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SATB1 Defines a Subtype of Cutaneous CD30+ Lymphoproliferative Disorders Associated with a T-Helper 17 Cytokine Profile

  • Author Footnotes
    10 These authors contributed equally to this work.
    Jingru Sun
    Footnotes
    10 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology and Venerology, Peking University First Hospital, Beijing, China

    Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, China
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  • Author Footnotes
    10 These authors contributed equally to this work.
    Shengguo Yi
    Footnotes
    10 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology and Venerology, Peking University First Hospital, Beijing, China

    Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, China
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  • Lei Qiu
    Affiliations
    Department of Dermatology and Venerology, Peking University First Hospital, Beijing, China

    Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, China
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  • Wenjing Fu
    Affiliations
    Department of Dermatology and Venerology, Peking University First Hospital, Beijing, China

    Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, China

    Department of Dermatology and Venerology, Binzhou Medical University Hospital, Binzhou, Shandong, China
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  • Anqi Wang
    Affiliations
    Department of Dermatology and Venerology, Peking University First Hospital, Beijing, China

    Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, China
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  • Fengjie Liu
    Affiliations
    Department of Dermatology and Venerology, Peking University First Hospital, Beijing, China

    Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, China
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  • Lin Wang
    Affiliations
    Department of Dermatovenereology, West China Hospital, Sichuan University, Chengdu, Sichuan, China
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  • Tingting Wang
    Affiliations
    Department of Dermatovenereology, West China Hospital, Sichuan University, Chengdu, Sichuan, China
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  • Hao Chen
    Affiliations
    Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, Jiangsu, China
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  • Lei Wang
    Affiliations
    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shanxi, China
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  • Marshall E. Kadin
    Affiliations
    Department of Dermatology and Skin Surgery, Boston University School of Medicine, Boston, Massachusetts, USA

    Roger Williams Medical Center, Providence, Rhode Island, USA

    Department of Pathology and Laboratory Medicine, Rhode Island Hospital, Providence, Rhode Island, USA
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  • Ping Tu
    Correspondence
    Ping Tu, Department of Dermatology and Venerology, Peking University First Hospital, No. 8 Xishiku Street, Beijing 100034, China.
    Affiliations
    Department of Dermatology and Venerology, Peking University First Hospital, Beijing, China

    Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, China
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  • Yang Wang
    Correspondence
    Correspondence: Yang Wang, Department of Dermatology and Venerology, Peking University First Hospital, No. 8 Xishiku Street, Beijing 100034, China.
    Affiliations
    Department of Dermatology and Venerology, Peking University First Hospital, Beijing, China

    Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, China
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  • Author Footnotes
    10 These authors contributed equally to this work.
Open ArchivePublished:March 03, 2018DOI:https://doi.org/10.1016/j.jid.2018.02.028
      Cutaneous CD30+ lymphoproliferative disorders (LPDs), including lymphomatoid papulosis (LyP) and primary cutaneous anaplastic large-cell lymphoma, comprise the second most common group of cutaneous T-cell lymphomas. Previously, we reported that special SATB1, a thymocyte-specific chromatin organizer, was overexpressed and promoted malignant T-cell proliferation in a portion of CD30+ LPDs. Here, we investigated the expression pattern of SATB1 in CD30+ LPDs with a large cohort of patient samples, and examined the potential of SATB1 as a molecular marker to classify CD30+ LPDs with differential clinicopathological behaviors. SATB1 expression was identified in the CD30+ anaplastic T cells in 11 of 12 (91.7%) lymphomatoid papulosis and 16 of 42 (38.1%) primary cutaneous anaplastic large-cell lymphoma cases. SATB1+ cases showed T-helper 17 polarization, together with more prominent epidermal hyperplasia and granulocytic infiltration. SATB1+ lesions responded better to combined treatment of methotrexate and interferon. SATB1 activated the expression of T-helper 17 cytokines while repressing T-helper 1–related genes. The heterogeneity in SATB1 expression across CD30+ LPDs was associated with the extent of promoter DNA methylation. Hence, SATB1 expression defines a subtype of CD30+ LPDs with characteristic pathobiology and prognosis. These data provide valuable insights into the heterogeneity of cutaneous T-cell malignancies, which may lead to individualized therapy in the future.

      Abbreviations:

      CTCL (cutaneous T-cell lymphoma), LPD (lymphoproliferative disorder), LyP (lymphomatoid papulosis), MTX (methotrexate), PCALCL (primary cutaneous anaplastic large-cell lymphoma), Th (T helper)

