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

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.

Cytomorphological similarities between feline viral leukemia, bovine enzootic leukosis and adult T-cell leukemia/lymphoma: A review

“…The method had the combination of two assays capable of detecting the virus: the in situ hybridization of ultrasensitive RNA and quantitative PCR targeting the tax gene (tax-qPCR). The diagnostic algorithm that combines these two assays successfully evaluated 94% (112/119) of the samples and distinguished ATL from non-ATL cases, including HTLV-1 carriers with 100% sensitivity and specificity (Takatori et al, 2020). The authors hope that, in the future, this new method may replace SBH.…”

“…Therefore, indirect immunofluorescence tests, radioimmunoprecipitation on polyacrylamide gel and Western blotting can be used as confirmatory tests. However, when they reach indeterminate results, there is a need to resort to more sensitive techniques, such as PCR (Bazarbachi et al, 2011;Melo et al, 2017) and Southern blot (Qayyum & Choi, 2014;Takatori et al, 2020). These techniques are performed by a few laboratories and are therefore generally not accessible.…”

“…For the execution of the Southern blot (SBH) there is a need for a large amount of DNA without degradation and the method cannot be performed from formalin-fixed paraffin-embedded (FFPE) tissue samples. In the recent study by Takatori et al (2020), a new diagnostic algorithm has been developed for the accurate identification of ATL using FFPE samples. The method had the combination of two assays capable of detecting the virus: the in situ hybridization of ultrasensitive RNA and quantitative PCR targeting the tax gene (tax-qPCR).…”

Classification and challenges in the histopathological diagnosis of peripheral T-cell lymphomas, emphasis on the WHO-HAEM5 updates

“…The prognosis is poor, regardless of the clinical presentation, and ranges from 1 year to 2 years after diagnosis. HTLV-1 serology testing is non-diagnostic of ATLL, and direct detection of viral transcripts in the neoplastic cells is critical for a definitive diagnosis in cases where the clinical and histopathological presentation is suggestive of different subtypes of T-cell lymphomas ( 15 , 16 ).…”

A Random Walk-Based Method to Identify Candidate Genes Associated With Lymphoma

“…For familial lymphoma cases, family pedigree analyses based on Sanger sequencing ( Liu et al, 2014 ), microarray analyses ( Hedvat et al, 2002 ), next generation target sequencing and whole genome wide sequencing ( Hung et al, 2018 ) on large familial samples are major traditional methods to identify potential pathogenic lymphoma associated genes or variants. As for sporadic lymphoma cases, to validate the molecular abnormalities associated with lymphoma, Southern blot analyses ( Sangueza et al, 1992 ), in situ hybridization ( Quintanilla-Martinez et al, 2009 ) and quantitative real-time PCR ( Takatori et al, 2021 ) are also applied to explore and confirm specific distribution of genetic abnormal arrangement associated with lymphoma. There are three advantages for traditional analyses: 1) Firstly, the accuracy of traditional experimental analyses is generally higher than statistical bioinformatics analyses; 2) Secondly, independent repeat experimental analyses are easier to perform at experimental level to validate the identified potential biomarkers; 3) Thirdly, results from experimental analyses were easier to be used for further functional exploration.…”