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Targeted next generation sequencing of breast implant‐associated anaplastic large cell lymphoma reveals mutations in JAK/STAT signalling pathway genes, TP53 and DNMT3A

Breast implant-associated anaplastic large cell lymphoma (BI-ALCL) is an uncommon neoplasm occurring in women with either cosmetic or reconstructive breast implants (Clemens et al, 2016). Until now, most studies have focused on defining the clinico-pathological features of BI-ALCL, leading to its inclusion as a new provisional entity, a subtype of anaplastic lymphoma kinase (ALK)-negative ALCL, in the revised World Health Organization classification of lymphoid malignancies (Swerdlow et al, 2016). BI-ALCL is characterized by the presence of CD30 large atypical lymphocytes frequently confined to the peri-implant seroma fluid. Nevertheless, solid infiltrating masses and cases pursuing an aggressive clinical course have been reported. The surgical and pathological staging system designed by Clemens et al (2016) suggests that BI-ALCL has a pattern of progression similar to that of solid tumours rather than non-Hodgkin lymphomas, and that the effusionand solid-types might represent different stages of the same disease rather than two distinct variants. The molecular pathogenesis and mechanisms of progression of BI-ALCL, however, remain largely unknown, thus limiting the identification of biomarkers that enable disease prognostication and optimal treatment. Hence, we performed targeted next generation sequencing of seven BI-ALCL, identified in the archives of three institutions over 7 years, to investigate the presence of underlying somatic mutations. Informed consent was obtained from patients and the study was performed in accordance with the Declaration of Helsinki. DNA extracted from micro-dissected tumour cells of formalin-fixed paraffin-embedded BI-ALCL samples (QIAamp DNA Mini kit; Qiagen, Germantown, MD, USA) was used to prepare DNA libraries (Sureselect kit; Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed on a HiSeq2500 (Illumina, San Diego, CA, USA) using a panel of 465 cancerassociated genes (Table SI). The sequence data were aligned to the human reference genome (hg19) and variants were identified using NextGENe (SoftGenetics, State College, PA, USA). The average read depth of the samples was 4009 (Table SII). Somatic mutations were identified by comparison of variants detected in lymphoma with those from matched constitutional DNA. Common variants (>1% frequency) present in the 1000 genomes database, and the database of Columbia University were removed. Somatic mutations were classified using the prior literature, and two different prediction algorithms (SIFT http://sift.bii.a-star.edu.sg and Polyphen-2 [PP2] http://genetics.bwh.harvard.edu/pph2/). The exonic somatic variants were confirmed by bidirectional Sanger sequencing using Big-Dye terminators v3.1 (Applied Biosystems, Carlsbad, CA, USA). The clinical and pathological features of the patients are summarized in Table I. Informative results were obtained in five of seven cases (Table SII); analysis failed in two cases due to the poor quality of DNA. Five somatic variants affecting four genes were identified in two cases: one intronic and four within coding regions (Fig 1 and Table SIII). A STAT3 missense variant (p.S614R) affecting the SH2 domain, which mediates STAT3 dimerization, was detected in one of these two BI-ALCLs. JAK/STAT signalling is implicated in cell proliferation, differentiation and apoptosis, and aberrant activation of STAT3 has been reported in several human cancers associated with persistent immune stimulation and/or inflammation. Notably, the gain-of-function mutation (S614R) was recently described in one BI-ALCL (Blombery et al, 2016), and has been reported in angioimmunoblastic T cell lymphomas, chronic lymphoproliferative disorders of natural killer cells, and T-cell large granular lymphocyte leukaemias (Odejide et al, 2014). Moreover, gain-of-function mutations in STAT3 have been reported in 18% of systemic ALK-negative ALCLs and 5% of cutaneous ALCLs (Crescenzo et al, 2015). An in vitro study using BI-ALCL-derived cell lines also showed activation of the JAK/STAT pathway through autocrine production of interleukin 6, suggesting a possible pathogenic mechanism (Lechner et al, 2012). A frameshift deletion causing a premature stop codon in SOCS1 (p.P83Rfs*20) was detected in the BI-ALCL harbouring the STAT3 mutation. SOCS1 is a negative feedback regulator of the JAK/STAT pathway. The p.P83Rfs*20 mutation deletes the C-terminal SOCS box domain and partially deletes the SH2 domain, which downregulates the kinase activity of JAK. Loss-of-function mutations of SOCS1, leading to constitutive activation of JAK/STAT signalling, have been described in B-cell lymphomas and in classical Hodgkin lymphomas (Mottok et al, 2009). Moreover, SOCS1 was found to be silenced by miR-155 in ALK-negative ALCL (Merkel et al, 2015). Mutations in STAT3 and SOCS1 suggest that deregulated activation of the JAK/STAT pathway may contribute to the development of BI-ALCL. A missense mutation of TP53 (p.D259Y) affecting the DNA binding domain was also observed in the Correspondence