Diagnostic Validity of Combining HTLV-1 Serology and Immunophenotyping in Adult T-Cell Leukemia

Adult T-cell leukemia (ATL) is heterogeneous and sometimes equivocal to other T-cell neoplasms. Detecting anti- HTLV-1 antibodies is significant for a first screening not only for HTLV-1 infection but also for the HTLV-1-related disorders of ATL and TSP/HAM. The purpose of the present study was to investigate the diagnostic validity of HTLV-1 serology in ATL. The serologic results by a gelatin particle agglutination (PA) assay were highly sensitive (100%) and specific (99.5%) for the results of polymerase chain reaction (PCR) assay in 666 healthy blood donors who live in an area endemic for the HTLV-1 virus. Of 7,536 hospitalized patients, 189 patients with ATL were serologically screened. There were 1,140 patients (15.2%) infected by chance with HTLV-1, showing specificity, sensitivity, positive predictive value (PV), and negative PV of 84.3%, 100%, 14.2%, and 100%, respectively. Since the low positive PV (14.2%) was useless, we tried combining the anti-HTLV-1 assay with the immunophenotyping necessary for the diagnosis of lymphoid neoplasms. This combination gave nearly 100% positive and negative PV, and could prove to be useful in diagnosing ATL with the probability of 98%, especially for epidemiologic studies

Induction of apoptosis by HBI-8000 in adult T-cell leukemia/lymphoma is associated with activation of Bim and NLRP3

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell malignancy caused by human T-cell lymphotropic virus 1. Treatment options for acute ATL patients include chemotherapy, stem cell transplantation, and recently the anti-chemokine (C-C motif) receptor 4 antibody, although most patients still have a poor prognosis and there is a clear need for additional options. HBI-8000 is a novel oral histone deacetylase inhibitor with proven efficacy for treatment of T-cell lymphomas that recently received approval in China. In the present study, we evaluated the effects of HBI-8000 on ATL-derived cell lines and primary cells obtained from Japanese ATL patients. In most cases HBI-8000 induced apoptosis in both primary ATL cells and cell lines. In addition, findings obtained with DNA microarray suggested Bim activation and, interestingly, the contribution of the NLR family, pyrin domain containing 3 (NLRP3) inflammasome pathway in HBI-8000-induced ATL cell death. Further investigations using siRNAs confirmed that Bim contributes to HBI-8000-induced apoptosis. Our results provide a rationale for a clinical investigation of the efficacy of HBI-8000 in patients with ATL. Although the role of NLRP3 inflammasome activation in ATL cell death remains to be verified, HBI-8000 may be part of a novel therapeutic strategy for cancer based on the NLRP3 pathway

RELEVANCE OF MOLECULAR TESTS FOR HTLV-1 INFECTION AS CONFIRMATORY TESTS AFTER THE FIRST SERO-SCREENING

The diagnosis of human T-cell leukemia virus type-1 (HTLV-1) infection has been widely examined by serologics. In the first screening tests, serological false negative and positive samples have been reduced thanks to advances in assay techniques that apply new emission agents and sensors. On the other hand, western blot (WB) remains problematic. For example, WB analysis yields many samples equivalent to antibody positive ones. To reduce the need for WB, an alternative testing strategy is required to detect HTLV-1 infection. Polymerase chain reaction (PCR) for the HTLV-1 provirus has recently been recommended for a final diagnosis of infection. However, although PCR is thought to be one element, the validation of detection performance for HTLV-1 infection between serological and molecular testing is not always clear. Thus, this study aimed to evaluate the accuracy and test the validity of an improved methodology for serological detection of HTLV-infection, as well as that of PCR. In conclusion, the high values of kappa-statistics are expected to deliver high quality in chemiluminescent enzyme immunoassay (or chemiluminescent immunoassay), while the problems with WB assays remain to be elucidated. As an alternative to WB, a combination of real-time qPCR and nested PCR is proposed as a suitable confirmatory test

High-resolution melting analysis for a reliable and two-step scanning of mutations in the tyrosine kinase domain of the chimerical bcr-abl gene.