      Introduction

      Primary cutaneous CD30+ lymphoproliferative disorders (LPDs) are the second most common form of cutaneous T-cell lymphoma (CTCL) and represent a spectrum of disorders including lymphomatoid papulosis (LyP), primary cutaneous anaplastic large-cell lymphoma (PCALCL), and borderline forms (
      • Slater D.N.
      The new World Health Organization-European Organization for Research and Treatment of Cancer classification for cutaneous lymphomas: a practical marriage of two giants.
      ,
      • Willemze R.
      • Jaffe E.S.
      • Burg G.
      • Cerroni L.
      • Berti E.
      • Swerdlow S.H.
      • et al.
      WHO-EORTC classification for cutaneous lymphomas.
      ). They are characterized by the phenotypic hallmark of CD30+ anaplastic T cells without anaplastic lymphoma kinase expression (
      • Kempf W.
      • Pfaltz K.
      • Vermeer M.H.
      • Cozzio A.
      • Ortiz-Romero P.L.
      • Bagot M.
      • et al.
      EORTC, ISCL, and USCLC consensus recommendations for the treatment of primary cutaneous CD30-positive lymphoproliferative disorders: lymphomatoid papulosis and primary cutaneous anaplastic large-cell lymphoma.
      ). Nearly 20% LyP patients progress to more aggressive lymphomas, including PCALCL. More than 20% of PCALCL patients could develop multifocal skin lesions and involve lymph nodes, with a poor prognosis. The CD30+ T cells in LyP share many characteristics with PCALCL cells, including anaplastic morphology, aneuploidy, cytogenetic abnormalities, aberrant phenotype, up-regulation of CD30, Fra2, and Id2 (
      • Mathas S.
      • Kreher S.
      • Meaburn K.J.
      • Johrens K.
      • Lamprecht B.
      • Assaf C.
      • et al.
      Gene deregulation and spatial genome reorganization near breakpoints prior to formation of translocations in anaplastic large cell lymphoma.
      ). The wide spectrum of clinical behaviors can lead to difficulties in making therapeutic decisions for patients with CD30+ LPDs. Five LyP variants (type A–E) are recognized without prognostic or therapeutic implication (
      • Swerdlow S.H.
      • Campo E.
      • Pileri S.A.
      • Harris N.L.
      • Stein H.
      • Siebert R.
      • et al.
      The 2016 revision of the World Health Organization classification of lymphoid neoplasms.
      ). The molecular events underlying CD30+ LPDs with distinct clinical behaviors remain unidentified.
      Previously, we reported that special SATB1, a thymocyte-specific chromatin organizer, was overexpressed in the CD30+ anaplastic T cells in CD30+ LPDs (
      • Wang Y.
      • Gu X.
      • Zhang G.
      • Wang L.
      • Wang T.
      • Zhao Y.
      • et al.
      SATB1 overexpression promotes malignant T-cell proliferation in cutaneous CD30+ lymphoproliferative disease by repressing p21.
      ). SATB1 is a nuclear matrix protein that plays a crucial role in T-cell development (
      • Krangel M.S.
      T cell development: better living through chromatin.
      ). It functions as a “genome organizer” with a cage-like “network” distribution circumscribing heterochromatin. By dynamically altering the organization and epigenetic status of the chromatin, SATB1 functions as a highly pleiotropic regulator of gene expression (
      • Cai S.
      • Lee C.C.
      • Kohwi-Shigematsu T.
      SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine genes.
      ). We have demonstrated that SATB1 overexpression is a critical mechanism promoting anaplastic T-cell proliferation in CD30+ LPDs (
      • Wang Y.
      • Gu X.
      • Zhang G.
      • Wang L.
      • Wang T.
      • Zhao Y.
      • et al.
      SATB1 overexpression promotes malignant T-cell proliferation in cutaneous CD30+ lymphoproliferative disease by repressing p21.
      ). Interestingly, as a master regulator of malignant behavior of CD30+ T cells, SATB1 was negative in a portion of PCALCLs, indicating that distinct molecular pathogenesis exists within CD30+ LPDs.
      In this study, with an independent, large cohort of patients, we systemically analyzed the molecular and clinicopathological features of the two subgroups of CD30+ LPDs based on SATB1 expression. Distinct molecular pathways were activated in CD30+ LPDs with differential SATB1 expression. SATB1+ cases demonstrated a T-helper (Th)-17 cytokine profile in anaplastic T cells, and showed marked epidermal hyperplasia with increased granulocyte infiltration. Moreover, SATB1 regulates the expression of genes favoring Th17 polarization. The differential expression of SATB1 in CD30+ LPDs was correlated with the extent of its promoter methylation. Of note, SATB1+ cases responded better to combined therapy of methotrexate (MTX) and IFN-α2b. Our findings demonstrated the intrinsic heterogeneity within CD30+ LPDs. SATB1 may serve as a useful marker in subtyping patients with varied molecular pathogenesis and prognosis, leading to individualized therapeutic strategies.

      Results

      Differential SATB1 expression in CD30+ LPDs

      To characterize the expression pattern of SATB1 in CD30+ LPDs, we investigated an independent cohort of 54 patients (including 12 LyP patients and 42 PCALCL patients). Lesional biopsies from each patient were subjected to immunohistochemistry with antibodies against CD30 and SATB1. Patient characteristics and the detailed staining results are listed in Supplementary Table S1 (online). Bright and diffuse nuclear SATB1 staining coincided with membranous CD30 staining on anaplastic T cells in 11 of 12 LyP (91.7%) and in 16 of 42 PCALCL (38.1%) specimens (Figure 1a). The 11 SATB1+ LyP cases included 10 type-A cases and 1 type-C case. On the contrary, 1 of 12 LyPs (8.3%) and 26 of 42 PCALCLs (61.9%) showed absence of SATB1 staining of anaplastic T cells, despite CD30 staining. The only SATB1-negative LyP case was consistent with the newly defined type E (angioinvasive) LyP (
      • Swerdlow S.H.
      • Campo E.
      • Pileri S.A.
      • Harris N.L.
      • Stein H.
      • Siebert R.
      • et al.
      The 2016 revision of the World Health Organization classification of lymphoid neoplasms.
      ). Our data showed that the anaplastic T cells in a minority of LyP and a significant proportion of PCALCL are negative for SATB1.
      Figure 1
      Figure 1Heterogeneity of SATB1 expression in cutaneous CD30+ LPDs and the underlying difference in transcriptome. (a) Paraffin-embedded tissue from LyP and PCALCL patients were stained with anti-CD30 and anti-SATB1 antibodies by immunohistochemistry. Bright and diffuse nuclear staining of SATB1 were seen in CD30+ anaplastic T cells in LyP and PCALCL-1, while the CD30+ anaplastic T cells in PCALCL-2 were negative for SATB1. Original magnification ×400, scale bar = 20 μm. (b) Varied SATB1 protein expressions were detected in seven CTCL lines with Western blot. (c) Hierarchical clustering of the 936 genes (Probability > 0.6, P < 0.05, and fold change >2) based on RNA sequencing data, demonstrating distinct transcriptome between SATB1+ and SATB1 PCALCLs. Pt., patient number. CTCL, cutaneous T-cell lymphoma; LPD, lymphoproliferative disorder; LyP, lymphomatoid papulosis; PCALCL, primary cutaneous anaplastic large-cell lymphoma.
      Previously, we showed that PCALCL cell lines Mac-1, Mac-2A, and Mac-2B highly expressed SATB1, and the expression levels increased upon disease progression (
      • Wang Y.
      • Gu X.
      • Zhang G.
      • Wang L.
      • Wang T.
      • Zhao Y.
      • et al.
      SATB1 overexpression promotes malignant T-cell proliferation in cutaneous CD30+ lymphoproliferative disease by repressing p21.
      ). Mac cell lines were established from a patient who progressed from LyP to PCALCL; the Mac-1 cell line was derived from an indolent course of disease, whereas the Mac-2A and Mac-2B cells were derived from separate rapidly growing skin tumors 3 years later in disease progression (
      • Davis T.H.
      • Morton C.C.
      • Miller-Cassman R.
      • Balk S.P.
      • Kadin M.E.
      Hodgkin's disease, lymphomatoid papulosis, and cutaneous T-cell lymphoma derived from a common T-cell clone.
      ). Because Mac cells are the only available PCALCL cell lines, we evaluated SATB1 expression in seven additional CTCL cell lines. While MJ highly expressed SATB1, Myla and PB2B expressed moderate level of SATB1, H9, Hut-78, HH, and Sz4 lacked SATB1 expression, indicating heterogeneity for SATB1 activity among CTCL cell lines (Figure 1b). Among those cell lines, Hut-78 and HH were also positive for surface CD30 (
      • Wang Y.
      • Gu X.
      • Zhang G.
      • Wang L.
      • Wang T.
      • Zhao Y.
      • et al.
      SATB1 overexpression promotes malignant T-cell proliferation in cutaneous CD30+ lymphoproliferative disease by repressing p21.
      ). Therefore, these results indicate that intrinsic heterogeneity regarding SATB1 expression status exists in cutaneous CD30+ LPDs and CD30+ CTCL cell lines.

