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

Abstract

Mycosis fungoides (MF), the most common cutaneous T cell lymphoma (CTCL) is a malignancy of skin-tropic memory T cells. Most MF cases present as early stage (stage I A/B, limited to the skin), and these patients typically have a chronic, indolent clinical course. However, a small subset of early-stage cases develop progressive and fatal disease. Because outcomes can be so different, early identification of this high-risk population is an urgent unmet clinical need. We evaluated the use of next-generation high-throughput DNA sequencing of the T cell receptor β gene (TCRB) in lesional skin biopsies to predict progression and survival in a discovery cohort of 208 patients with CTCL (177 with MF) from a 15-year longitudinal observational clinical study. We compared these data to the results in an independent validation cohort of 101 CTCL patients (87 with MF). The tumor clone frequency (TCF) in lesional skin, measured by high-throughput sequencing of the TCRB gene, was an independent prognostic factor of both progression-free and overall survival in patients with CTCL and MF in particular. In early-stage patients, a TCF of >25% in the skin was a stronger predictor of progression than any other established prognostic factor (stage IB versus IA, presence of plaques, high blood lactate dehydrogenase concentration, large-cell transformation, or age). The TCF therefore may accurately predict disease progression in early-stage MF. Early identification of patients at high risk for progression could help identify candidates who may benefit from allogeneic hematopoietic stem cell transplantation before their disease becomes treatment-refractory.

Figure 1.. High throughput TCRB sequencing in 309 patients with cutaneous T cell lymphomas

A. Clinical diagnosis in 309 patients with cutaneous T cell lymphomas in the discovery and validation sets. Other: CD30+ lymphoproliferative disorder and CD8+ aggressive epidermotropic cutaneous T cell lymphoma. 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 Sézary syndrome (26). B. TCRBV gene family usage by the malignant clone in 309 cases of primary cutaneous T cell lymphomas 3. Example of the measurement of the malignant clone frequency in skin in two patients with stage IB mycosis fungoides. Clinical pictures and 3D histograms of the TCRB sequencing data in lesional skin in two patients with stage IB mycosis fungoides. On the upper panel, the 3D histogram shows the presence of a tumor clone frequency of 8% (18,131 reads). This patient showed no evidence of disease progression after 4 years follow-up. On the lower panel, the 3D histogram shows a tumor clone frequency 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-eosin sections of lesional skin biopsies in 4 patients with cutaneous T cell lymphoma and various malignant clone frequencies and outcomes, magnification x10 Upper left. Malignant clone 32% of the T cells. Progression after 2 years Upper right. Malignant clone 41% of the T cells. Progression after 2 months Lower left. Malignant clone 6% of the T cells. No progression in 8 years Lower right. Malignant clone 6% of the T cells. No progression in 9 years E. Malignant clone frequency according to the extent of body surface area involved in patients with mycosis fungoides Medians are indicated by horizontal bars and comparisons are carried out using Mann-Whitney U-test, *p<0.05 considered significant.

Figure 2.. The tumor clone frequency in skin as predictor of progression-free and overall survival in patients with cutaneous T cell lymphomas.

A. Kaplan-Meier estimates of progression-free (left panel) and overall survival (right) in 208 patients with cutaneous T cell lymphomas in the discovery set, according to the tumor clone frequency in skin (<25% versus >25% of the total T cells in skin). B. Kaplan-Meier estimates of progression-free survival in 101 patients with cutaneous T cell lymphomas in the validation set, according to the tumor clone frequency in skin (<25% versus >25% of the total T cells in skin). C. Kaplan-Meier estimates of progression-free (left panel) and overall survival (right) in 177 patients with mycosis fungoides in the discovery set, according to the tumor clone frequency in skin (<25% versus >25% of the total T cells in skin). D. Kaplan-Meier estimates of progression-free survival in 87 patients with mycosis fungoides in the validation set, according to the tumor clone frequency in skin (<25% versus >25% of the total T cells in skin). p-values in A-D are estimated by Cox univariable analysis. E. Kaplan-Meier estimates of progression-free (left panel) and overall survival (right) in 22 patients with Sézary syndrome in the discovery set, according to the tumor clone frequency in skin (<25% versus >25% of the total T cells in skin).

Figure 3.. The tumor clone frequency in skin as predictor of progression-free and overall survival in patients with early-stage mycosis fungoides

A. Kaplan-Meier estimates of progression-free (left) and overall survival (right) in 141 patients with early-stage (IA to IIA) mycosis fungoides in the discovery set, according to the tumor clone frequency (<25% versus >25% of the total T cells in skin). B. Kaplan-Meier estimates of progression-free survival in 69 patients with early-stage (IA to IIA) mycosis fungoides in the validation set, according to the tumor clone frequency (<25% versus >25% of the total T cells in skin). C. Kaplan-Meier estimates of progression-free (left) and overall survival (right) in 70 patients with stage IB mycosis fungoides in the discovery set, according to the tumor clone frequency (<25% versus >25% of the total T cells in skin, upper panels) or to the presence of plaques (lower panels). D. Kaplan-Meier estimates of progression-free survival in 42 patients with stage IB mycosis fungoides in the validation set, according to the tumor clone frequency (<25% versus >25% of the total T cells in skin, upper panel) or to the presence of plaques (lower panel). p-values in A-D are estimated by Cox univariable analysis. E. Dot plot and linear regression of the time to progression/death according to the tumor clone frequency in 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 tumor clone frequency in skin (>25%) in patients with stage IB mycosis fungoides 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 skin among progressors. The specificity is defined as the percentage of patients with a malignant clone <25% of T cells in skin among nonprogressors.

Figure 4.. Samples with a high tumor clone frequency are not associated with a decreased anti-tumor immune response

A. Example of CD8+ and granzyme immunostaining in lesional skin in 2 lesional CTCL skin biopsies. B. Percentage of CD8+ T cell % (left) and granzyme B positive cell % in lesional skin of CTCL patients with a low tumor clone frequency (<10% T cells) and high tumor clone frequency (>30% T cells). (Mann Whitney U-test, *p<0.05 **p<0.01). C. Reactive T cell clonality (left) and entropy (right) in lesional skin of CTCL patients with a low tumor clone frequency (<10% T cells) and high tumor clone frequency (>30% T cells) (Mann Whitney U-test, *p<0.05).

Figure 5.. A high tumor clone frequency in 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 3 different clusters of patients. Intensity expression values in the heatmap are expressed as log2 fold changes compared to the average expression of each gene in the whole study group. The tumor clone frequency in each sample is represented by a colour scale at the bottom of the heatmap. B. Dot plots of the T cell percentages of nucleated cells) in patients in cluster 1, 2 and 3. Medians were compared by Mann-Whitney U-test with Bonferroni adjustment for multiple testing, * p<0.05 **p<0.01 C. Dot plots of the tumor clone frequency (TCF) in patients in cluster 1, 2 and 3. Means were compared by Mann-Whitney U-test with Bonferroni adjustment for multiple testing, * p<0.05 **p<0.01 D. Kaplan-Meier estimates of progression-free survival in 157 patients with cutaneous T cell lymphomas 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 ***p<0.001 E. Whole exome sequencing data of microdissected skin T cells in patients with mycosis fungoides. Number of somatic mutations according to the clinical stage (left). Mann-Whitney U-test, * p<0.05 Number of somatic mutations according to the malignant clone frequency in skin (right). Spearman correlation, p<0.05 considered significant