A new diagnostic algorithm using biopsy specimens in adult T-cell leukemia/lymphoma: combination of RNA in situ hybridization and quantitative PCR for HTLV-1

Mitsuyoshi Takatori,Shugo Sakihama,Megumi Miyara,Naoki Imaizumi,Takashi Miyagi,Kazuiku Ohshiro,Iwao Nakazato,Masaki Hayashi,Junpei Todoroki,Satoko Morishima,Hiroaki Masuzaki,Takuya Fukushima,Kennosuke Karube

doi:10.1038/s41379-020-0635-8

Abstract

Histopathological distinction between adult T-cell leukemia/lymphoma (ATLL) and other T-cell neoplasms is often challenging. The current gold standard for the accurate diagnosis of ATLL is the Southern blot hybridization (SBH) assay, which detects clonal integration of human T-cell leukemia virus type I (HTLV-1) provirus. However, SBH cannot be performed with small biopsy or formalin-fixed paraffin-embedded (FFPE) tissue samples because this assay requires a large amount of DNA without degradation. Here we developed a new diagnostic algorithm for the accurate diagnosis of ATLL using FFPE samples. This method combines two HTLV-1 detection assays, namely, ultrasensitive RNA in situ hybridization using RNAscope for HTLV-1 bZIP factor (HBZ-RNAscope), and quantitative PCR targeting the tax gene (tax-qPCR). We analyzed 119 FFPE tissue specimens (62 ATLL, and 57 non-ATLL, including 41 HTLV-1 carriers) and compared them with the SBH results using the corresponding fresh-frozen samples. As a result, tax-qPCR had a higher ATLL identification rate than HBZ-RNAscope (88% [52/59], and 63% [39/62], respectively). However, HBZ-RNAscope clearly visualized the localization of HTLV-1-infected tumor cells and its identification rate increased to 94% (17/18) when the analysis was limited to samples up to 2 years old, indicating its usefulness in the daily diagnosis. The diagnostic algorithm combining these two assays successfully evaluated 94% (112/119) of samples and distinguished ATLL from non-ATLL cases including HTLV-1 carriers with 100% sensitivity and specificity. This method is expected to replace SBH and increase the accuracy of the diagnosis of ATLL.

Highlights

We carried out HTLV-1 bZIP factor (HBZ)-RNAscope and quantified HTLV-1 proviral load (PVL) using formalin-fixed paraffin-embedded (FFPE) samples from Adult T-cell leukemia/lymphoma (ATLL) patients and nonATLL HTLV-1 carriers, and established a diagnostic algorithm applicable to FFPE samples with 100% sensitivity and specificity (Fig. 5)
In the application of tax-qPCR to FFPE samples, we set a cutoff value for HBB gene as an internal control, which was not used in the previous study with fresh peripheral blood mononuclear cells (PBMCs) [24]
This eliminated the potential effects of nucleic acid degradation in FFPE samples due to various factors such as time between surgery and formalin fixation, type of formalin, time of fixation, and storage period [27, 28]; this is in contrast to nucleic acids derived from raw or frozen specimens, which are usually of sufficient quality for PCR analysis

Summary

Introduction

As large amounts of DNA (as much as 10–15 μg per sample) are needed for SBH [6], raw or frozen peripheral blood mononuclear cells (PBMCs), or fresh-frozen tissue specimens, are typically used [5]. It is as such impossible to perform SBH with small biopsy or formalin-fixed paraffin-embedded (FFPE) samples, which are commonly used for pathological diagnoses. It is possible to overlook peripheral T-cell lymphoma or anaplastic large cell lymphoma, which histologically mimic ATLL, in patients with HTLV-1 infection [7]

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Conclusion