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International audience; Background: Mastocytosis is characterized by the accumulation/proliferation of abnormal mast cells. The frequency ofisolated cutaneous involvement in adults with mastocytosis has not been fully determined. The main objective of ourstudy was to assess the frequency of isolated cutaneous mastocytosis (CM) in adults with mastocytosis skin lesions.The second objective was to compare the clinical, histological, biological and imaging features in patients with isolatedCM and patients with systemic mastocytosis (SM).Methods: We included all patients with histology-proven mastocytosis skin lesions between January 2009 and Decem-ber 2017. The mastocytosis diagnosis was made according to the international diagnostic criteria. All data were col-lected from a dedicated specific case report.Results: Among 160 patients with mastocytosis skin lesions, 25 patients had isolated CM (15.6%), 105 had SM and 30(18.7%) patients had undetermined mastocytosis. Skin KIT mutation (OR: 51.9, 95% CI: 3.9–678, P = 0.001) and highbone marrow tryptase (OR: 97.4, 95% CI: 10.3–915, P = 0.001) were strong predictors of SM. The prevalence of osteo-porosis was higher in the SM population than in the isolated CM population. Moreover, a decrease in bone mineral den-sity over a short period of follow-up (1–2 years) was associated with SM. There were no differences between the twogroups regarding the frequency of mast cell activation symptoms, the presentation of skin lesions, the number of mastcells in the dermis and the level of serum tryptase. We propose considering the KIT mutation status and bone marrowtryptase levels to aid the diagnosis of isolated CM in adult mastocytosis patients.Conclusion: Only a small minority of adults with mastocytosis skin lesions has isolated cutaneous involvement. In18.7% of mastocytosis cases, even complete workup does not allow for a precise classification of patients.Received: 1 January 2019; Accepted: 9 April 2019

BACKGROUND: Skin involvement is frequent in ALK-positive anaplastic large cell lymphomas. The role of an insect bite as a triggering event has been postulated but not well documented. DESIGN AND METHODS: We retrospectively investigated five cases of ALK-positive anaplastic large cell lymphoma who presented with skin lesions occurring after an insect bite. Biopsies were immunostained with antibodies against CD30, ALK, T- and B-cell antigens. RESULTS: Persistent skin lesions developed after solitary insect bites in three patients and after multiple bites in two. Regional lymphadenopathy developed within weeks after the bite in three cases. In four cases the correct diagnosis was delayed due to misinterpretation of the findings as a reactive infiltrate in the skin (n=2) or lymph nodes (n=2); all cases subsequently showed small numbers of cells with nuclear and cytoplasmic staining for ALK. The final diagnoses were lymphohistiocytic variant (n=3) and composite common/small cell type (n=2) anaplastic large cell lymphoma. The patients were treated and three were alive at the last follow-up. Two patients died, one of pneumonia and the other of disseminated disease. CONCLUSIONS: In these cases the sequence of events between the insect bites and the occurrence of both skin lesions and satellite lymphadenopathy suggest a direct relationship between the bite and the presentation with anaplastic large cell lymphoma. We postulate that insect bite-associated antigens could result in an influx of T lymphocytes, some bearing the t(2;5). The subsequent release of cytokines at the site of the bite could act as a 'second hit', eliciting activation of the latter cells, which would then express the oncogenic NPM-ALK protein and undergo uncontrolled proliferation.

In a previous study, we showed that the high level of protein tyrosine phosphorylation present in lymphomas containing an anaplastic lymphoma kinase (ALK) can be demonstrated in routinely processed paraffin tissue sections using immunolabelling techniques. In the present study we investigated whether oncogenic tyrosine kinase activation also occurs in other categories of lymphoma by staining 145 cases of lymphoma covering those tumours with a range of different subtypes including those with morphological similarity to ALK-positive anaplastic large cell lymphoma (ALCL). Twelve cases of the borderline malignant disorder lymphomatoid papulosis were also studied. Twenty seven of the 28 cases of ALK-positive ALCL showed the extensive cytoplasmic labelling for phosphotyrosine in the neoplastic cells. The remaining case containing moesin-ALK exhibited membrane-associated phosphotyrosine expression. There was no nuclear phosphotyrosine labelling in any of the ALK-positive ALCL, even though ALK was present within the cell nuclei in 23 of the tumours. Variable degrees of phosphotyrosine labelling, usually membrane-restricted, were observed in 7/40 cases of ALK-negative ALCL, 9/29 cases of diffuse large B-cell lymphoma, 3/6 cases of mediastinal B-cell lymphoma, 2/7 cases of Hodgkin's lymphoma, 3/6 cases of peripheral T-cell lymphomas unspecified, 4/6 cases of B-cell chronic lymphocytic leukaemia, 2/6 cases of follicular lymphomas and 2/12 cases of lymphomatoid papulosis studied. However none of these phosphotyrosine-positive cases showed the strong cytoplasmic labelling comparable to that seen in ALK-positive lymphoma. We conclude that activation of a tyrosine kinase is probably not a major oncogenic event in lymphomas other than ALK-positive ALCL.

