Primary mediastinal B-cell lymphoma (PMBL) is a specific subtype of diffuse large B-cell lymphoma with a distinctive clinical course and a specific genetic profile.1,2 Several prior studies have provided key molecular–genetic aberrations;1,3 however, a targeted next-generation sequencing-based study of relevant oncogenes in all currently available PMBL cell lines has not been performed. Here we report previously unrecognized mutations in known hot-spot regions of the KIT oncogene in ~12.5% of PMBL tumors. Interestingly, activating mutations in the proto-oncogene KIT have already been described in a variety of malignancies, including mast cell leukemia, acute myelogenous leukemia, gastrointestinal stromal tumors, as well as several other solid tumors,4 where they carry, in part, therapeutic relevance due to clinically effective inhibitors.5,6
We performed next-generation sequencing-based screening of all three available PMBL cell lines using a panel of 50 relevant oncogenes (see Supplementary material and Supplementary Table 1). The full list of sequence alterations is provided in Supplementary Table 2. Notably, we found KIT mutations in two of three PMBL cell lines that we confirmed by Sanger sequencing (Figure 1). Given that these alterations were present at previously described hot spots, we further determined the mutation frequency in primary tumor samples using our PMBL cohort.7 We found three missense mutations in 30 tumor samples (10%). One of these samples (PMBL.1) was the parental tumor, from which we derived the Med-B1 cell line,8 thereby confirming that the KIT mutation is indeed present in the primary tumor and is not a result of genomic cell culture permutation. DNA analysis from microdissected non-tumor tissue in two of the mutated tumor samples showed wild-type sequence and confirmed the mutations as somatic.
Regarding the prevalence of KIT mutations in PMBL, we had to take the duplicate sample Med-B1 and parental tumor (PMBL.1) into account; we found a total of five mutations in 32 tested samples and determined a mutation frequency of ~15.6% in our cohort. When further restricting these calculations to missense mutations, we excluded sample PMBL.29 that harbored only a sense mutation (c.2394 C>T; p.I798I); the overall frequency of missense KIT mutations in PMBL (4 of 32 different samples) surmounts to ~12.5% (Figure 1). Besides this relatively high prevalence of KIT mutations, interestingly, all detected KIT missense mutations in PMBL are rare. Three mutations have been previously reported in other tumors (Table 1), and affect amino acids located in the activation loop (p.809–823) of the kinase domain (Supplementary Figure 1). One mutation (p.C788Y) is located in the kinase domain and has not been reported before. Notably, all four KIT missense mutations in PMBL are classified as ‘damaging’ by several in silico prediction tools and have therefore a possible impact on protein function (Table 1). This is also suggested by previous functional studies investigating missense mutations at these genetic hotspot regions of KIT.9, 10, 11 Thus, our findings collectively suggest the possibility of specific diagnostic, prognostic and therapeutic implications of KIT mutations in PMBL.
Notably, a recent genome-wide study delineated the mutational landscape in PMBL,3 but missed the striking frequency of over 10% recurrent KIT mutations in PMBL. Arguably, this genome-wide study was performed according to very high standards, sufficient coverage and expert bioinformatics analysis. Nonetheless, their approach was to initially sequence two PMBL patient samples, and further experiments—including targeted sequencing of one identified gene in a larger number of samples—were made based on the findings in these two patient samples. Thus, despite the advents of genome-wide analyses, the imposed sampling bias precluded identification of recurrent KIT mutations in PMBL. Pointing this out should in no way compromise the importance of revealing the mutational spectrum of PMBL; however, our data paradigmatically prove the existence of false negatives in these large-scale data sets.12, 13, 14 Furthermore, our data—albeit at a much smaller sequencing scale—emphasize targeted re-sequencing as an effective approach to perform high-throughput genotyping in tumor samples.14,15 In summary, we report for the first time that ~12.5% of PMBL samples harbor mutations in the KIT oncogene.
Barth TF, Leithauser F, Joos S, Bentz M, Moller P . Mediastinal (thymic) large B-cell lymphoma: where do we stand? Lancet Oncol 2002; 3: 229–234.
