The evolution of clonality testing in the diagnosis and monitoring of hematological malignancies

Abstract

Currently, distinguishing between benign and malignant lymphoid proliferations is based on a combination of clinical characteristics, cyto/histomorphology, immunophenotype and the identification of well-defined chromosomal aberrations. However, such diagnoses remain challenging in 10-15% of cases of lymphoproliferative disorders, and clonality assessments are often required to confirm diagnostic suspicions. In recent years, the development of new techniques for clonality detection has allowed researchers to better characterize, classify and monitor hematological neoplasms. In the past, clonality was primarily studied by performing Southern blotting analyses to characterize rearrangements in segments of the IG and TCR genes. Currently, the most commonly used method in the clinical molecular diagnostic laboratory is polymerase chain reaction (PCR), which is an extremely sensitive technique for detecting nucleic acids. This technique is rapid, accurate, specific, and sensitive, and it can be used to analyze small biopsies as well as formalin-fixed paraffin-embedded samples. These advantages make PCR-based approaches the current gold standard for IG/TCR clonality testing. Since the completion of the first human genome sequence, there has been a rapid development of technologies to facilitate high-throughput sequencing of DNA. These techniques have been applied to the deep characterization and classification of various diseases, patient stratification, and the monitoring of minimal residual disease. Furthermore, these novel approaches have the potential to significantly improve the sensitivity and cost of clonality assays and post-treatment monitoring of B- and T-cell malignancies. However, more studies will be required to demonstrate the utility, sensitivity, and benefits of these methods in order to warrant their adoption into clinical practice. In this review, recent developments in clonality testing are examined with an emphasis on highly sensitive systems for improving diagnostic workups and minimal residual disease assessments.

Characterization of clonal immunoglobulin heavy (IGH) V-D-J gene rearrangements and the complementarity-determining region in South Indian patients with precursor B-cell acute lymphoblastic leukemia

“…Although the techniques such as Southern blotting and heteroduplex analysis have been used as gold-standard methods for the detection of clonal gene rearrangements, recent technologies such as next-generation sequencing and analysis of next-generation sequencing data by high-throughput and deep sequencing using IMGT/HighV QUEST analysis [2627] are revolutionizing disease diagnostics for detecting the clonal repertoire of IG and T-cell receptor V-(D)-J sequences and precisely identifying the clonal evolution of the disease at the molecular level.…”

Emerging diagnostic tests for vitreoretinal lymphoma: a review

“…Cytological analyses are typically combined with immunophenotyping, which identifies the tumour cell as a B cell, but also indicates light‐chain restriction to kappa or lamda . As well summarized by Arber and Gazzola et al ., molecular confirmation of the clonal nature of a lymphoma (i.e. that all tumour cells derive from one lymphocyte) is an established method for diagnosing tumours that are not readily identified on the standard features, including clinical presentation, cytological or histological findings and immunophenotype.…”

“…In theory, PCR using primers that amplify within the junctional region gives multiple amplicons when the cell population is reactive and polyclonal, versus single amplicons when the cell population is neoplastic and monoclonal. However, there are important considerations for the design of tests and interpretation of results, including reduced sensitivity associated with a polyclonal background, and reduced specificity when small numbers of cells in a sample result in a pseudoclonality pattern; thus analyses should be duplicated and multiple targets should be included …”

“…However, there are important considerations for the design of tests and interpretation of results, including reduced sensitivity associated with a polyclonal background, and reduced specificity when small numbers of cells in a sample result in a pseudoclonality pattern; thus analyses should be duplicated and multiple targets should be included. 11 The first application of the technology to vitreoretinal lymphoma was by Katai et 20 performed two PCR analyses with different primer sets on 28 paraffin-embedded material or fresh vitreous from cases with a differential diagnosis that included vitreoretinal lymphoma, plus normal vitreous taken at macular hole surgery: a clonal rearrangement was detected in 16 lymphomas with early histopathological confirmation, and importantly, in four additional lymphomas that were initially undiagnosed and recognized only in followup. With both primer sets, sensitivity was 0.95higher than cytopathologyand specificity was 1.00.…”

Determinación de la clonalidad en tejidos humanos

“…Este es un problema en general invisible en la literatura pues a la larga el patólogo o los médi-cos tratantes se ven obligados a tomar una decisión. Además, a veces es necesario determinar si dos lesiones presentes en el mismo paciente tienen el mismo origen o son diferentes (7,8).…”