      Distinct gene expression profiles in PCALCLs with differential SATB1 expression

      SATB1 is a global gene regulator that determines cell fate in T cells (
      • Krangel M.S.
      T cell development: better living through chromatin.
      ). To elucidate the underlying molecular events in CD30+ LPDs with differential SATB1 expression, we subjected four SATB1+ and three SATB1 PCALCL skin biopsies to second-generation RNA sequencing (#GSE109620). While no difference in single-nucleotide variation, alternative splicing, or recurrent gene fusion was found, distinct gene expression profiles were identified between SATB1+ and SATB1 samples. With the criteria of Probability >0.6, P < 0.05, and fold change >2, there were 936 genes differentially expressed between SATB1+ and SATB1 PCALCLs. Four hundred and seventy-three genes were dominantly up-regulated in the SATB1+ group, and 463 genes were overexpressed in the SATB1 group (Figure 1c). Pathway analysis by Genomatix Pathway System revealed distinct enriched molecular pathways in SATB1+ versus SATB1 cases (Table 1). Immune response pathways and cytokine signaling pathways were enriched in SATB1+ cases. Among them, 4 out of 21 enriched pathways were related to Th17 differentiation, including JAK-STAT molecular variation 1, JAK-STAT molecular variation 2, IL-6–mediated signaling events, and IL-23–mediated signaling events. Genes inducing Th17 differentiation (IL6, TGFBR1, IL22RA2, STAT3, IRF4, IL23A, and RORC), as well as genes encoding the prototype Th17 cytokines (IL17A, IL17F, and IL22) were up-regulated in SATB1+ cases. Other pathways enriched in SATB1+ cases included those regulating cell–cell interaction (COL6A3, LAMB1), cell–matrix adhesion (PLAT, PLAUR), and cytokine signaling (IL4R, IL13, and IL13RA1). In SATB1 PCALCLs, the majority of enriched pathways were involved in cell cycle control and cell mitosis (Table 1).
      Table 1Molecular pathways enriched in SATB1+ and SATB1 primary cutaneous anaplastic large-cell lymphomas
      VariableP Value
      Signaling pathways enriched in SATB1+ cases
       Beta1 integrin cell surface interactions1.87 × 10–11
       JAK_STAT_MolecularVariation_25.66 × 10–7
       JAK_STAT_MolecularVariation_14.13 × 10–6
       Amb2 Integrin signaling2.49 × 10–5
       IL4-mediated signaling events5.78 × 10–5
       AP-1 transcription factor network1.44 × 10–4
       Fc-epsilon receptor I signaling in mast cells2.32 × 10–4
       IL-6–mediated signaling events3.03 × 10–4
       Filopodium formation (Integrin signaling pathway)3.40 × 10–4
       Syndecan-4–mediated signaling events8.20 × 10–4
       IL-13 signaling pathway (JAK1 TYK2 STAT6)1.01 × 10–3
       Validated transcriptional targets of AP1 family members Fra1 and Fra21.72 × 10–3
       Cell to cell adhesion signaling2.21 × 10–3
       ATF-2 transcription factor network2.63 × 10–3
       Signaling events mediated by PTP1B3.41 × 10–3
       Erk and Pi-3 kinase are necessary for collagen binding in corneal epithelia4.45 × 10–3
       Eicosanoid metabolism4.83 × 10–3
       Urokinase-type plasminogen activator (uPA) and uPA receptor–mediated signaling6.37 × 10–3
       Cytokine receptor degradation signaling7.38 × 10–3
       IL-23–mediated signaling events9.28 × 10–3
       GM-CSF–mediated signaling events9.28 × 10–3
      Signaling pathways enriched in SATB1 cases
       Aurora B signaling7.49 × 10–16
       PLK1 signaling events1.18 × 10–6
       E2F transcription factor network5.17 × 10–6
       CDK regulation of DNA replication4.71 × 10–5
       FOXM1 transcription factor network1.73 × 10–4
       Validated targets of C-MYC transcriptional activation3.58 × 10–4
       Role of RAN in mitotic spindle regulation6.62 × 10–4
       ATR signaling pathway7.61 × 10–4
       Aurora A signaling1.21 × 10–3
       Mechanisms of transcriptional repression by DNA methylation2.41 × 10–3
       BTG family proteins and cell cycle regulation4.47 × 10–3
       Aurora C signaling4.62 × 10–3
      These data demonstrated that the molecular differences between SATB1+ and SATB1 PCALCLs lie in the transcriptome level; distinct molecular pathways dominate cases with differential SATB1 expression.

      Th17-related genes are up-regulated in SATB1+ CD30+ LPD skin samples and cell lines

