Utility of flow cytometry and gene rearrangement analysis in tissue and blood of patients with suspected cutaneous T‑cell lymphoma

Abstract

Cutaneous T-cell lymphoma (CTCL) is difficult to diagnose at an early stage. Current diagnostic tools include clinical examination, histomorphologic analysis, immunohistochemistry, flow cytometry of peripheral blood and/or lesional tissue, and evaluation of T-cell receptor (TCR) clonality by gene rearrangement analysis (TCRGR). Advances in genomic sequencing, including high-throughput sequencing (HTS), can be used to identify specific clones of rearranged TCR genes. Even with all of these tools, CTCL can take as long as a decade to definitively diagnose, potentially delaying treatment options and causing patient anxiety. This study aimed to evaluate the performance of the various ancillary testing modalities used to diagnose early-stage CTCL. In a subset of patients the performance of HTS was compared to flow cytometry and conventional TCRGR analysis via polymerase chain reaction (PCR). Fifty-five patients, with a total of 68 skin biopsies and peripheral blood draws, were evaluated using flow cytometry, PCR-TCRGR, and HTS-TCRGR to determine the sensitivity and specificity of each ancillary test. In tissue biopsies, flow cytometry (64%), PCR (71%) and HTS (69%) shared similar sensitivities; flow cytometry had the highest specificity (93%), followed by HTS (86%) and PCR (76.9%). However, flow cytometry and PCR had insufficient DNA quantities in 29 and 15% of the specimens, respectively. Peripheral blood testing was less sensitive than tissue testing (flow cytometry 14%, PCR 41%, HTS 33%); in peripheral blood, HTS was the most specific test (flow cytometry, 70%; PCR, 62.5%; and HTS, 100%). HTS is a highly specific molecular test for identifying CTCL in peripheral blood and skin biopsy specimens; however, our findings suggest a need for a continued search for more sensitive tests for early-stage CTCL.

Real-Life Barriers to Diagnosis of Early Mycosis Fungoides: An International Expert Panel Discussion

“…However, there may be few malignant cells in early-stage MF with little sign of clonality, rendering PCR testing unreliable in a significant fraction of early cases [41,44]. High-throughput, next-generation TCR skin biopsy gene sequencing or peripheral blood samples is an emerging technique with potentially higher specificity than PCR-based techniques for differentiating MF from BIDs [44][45][46].…”

The diagnosis and management of suspected lymphoma in general practice

“…4 In addition, FC may not accurately pick up T-cell clones and T-cell rearrangement studies are often needed for diagnosis of T-cell lymphoma, though this is typically managed by the pathologist and not the ordering provider. 82…”

“…Providers should monitor all patients with negative biopsy results for persistent signs and symptoms of lymphoma and pursue larger‐volume biopsy if clinical suspicion for lymphoma remains high 4 . In addition, FC may not accurately pick up T‐cell clones and T‐cell rearrangement studies are often needed for diagnosis of T‐cell lymphoma, though this is typically managed by the pathologist and not the ordering provider 82 …”

Cutaneous T‐cell lymphomas: 2021 update on diagnosis, risk‐stratification, and management

“…42 Recent studies have suggested that next generation sequencing (NGS) may be more sensitive and/or specific for assessing T-cell clonality in MF/SS, but NGS is not yet widely available. [43][44][45] The extent to which MF/SS may be preceded by a pre-malignant state, analogous to monoclonal B-cell lymphocytosis (MBL) or monoclonal gammopathy of undetermined significance (MGUS), is debatable and poorly defined. 46 The malignant lymphocytes in MF/SS are usually CD3 + CD4 + and CD8 À , but frequently lose the expression of other pan-T-cell antigens.…”

“…Moreover, some MF cases may not have a detectable T‐cell clone 42 . Recent studies have suggested that next generation sequencing (NGS) may be more sensitive and/or specific for assessing T‐cell clonality in MF/SS, but NGS is not yet widely available 43–45 . The extent to which MF/SS may be preceded by a pre‐malignant state, analogous to monoclonal B‐cell lymphocytosis (MBL) or monoclonal gammopathy of undetermined significance (MGUS), is debatable and poorly defined 46 .…”