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High-throughput sequencing of the T cell receptor β gene identifies aggressive early-stage mycosis fungoides

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Science Translational Medicine  09 May 2018:
Vol. 10, Issue 440, eaar5894
DOI: 10.1126/scitranslmed.aar5894
  • Fig. 1 High-throughput TCRB sequencing in 309 patients with CTCLs.

    (A) Clinical diagnosis in 309 patients with CTCLs in the discovery and validation sets. Other: CD30+ lymphoproliferative disorder and CD8+ aggressive epidermotropic CTCL. Pre-Sézary refers to the evidence of blood abnormalities (B1; elevated absolute CD4+ T cell count or CD4+/CD8+ T cell ratio) that do not meet the criteria for stage B2 or SS (26). (B) TCRBV gene family usage by the malignant clone in 309 cases of primary CTCLs (C) Example of the measurement of the malignant clone frequency in the skin in two patients with stage IB MF. Three-dimensional (3D) histograms of the TCRB sequencing data in lesional skin in two patients with stage IB MF. On the upper panel, the 3D histogram shows the presence of a TCF of 8% (18,131 reads). This patient showed no evidence of disease progression after 4 years of follow-up. On the lower panel, the 3D histogram shows a TCF of 37% (264,252 reads; the y axis has been cut at 80,000). This patient died of disease progression after 28 months. (D) Hematoxylin and eosin sections of lesional skin biopsies in four patients with CTCL and various malignant clone frequencies and outcomes. Scale bars, 100 μm. Upper left: TCF (32% of the T cells) with progression after 2 years. Upper right: TCF (41% of the T cells) with progression after 2 months. Lower left: TCF (6% of the T cells) with no progression in 8 years. Lower right: TCF (6% of the T cells) with no progression in 9 years. (E) TCF according to the extent of body surface area involved in patients with MF. Medians are indicated by horizontal bars, and comparisons are carried out using Mann-Whitney U test; *P < 0.05 is considered significant. NS, not significant.

  • Fig. 2 The TCF in the skin as predictor of PFS and OS in patients with CTCLs.

    (A) Kaplan-Meier estimates of PFS (left) and OS (right) in 208 patients with CTCLs in the discovery set, according to the TCF in the skin (≤25% versus >25% of the total T cells in the skin). (B) Kaplan-Meier estimates of PFS in 101 patients with CTCLs in the validation set, according to the TCF in the skin (≤25% versus >25% of the total T cells in the skin). (C) Kaplan-Meier estimates of PFS (left) and OS (right) in 177 patients with MF in the discovery set, according to the TCF in the skin (≤25% versus >25% of the total T cells in the skin). (D) Kaplan-Meier estimates of PFS in 87 patients with MF in the validation set, according to the TCF in the skin (≤25% versus >25% of the total T cells in the skin). P values in (A) to (D) are estimated by Cox univariable analysis. (E) Kaplan-Meier estimates of PFS (left) and OS (right) in 22 patients with SS in the discovery set, according to the TCF in the skin (≤25% versus >25% of the total T cells in the skin).

  • Fig. 3 The TCF in the skin as predictor of PFS and OS in patients with early-stage MF.

    (A) Kaplan-Meier estimates of PFS (left) and OS (right) in 141 patients with early-stage (IA to IIA) MF in the discovery set, according to the TCF (<25% versus >25% of the total T cells in the skin). (B) Kaplan-Meier estimates of PFS in 69 patients with early-stage (IA to IIA) MF in the validation set, according to the TCF (≤25% versus >25% of the total T cells in the skin). (C) Kaplan-Meier estimates of PFS (left) and OS (right) in 70 patients with stage IB MF in the discovery set, according to the TCF (≤25% versus >25% of the total T cells in the skin; top) or to the presence of plaques (bottom). (D) Kaplan-Meier estimates of PFS in 42 patients with stage IB MF in the validation set, according to the TCF (≤25% versus >25% of the total T cells in the skin; top) or to the presence of plaques (bottom). P values in (A) to (D) are estimated by Cox univariable analysis. (E) Dot plot and linear regression of the time to progression/death according to the TCF in the skin in stage IB patients from the discovery and validation sets who experienced disease progression during the follow-up. Pearson’s correlation coefficient and P value are indicated. (F) Receiver operating characteristic curve of the TCF in the skin (>25%) in patients with stage IB MF in the discovery and validation sets for 5-year progression or death. Progressors are patients who progressed or died within 5 years after the test. Nonprogressors are patients with at least 5 years of follow-up and no event of death or progression in 5 years. The sensitivity is defined as the percentage of patients with a malignant clone (>25% of T cells in the skin) among progressors. The specificity is defined as the percentage of patients with a malignant clone (≤25% of T cells in the skin) among nonprogressors.