The t(2;5) generates a chimeric NPM-ALK transcript encoded by the nucleophosmin NPM gene fused to the anaplastic lymphoma kinase gene ALK. Using a reverse transcriptase nested polymerase chain reaction assay we have detected NPM-ALK transcripts within CD30+ primary cutaneous lymphoma and lymphomatoid papulosis (LP). The t(2;5) was identified in 4 out of 9 CD30+ anaplastic lymphomas and in 1 out of 4 CD30+ pleomorphic lymphomas. Moreover, the t(2;5) was detected in 3 out of 10 LPs. All NPM-ALK-positive lymphomas and 1 NPM-ALK-positive LP exhibited a clonal rearrangement of the T cell receptor gamma-chain gene. The t(2;5) was detected in 2 cases of LP without other evidence for a clonal lymphoid population. To identify cells carrying the t(2;5) translocation, we used immunohistochemistry to detect the ALK-encoded p80 protein and in situ hybridization for the specific detection of NPM-ALK transcripts. Both p80 protein and NPM-ALK transcripts were expressed by anaplastic or large CD30+ lymphoma cells with positive NPM-ALK amplification. The presence of t(2;5) in a subset of CD30+ cutaneous lymphoma and LP may indicate a common pathogenesis with a subset of anaplastic nodal lymphoma.

Patients diagnosed with anaplastic large cell lymphoma (ALCL) are still treated with toxic multi-agent chemotherapy and as many as 25-50% of patients relapse. To understand disease pathology and to uncover novel targets for therapy, we performed whole-exome sequencing of anaplastic lymphoma kinase (ALK)+ ALCL, as well as gene-set enrichment analysis. This revealed that the T-cell receptor and Notch pathways were the most enriched in mutations. In particular, variant T349P of NOTCH1, which confers a growth advantage to cells in which it is expressed, was detected in 12% of ALK+ and ALK– ALCL patients’ samples. Furthermore, we demonstrated that NPM-ALK promotes NOTCH1 expression through binding of STAT3 upstream of NOTCH1. Moreover, inhibition of NOTCH1 with γ- secretase inhibitors or silencing by short hairpin RNA leads to apoptosis; cotreatment in vitro with the ALK inhibitor crizotinib led to additive/synergistic antitumor activity suggesting that this may be an appropriate combination therapy for future use in the circumvention of ALK inhibitor resistance. Indeed, crizotinib-resistant and -sensitive ALCL were equally sensitive to γ-secretase inhibitors. In conclusion, we show a variant in the extracellular domain of NOTCH1 that provides a growth advantage to cells and confirm the suitability of the Notch pathway as a second-line druggable target in ALK+ ALCL.

The regulatory microRNA miR-150 is involved in the development of hemopathies and is downregulated in T-lymphomas, such as anaplastic large-cell lymphoma (ALCL) tumors. ALCL is defined by the presence or absence of translocations that activate the anaplastic lymphoma kinase (ALK), with nucleophosmin-ALK (NPM-ALK) fusions being the most common. Here, we compared samples of primary NPM-ALK(+) and NPM-ALK(–) ALCL to investigate the role of miR-150 downstream of NPM-ALK. Methylation of the MIR150 gene was substantially elevated in NPM-ALK(+) biopsies and correlated with reduced miR-150 expression. In NPM-ALK(+) cell lines, DNA hypermethylation–mediated miR-150 repression required ALK-dependent pathways, as ALK inhibition restored miR-150 expression. Moreover, epigenetic silencing of miR-150 was due to the activation of STAT3, a major downstream substrate of NPM-ALK, in cooperation with DNA methyltransferase 1 (DNMT1). Accordingly, miR-150 repression was turned off following treatment with the DNMT inhibitor, decitabine. In murine NPM-ALK(+) xenograft models, miR-150 upregulation induced antineoplastic activity. Treatment of crizotinib-resistant NPM-ALK(+) KARPAS-299-CR06 cells with decitabine or ectopic miR-150 expression reduced viability and growth. Altogether, our results suggest that hypomethylating drugs, alone or in combination with other agents, may benefit ALK(+) patients harboring tumors resistant to crizotinib and other anti-ALK tyrosine kinase inhibitors (TKIs). Moreover, these results support further work on miR-150 in these and other ALK(+) malignancies.