Use of PLS3, Twist, CD158k/KIR3DL2, and NKp46 gene expression combination for reliable Sézary syndrome diagnosis

To the editor:

Several publications, including ours, reported that T-plastin (PLS3) is specifically expressed in patients with Sézary syndrome (SS), the erythrodermic and leukemic form of cutaneous T-cell lymphoma (CTCL).^1-5  Besides PLS3, several other molecular SS-restricted markers, including the transcription factor Twist,⁶  have been identified by gene expression analysis on whole peripheral blood mononuclear cells. We also previously reported SS malignant cells characterization by their membrane expression of CD158k/KIR3DL2^7-9  and CD335/NKp46.¹⁰  Here we investigated whether PLS3, Twist, KIR3DL2, and NKp46 gene expression profiling by quantitative PCR (qRT-PCR) can be used for an efficient molecular SS diagnosis. A well-defined unicentric cohort of 81 patients (47 males, 34 females, with a median age of 69.5 years [22-88]), all with SS diagnosed according to international criteria including a median absolute number of Sézary cells of 4740 (1171-62 240) and a T-cell clone characterized by a specific Vβ expression (median 87% of CD3⁺ T-cells [22-99.5]), was investigated. Quantification of mRNA expression profiling was established in CD4⁺ purified T cells from SS patients'blood samples by positive sorting (Miltenyi) and use of SYBR Green PCR Core (ABI PRISM7300 Real-time PCR System) with specific primer pairs (available upon request). For each marker, an mRNA level threshold was established using qPCR mean values (± SEM) of PLS3, Twist, KIR3DL2, and NKp46 mRNA levels detected in blood CD4⁺-purified T cells from healthy donors (Table 1). A threshold of 95% significance was set up for each marker and any value less than the respective threshold was considered as negative. As positive controls were used mRNA mean levels detected in SS HuT-78 and CD56⁺ NK/T cell lines. Our results show that CD4⁺-purified T cells from SS patients expressed significant PLS3, Twist, KIR3DL2, and NKp46 mRNA mean levels (Table 1) and demonstrated that qRT-PCR data accurately classified 100% of 81 SS patients. As schematized in Figure 1, a combination of the 4 markers was observed in 20% of CD4⁺-purified T cell SS patients' samples; 53% of patients' samples exhibited mRNA combination of 3 markers, mainly PLS3+Twist+KIR3DL2 (98%), 20% showed a 2 marker combination (9% Twist+KIR3DL2, 6% PLS3+Twist, 5% PLS3+KIR3DL2) and 7% did express only one marker (4%Twist,2%PLS3,1%NKp46). Our data clearly indicate tha t combination of Twist and PLS3 or KIRD3DL2 positive mRNA values from CD4⁺-isolated T-cells accounted for the diagnosis of 98% SS patients, and designed Twist as the strongest biologic SS marker from the combination with positivity in 91% SS patients. It is noteworthy that mRNA extracts from whole peripheral blood mononuclear cells (PBMCs) gave mean values (± SEM, n = 48) of 620 ± 427 and 538 ± 219, respectively, and thus can be used in routine for PLS3 and Twist mRNA detection using identical thresholds than those for mRNA detection in CD4⁺ isolated T cells. It should be mentioned that KIR3DL2 and NKp46 must be detected carefully in whole PBMC mRNA extracts since circulating NK cells should hamper distinction between healthy donors and SS patients, but both of these transmembrane receptors could contribute to ascertain the SS diagnosis by multi-color flow cytometry analysis.

In conclusion, we show for the first time that our combination of 4 biomarkers in PCR allows easy diagnosis of SS in 100% of cases.