  • Fig. 4 Samples with a high TCF are not associated with a decreased antitumor immune response.

    (A) Example of CD8+ and granzyme immunostaining in lesional skin in two lesional CTCL skin biopsies. DAPI, 4′,6-diamidino-2-phenylindole. (B) CD8+ T cell percentage (left) and granzyme B–positive cell percentage in lesional skin of CTCL patients with a low TCF (<10% T cells) and high TCF (>30% T cells) (*P < 0.05 and **P < 0.01, Mann-Whitney U test). (C) Reactive T cell clonality (left) and entropy (right) in lesional skin of CTCL patients with a low TCF (<10% T cells) and high TCF (>30% T cells) (*P < 0.05, Mann-Whitney U test).

  • Fig. 5 A high TCF in the skin is associated with a distinct gene expression profile and a higher number of somatic mutations.

    (A) Unsupervised analysis by hierarchical clustering (complete linkage) according to the expression of 78 genes in 157 patients reveals three different clusters of patients. Intensity expression values in the heat map are expressed as log2 fold changes compared to the average expression of each gene in the whole study group. The TCF in each sample is represented by a color scale at the bottom of the heat map. (B) Dot plots of the T cells (frequency of nucleated cells) in patients in clusters 1 to 3. Medians were compared by Mann-Whitney U test with Bonferroni adjustment for multiple testing (*P < 0.05 and **P < 0.01). (C) Dot plots of the TCF in patients in clusters 1 to 3. Means were compared by Mann-Whitney U test with Bonferroni adjustment for multiple testing (*P < 0.05 and **P < 0.01). (D) Kaplan-Meier estimates of PFS in 157 patients with CTCLs in the training group, according to the gene expression clustering. Log-rank test with Bonferroni adjustment for multiple testing (*P < 0.05, **P < 0.01, and ***P < 0.001). (E) Whole-exome sequencing data of microdissected skin T cells in patients with MF. Left: Number of somatic mutations according to the clinical stage. *P < 0.05, Mann-Whitney U test. Right: Number of somatic mutations according to the malignant clone frequency in the skin. P < 0.05 by Spearman correlation is considered significant.

  • Table 1 Uni- and multivariable analysis on PFS and OS in 177 patients with MF in the discovery set.

    Treatments used before first evidence of progression, death, or censoring. Phototherapy includes PUVA (psoralen + ultraviolet A) and UVB therapy. Radiation therapy includes electron-beam therapy and brachytherapy. Systemic treatments include interferon-α, oral bexarotene, folate inhibitors, systemic histone deacetylase inhibitors, and monoclonal antibodies. The multivariable model was stratified on LDH levels and the use of systemic treatments. CI, confidence interval.

    Discovery set (n = 177)
    Progression-free survivalOverall survival
    UnivariableMultivariableUnivariableMultivariable
    HR95% CIPHR95% CIPHR95% CIPHR95% CIP
    Tumor clone frequency>15%2.11.3–3.50.0022.51.4–4.70.003
    >25%4.02.4–6.8<0.0013.31.9–5.9<0.0014.82.6–9.0<0.0015.12.5–10<0.001
    >35%4.62.6–8.1<0.0015.22.7–10<0.001
    LDH levelsElevated versus normal2.31.2–4.30.0083.21.6–6.50.001
    Large-cell transformationPresence3.41.8–6.4<0.0011.80.9–3.70.13.31.6–7.00.0011.30.5–3.00.6
    Age>60 years2.21.3–3.70.0022.31.3–4.00.0033.41.7–6.70.00053.51.7–7.4<0.001
    TreatmentsPhototherapy0.90.5–1.50.70.80.4–1.50.5
    Radiation therapy1.91.1–3.40.021.40.7–2.50.31.80.9–3.80.1
    Systemic treatments3.42.1–5.5<0.0013.92.1–7.1<0.001
    PCRClonal pattern1.50.7–3.40.31.00.5–4.20.4
    GenderMale versus female1.50.9–2.50.11.20.7–2.40.5
    FolliculotropismPresence1.50.9–2.50.11.20.6–2.30.6
  • Table 2 Prognostic factors of PFS in 141 patients with early-stage disease from the training set and 69 patients in the validation set.
    Progression-free survival
    Discovery
    set (n = 141)
    Validation
    set (n = 69)
    HR95% CIPHR95% CIP
    TCF of >25%4.92.5–9.7<0.001103.4–31<0.001
    Stage (IB versus IA)2.51.3–4.90.0086.41.5–280.01
    Presence of plaques2.21.1–4.20.021.90.8–4.40.15
    Elevated LDH levels1.20.4–3.10.81.00.3–3.41
    Large-cell transformation1.50.4–6.40.61.60.2–120.6
    Age (>60 years)2.01.0–3.70.041.30.5–3.00.6
    CLIPI score*
    Intermediate risk (versus low)2.21.0–5.00.050.40.1–2.00.2
    High risk (versus low)3.51.6–7.70.0022.30.9–5.90.07