For relevant imatinib therapy against Philadelphia (Ph)-positive leukemias, it is essential to monitor mutations in the chimerical bcr-abl tyrosine kinase domain (TKD). However, there is no universally acceptable consensus on how to efficiently identify mutations in the target TKD. Recently, high-resolution melting (HRM) technology was developed, which allows gene scanning using an inexpensive generic heteroduplex-detecting dsDNA-binding dye. This study aimed to validate the introduction of HRM in a practical clinical setting for screening of mutations in sporadic sites of the chimerical bcr-abl TKD. All chimerical and wild-type abl TKD regions selectively amplified were used for HRM assays and direct sequencing. The HRM test had approximately 5-90% detection sensitivity for mutations. In contrast to mixture samples with mutant and wild-type cells, all mutant cell samples had indeterminate melting curves equivalent to those of the wild-type due to formation of only a homodulex. This issue was improved by the addition of exogenous wild-type DNA after PCR. Subsequently, HRM results gave a high accordance rate of 97.8% (44/45 samples) compared to the sequencing data. The discordant results in one appear to be due to unsuccessful amplification. Thus, HRM may be considered to be suitable for reliable scanning of mutations in the chimerical abl TKD in a clinical setting

Aberrant overexpression of membrane-associated mucin contributes to tumor progression in adult T-cell leukemia/lymphoma cells.

Aberrant overexpression of membrane-associated mucin (MUC1) is implicated in the pathogenesis of cancer, particularly of adenocarcinomas. Adult T-cell leukemia/lymphoma (ATL), an aggressive neoplasm etiologically associated with human T-lymphotropic virus type-1 (HTLV-1), exhibits invasive tropism into various organs, resulting in disease progression and resistance to treatment. In the present study, we showed that MUC1 is overexpressed exclusively in cells of ATL among hematological malignancies. Furthermore, increased expression of MUC1 correlated with a poor prognosis, suggesting MUC1 to be a prognostic marker in ATL. Various functional analyses with knockdown experiments using a specific siRNA for MUC1 revealed that MUC1 is involved in cell growth, cell aggregation, and resistance to apoptosis. Although it has been shown that the anti-adhesive properties of MUC1 facilitate migration and metastasis of tumor cells, our findings indicated that MUC1 contributes to cell-cell adhesion. Mucins thus seem to play a role in the pathogenesis and/or progression of ATL

A Relevant Reference Gene and Normalization for mRNA Real-Time PCR Quantification in Specimens with Distinct Cell Types and Variant Integrity

Variability of clinical samples with respect to cell types and quality makes it indispensable to normalize mRNA quantification by real-time reverse transcription-polymerase chain reaction (RT-PCR). The objective of the present study was to elucidate the influence of the difference in RNA integrity and the expression status of control genes commonly used. To compare the expression status of GAPDH, β-actin, PBGD and 18S rRNA in different cell samples, real-time RT-PCR by the LightCycler Technology was applied. The relevance of the above-mentioned normalization by control gene was evaluated through the practical measurement of survivin using normal lymphocytes from 19 healthy donors, adult T-cell leukemia (ATL) cells from 30 patients with ATL and 27 cell line cells. The mRNA integrity was found to be tolerable for absolute quantification when the preservation rate of 28S and 18S rRNA within total RNA was at least 30%. The expression status of control genes was prominently variable with the expression difference of 4-fold in β-actin, 20-fold in GAPDH, 30-fold in 18S rRNA and 66-fold in PBGD among the different cell types, normal lymphocytes, chronic and acute ATL cells, ATL cell lines, other hematopoietic cell lines and solid tumor cell lines. The survivin mRNA data normalized by such a control gene were influenced by the expression variability of the control genes. These results indicate that the control genes can be used to correct for sample-to-sample variation within the same cell types, but they are not always relevant for normalization in the distinct cell types