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The mutational landscape of cutaneous T cell lymphoma and Sézary syndrome

Abstract

Sézary syndrome is a leukemic and aggressive form of cutaneous T cell lymphoma (CTCL) resulting from the malignant transformation of skin-homing central memory CD4+ T cells. Here we performed whole-exome sequencing of tumor-normal sample pairs from 25 patients with Sézary syndrome and 17 patients with other CTCLs. These analyses identified a distinctive pattern of somatic copy number alterations in Sézary syndrome, including highly prevalent chromosomal deletions involving the TP53, RB1, PTEN, DNMT3A and CDKN1B tumor suppressors. Mutation analysis identified a broad spectrum of somatic mutations in key genes involved in epigenetic regulation (TET2, CREBBP, KMT2D (MLL2), KMT2C (MLL3), BRD9, SMARCA4 and CHD3) and signaling, including MAPK1, BRAF, CARD11 and PRKG1 mutations driving increased MAPK, NF-κB and NFAT activity upon T cell receptor stimulation. Collectively, our findings provide new insights into the genetics of Sézary syndrome and CTCL and support the development of personalized therapies targeting key oncogenically activated signaling pathways for the treatment of these diseases.

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Figure 1: Somatic copy number variants and mutations in Sézary syndrome and CTCL.
Figure 2: Somatic alterations in Sézary syndrome and CTCL.
Figure 3: Functional characterization of Sézary syndrome CARD11 linker domain alterations.
Figure 4: Functional characterization of Sézary syndrome cGKIβ alterations.

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Acknowledgements

This work was supported by a Leukemia and Lymphoma Society Translational Research Grant (A.F.), a Herbert Irving Comprehensive Cancer Center interprogrammatic pilot project grant (A.F. and R.R.) and Dutch Cancer Society grant UL2013-6104 (C.P.T. and M.H.V.). A.C.d.S.A. is supported by a Lady Tata Memorial Trust fellowship.

Author information

Authors and Affiliations

Authors

Contributions

A.C.d.S.A. performed functional assays. F.A. and H.K. performed exome and copy number analyses. E.M.-E., J.G., C.P.T. and M.H.V. contributed clinical samples. R.R. directed sequencing analyses. A.F. and T.P. designed the study, directed and supervised research, and wrote the manuscript.

Corresponding authors

Correspondence to Raul Rabadan, Adolfo Ferrando or Teresa Palomero.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 DNMT3A and TP53 expression analysis in samples from patients with Sézary syndrome.

(a,b) Quantitative RT-PCR analysis of CD4+ T cells isolated from the peripheral blood of individuals with benign erythroderma lesions and representative cases of Sézary syndrome harboring 2p23.3 deletions (a) or 17p13.1 deletions (b). The bar graphs in a and b show the mean values from three technical triplicates in the quantitative PCR reaction; error bars, s.d. P values were calculated using Student's t test. Gray boxes indicate heterozygous deletions; solid boxes indicate homozygous deletions. CNV, copy number variation.

Supplementary Figure 2 Signatures of mutational processes in Sézary syndrome.

Analysis of mutational processes showed the presence of a mutational signature characterized by C>T substitutions at NpCpG trinucleotides, as well as a high frequency of C>A substitutions at CpCpN trinucleotides and C>T substitutions at CpCpN and TpCpN trinucleotides.

Supplementary Figure 3 Functional characterization of CTCL MAPK1 mutations.

(a) Schematic of the structure of the MAPK1 (ERK2) protein. The positions of the MAPK1 alterations identified in CTCL samples are indicated with solid circles; the positions of recurrent alterations in COSMIC (p.Glu322Lys) identified in solid tumors are indicated with open circles. (b,c) Immunoblot analysis (b) and quantification (c) of ERK phosphorylation in HEK293T cells expressing wild-type V5-MAPK1, V5-MAPK1 E322K and V5-MAPK1 E322A. WT, wild type.

Supplementary Figure 4 Comparative functional analysis of Sézary syndrome CARD11 linker domain mutations and DLBCL CARD11 coiled-coil domain mutations.

(a) Schematic of the structure of the CARD11 protein. The positions of the CARD11 alterations identified in the linker domain are indicated with black circles, and the positions of three examples of CARD11 alterations identified in DLBCL are indicated with blue circles. (b) NF-κB luciferase reporter activity in HEK293T cells transfected with V5-CARD11 wild type, mutants (S615F, E626K, D230N, L215P, M183L) or empty vector. (c) NF-κB–dependent GFP reporter activity in non-stimulated Jurkat cells expressing CARD11 wild type, CARD11 mutants (S615F, E626K, D230N, L215P, M183L) or empty vector. Bar graphs indicate the percentage of GFP-positive cells analyzed by flow cytometry. Data are representative of three independent experiments. (d) NF-κB–dependent GFP reporter activity after stimulation for 6 h with 1 μg/ml ionomycin and 0.2 nM PMA in Jurkat cells expressing CARD11 wild type, CARD11 mutants (S615F, E626K, D230N, L215P, M183L) or empty vector. Bar graphs indicate mean GFP intensity measured by flow cytometry across three replicates. (e) Analysis of the levels of V5-CARD11 protein in a Jurkat NF-κB–GFP reporter cell line infected with lentiviruses driving the expression of CARD11 wild type, CARD11 mutants (S615F, E626K, D230N, L215P, M183L) or empty vector. The bar graphs in bd show mean values; error bars, s.d. P values were calculated using Student's t test. WT, wild type.

Supplementary Figure 5 Activated signaling pathways in CTCL cell lines.

(a) Immunoblot analysis of STAT3, ERK1/2 and JNK phosphorylation. (b) Analysis of nuclear NF-κB after subcellular fractionation in CTCL cell lines.

Supplementary Figure 6 Antitumor activity of signaling inhibitors in CTCL.

Proliferation analysis of CTCL cell lines (HH, HUT78, HUT102, SeAX) after treatment with the indicated compounds for 72 h.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1, 2 and 8. (PDF 2339 kb)

Supplementary Table 3

CNVs in Sézary syndrome samples. (XLSX 201 kb)

Supplementary Table 4

CNVs in CTCL samples. (XLSX 29 kb)

Supplementary Table 5

Number of nonsynonymous somatic mutations per sample. (XLSX 14 kb)

Supplementary Table 6

SNVs in Sézary syndrome samples. (XLSX 154 kb)

Supplementary Table 7

SNVs in CTCL samples. (XLSX 99 kb)

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da Silva Almeida, A., Abate, F., Khiabanian, H. et al. The mutational landscape of cutaneous T cell lymphoma and Sézary syndrome. Nat Genet 47, 1465–1470 (2015). https://doi.org/10.1038/ng.3442

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