      To validate the results from RNA sequencing, we analyzed the expression status of differentially expressed genes in major molecular pathways with RNA samples from an independent cohort of patients (SATB1+: n = 5, SATB1: n = 4), and confirmed up-regulation of Th17 differentiation–related genes in SATB1+ cases, including SATB1, IL17F, IL17A, IL22, IL6, IL22RA2, TGFBR1, IRF4, STAT3, IL23A, and RORC (Figure 2a). Cell mitosis–related genes PLK1, E2F1, E2F2, AURKA, and AURKB were confirmed to be up-regulated in SATB1 cases, although only PLK1 showed a significant difference (Figure 2b). To confirm the activation of Th17 differentiation in SATB1+ cases, we performed immunostaining of IL-17F, IL-22, RORγt (encoded by RORC), STAT3, and p-STAT3 on all 54 cases of CD30+ LPDs (Figure 2c). Nineteen out of 27 (70.4%) SATB1+ cases showed positive staining of IL-17F in the cytoplasm of anaplastic T cells, while only 5 out of 27 (18.5%) SATB1 cases showed IL-17F staining. Consistently, all 10 cases with positive cytoplasmic IL-22 staining of anaplastic T cells belonged to the SATB1+ group. Moreover, 15 out of 25 (60%) SATB1+ cases were positive for RORγt staining of anaplastic T cells, while only 2 out of 27 SATB1 cases showed positive RORγt staining. The majority of cases with positive nuclear STAT3 and p-STAT3 staining on anaplastic T cells were in the SATB1+ group, although only a portion of SATB1+ cases showed positive STAT3 and p-STAT3 staining. Representative staining pictures were shown in Figure 2d.
      Figure 2
      Figure 2Th17 differentiation was activated in SATB1+ CD30+ LPDs. (a) Th17 differentiation–related genes were up-regulated in SATB1+ CD30+ LPDs. (b) Cell mitosis–related genes were up-regulated in SATB1 CD30+ LPDs. (c) Summary of immunohistochemistry staining results of CD30, SATB1, IL-17F, IL-22, RORγt, STAT3, and p-STAT3 in 54 CD30+ LPDs. N/A, not available. (d) Representative cases showed IL-17F, IL-22, RORγt, STAT3, and p-STAT3 staining on the anaplastic T cells in SATB1+ PCALCL, while SATB1 PCALCL showed negative staining for the above proteins. Original magnification ×400, scale bar = 20 μm. (e) SATB1+ CTCL cell lines (Mac-1 and Mac-2A) highly expressed Th17-related genes, while SATB1 CTCL cell lines (Hut-78 and HH) highly expressed cell mitosis–related genes. Each experiment was repeated 2 times with 3 biological replicates. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. ns, no significance. (f) Western blot showed high STAT3, p-STAT3, RORγt, and IL23R expression in SATB1+ CTCL cell lines (Mac-1 and Mac-2A) compared to SATB1 CTCL cell lines (Hut-78 and HH). CTCL, cutaneous T-cell lymphoma; LPD, lymphoproliferative disorder; PCALCL, primary cutaneous anaplastic large-cell lymphoma; Th, T helper.
      To evaluate the representativeness of the SATB1+ and SATB1 CD30+ CTCL cell lines, we analyzed genes differentially expressed between SATB1+ versus SATB1 cases on Mac-1, Mac-2A, Hut-78, and HH cells. In line with the phenotype identified in clinical samples, SATB1+ cell lines Mac-1 and Mac-2A demonstrated higher expression of Th17-related genes, including IL17F, IL22, IL6, RORC, and IRF4, while SATB1 cell lines Hut-78 and HH demonstrated high expression of cell cycle control genes, including PLK1, E2F1, E2F2, AURKA, and AURKB (Figure 2e). Moreover, Mac-1 and Mac-2A cells expressed high levels of STAT3, p-STAT3, RORγt, and IL23R compared to Hut-78 and HH cells, consistent with differences observed in clinical samples (Figure 2f). These results indicate that the four cell lines can at least partially represent the molecular profiles of SATB1+ versus SATB1 clinical samples, and may serve as in vitro models for SATB1+/– CD30+ LPDs.
      Collectively, these results confirm that distinct molecular pathways are activated in SATB1+ versus SATB1 CD30+ LPDs, and that Th17 cytokine profile predominates in SATB1+ CD30+ LPDs.

      Clinicopathological analyses demonstrate Th17 activation associated phenotypes in SATB1+ CD30+ LPDs

