Signal transducer and activator of transcription-3 (STAT3) is a dimerizing transcription factor and oncogene involved in cytokine growth signaling and cell survival pathways.1 While inactivating STAT3 mutations have been identified in hyper-IgE syndrome,2 activating mutations are seen in specific T-cell and NK-cell malignancies such as T-cell large granular lymphocytic leukemia (T-LGL), chronic NK lymphoproliferative disorders, and CD30 T-cell lymphomas.53 Though the frequencies and role of STAT3 mutations in T- and NK-cell malignancies have been well-studied, the significance of STAT3 mutations in B-cell malignancies is uncertain.
As such, we read with great interest the recent Haematologica paper by Couronne et al.6 that studied the role of the STAT3 activating mutation Y640F in an in vitro mouse model and demonstrated that expression of this mutated protein resulted in myeloproliferative neoplasms in mice. In addition, the mutational status of STAT3 in numerous hematologic neoplasms was assessed and mutations were seen in 4 cases of T-cell lymphomas and 2 cases of diffuse large B-cell lymphoma (DLBCL). While these DLBCL cases were identified as mutated, the significance of such mutations in these cases of DLBCL remains unknown. Given these recent findings, we strove to further advance the understanding of STAT3 mutations in B-cell malignancies by analyzing 143 B-cell lymphomas.
We sequenced exons 19–24 of STAT3, where all known activating mutations have been identified, and included cases of: diffuse large B-cell lymphoma not otherwise specified (DLBCL-NOS; n=48), follicular lymphoma (FL; n=24), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; n=24), mantle cell lymphoma (MCL; n=14), marginal zone lymphoma (MZL; n=12), Burkitt lymphoma (n=10), primary mediastinal large B-cell lymphoma (PMLBL; n=4), classical Hodgkin lymphoma (CHL; n=1), Epstein Barr virus-positive diffuse large B-cell lymphoma of the elderly (EBV+ DLBCL; n=2), B-cell lymphoma unclassifiable with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (BCLU, DLBCL/B; n=3), and B-cell lymphoma unclassifiable with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma (BCLU, DLBCL/CHL; n=1). Genomic DNA was isolated from May-Grunwald-Giemsa stained slides (n=7), frozen whole blood (n=11), or formalin fixed paraffin embedded (FFPE) tissues (n=125) using a Qiagen (Louisville, KY, USA) DNeasy Blood & Tissue Kit as previously described.4
Of these 143 lymphomas, STAT3 mutations were identified in 3 of 48 DLBCL, NOS cases (6%) and in 2 of 3 cases of BCLU, DLBCL/B (67%). Mutations were all localized to the SH2 domain of STAT3. In the cases of DLBCL, NOS that contained STAT3 mutations, the histomorphological and immunophenotypic features were similar (Table 1 and Figure 1). Neoplastic cells showed anaplastic morphologies with high mitotic indices of more than 10 mitoses/5 high power fields (HPF) (Table 1). All cases were CD20 positive and CD30 expression was seen in a subset of the anaplastic cells (>20% of cells) in all 3 DLBCL. Anaplastic lymphoma kinase (ALK) protein expression was not detected by immunohistochemistry in these cases. Of all cases of DLBCL, NOS with CD30 malignant cells, patients with STAT3 mutations represented 17% of such cases (3 of 18 cases). No significant association with anatomic site, sex, or age was observed.
Of the 2 BCLU, DLBCL/B cases with STAT3 mutations, one had a MYC translocation (case 4) whilst the other positive case (case 5) had both an MYC and BCL-6 translocation (Figure 1 and Table 1). Both cases had similar morphological features with high mitotic rates (>10 mitoses/5HPF) and a subset of anaplastic multinucleated large lymphoid cells; however, unlike mutated STAT3 DLBCL, NOS, neither of these were CD30 positive.