Rosenwald A, Wright G, Leroy K, Yu X, Gaulard P, Gascoyne RD et al. Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma. J Exp Med 2003; 198: 851–862.
Gunawardana J, Chan FC, Telenius A, Woolcock B, Kridel R, Tan KL et al. Recurrent somatic mutations of PTPN1 in primary mediastinal B cell lymphoma and Hodgkin lymphoma. Nat Genet 2014; 46: 329–335.
Lennartsson J, Jelacic T, Linnekin D, Shivakrupa R . Normal and oncogenic forms of the receptor tyrosine kinase kit. Stem Cells 2005; 23: 16–43.
Demetri GD, von Mehren M, Blanke CD, Van den Abbeele AD, Eisenberg B, Roberts PJ et al. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med 2002; 347: 472–480.
Guo J, Si L, Kong Y, Flaherty KT, Xu X, Zhu Y et al. Phase II, open-label, single-arm trial of imatinib mesylate in patients with metastatic melanoma harboring c-Kit mutation or amplification. J Clin Oncol 2011; 29: 2904–2909.
Nagel PD, Feld FM, Weissinger SE, Stenzinger A, Moller P, Lennerz JK . Absence of BRAF and KRAS hotspot mutations in primary mediastinal B-cell lymphoma. Leuk Lymphoma 2014; e-pub ahead of print 4 February 2014.
Moller P, Brüderlein S, Strater J, Leithauser F, Hasel C, Bataille F et al. MedB-1, a human tumor cell line derived from a primary mediastinal large B-cell lymphoma. Int J Cancer 2001; 92: 348–353.
Furitsu T, Tsujimura T, Tono T, Ikeda H, Kitayama H, Koshimizu U et al. Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product. J Clin Invest 1993; 92: 1736–1744.
Heinrich MC, Maki RG, Corless CL, Antonescu CR, Harlow A, Griffith D et al. Primary and secondary kinase genotypes correlate with the biological and clinical activity of sunitinib in imatinib-resistant gastrointestinal stromal tumor. J Clin Oncol 2008; 26: 5352–5359.
Yang Y, Letard S, Borge L, Chaix A, Hanssens K, Lopez S et al. Pediatric mastocytosis-associated KIT extracellular domain mutations exhibit different functional and signaling properties compared with KIT-phosphotransferase domain mutations. Blood 2010; 116: 1114–1123.
Lawrence MS, Stojanov P, Mermel CH, Robinson JT, Garraway LA, Golub TR et al. Discovery and saturation analysis of cancer genes across 21 tumour types. Nature 2014; 505: 495–501.
Rao DC, Gu C . False positives and false negatives in genome scans. Adv Genet 2001; 42: 487–498.
Rehm HL, Bale SJ, Bayrak-Toydemir P, Berg JS, Brown KK, Deignan JL et al. ACMG clinical laboratory standards for next-generation sequencing. Genet Med 2013; 15: 733–747.
Endris V, Penzel R, Warth A, Muckenhuber A, Schirmacher P, Stenzinger A et al. Molecular diagnostic profiling of lung cancer specimens with a semiconductor-based massive parallel sequencing approach: feasibility, costs, and performance compared with conventional sequencing. J Mol Diagn 2013; 15: 765–775.
We appreciate the support of all participating patients and thank P Kuhn and C Welke from the Comprehensive Cancer Center Ulm and W Kugler for administrative support. We thank Dr U Kostezka, K Dorsch, Y Nerbas, L Moser, E Kelsch, K Steinhauser, K Lorenz and J Kiedaisch for expert technical assistance. In addition, we thank Dr U Gerstenmaier for help with genotyping. The Else-Kröner Foundation supports JKL. The National Center of Tumor Diseases (NCT) and the German Consortium for Translational Cancer Research (DKTK) supports WW.
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on Blood Cancer Journal website
About this article
Cite this article
Nagel, P., Stenzinger, A., Feld, F. et al. KIT mutations in primary mediastinal B-cell lymphoma. Blood Cancer Journal 4, e241 (2014) doi:10.1038/bcj.2014.61