Research ArticleCancer

TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL

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Science Translational Medicine  07 Oct 2015:
Vol. 7, Issue 308, pp. 308ra158
DOI: 10.1126/scitranslmed.aaa9122
  • Fig. 1. High-throughput TCRβ CDR3 region sequencing identifies expanded T cell clones and discriminates CTCL from benign inflammatory skin disorders.

    DNA was isolated from the lesional skin of patients with CTCL, psoriasis, eczematous dermatitis (ED), allergic contact dermatitis (ACD), and from the skin of healthy individuals (Nml skin) and subjected to high-throughput TCRβ sequencing. (A) Clonality of lesional skin T cells increased with advanced stage of CTCL. The mean and SEM of clonality scores of 8 stage IA biopsies, 18 stage IB biopsies, 4 stage IIA biopsies, 8 stage IIB biopsies, 2 stage III biopsies, and 5 stage IV biopsies are shown. (B and C) TCR sequencing identified expanded populations of clonal malignant T cells in CTCL skin lesions. The V versus J gene usages of T cells from a lesional skin sample are shown (B). The green peak includes the clonal malignant T cell population, as well as other benign T cells that share the same V and J gene usage. The individual T cell clone sequence is shown (C), with detailed information on the CDR3 amino acid sequence and V and J gene usage. The nine most frequent benign infiltrating T cell sequences are also shown. In this patient, the malignant T cell clone made up 10.3% of the total T cell population in lesional skin. (D and E) The most frequent T cell clone expressed as a percentage of total T cells did not completely discriminate CTCL from patients with benign inflammatory skin disease. The most frequent T cell sequences expressed as a percentage of total T cells are shown for individual samples (D) and aggregate data (E). ns, not significant. (F and G) The most frequent T cell clone expressed as the fraction (fract) of total nucleated cells successfully discriminates CTCL from benign inflammatory skin diseases. The most frequent T cell sequence expressed as a fraction of total nucleated cells is shown for individual samples (F) and aggregate data (G). This analysis allowed the discrimination of CTCL from benign inflammatory skin diseases and healthy skin. Forty-six CTCL skin lesions, lesional skin from 23 patients with psoriasis, 11 patients with eczematous dermatitis, and 12 patients with contact dermatitis, and the skin of 6 healthy donors were evaluated [test: Kruskal-Wallis one-way analysis of variance (ANOVA) with a Bonferroni-Dunn’s post test].

  • Fig. 2. High-throughput sequencing of the TCRγ CDR3 regions also discriminates CTCL from benign inflammatory skin diseases.

    (A) Skin samples were subjected to deep sequencing of both the TCRγ and TCRβ CDR3 regions. The top TCRβ sequence and the sum of the top two most frequent TCRγ sequences (divided by two because most T cells have two rearranged TCRγ genes) were expressed as the fraction of total nucleated cells, and the results from TCRγ and TCRβ sequencing are compared. In general, there was close concordance between TCRγ and TCRβ sequencing results. The exception was one patient with a known γδ T cell malignancy, in whom TCRγ sequencing identified a malignant clone but TCRβ did not, consistent with the known lack of TCR Vβ gene rearrangement in γδ T cells. (B and C) The sum of the top two TCRγ sequences, divided by two and expressed as a fraction of total nucleated cells discriminates CTCL from benign inflammatory skin diseases. Individual (B) and aggregate (C) data are shown. Forty-six CTCL skin lesions, lesional skin from 23 patients with psoriasis and 11 patients with eczematous dermatitis, and the skin of 6 healthy donors were evaluated (test: Kruskal-Wallis one-way ANOVA with a Bonferroni-Dunn’s post test).

  • Fig. 3. HTS diagnoses CTCL in patients negative for clonality by conventional TCRγ PCR.

    (A) HTS and TCRγ PCR were carried out on skin biopsies from 39 CTCL patients; HTS identified clones in 39 of 39 CTCL patients compared to TCRγ PCR, which identified clonal populations in 27 of 39 samples. The stages of the 10 CTCL patients with clones detected by HTS but negative for clonality by TCRγ PCR are shown. (B and C) Failure of TCRγ PCR to detect clonality was not related to the predominance of the malignant T cell clone within the skin sample. The top TCRβ clone, expressed as the fraction of nucleated cells, is shown. Individual (B) and aggregate (C) data are shown along with psoriasis samples, which are included for comparison. Thirty-nine CTCL skin lesions and lesional skin from 23 patients with psoriasis are shown (test: Kruskal-Wallis one-way ANOVA with a Bonferroni-Dunn’s post test). (D) Clinical photos and HTS results are shown for patient (Pt) 541, who had pathology-proven stage IB CTCL but in whom TCRγ PCR did not detect a clonal population. (E) In patient 347, TCRγ PCR was negative and pathology was equivocal, but HTS demonstrated a clear malignant clone. (F to I) In patient 551, four skin biopsies were sent for HTS, and three were also studied by TCRγ PCR. All were negative for clonality by TCRγ PCR, but four of four were positive for clonality by HTS. TCRγ PCR results are shown for two biopsies; the third is included in fig. S3. Asterisks indicate peaks noted by the pathologist within the expected areas, but none were judged significant enough to designate as a clonal population (F and G). TCR Vβ demonstrated the presence of two distinct Vβ clonal sequences, denoting the presence of either a single malignant clone with two rearranged TCRβ alleles or two separate malignant T cell clones (H). TCRγ HTS demonstrated the presence of four dominant γ chain clones in all four biopsies (black circles), confirming that there were two clonal malignant T cell populations in this patient, each with one rearranged TCRβ allele and two rearranged TCRγ alleles. The five most frequent benign T cell TCRγ sequences are also shown for comparison (white circles) (I).