      To characterize the clinicopathological features in SATB1+ versus SATB1 CD30+ LPDs, we retrospectively reviewed the clinicopathological data of the 54 CD30+ LPD patients, in which 27 patients had lesions that were SATB1+ and 27 patients had SATB1 lesions. The median age of these 54 patients was 54.5 years (range from 9 to 87 years), and the median follow-up period was 24.8 months (range from 1 to 71 months). Regarding disease diagnosis, the majority of LyP cases were SATB1+, thus patients were more likely to be diagnosed with LyP than PCALCL in SATB1+ subgroup (P = 0.043). Considering the distinct clinical and pathological features between LyP and PCALCL, we excluded all 12 LyP cases in the following clinicopathological and therapeutic outcome analyses.
      Analysis on clinical features showed that while 45.5% of SATB1+ PCALCLs presented as multiple rather than solitary skin tumors, a greater proportion of SATB1 PCALCLs (78.9%) presented with multiple skin tumors with marginal significance (P = 0.061) (Table 2). There were no significant differences between the two subgroups in demographics, disease duration, previous history of LyP, size of the lesions, or lymph node involvement (Supplementary Table S2 online).
      Table 2Clinical and pathological features with significant differences between SATB1+ and SATB1 primary cutaneous anaplastic large-cell lymphomas
      VariableSATB1+, n (%)SATB1, n (%)Total, nP Value
      Clinical characteristics of PCALCLs (n = 30)
       No. of lesions0.061
      Solitary6 (54.5)4 (21.1)10
      Multiple5 (45.5)15 (78.9)20
      Pathological characteristics of PCALCLs (n = 42)
       Epidermal hyperplasia0.010
      Present11 (73.3)7 (29.2)18
      Not present4 (26.7)17 (70.8)21
       Epidermotropism0.028
      Present14 (93.3)14 (58.3)28
      Not present1 (0.07)10 (41.7)11
      Vasculitic changes
       Hyalinized/damaged vessel walls0.002
      Present6 (37.5)0 (0)6
      Not present10 (62.5)26 (100)36
       Dermal vessel dilatation0.020
      Present9 (56.3)5 (19.2)14
      Not present7 (43.7)21 (80.8)28
       Extravasated red blood cells0.016
      Present7 (43.7)2 (7.7)9
      Not present9 (56.3)24 (92.3)33
      Granulocyte infiltration
       Eosinophils0.004
      Present9 (56.3)3 (11.5)12
      Not present7 (43.7)23 (88.5)30
       Neutrophils
      Present11 (68.8)1 (3.8)120.000
      Not present5 (31.2)25 (96.2)30
      Therapeutic outcomes of PCALCLs (n = 22)
       Response to MTX/IFN0.054
      Complete remission4 (57.1)2 (13.3)6
      No complete remission3 (42.9)13 (86.7)16
      Abbreviations: MTX, methotrexate; PCALCL, primary cutaneous anaplastic large-cell lymphoma.
      Histologically, within all characteristics we analyzed (Table 2, Supplementary Table S2), there were no differences in infiltration pattern, lymphocyte size, large cell density, large cell morphology, number of cell mitoses or Ki-67 labeling (Figure 3a). In contrast, epidermal hyperplasia was more prominent in SATB1+ PCALCLs (P = 0.010). Pseudo-epitheliomatous hyperplasia was observed in SATB1+ cases (Figure 3a). Other than that, SATB1+ PCALCLs demonstrated significantly more epidermotropism (P = 0.028), eosinophil infiltration (P = 0.004), neutrophil infiltration (P = 0.000), hyalinized vessel walls (P = 0.002), dermal vessel dilatation (P = 0.020), and red blood cell extravasation (P = 0.016) (Figure 3a). These features mimic the histological manifestations of LyP, in which most cases were SATB1+. These features also resemble the phenotype observed in Th17-associated skin inflammation, such as psoriasis, as a result of IL-17 and IL-22 expression (
      • Korn T.
      • Bettelli E.
      • Oukka M.
      • Kuchroo V.K.
      IL-17 and Th17 Cells.
      ).
      Figure 3
      Figure 3Differences in histology, therapeutic response, molecular features, and promoter methylation status between SATB1+ and SATB1 CD30+ LPDs. (a) Representative cases showed that epidermal hyperplasia, epidermotropism, red blood cell extravasation, and granulocytes infiltration were more prominent in SATB1+ case compared to those in SATB1 case. Ki-67 staining showed little difference between SATB1+ case and SATB1 case. (SATB1+: 82%, SATB1: 83%). Original magnification ×200, scale bar = 20 μm. (b) MTS-based cell viability assay showed that Mac-1, Mac-2A, Hut-78, and HH cell lines are equally sensitive to MTX (10 μM) treatment, whereas Mac-1, Mac-2A are more sensitive to IFN-α2b (2000 U/ml and 10,000 U/ml) treatment compared to Hut-78 and HH cell lines. Each experiment was repeated two times with three biological replicates. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. ns, no significance. (c) The Venn diagram showed that 121 differentially expressed genes between SATB1+ and SATB1 CD30+ LPDs were SATB1 downstream genes. (d) Quantitative methylation analysis on CpG sites in the specific region of SATB1 promoter on SATB1+ (n = 8) versus SATB1 (n = 8) cases. P < 0.001. Methylation level 1 represents 100% methylated CpG dinucleotides on this site. (e) SATB1 mRNA expression was up-regulated in Mac-1, Mac-2A, Hut-78, and HH cells treated with a DNA demethylation reagent 5-aza-2′-deoxycytidine (250 μM). LPD, lymphoproliferative disorder; PCALCL, primary cutaneous anaplastic large-cell lymphoma.
      Of note, although no significant difference in disease prognosis in terms of progression-free survival were observed between the two groups of PCALCL cases, there is a trend that patients with positive SATB1 staining responded better to combined low-dose MTX and IFN-α2b treatment, which is the conventional initiation therapy for PCALCLs in our center. While 57.1% SATB1+ cases achieved complete remission after MTX and IFN-α2b therapy, only 13.3% SATB1 cases achieved complete remission with the same therapeutic regimen (P = 0.054) (Table 2, Supplementary Table S2). Though the limited number of cases did not allow multivariate analyses, we performed Fisher’s exact test to exclude the impact of the number of lesions on the response to combined MTX and IFN-α2b therapy (P = 1.000) (Supplementary Table S2). Because all our patients underwent combined therapy, to elucidate which drug made the most difference, we evaluated the sensitivities of PCALCL cells to MTX or IFN-α2b in our in vitro model. Mac-1, Mac-2A, Hut-78, and HH cell lines were incubated with 10 μM MTX, 2000 U/ml IFN-α2b, or 10,000 U/ml IFN-α2b (Figure 3b) for 3 days. Interestingly, while all four cell lines were equally sensitive to MTX, the growth inhibition by IFN-α2b was more obvious in SATB1+ cell lines than SATB1 cell lines, consistent with the clinical observations. Although the action mechanism of MTX and IFN-α2b in CD30+ LPDs had not been fully elucidated, we postulated that the different responses to IFN-α2b between cells with differential SATB1 expression may be attributed to IFN-α2b–mediated STAT3 inactivation (
      • Asmana Ningrum R.
      Human interferon alpha-2b: a therapeutic protein for cancer treatment.
      ,
      • Kirkwood J.M.
      • Farkas D.L.
      • Chakraborty A.
      • Dyer K.F.
      • Tweardy D.J.
      • Abernethy J.L.
      • et al.
      Systemic interferon-alpha (IFN-alpha) treatment leads to Stat3 inactivation in melanoma precursor lesions.
      ,
      • O'Shea J.J.
      • Holland S.M.
      • Staudt L.M.
      JAKs and STATs in immunity, immunodeficiency, and cancer.
      ,
      • Thomas S.
      • Fisher K.
      • Snowden J.
      • Danson S.
      • Brown S.
      • Zeidler M.
      Effect of methotrexate on JAK/STAT pathway activation in myeloproliferative neoplasms.
      ,
      • Thomas S.
      • Fisher K.H.
      • Snowden J.A.
      • Danson S.J.
      • Brown S.
      • Zeidler M.P.
      Methotrexate is a JAK/STAT pathway inhibitor.
      ).
      Therefore, SATB1+ PCALCLs demonstrated characteristic histological features consistent with Th17 differentiation; these cases are more sensitive to treatment with low-dose MTX and IFN-α2b.

      SATB1 up-regulates genes favoring Th17 differentiation

      To explore how much the difference between SATB1+ and SATB1 cases can be attributed to SATB1 expression, we compared the differentially expressed genes between SATB1+ and SATB1 PCALCLs (1,833 genes) with genes affected by SATB1 silencing in PCALCL cell lines Mac-1 (1,844 genes, from our previous study [
      • Wang Y.
      • Gu X.
      • Zhang G.
      • Wang L.
      • Wang T.
      • Zhao Y.
      • et al.
      SATB1 overexpression promotes malignant T-cell proliferation in cutaneous CD30+ lymphoproliferative disease by repressing p21.
      ], data accessible at the National Center for Biotechnology Information Gene Expression Omnibus database, #GSE50916). As seen in Figure 3c, 121 out of 1,833 genes with differential expression between SATB1+ and SATB1 cases were SATB1 downstream genes. Among those, Th17 lineage genes IL17F and IL23A, which were overexpressed in SATB1+ cases, were down-regulated upon SATB1 silencing. Negative regulators of Th17 differentiation, including SOCS1, IL12RB1, IFNG, IFNGR1, STAT1, and STAT4, which were overexpressed in SATB1 cases, were up-regulated upon SATB1 silencing (Supplementary Figure S1 online). SOCS1 plays a significant role in negatively regulating the JAK-STAT signaling pathway, thus inhibiting Th17 cytokines’ formation. IFNG, IFNGR1, STAT1, and IL12RB1 are involved in Th1 differentiation, indirectly inhibiting Th17 polarization. STAT4 is a key regulator in Th2 formation, negatively correlated with Th17 differentiation (
      • Muranski P.
      • Restifo N.P.
      Essentials of Th17 cell commitment and plasticity.
      ). Therefore, SATB1 positively regulates expression of Th17-related genes, and negatively regulates genes antagonizing Th17 differentiation.

