CD164 identifies CD4+ T cells highly expressing genes associated with malignancy in Sézary syndrome: the Sézary signature genes, FCRL3, Tox, and miR-214

Sézary syndrome (SS), a leukemic variant of cutaneous T-cell lymphoma (CTCL), is associated with a significantly shorter life expectancy compared to skin-restricted mycosis fungoides. Early diagnosis of SS is, therefore, key to achieving enhanced therapeutic responses. However, the lack of a biomarker(s) highly specific for malignant CD4+ T cells in SS patients has been a serious obstacle in making an early diagnosis. We recently demonstrated the high expression of CD164 on CD4+ T cells from Sézary syndrome patients with a wide range of circulating tumor burdens. To further characterize CD164 as a potential biomarker for malignant CD4+ T cells, CD164+ and CD164-CD4+ T cells isolated from patients with high-circulating tumor burden, B2 stage, and medium/low tumor burden, B1-B0 stage, were assessed for the expression of genes reported to differentiate SS from normal controls, and associated with malignancy and poor prognosis. The expression of Sézary signature genes: T plastin, GATA-3, along with FCRL3, Tox, and miR-214, was significantly higher, whereas STAT-4 was lower, in CD164+ compared with CD164-CD4+ T cells. While Tox was highly expressed in both B2 and B1-B0 patients, the expression of Sézary signature genes, FCRL3, and miR-214 was associated predominantly with advanced B2 disease. High expression of CD164 mRNA and protein was also detected in skin from CTCL patients. CD164 was co-expressed with KIR3DL2 on circulating CD4+ T cells from high tumor burden SS patients, further providing strong support for CD164 as a disease relevant surface biomarker

Multiplex RT-PCR for the Detection of Leukemia-Associated Translocations : Validation and Application to Routine Molecular Diagnostic Practice

The aim of this study was to validate the application of a commercially available multiplex reverse transcription polymerase chain reaction (RT-PCR) assay [Hemavision-7 System] for the seven most common leukemia translocations for routine molecular diagnostic hematopathology practice. A total of 98 samples, comprising four groups, were evaluated: Group 1, 16 diagnostic samples molecularly positive by our existing laboratory-developed assays for PML-RARα/t (15;17) or BCR-ABL/t (9;22); Group 2, 51 diagnostic samples negative by our laboratory-developed assays for PML-RARα/t (15;17) or BCR-ABL/t (9;22); Group 3, 21 prospectively analyzed diagnostic cases, without prior molecular studies; and Group 4, 10 minimal residual disease (MRD) samples. Analysis of the two previously studied cohorts (Groups 1 and 2) confirmed the diagnostic sensitivity and specificity of the multiplex assay with regard to these two translocations. Additionally, however, in the “negative” Group (Group 2) the assay revealed three unanticipated translocations (CBFβ-MYH11, BCR-ABL, and MLL-AF4), two of which were confirmed on cytogenetics. Analysis of the prospective cohort demonstrated that the assay was cost-effective and amenable to standard laboratory practice, with an identically sensitive MRD detection rate to that of our laboratory-developed tests. Virtually all of the results obtained were consistent with the phenotype and karyotype by conventional methods. This study illustrates the utility of a kit-based multiplex RT-PCR assay for the molecular diagnosis and monitoring of leukemias and reinforces the complementary roles of molecular testing and cytogenetics in diagnostic hematopathology

CD164 and FCRL3 Are Highly Expressed on CD4+CD26 − T Cells in Sézary Syndrome Patients

Sézary syndrome (SS) cells express cell surface molecules also found on normal activated CD4 T cells. In an effort to find a more specific surface marker for malignant SS cells, a microarray analysis of gene expression was performed. Results showed significantly increased levels of mRNA for CD164, a sialomucin found on human CD34+ hematopoietic stem cells, and FCRL3, a molecule present on a subset of human natural T regulatory cells. Both markers were increased in CD4 T cells from SS patients compared with healthy donors (HD). Flow cytometry studies confirmed the increased expression of CD164 and FCRL3 primarily on CD4+CD26- T cells of SS patients. Importantly, a statistically significant correlation was found between an elevated percentage of CD4+CD164+ T cells and an elevated percentage of CD4+CD26- T cells in all tested SS patients but not in patients with mycosis fungoides and atopic dermatitis or HD. FCRL3 expression was significantly increased only in patients with high tumor burden. CD4+CD164+ cells displayed cerebriform morphology and their loss correlated with clinical improvement in treated patients. Our results suggest that CD164 can serve as a marker for diagnosis and for monitoring progression of cutaneous T-cell lymphoma (CTCL)/SS and that FCRL3 expression correlates with a high circulating tumor burden