In Situ Hybridization of HBZ and Tax in FFPE Samples from ATLL Patients and Its Association with Clinicopathological Characteristics
Authors: Yamada Kyohei , Miyoshi Hiroaki , Takeuchi Mai , Asano Naoko , Shimono Joji , Arakawa Fumiko , Nakashima Kazutaka , Sato Kensaku , Seto Masao , Ohshima Koichi 
Published: October 2019

Abstract
Introduction
Adult T-cell leukemia/lymphoma (ATLL) is a mature T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). Many studies on HTLV-1-related mRNA including HTLV-1 bZIP factor (HBZ) and Tax have been performed, mainly using cell lines, patient-derived cells, and mice. However, there are scant data concerning HTLV-1-related mRNA in formalin-fixed, paraffin-embedded (FFPE) tissue samples. We detected HBZ and Tax mRNA on FFPE tissue samples using in situ hybridization (ISH), and investigated the association with clinicopathological characteristics.
Materials and methods
Eighty-seven biopsy samples from newly diagnosed ATLL patients were examined. The same samples were used in a previous study (Miyoshi et al. Blood, 2016). Use of patient materials and clinical information was approved by the Ethical Committee of Kurume University, in accordance with the Declaration of Helsinki.
ISH was performed on FFPE tissue samples using RNAscope 2.5 HD Reagent Kit-BROWN (Advanced Cell Diagnostics, Hayward, CA). HBZ-and Tax-specific probes were used. MT-4 (HTLV-1 immortalized cell line) and Jurkat (T-cell acute lymphoblastic leukemia cell line) were used as positive and negative controls, respectively. Dot-like signals were counted at high magnification (40 diameters) on 10 randomly selected fields and the number of signals per 1,000 ATLL cells were calculated. The results were reviewed by two experienced hematopathologists. High expression was indicated when more than the median value of HBZ or Tax signals was stained. The antibodies for immunohistochemistry targeted CD4, CD30, Ki-67(MIB-1), CCR4, FoxP3, GATA3, IRF4, HLA class I, β2-microglobulin (β2M), PD-1, and PD-L1.
Clinicopathological characteristics of ATLL patients were compared by Fisher's exact test (2-sided), Mann-Whitney's U test, and Spearman's rank correlation analysis. Overall survival was estimated by the Kaplan-Meier method and compared by the Log-rank test. P< .05 was considered statistically significant. EZR ver. 1.32 was used for all statistical analyses.
Results
In the histogram and scatter plot of HBZ-ISH and Tax-ISH, the median values of HBZ signals and Tax signals were 806/1000 ATLL cells (range 0.4 - 4013.1) and 5.0/1000 ATLL cells (range 0.1 - 891.2), respectively (Figure 1). Representative samples of HBZ-ISH and Tax-ISH are presented in Figure 2A and 2B, respectively.
Notably, the high-expression group of HBZ displayed significant reductions in skin lesions (P = .025), Ann Arbor stage (P = .021), and peripheral blood involvement (P = .028). The high-expression group of Tax displayed significant increases in lactate dehydrogenase activity (P = .0020), splenomegaly (P = .0070), CD 30 (P = .015), and PD-1 in tumor-infiltrating lymphocytes (PD-1_TIL; P <.0010).
Weak but significant positive correlation was observed between HBZ and IRF4 (ρ = .33; P = .029), between HBZ and PD-L1 (ρ = -. 28; P = .010), between Tax and CD4 (ρ = .41; P = .0074), between Tax and CD30 (ρ = .26; P = .016), between Tax and GATA 3 (ρ = .26; P = .014), between Tax and PD-1 in ATLL cells (ρ = .25; P = .0074), between Tax and PD-L1 in ATLL cells (ρ =. 32; P = .0026), and between Tax and PD-1_TIL (ρ = .37; p = .0026) (Figure 3). There was almost no correlation between HBZ and Tax (ρ = .052; P = .63). There were no significant differences between HBZ or Tax and OS (Log-rank P = .21 and .95, respectively).
Conclusion
The RNA scope assay is useful for detecting low expressing mRNA in FFPE tissue samples. Our data suggest that ISH of HBZ and Tax can detect differences in clinicopathological characteristics of ATLL patients. In clinical and cell samples, HBZ and Tax might be involved in the pathogenesis of ATLL.
Disclosures
No relevant conflicts of interest to declare.

Disclosures
Asano: Takeda: Honoraria; Chugai Pharma: Honoraria; Celgene: Honoraria.