An ATL cell line with an IgH pseudo-rearranged band pattern by southern blotting: a pitfall of genetic diagnosis.

Adult T-cell leukemia (ATL), which is a mature T-cell malignancy that develops from human T-cell leukemia virus type-1 (HTLV-1)-infected T-cells, is diagnosed based on morphologic, immunophenotypic, serologic, and genetic characteristics. In particular, Southern blot hybridization (SBH) and polymerase chain reaction analyses for antigen receptor genes and the retrovirus of HTLV-1 provide a diagnostic hallmark for the clonality of leukemic cells and the causative agent of the disease. We report here a case of an ATL cell line, designated as SO4 cells, established from primary ATL cells presenting with an irrational genetic abnormality of the immunoglobulin heavy chain (IgH)-rearranged band in spite of harboring a clonally rearranged T-cell receptor gene and a clonally integrated provirus of HTLV-1 within their genomic DNA. Moreover, fluorescence in situ hybridization analysis using the IgH (14q32) dual-color break-apart probe revealed 3 pair-signals of colocalizing red and green spots, implying 2 intact and 1 amplified 14q32 regions without translocation, where the region contains the IgH gene locus. Although the exact mechanism remains to be elucidated, some alteration of a portion of the amplified 14q32 region seems to have a role in the false-positive band pattern in the SBH. The SO4 cells, in the hematology laboratory, will provide a lesson about the pitfalls of genetic tests for mature T-cell neoplasms and contribute to the genetic elucidation of leukomogenesis as an in vitro model.

A novel plasmacytoid dendritic cell line, CAL-1, established from a patient with blastic natural killer cell lymphoma.

Blastic natural killer (NK) cell lymphoma corresponding to CD4+CD56+ malignancies is a novel disease entity, according to the results of clinical, morphologic, and immunologic studies. It is especially noteworthy that this disease likely arises from plasmacytoid dendritic cells (pDCs), described previously as plasmacytoid T-cells, which have an important role in innate and adaptive immunity. However, the exact relationship between the tumor cells and pDCs remains to be elucidated. We encountered a patient with typical blastic NK cell lymphoma, which later converted to leukemic manifestations, and tried to establish a cell line using the leukemic cells. We succeeded in establishment of a novel cell line, CAL-1, which originated from the primary malignant cells. The genetic and phenotypic features of CAL-1 cells bear a similarity to those of pDCs, namely, plasmacytoid morphology at light and electron microscopy; negative results for CD11c and lineage-associated markers of CD3, CD14, CD19, and CD16; positive results for HLA-DR, CD4, CD56, CD45RA, and CD123; and negative results for TCR and IgH gene rearrangements. An interesting finding was that CAL-1 cells change morphologically into the mature DC appearance with many long dendrites after short-term culture in the presence of granulocyte-macrophage colony-stimulating factor and interleukin 3. CAL-1 cells can secrete tumor necrosis factor alpha but not interferon alpha. Thus although they do not share in part phenotypic and functional features with their normal counterparts, CAL-1 cells mostly exhibit a striking pDC phenotype. We describe the first novel pDC cell line of CAL-1. This cell line should open the opportunity for study not only of CD4+CD56+ tumor cells but also of pDCs in vitro.

Clinical relevance of survivin as a biomarker in neoplasms, especially in adult T-cell leukemias and acute leukemias.

Survivin has been identified as one of the top 4 transcripts among 3.5 million human transcriptomes uniformly up-regulated in cancer tissues but not in normal tissues. Therefore, we quantitatively determined the messenger RNA (mRNA) expression profile for survivin by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) technique in 113 patients with leukemias, such as adult T-cell leukemia (ATL), acute lymphoid leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia in crisis, and chronic lymphocytic leukemia (CLL), and in 25 cell lines, including 7 ATL cell lines and 15 solid-tumor cell lines. Furthermore, we examined whether the plasma level of survivin protein as measured by enzyme-linked immunosorbent assay (ELISA) substituted for mRNA expression by PCR quantification. Gene expression was quantitatively confirmed to be up-regulated in approximately 90% of ATL and acute leukemia cases and in all of the cell lines tested, whereas it was down-regulated in almost all cases of CLL. Furthermore, with respect to the interpretation of the gene expression findings, attention was paid to standardization with a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in the real-time PCR quantification, because the variability in GAPDH expression among the different cell types was significant. GAPDH expression was relatively low in ATL cells and high in ALL and AML cells. The rates of increase in the levels of survivin protein in the plasma of ATL patients and in the supernatants from in vitro cultures of solid-tumor cell lines were low compared with rates of increase of the mRNA and protein level in the cells, suggesting that the protein levels in plasma do not always reflect survivin expression in tumor cells. Our findings indicate the potential clinical relevance of survivin quantified by real-time PCR but not for the protein level in plasma as determined by ELISA, especially in cases of ATL and acute leukemias.