Structural and diffusion weighted MRI demonstrates responses to ibrutinib in a mouse model of follicular helper (Tfh) T-cell lymphoma

Abstract

Recent analyses of the genetics of peripheral T-cell lymphoma (PTCL) have shown that a large proportion of cases are derived from normal follicular helper (Tfh) T-cells. The sanroque mouse strain bears a mutation that increases Tfh cell number and heterozygous animals (Roquinsan/+) develop lymphomas similar to human Tfh lymphoma. Here we demonstrate the usefulness of Roquinsan/+ animals as a pre-clinical model of Tfh lymphoma. Long latency of development and incomplete penetrance in this strain suggests the lymphomas are genetically diverse. We carried out preliminary genetic characterisation by whole exome sequencing and detected tumor specific mutations in Hsp90ab1, Ccnb3 and RhoA. Interleukin-2-inducible kinase (ITK) is expressed in Tfh lymphoma and is a potential therapeutic agent. A preclinical study of ibrutinib, a small molecule inhibitor of mouse and human ITK, in established lymphoma was carried out and showed lymphoma regression in 8/12 (67%) mice. Using T2-weighted MRI to assess lymph node volume and diffusion weighted MRI scanning as a measure of function, we showed that treatment increased mean apparent diffusion coefficient (ADC) suggesting cell death, and that change in ADC following treatment correlated with change in lymphoma volume. We suggest that heterozygous sanroque mice are a useful model of Tfh cell derived lymphomas in an immunocompetent animal.

Fig 1. ITK expression in Roquin^san/+ mice.

(A) Western blot showing ITK expression in sorted CD4⁺ T-cells from wildtype (WT) or Roquin^san/+ (San/+) and from whole lymph node. In order to demonstrate specificity of the antibody for mouse ITK and not BTK three mouse B-cell lines are included (Bal-17, WEHI231 and A20). GAPDH is loading control. (B) Purified splenic CD4⁺ T-cells from Roquin^san/+ (black bars) and wild-type (grey bars) animals were stimulated with anti-CD3 and anti-CD28 antibodies in the presence or absence of ibrutinib. ATP luminescence is shown relative to that of unstimulated cells. Bars show mean±SEM. n = 7 Roquin^san/+ and n = 10 wildtype. Ibrutinib caused significant (paired t-test) reduction in ATP luminescence (Roquin^san/+ **P = 0.0016 and wildtype **P = 0.003).

Fig 2. Ibrutinib causes repression of lymphoma growth.

(A) T2 weighted MRI scans showing spontaneous regression and progression over the course of treatment with vehicle. (B) T2 weighted MRI scans showing exemplar slices from animals responding to ibrutinib or with stable disease. (C) Waterfall plot showing change in enlarged lymph node volume in mice treated with vehicle. Horizontal dotted line indicates mean change in lymph node size in the group of vehicle treated animals over the treatment period. n = 8. (D) Waterfall plot showing change in enlarged lymph node volume in mice treated with ibrutinib. Light grey columns indicate treatment with ibrutinib for 1 or 2 weeks and dark grey for 3, 4 or 7 weeks. Horizontal dotted line indicates mean change in lymph node size in the group of ibrutinib treated animals over the treatment period. n = 12.

Fig 3. Functional imaging: Change in ADC correlates with change in lymphoma volume.

Pre-treatment MRI scans from a representative mouse showing (A) T2-weighted, (B) diffusion-weighted and (C) ADC map. Corresponding post-treatment MRI showing (D) T2-weighted, (E) diffusion-weighted and (F) ADC map. (G) Treatment with ibtrutinib resulted in a significant increase in ADC (paired t-test, P < 0.05). (H) Change in lymph node volume after treatment (Δ Volume) measured by T2-weighted MRI and change in ADC after treatment (Δ ADC) measured by diffusion-weighted MRI were inversely correlated (R² = 0.80, P = 0.016). n = 6 lymph nodes in 4 mice. (I) Change in lymph node volume after treatment (Δ Volume) correlates with pre-treatment ADC (R² = 0.93, P = 0.036). n = 4 lymph nodes in 4 mice.