      SATB1 expression status is associated with the extent of promoter DNA methylation

      An increasing number of studies indicate expression of SATB1 can be regulated by epigenetic mechanisms (
      • Beyer M.
      • Thabet Y.
      • Muller R.U.
      • Sadlon T.
      • Classen S.
      • Lahl K.
      • et al.
      Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation.
      ,
      • Di Leva G.
      • Piovan C.
      • Gasparini P.
      • Ngankeu A.
      • Taccioli C.
      • Briskin D.
      • et al.
      Estrogen mediated-activation of miR-191/425 cluster modulates tumorigenicity of breast cancer cells depending on estrogen receptor status.
      ). There are several CpG dinucleotide–rich regions around and upstream of SATB1 transcription start site locus (
      • Beyer M.
      • Thabet Y.
      • Muller R.U.
      • Sadlon T.
      • Classen S.
      • Lahl K.
      • et al.
      Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation.
      ). We previously demonstrated with PCALCL cell lines that methylation status of a specific CpG-rich region on SATB1 promoter (–733 to –297) is correlated with SATB1 expression levels (
      • Wang Y.
      • Gu X.
      • Zhang G.
      • Wang L.
      • Wang T.
      • Zhao Y.
      • et al.
      SATB1 overexpression promotes malignant T-cell proliferation in cutaneous CD30+ lymphoproliferative disease by repressing p21.
      ). To validate whether this is responsible for the differential SATB1 expression in CD30+ LPDs, we subjected 16 PCALCL skin biopsies (8 SATB1+ and 8 SATB1, independent from the cases described in previous study) to bisulfite sequencing followed by mass spectrometry–based methylation quantification. As expected, SATB1+ cases showed marked demethylation on this CpG-rich region, while SATB1 cases were highly methylated on this region (Figure 3d, P < 0.001). Moreover, SATB1 expression was remarkably up-regulated upon treatment of all four cell lines with DNA methylation inhibitor 5-aza-2′-deoxycytidine (Figure 3e). The expression of IL17F, IL17A, IL22, IL6, STAT3, RORC, and IRF4 were also up-regulated under 5-aza-2′-deoxycytidine treatment (Supplementary Figure S2 online). It remains to be elucidated whether this transcriptional activation of Th17 cytokines is a consequence of SATB1 up-regulation or the direct effects of DNA demethylation. Collectively, these results indicate that the differential SATB1 expression status is correlated with the extent of SATB1 promoter methylation in CD30+ LPDs.

