-occurring genetic aberrations were found in 74.4% of EGFR p.T790-positive samples (n = 124). Mutations in TP53 were the most frequent aberrations detected (44.5%; n = 53) and had no significant impact on third-generation EGFR TKI treatment. Mesenchymal-epithelial transition factor (MET) amplifications were found in 5% of samples (n = 6) and reduced efficacy of third-generation EGFR TKIs significantly (eg, median progression-free survival, 1.0 months; 95% CI, 0.37 to 1.72 v 8.2 months; 95% CI, 1.69 to 14.77 months; P ≤ .001). Genetic changes in the 29 samples with AR to third-generation EGFR TKIs were found in EGFR (eg, p.T790M loss, acquisition of p.C797S or p.G724S) or in other genes (eg, MET amplification, KRAS mutations). CONCLUSION: Additional genetic aberrations are frequent in EGFR-mutant lung cancer and may mediate innate and AR to third-generation EGFR TKIs. MET amplification was strongly associated with primary treatment failure and was a common mechanism of AR to third-generation EGFR TKIs. Thus, combining EGFR inhibitors with TKIs targeting common mechanisms of resistance may delay AR.

Paraproteinaemia in Primary Cutaneous Marginal Zone Lymphoma.

Primary cutaneous marginal zone lymphomas (PCMZL) frequently exhibit lymphoplasmacytoid/plasmacytic differentiation, implying the capacity to produce monoclonal immunoglobulins. As these paraproteins are secreted, and thus are measurable in blood and urine, they may correlate with disease burden and serve as tumour markers reflecting therapeutic response. This study retrospectively analysed the records of 23 patients with PCMZL. During treatment and follow-up, laboratory tests, including full blood count, lactate dehydrogenase, serum protein electrophoresis and turbidimetric analyses, were conducted. Thirty-nine percent of cases showed a suspicious serum protein electrophoresis in terms of paraproteinaemia. In 44% of cases the heavy and light chain restriction in tissue samples correlated with serological findings. Altogether, 89% of the PCMZL patients with paraproteinaemia eventually experienced a relapse, in contrast to 62% of the group without paraproteinaemia. This study analysed the incidence and clinical implications of paraproteinaemia in patients with PCMZL. A clear correlation was found between paraproteinaemia, tumour relapse and therapeutic intervention.

Prevalence and Characteristics of Persistent Clonal T Cell Large Granular Lymphocyte Expansions in Rheumatoid Arthritis: A Comprehensive Analysis of 529 Patients.

OBJECTIVE: Up to one-third of patients with T cell large granular lymphocyte (T-LGL) leukemia display symptoms of rheumatoid arthritis (RA). In Crohn's disease and psoriasis, treatment with tumor necrosis factor (TNF) inhibitors is associated with hepatosplenic γδ T cell lymphoma and with clonal expansion of γδ T cells, respectively. This study was undertaken to determine the prevalence of clonal T-LGL cells in patients with RA and define risk factors for this rare hematologic malignancy. METHODS: A total of 529 RA patients were recruited between November 2013 and August 2015. Eight-color flow cytometry (fluorescence-activated cell sorting [FACS]) was performed to screen for aberrant T cell populations of LGLs. Molecular analysis of the T cell receptor was used to confirm the diagnosis in patients with suggestive FACS findings. Electronic patient files were used to determine risk factors. Patients with clonal populations were monitored prospectively for up to 4 years. RESULTS: The median patient age was 61 years, and 74% were female. The median duration of RA was 12 years. The median Disease Activity Score in 28 joints was 2.8, and 69.9% of patients had ever been treated with biologic disease-modifying antirheumatic drugs. We identified clonal T-LGL expansions in 19 patients, equaling a prevalence of 3.6%. The T-LGL cell clone was constant over time in most patients and was significantly associated with the duration of the exposure to TNF-blocking agents (P = 0.01). No other risk factors could be detected. CONCLUSION: RA patients with long-term exposure to TNF-blocking agents were at a greater risk of developing clonal expansions of LGLs. This finding may prompt clinicians to refrain from using these substances in RA patients with known T cell aberrations.

Panel Sequencing Shows Recurrent Genetic FAS Alterations in Primary Cutaneous Marginal Zone Lymphoma.

Primary cutaneous marginal zone lymphoma (PCMZL) represents an indolent subtype of non-Hodgkin lymphoma that is clinically characterized by slowly growing skin tumors with a very low propensity for systemic dissemination. The underlying genetic basis of PCMZL has not been comprehensively elucidated. To gain deeper insight into the molecular pathogenesis of PCMZL, we performed hybridization-based panel sequencing of 38 patients with well-characterized PCMZL. In 32 of the 38 patients, we identified genetic alterations within 39 selected target genes. The most frequently detected alterations (24/38 patients, 63.2%) affected the FAS gene, of which 22 patients harbored alterations, which affect the functionally relevant death domain of the apoptosis-regulating FAS/CD95 protein in a dominant-negative manner. In addition, we identified highly recurrent mutations in three other genes, namely SLAMF1, SPEN, and NCOR2. Our molecular data suggest that apoptosis defects provide the molecular basis of the observed clinical features of PCMZL, which commonly presents with only slowly growing skin tumors, reflecting its invariably indolent behavior. From a diagnostic point of view, highly recurrent FAS mutations in PCMZL presumably separate this indolent lymphoma entity from pseudolymphoma, and this adds adjunctive discriminatory features at a molecular level.

SYK expression in monomorphic epitheliotropic intestinal T-cell lymphoma.

Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), formerly known as type II enteropathy associated T-cell lymphoma (type II EATL), is a rare, aggressive primary intestinal T-cell lymphoma with a poor prognosis and an incompletely understood pathogenesis. We collected 40 cases of MEITL and 27 cases of EATL, formerly known as type I EATL, and comparatively investigated the T-cell receptor (TCR) itself and associated signaling molecules using immunohistochemistry, amplicon deep sequencing and bisulfite pyrosequencing. The TCR showed both an αß-T-cell origin (30%) and a γδ-T-cell derivation (55%) resulting in a predominant positive TCR phenotype in MEITL compared with the mainly silent TCR phenotype in EATL (65%). The immunohistochemical expression of the spleen tyrosine kinase (SYK) turned out to be a distinctive feature of MEITL (95%) compared with EATL (0%). Aberrant SYK overexpression in MEITL is likely caused by hypomethylation of the SYK promoter, while no common mutations in the SYK gene or in its promoter could be detected. Using amplicon deep sequencing, mutations in DNMT3A, IDH2, and TET2 were infrequent events in MEITL and EATL. Immunohistochemical expression of linker for activation of T-cells (LAT) subdivided MEITL into a LAT expressing subset (33%) and a LAT silent subset (67%) with a potentially earlier disease onset in LAT-positive MEITL.