We also assessed the protein expression of STAT3 in the 3 cases of mutated DLBCL, NOS and 2 cases of mutated BCLU, DLBCL/B. In the cases of DLBCL, NOS, STAT3 was over-expressed in the nucleus of malignant cells in these mutated cases and visibly up-regulated in the CD30 cells (Figure 1). However, in the STAT3 mutated BCLU, DLBCL/B, the protein was not over-expressed by immunohistochemistry. Furthermore, overexpression of STAT3 was not specific to B-cell lymphoma cases with STAT3 mutations. Nuclear overexpression of STAT3 was also seen in 14 of 39 other DLBCL, as well as 3 of 8 FL, 2 of 7 CLL/SLL, 2 of 10 Burkitt lymphomas (BL), and one of 5 MZL; 2 cases of PMLBL had no evidence for STAT3 overexpression. Interestingly, one case of classical Hodgkin lymphoma showed distinctive nuclear overexpression of STAT3 in malignant cells; malignant cells constituted more than 20% of the tumor, though STAT3 was not mutated in this case.
While Couronne et al. identified rare cases of DLBCL with STAT3 mutations recently,6 our results demonstrate a phenotypic-genotypic link between the mutational status of STAT3 and morphology of cells in these aggressive B-cell lymphomas. While only 3 of 48 of our cases of DLBCL, NOS were positive for a STAT3 mutation (6% of DLBCL cases), the frequency of STAT3 mutations specifically in CD30 DLBCL (3 of 18 cases; 17%) is higher than by chance alone (P=0.047). In addition, though we were only able to sequence 3 cases of BCLU, DLBCL/B, 2 of these cases harbored STAT3 SH2 domain mutations. Similarly to the cases of STAT3 mutated DLBCL, NOS, in the 2 cases of STAT3 mutated BCLU, DLBCL/B, these both contained a subset of anaplastic multinucleated cells; though CD30 and STAT3 proteins were not over-expressed. One possible explanation for the absence of STAT3 protein overexpression in mutated cases of BCLU, DLBCL/B, concerns the relationship between MYC and STAT3 transcriptional protein pathways. STAT3 is known to up-regulate MYC protein expression and it is possible that the increased activity of MYC in these cases with MYC translocations, results in a feed-back loop which suppresses visible overexpression of the STAT3 protein.97 However, further in vitro experiments are necessary to test such a hypothesis. Finally, we have also demonstrated that these mutations are not seen in lower grade B-cell lymphomas such as CLL/SLL, FL and MZL.
In addition, STAT3 was also over-expressed in a subset of other B-cell malignancies without these mutations: 14 of 39 DLBCL, 3 of 8 cases of FL, 2 of 7 cases of CLL/SLL, and one of 5 cases of MZL. These findings are in agreement with the studies of other groups that have noted increased activation of STAT3 in a subset of B-cell lymphomas.1310 Why some cases of DLBCL show protein overexpression of STAT3 but harbor no STAT3 mutation is not entirely clear; however, it is certainly feasible that there are other mechanisms to increase STAT3 activity via upstream protein regulators.
In some studies, STAT3 overexpression has been correlated with poor prognosis.1410 Whether cases with STAT3 DNA mutations, as seen here, are associated with poor prognosis is unclear, and larger patient cohorts are necessary to evaluate this hypothesis. However, it is interesting to note that of the 4 cases of PMLBL, a CD30 large B-cell lymphoma that responds favorably to chemotherapy, none had STAT3 mutations, and, in addition, the STAT3 protein was not over-expressed in 2 cases of PMLBL analyzed.
Finally, our results expand on the mechanistic relationship between STAT3 and morphological anaplasia which has been noted by others, including Chiarle et al.,15 whereby STAT3 overexpression results in increased morphological anaplasia, and overall increased proliferation and cell survival.15 In addition, given the recent development of STAT3 inhibitors and their current clinical testing, the significance of our findings and the potential therapeutic implications in these cases with STAT3 activating mutations is an area for promising future research.