    *The CLIPI was calculated on the basis of the presence of the following factors: age (>60 years), male sex, plaques, folliculotropism, and clinical adenopathy N1/Nx; low risk = 0 to 1, intermediate risk = 2, and high risk = 3 to 5 prognostic factors.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/10/440/eaar5894/DC1

      Fig. S1. Clinical pictures of two patients with stage IB MF.

      Fig. S2. TCR Vβ high-throughput sequencing allows specific quantification of the frequency of the malignant T cell clone within a Vβ gene family.

      Fig. S3. Continuous relationship between the TCF and the HRs for PFS and OS.

      Fig. S4. Prognostic value of the CLIPI in early-stage MF.

      Fig. S5. TCF in patches versus plaques.

      Fig. S6. Prognosis in early-stage patients according to body surface area involved and the presence of plaques.

      Fig. S7. Prognosis in stage IA patients.

      Fig. S8. Reproducibility of the TCF as measured by high-throughput sequencing of the TCRβ gene in different lesions in the same patient.

      Fig. S9. PFS and OS in pretreated and treatment-naïve early-stage MF patients.

      Table S1. ISCL/EORTC classification and staging of MF and SS.

      Table S2. Clinical characteristics of 208 patients with CTCL in the discovery set.

      Table S3. Clinical characteristics of 101 patients with CTCL in the validation set.

      Table S4. Multivariable analysis on PFS in early-stage patients.

    • Supplementary Material for:

      High-throughput sequencing of the T cell receptor β gene identifies aggressive early-stage mycosis fungoides

      Adele de Masson, John T. O'Malley, Christopher P. Elco, Sarah S. Garcia, Sherrie J. Divito, Elizabeth L. Lowry, Marianne Tawa, David C. Fisher, Phillip M. Devlin, Jessica E. Teague, Nicole R. Leboeuf, Ilan R. Kirsch, Harlan Robins, Rachael A. Clark,* Thomas S. Kupper*

      *Corresponding author. Email: tkupper{at}bwh.harvard.edu (T.S.K.); rclark{at}bwh.harvard.edu (R.A.C.)

      Published 9 May 2018, Sci. Transl. Med. 10, eaar5894 (2018)
      DOI: 10.1126/scitranslmed.aar5894

      This PDF file includes:

      • Fig. S1. Clinical pictures of two patients with stage IB MF.
      • Fig. S2. TCR Vβ high-throughput sequencing allows specific quantification of the frequency of the malignant T cell clone within a Vβ gene family.
      • Fig. S3. Continuous relationship between the TCF and the HRs for PFS and OS.
      • Fig. S4. Prognostic value of the CLIPI in early-stage MF.
      • Fig. S5. TCF in patches versus plaques.
      • Fig. S6. Prognosis in early-stage patients according to body surface area involved and the presence of plaques.
      • Fig. S7. Prognosis in stage IA patients.
      • Fig. S8. Reproducibility of the TCF as measured by high-throughput sequencing of the TCRβ gene in different lesions in the same patient.
      • Fig. S9. PFS and OS in pretreated and treatment-naïve early-stage MF patients.
      • Table S1. ISCL/EORTC classification and staging of MF and SS.
      • Table S2. Clinical characteristics of 208 patients with CTCL in the discovery set.
      • Table S3. Clinical characteristics of 101 patients with CTCL in the validation set.
      • Table S4. Multivariable analysis on PFS in early-stage patients.

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