      Discussion

      Intrinsic heterogeneity has been reported in cutaneous CD30+ LPDs. Chromosomal rearrangements involving the DUSP22-IRF4 locus on 6p25.3 were identified in 28% of PCALCLs and a minority of LyPs (
      • Fauconneau A.
      • Pham-Ledard A.
      • Cappellen D.
      • Frison E.
      • Prochazkova-Carlotti M.
      • Parrens M.
      • et al.
      Assessment of diagnostic criteria between primary cutaneous anaplastic large-cell lymphoma and CD30-rich transformed mycosis fungoides; a study of 66 cases.
      ,
      • Feldman A.L.
      • Dogan A.
      • Smith D.I.
      • Law M.E.
      • Ansell S.M.
      • Johnson S.H.
      • et al.
      Discovery of recurrent t(6;7)(p25.3;q32.3) translocations in ALK-negative anaplastic large cell lymphomas by massively parallel genomic sequencing.
      ,
      • Karai L.J.
      • Kadin M.E.
      • Hsi E.D.
      • Sluzevich J.C.
      • Ketterling R.P.
      • Knudson R.A.
      • et al.
      Chromosomal rearrangements of 6p25.3 define a new subtype of lymphomatoid papulosis.
      ,
      • Onaindia A.
      • Montes-Moreno S.
      • Rodriguez-Pinilla S.M.
      • Batlle A.
      • Gonzalez de Villambrosia S.
      • Rodriguez A.M.
      • et al.
      Primary cutaneous anaplastic large cell lymphomas with 6p25.3 rearrangement exhibit particular histological features.
      ). NPM1-TYK2, NFkB2-ROS-1, and NFkB2-TYK2 gene fusions were found in scattered cases of CD30+ LPDs. In this study, we did not find any of these gene translocations or fusions in the seven cases of PCALCL undergoing RNA sequencing, indicating that the expression status of SATB1 is independent from these gene fusion events. Our study revealed distinct SATB1 expression status in cutaneous CD30+ LPDs cases, which can easily differentiate CD30+ LPDs into SATB1+ and SATB1 subgroups by immunohistochemistry. The molecular difference between SATB1+ and SATB1 cases are at the transcriptome level, consistent with the role of SATB1 as a global gene regulator (
      • Han H.J.
      • Russo J.
      • Kohwi Y.
      • Kohwi-Shigematsu T.
      SATB1 reprogrammes gene expression to promote breast tumour growth and metastasis.
      ).
      SATB1+ cases comprised the majority of LyPs and 38.1% of PCALCLs in our cohort. These cases demonstrated activation of inflammatory response–related molecular pathways, especially pathways regulating Th17 differentiation. Key Th17 cytokines IL-17, IL-22, as well as Th17 transcription factors RORγt and STAT3 were identified in anaplastic T cells in the majority of SATB1+ cases. In line with our findings at the molecular level, histology of SATB1+ cases demonstrated features of Th17 activation: IL-22 is a key regulator for epidermal hyperplasia (
      • Depianto D.
      • Kerns M.L.
      • Dlugosz A.A.
      • Coulombe P.A.
      Keratin 17 promotes epithelial proliferation and tumor growth by polarizing the immune response in skin.
      ,
      • Guitart J.
      • Martinez-Escala M.E.
      • Deonizio J.M.
      • Gerami P.
      • Kadin M.E.
      CD30(+) cutaneous lymphoproliferative disorders with pseudocarcinomatous hyperplasia are associated with a T-helper-17 cytokine profile and infiltrating granulocytes.
      ); granulocytic infiltration and increased vascularization were related to IL-17 secretion (
      • Korn T.
      • Bettelli E.
      • Oukka M.
      • Kuchroo V.K.
      IL-17 and Th17 Cells.
      ). The more prominent inflammatory background in SATB1+ CD30+ LPDs results in more damaged vessel walls, and increased extravasated red blood cells. Moreover, epidermotropism was more obvious in SATB1+ cases, probably due to the up-regulated skin homing capacity of malignant T cells via inflammatory stimuli (
      • Girardi M.
      • Heald P.W.
      • Wilson L.D.
      The pathogenesis of mycosis fungoides.
      ). Previously,
      • Guitart J.
      • Martinez-Escala M.E.
      • Deonizio J.M.
      • Gerami P.
      • Kadin M.E.
      CD30(+) cutaneous lymphoproliferative disorders with pseudocarcinomatous hyperplasia are associated with a T-helper-17 cytokine profile and infiltrating granulocytes.
      reported a case series of CD30+ LPDs with pseudocarcinomatous hyperplasia and infiltrating granulocytes, which demonstrated a Th17 cytokine profile. Here, with a large cohort of CD30+ LPDs, we demonstrated that the differential expression status of SATB1 was responsible for this distinct morphology and phenotype. It is noteworthy that the inflammatory cells may contribute to the transcriptional profile observed in SATB1+ cases. Therefore, the contribution of SATB1 to the inflammatory response in SATB1+ cases is being further investigated.
      Several studies have demonstrated IL-17 expression on malignant T cells in CTCL, including mycosis fungoides and Sézary syndrome (
      • Fontao L.
      • Brembilla N.C.
      • Masouye I.
      • Kaya G.
      • Prins C.
      • Dupin N.
      • et al.
      Interleukin-17 expression in neutrophils and Th17 cells in cutaneous T-cell lymphoma associated with neutrophilic infiltrate of the skin.
      ,
      • Krejsgaard T.
      • Ralfkiaer U.
      • Clasen-Linde E.
      • Eriksen K.W.
      • Kopp K.L.
      • Bonefeld C.M.
      • et al.
      Malignant cutaneous T-cell lymphoma cells express IL-17 utilizing the Jak3/Stat3 signaling pathway.
      ,
      • Krejsgaard T.
      • Litvinov I.V.
      • Wang Y.
      • Xia L.
      • Willerslev-Olsen A.
      • Koralov S.B.
      • et al.
      Elucidating the role of interleukin-17F in cutaneous T-cell lymphoma.
      ). IL-17 was reported to act as a positive regulator for tumor progression, which stimulates the secretion of angiogenic factors, thus promoting tumor vascularization (
      • Murugaiyan G.
      • Saha B.
      Protumor vs antitumor functions of IL-17.
      ). Malignant T cells display a considerable level of plasticity in their production of cytokines and expression of regulatory molecules (
      • Krejsgaard T.
      • Litvinov I.V.
      • Wang Y.
      • Xia L.
      • Willerslev-Olsen A.
      • Koralov S.B.
      • et al.
      Elucidating the role of interleukin-17F in cutaneous T-cell lymphoma.
      ,
      • Muranski P.
      • Restifo N.P.
      Essentials of Th17 cell commitment and plasticity.
      ). Our findings that anaplastic T cells in CD30+ LPDs spontaneously produce IL-17 and IL-22 support the concept of malignant T-cell plasticity. Our data demonstrate the role of SATB1 in regulation of Th17 cytokine expression of malignant T cells. This is consistent with SATB1’s pivotal function in T-cell development (
      • Krangel M.S.
      T cell development: better living through chromatin.
      ). SATB1 expression in normal CD4+ T cells varied with their activation status. SATB1 is highly expressed in Th2 cells and acts as a key factor in Th2 cell activation by regulating the coordinated expression of IL-4, IL-5, and IL-13 (
      • Ahlfors H.
      • Limaye A.
      • Elo L.L.
      • Tuomela S.
      • Burute M.
      • Gottimukkala K.V.P.
      • et al.
      SATB1 dictates expression of multiple genes including IL-5 involved in human T helper cell differentiation.
      ). However, anergic CD4+ T cells and regulatory T cells showed down-regulation of SATB1, compared to naïve and activated T cells (
      • Beyer M.
      • Thabet Y.
      • Muller R.U.
      • Sadlon T.
      • Classen S.
      • Lahl K.
      • et al.
      Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation.
      ), indicating a pluripotent function of SATB1 in T-cell activation and differentiation. SATB1 has been shown to be critical for Th2, regulatory T, and natural killer T-cell differentiation (
      • Beyer M.
      • Thabet Y.
      • Muller R.U.
      • Sadlon T.
      • Classen S.
      • Lahl K.
      • et al.
      Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation.
      ,
      • Burute M.
      • Gottimukkala K.
      • Galande S.
      Chromatin organizer SATB1 is an important determinant of T-cell differentiation.
      ,
      • Kakugawa K.
      • Kojo S.
      • Tanaka H.
      • Seo W.
      • Endo T.A.
      • Kitagawa Y.
      • et al.
      Essential roles of SATB1 in specifying T lymphocyte subsets.
      ). However, in our results, SATB1 expression did not affect these cytokines in malignant T cells, indicating distinct roles of SATB1 in benign versus malignant T cells. SATB1 seems not to directly regulate the transcription of RORC or STAT3. The mechanism by which SATB1 regulates IL-17 cytokine expression in malignant T cells warrants further investigation.
      Heterogeneity in SATB1 expression exists in other peripheral T cell lymphomas.
      • Eckerle S.
      • Brune V.
      • Doring C.
      • Tiacci E.
      • Bohle V.
      • Sundstrom C.
      • et al.
      Gene expression profiling of isolated tumour cells from anaplastic large cell lymphomas: insights into its cellular origin, pathogenesis and relation to Hodgkin lymphoma.
      reported that in systemic ALCLs, SATB1 expression status was highly correlated with anaplastic lymphoma kinase expression. Anaplastic lymphoma kinase–positive ALCL strongly expressed SATB1, while anaplastic lymphoma kinase–negative cases lacked SATB1 staining. In accordance, STAT3, RORC, and IL17A are highly expressed in SATB1+ cases (Supplementary Figure S3 online, information extracted from #GSE14879) (
      • Eckerle S.
      • Brune V.
      • Doring C.
      • Tiacci E.
      • Bohle V.
      • Sundstrom C.
      • et al.
      Gene expression profiling of isolated tumour cells from anaplastic large cell lymphomas: insights into its cellular origin, pathogenesis and relation to Hodgkin lymphoma.
      ). These data suggested that the association between SATB1 and Th17 cytokines is not limited to PCALCLs.
      Although we did not observe difference in long-term prognosis in two groups of PCALCL patients due to the limited follow-up period, our data showed that SATB1+ CD30+ LPDs responded better to treatment with low-dose MTX and IFN-α2b. Low-dose MTX and IFN-α2b were first-line treatment for CD30+ LPDs in our center and others (
      • Kempf W.
      • Pfaltz K.
      • Vermeer M.H.
      • Cozzio A.
      • Ortiz-Romero P.L.
      • Bagot M.
      • et al.
      EORTC, ISCL, and USCLC consensus recommendations for the treatment of primary cutaneous CD30-positive lymphoproliferative disorders: lymphomatoid papulosis and primary cutaneous anaplastic large-cell lymphoma.
      ), with moderate side effects and low cost. With in vitro cell models, we demonstrated that this effect may be due to a differential response to IFN-α2b. This is consistent with the anti-tumor effects of IFN-α that regulates STAT3 activation (
      • Kirkwood J.M.
      • Farkas D.L.
      • Chakraborty A.
      • Dyer K.F.
      • Tweardy D.J.
      • Abernethy J.L.
      • et al.
      Systemic interferon-alpha (IFN-alpha) treatment leads to Stat3 inactivation in melanoma precursor lesions.
      ,
      • Zitvogel L.
      • Galluzzi L.
      • Kepp O.
      • Smyth M.J.
      • Kroemer G.
      Type I interferons in anticancer immunity.
      ). However, further prospective studies with better-controlled conditions are warranted.
      To further examine the correlation between SATB1 staining and the disease phenotype we observed, a small validation cohort with eight additional PCALCL cases (3 SATB1+ and 5 SATB1) were obtained at our center. The histology features, Th17 cytokine staining results, and therapeutic outcomes were highly consistent with our initial findings (Supplementary Table S3, Supplementary Figure S4 online). Nevertheless, multicentered validation studies with a large number of patents should be performed in the future.
      The causes of the differential expression of SATB1 in cutaneous CD30+ LPDs remain largely unknown. We demonstrated the correlation between SATB1 expression status with its promoter methylation, indicating a distinct epigenetic milieu existed in the two groups of CD30+ LPDs. Previous reports showed that activation of STAT3 and IL-17 could be induced by Staphylococcal enterotoxin A from the infected skin of patients with mycosis fungoides (
      • Willerslev-Olsen A.
      • Krejsgaard T.
      • Lindahl L.M.
      • Litvinov I.V.
      • Fredholm S.
      • Petersen D.L.
      • et al.
      Staphylococcus enterotoxin A (SEA) stimulates STAT3 activation and IL-17 expression in cutaneous T-cell lymphoma.
      ). It is possible that SATB1 may be involved in this process, and Staphylococcal enterotoxin A may contribute to the pathogenesis of SATB1+ CD30+ LPDs. Further studies exploring the cause of SATB1 expression may lead to identification of distinct origins of these anaplastic T cells.
      In conclusion, our study indicates that SATB1 can serve as a promising biomarker to classify CD30+ LPDs, with different molecular mechanism, clinical, and histopathological features. SATB1 represents a sensitive and practical immunohistochemistry marker indicating different therapeutic outcomes in cutaneous CD30+ LPDs, which provides a rationale for studying the implications of SATB1 staining further in other types of anaplastic lymphoma kinase–negative ALCL. This may lead to insight into the origin of this disease and individualized therapeutic strategies.