- Silva CM. Role of STATs as downstream signal transducers in src family kinase-mediated tumorigenesis. Oncogene. 2004; 23(48):8017-23. PubMedhttps://doi.org/10.1038/sj.onc.1208159Google Scholar
- Minegishi Y, Saito M. Molecular mechanisms of the immunological abnormalities in hyper-IgE syndrome. Ann NY Acad Sci. 2011; 1246:34-40. PubMedhttps://doi.org/10.1111/j.1749-6632.2011.06280.xGoogle Scholar
- Jerez A, Clemente MJ, Makishima H. STAT3 mutations unify the pathogenesis of chronic lymphoproliferative disorders of NK cells and T-cell large granular lymphocyte leukemia. Blood. 2012; 120(15):3048-57. PubMedhttps://doi.org/10.1182/blood-2012-06-435297Google Scholar
- Ohgami RS, Ma L, Merker JD, Martinez B, Zehnder JL, Arber DA. STAT3 mutations are frequent in CD30+ T-cell lymphomas and T-cell large granular lymphocytic leukemia. Leukemia. 2013; 27(11):2244-7. PubMedhttps://doi.org/10.1038/leu.2013.104Google Scholar
- Koskela HL, Eldfors S, Ellonen P. Somatic STAT3 mutations in large granular lymphocytic leukemia. N Engl J Med. 2012; 366(20):1905-13. PubMedhttps://doi.org/10.1056/NEJMoa1114885Google Scholar
- Couronne L, Scourzic L, Pilati C. STAT3 mutations identified in human hematological neoplasms induce myeloid malignancies in a mouse bone marrow transplantation model. Haematologica. 2013; 98(11):1748-52. PubMedhttps://doi.org/10.3324/haematol.2013.085068Google Scholar
- Valera A, Colomo L, Martinez A. ALK-positive large B-cell lymphomas express a terminal B-cell differentiation program and activated STAT3 but lack MYC rearrangements. Mod Pathol. 2013; 26(10):1329-37. PubMedhttps://doi.org/10.1038/modpathol.2013.73Google Scholar
- Kiuchi N, Nakajima K, Ichiba M. STAT3 is required for the gp130-mediated full activation of the c-myc gene. J Exp Med. 1999; 189(1):63-73. PubMedhttps://doi.org/10.1084/jem.189.1.63Google Scholar
- Cartwright P, McLean C, Sheppard A, Rivett D, Jones K, Dalton S. LIF/STAT3 controls ES cell self-renewal and pluripotency by a myc-dependent mechanism. Development. 2005; 132(5):885-96. PubMedhttps://doi.org/10.1242/dev.01670Google Scholar
- Wu ZL, Song YQ, Shi YF, Zhu J. High nuclear expression of STAT3 is associated with unfavorable prognosis in diffuse large B-cell lymphoma. J Hematol Oncol. 2011; 4(1):31. PubMedhttps://doi.org/10.1186/1756-8722-4-31Google Scholar
- Ding BB, Yu JJ, Yu RY. Constitutively activated STAT3 promotes cell proliferation and survival in the activated B-cell subtype of diffuse large B-cell lymphomas. Blood. 2008; 111(3):1515-23. PubMedhttps://doi.org/10.1182/blood-2007-04-087734Google Scholar
- Hazan-Halevy I, Harris D, Liu Z. STAT3 is constitutively phosphorylated on serine 727 residues, binds DNA, and activates transcription in CLL cells. Blood. 2010; 115(14):2852-63. PubMedhttps://doi.org/10.1182/blood-2009-10-230060Google Scholar
- Daigle D, Megyola C, El-Guindy A. Upregulation of STAT3 marks burkitt lymphoma cells refractory to epstein-barr virus lytic cycle induction by HDAC inhibitors. J Virol. 2010; 84(2):993-1004. PubMedhttps://doi.org/10.1128/JVI.01745-09Google Scholar
- Huang X, Meng B, Iqbal J. Activation of the STAT3 signaling pathway is associated with poor survival in diffuse large B-cell lymphoma treated with R-CHOP. J Clin Oncol. 2013; 31(86):4520-8. PubMedhttps://doi.org/10.1200/JCO.2012.45.6004Google Scholar
- Chiarle R, Voena C, Ambrogio C, Piva R, Inghirami G. The anaplastic lymphoma kinase in the pathogenesis of cancer. Nat Rev Cancer. 2008; 8(1):11-23. PubMedhttps://doi.org/10.1038/nrc2291Google Scholar