  • Fig. 4. HTS discriminates CTCL recurrences from benign inflammation, provides accurate assessment of responses to therapy, and facilitates early diagnosis of disease recurrence in both the skin and blood of patients with CTCL.

    (A to D) HTS distinguishes CTCL recurrences from benign inflammatory disease. Patient 247 had a history of stage IB CTCL, subsequently developed leukemic involvement, and was treated with low-dose alemtuzumab. Before therapy, TCR Vβ HTS demonstrated a clear malignant clone in blood (A). The patient initially improved on alemtuzumab and then developed the skin eruption shown (B). Histopathology of the lesional skin was suggestive of a drug hypersensitivity reaction, but a T cell dyscrasia could not be ruled out. HTS of blood and lesional skin showed clearance of the malignant T cell clone from blood and skin, confirming that this was a benign inflammatory dermatitis (C). Bactrim was discontinued, and the eruption completely resolved with topical steroids and narrowband UVB therapy. Diverse populations of T cells remain within the skin of alemtuzumab-treated patients. TCR Vβ HTS of the skin of patient 247 while on alemtuzumab is shown. This patient had no circulating T or B cells, but a diverse population of T cells remained in skin (D). tx, treatment. (E and F) HTS allows longitudinal observation of disease activity over time and assesses responses to therapy. Patient 409, stage IIA CTCL, was studied by HTS in 2012, after treatment with electron beam and brachytherapy, and again in 2014, after initiation of gemcitabine. HTS demonstrated an identical clonal T cell population in the skin at both time points, reduced but still frequent after gemcitabine therapy. The malignant clone (black circles) and the three most frequent benign T cell clones (white circles) are shown. (G and H) HTS provides an early diagnosis of disease recurrence. Patient 425 had recalcitrant stage IIB CTCL with CD30+ large-cell transformation. She underwent SCT and appeared well until she developed a new right chest lesion 10 months after SCT (G). HTS before SCT demonstrated the presence of a malignant T cell clone (H). Biopsy of the lesion post-SCT demonstrated recurrence of the same malignant T cell clone in the skin. The malignant T cell clone (black circles) and the three most frequent benign T cell clones (white circles) are shown. Subsequent withdrawal of systemic immunosuppression and narrowband UVB therapy induced a complete remission. (I) HTS allows accurate assessment of peripheral blood disease. HTS studies of 4 patients without CTCL, 12 patients with MF without evidence of blood disease (MF), and 7 patients with leukemic disease (L-CTCL) with known blood involvement are shown (test: Kruskal-Wallis one-way ANOVA with a Bonferroni-Dunn’s post-test).

  • Fig. 5. In patients with skin-limited disease and no clinical involvement of peripheral blood, HTS demonstrates hematogenous spread of small numbers of malignant T cells.

    (A to C) Patient 539, stage IIIB CTCL, had no evidence of peripheral blood disease by clinical flow analysis but was experiencing both patchy [LCT (large-cell transformation) on histopathology] and nodular skin (MF on histopathology) skin lesions (A). The patient improved after local radiation therapy but subsequently developed a new tumor at a previously uninvolved site 5 months later. HTS studies from all three biopsies are shown (B). Clinical flow was negative for blood involvement on both dates. HTS demonstrated the same clonal T cell population in all three skin biopsies, and analysis of the blood demonstrated the presence of small numbers of malignant T cells in the peripheral blood at the time new skin lesions were developing. (D and E) Patient 418 had a long-standing stage IIB folliculotropic CTCL (D) with recent thickening of existing skin lesions and new areas of disease (E). Clinical flow analyses were negative for peripheral blood involvement. (F) HTS studies of the skin and blood during the development of the skin lesions demonstrated a clear malignant T cell clone in lesional skin that was also found in low numbers in peripheral blood. (G and H) Patient 317 had a long-standing history of large-plaque parapsoriasis since childhood for over 40 years. The diagnosis of MF was finally made in 2007 (G). In 2014, he presented with a 1.5-year history of worsening disease with new areas of involvement. Clinical flow analyses showed no evidence of peripheral blood disease. HTS demonstrated a malignant T cell clone in the skin, and small numbers were also demonstrated in peripheral blood.