      Material and Methods

      Patient recruitment and clinical features

      Lesional skin biopsies were obtained from 54 patients with CD30+ LPDs (12 LyP, 42 PCALCL) recruited from the Skin Lymphoma Clinic of Peking University First Hospital (n = 36), Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College (n = 8), Department of Dermatovenereology, West China Hospital, Sichuan University (n = 5), and Department of Dermatology, Xijing Hospital, Fourth Military Medical University (n = 5), with approval from the Clinical Ethics Board of each institution, in accordance with the Declaration of Helsinki principles. The validation cohort of eight PCALCL skin biopsies were obtained from the Skin Lymphoma Clinic of Peking University First Hospital (n = 4), Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College (n = 1), Department of Dermatovenereology, West China Hospital, Sichuan University (n = 3), with approval from the Clinical Ethics Board of each institution, in accordance with the Declaration of Helsinki principles. Written informed consents were obtained from all of the patients. All patients were diagnosed on previously described criteria (
      • Kempf W.
      • Pfaltz K.
      • Vermeer M.H.
      • Cozzio A.
      • Ortiz-Romero P.L.
      • Bagot M.
      • et al.
      EORTC, ISCL, and USCLC consensus recommendations for the treatment of primary cutaneous CD30-positive lymphoproliferative disorders: lymphomatoid papulosis and primary cutaneous anaplastic large-cell lymphoma.
      ). All follow-ups were performed by cutaneous lymphoma specialists in the Skin Lymphoma Clinic. Each patient was reassessed every 1–6 months in the clinic. Disease progression in progression-free survival was defined as progression to a more advanced TNMB classification or death owing to disease (
      • Agar N.S.
      • Wedgeworth E.
      • Crichton S.
      • Mitchell T.J.
      • Cox M.
      • Ferreira S.
      • et al.
      Survival outcomes and prognostic factors in mycosis fungoides/Sézary syndrome: validation of the revised International Society for Cutaneous Lymphomas/European Organisation for Research and Treatment of Cancer staging proposal.
      ).
      Detailed methodology regarding cell culture and treatment, RNA sequencing, immunohistochemistry, and bisulfite sequencing are listed in the Supplementary Materials (online).

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This study was supported by grants from National Nature Science Foundation of China (81572674 [YW] and 81372914 [PT]), Beijing Municipal Natural Science Foundation (7162191 [YW]), Beijing Science and Technology Plan (Z151100004015091 [YW]), and Youth Research Project of Peking University First Hospital (2017QN30 [JS]). MEK is supported by the Drs. Martin and Dorothy Spatz Charitable Foundation.

      Supplementary Material

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      Linked Article

      • SATB1 Is a Pivotal Epigenetic Biomarker in Cutaneous T-Cell Lymphomas
        Journal of Investigative DermatologyVol. 138Issue 8
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          The SATB1 protein has been the focus of two recent studies of cutaneous T-cell lymphomas. Fredholm et al. observed a stage-related decrease of SATB1 expression in epidermotropic cutaneous T-cell lymphomas. SATB1 was negatively regulated by STAT5 through microRNA-155, which in turn triggered enhanced expression of T helper type 2 cytokines such as IL-5 and IL-9. In parallel, Sun et al. found that SATB1 expression was up-regulated by promoter demethylation in a subset of cutaneous anaplastic lymphoma and was associated with T helper type 17 polarization in patients with better therapeutic responses.
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