  • Fig. 6. CTCL is a malignancy derived from mature T cells.

    (A and B) HTS allows comprehensive study of both malignant and benign T cells. The TCR Vβ HTS profile is shown for patient 541; both the malignant clone and benign infiltrating T cells in the skin lesions are evaluated by this technique (A). CDR3 length analysis, similar to a spectrotype graph, provides a rapid assay of T cell diversity via HTS. The results of TCRγ HTS of lesional skin from a patient with stage III CTCL are shown. The CDR3 lengths of all T cells (left panel, including the malignant clone) and benign infiltrating T cells only (right panel) are shown. The two rearranged TCRγ allele sequences of the malignant clone are indicated by asterisks. A healthy diverse population of benign infiltrating T cells was present (B). (C and D) HTS demonstrates that CTCL is a malignancy of mature T cells. Previous studies demonstrated that mature αβ T cells have on average 1.8 rearranged TCRγ alleles (14). We studied 33 CTCL patients with malignant αβ T cell clones by TCRγ HTS. (E) Twenty-seven patients had two rearranged TCRγ alleles, as evidenced by the presence of two similarly frequent TCRγ sequences (C and E), and six patients had a single rearranged TCRγ allele, as evidenced by only a single high-frequency TCRγ sequence (D and E). T cells therefore had on average 1.8 rearranged TCRγ alleles, a proportion characteristic of mature T cells. (F to H) TCR Vβ HTS provides an unparalleled opportunity to isolate and study malignant clonal T cells. Identification of the TCR Vβ chain by HTS and subsequent immunostaining using commercially available TCR Vβ antibodies allows study of clonal malignant T cells (F). Immunostaining of malignant T cells with TCR Vβ–specific antibodies in two patients with stage IIB MF are shown (G and H). The patient shown in (H) had a high abundant clone and larger cells as a result of large-cell transformation. Scale bars, 100 μm.

  • Fig. 7. Malignant T cells in MF are localized to the dermoepidermal junction.

    A clinical image of disease (left panel) and immunostained cryosections for the same patient are shown. Clonal malignant T cells (red) localize to the dermoepidermal junction. The junction is indicated with a broken line in the last panel. Similar results were obtained in three additional MF patients. Scale bars, 100 μm. DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 8. Malignant T cells in L-CTCL are localized to the dermis.

    A clinical image of disease (left panel) and immunostained cryosections for the same patient are shown. Clonal malignant T cells (red) are localized in the dermis. The basement membrane is indicated with a broken line in the last panel; the dermis is located below this line. Similar results were obtained in three additional L-CTCL patients. Scale bars, 100 μm.

  • Table 1. Stages of CTCL patient samples.
    Stage
    IA
    Stage
    IB
    Stage
    IIA
    Stage
    IIB
    Stage
    III
    Stage
    IV
    Total
    Blood35152824
    Skin1119582550

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/308/308ra158/DC1

    Fig. S1. HTS studies of blood in patients with circulating expanded benign T cell clones and in the skin lesions of patients with PLEVA.

    Fig. S2. Comparison of TCRγ PCR studies carried out on DNA from fixed versus frozen skin biopsies.

    Fig. S3. TCRγ chain PCR results for the third biopsy from patient 551.

    Fig. S4. A schematic of the approach used by Adaptive Biotechnologies to minimize and control for PCR and sequencing bias.

    Primary data tables

  • Supplementary Material for:

    TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL

    Ilan R. Kirsch, Rei Watanabe, John T. O'Malley, David W. Williamson, Laura-Louise Scott, Christopher P. Elco, Jessica E. Teague, Ahmed Gehad, Elizabeth L. Lowry, Nicole R. LeBoeuf, James G. Krueger, Harlan S. Robins, Thomas S. Kupper,* Rachael A. Clark*

    *Corresponding author. E-mail: rclark1{at}partners.org (R.A.C.); tkupper{at}partners.org (T.S.K.)

    Published 7 October 2015, Sci. Transl. Med. 7, 308ra158 (2015)
    DOI: 10.1126/scitranslmed.aaa9122

    This PDF file includes:

    • Fig. S1. HTS studies of blood in patients with circulating expanded benign T cell clones and in the skin lesions of patients with PLEVA.
    • Fig. S2. Comparison of TCRγ PCR studies carried out on DNA from fixed versus frozen skin biopsies.
    • Fig. S3. TCRγ chain PCR results for the third biopsy from patient 551.
    • Fig. S4. A schematic of the approach used by Adaptive Biotechnologies to minimize and control for PCR and sequencing bias.
    • Primary data tables

    [Download PDF]

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