The MEC1 and MEC2 lines represent two CLL subclones in different stages of progression towards prolymphocytic leukemia.

The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL) patient at different stages of progression to prolymphocytoid transformation (PLL). This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells.

Human T-lymphotropic virus type I provirus and T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is a rare type of post-thymic T-cell neoplasm, the etiology of which is unknown. Patients with T-PLL have been found to be seronegative for human T-lymphotropic virus type-I (HTLV-I), and their leukemia cells do not retain monoclonally integrated HTLV-I provirus. Recently, we have demonstrated the presence of defective HTLV-I provirus by polymerase chain reaction in the DNA extracted from peripheral blood cells or affected lymph nodes of T-PLL patients. Although there is a possibility, from our observation, that an alternative mechanism is operating in HTLV-associated leukemogenesis, it is still unknown whether and how HTLV-I can contribute to the leukemogenesis of T-PLL. In this review, we describe controversial issues and discuss a role of HTLV-I in the leukemogenesis of T-PLL.

Defective human T-lymphotrophic virus type I provirus in T-cell prolymphocytic leukaemia.

T-cell prolymphocytic leukaemia (T-PLL) is a rare form of post-thymic T-cell neoplasm, the aetiology of which remains unknown. We examined human T-lymphotropic virus (HTLV) provirus in five HTLV-I/II seronegative patients with T-PLL. Southern blotting did not show monoclonal integration of the HTLV-I genome in any of the DNA samples. However, two of the five DNA samples contained an HTLV-I tax sequence. Other sets of oligonucleotide primers for HTLV-I gag, pol, env and LTR regions were all negative. HTLV-I tax gene expression and p40tax antibody were not detected in samples from cases with HTLV-I tax sequence. Our findings suggest that there may be alternative mechanisms involved in HTLV-associated leukaemogenesis, in which HTLV-I genome insertion triggers T-PLL but the deletion of various regions of the integrated provirus subsequently prevents active replication and the expression of the virus.

Deleted HTLV provirus in peripheral blood cells of a patient with T-cell prolymphocytic leukaemia.

We describe the first case of T-cell prolymphocytic leukaemia (T-PLL) in which the peripheral blood cells contained a human T-lymphotropic virus (HTLV) related tax sequence. Serum screening tests for anti-HTLV-I/II antibodies were negative. Polymerase chain reaction disclosed the presence of an HTLV-I tax sequence in the peripheral blood. Other sets of oligonucleotide primers for HTLV-I gag, pol, env and the long terminal repeat regions and for the HTLV-II pol region were negative in the DNA of the cells. Although patients with T-PLL have been reported to be seronegative for HTLV-I, our findings point to the possibility that HTLV-I infection might be involved in the aetiology of at least some cases of T-PLL and that there may be alternative mechanisms involved in HTLV-associated leukaemogenesis.

The human T-cell lymphotropic viruses types I/II are not involved in T prolymphocytic leukemia and large granular lymphocytic leukemia.

The possible involvement of the human T lymphotropic viruses type I and II (HTLV-I and -II) in lymphoproliferative disorders of mature T cells other than adult T cell leukemia/lymphoma (ATLL) has been controversial. Most studies have focused primarily on the cutaneous T cell lymphomas. However, skin involvement is a frequent feature of T prolymphocytic leukemia (T-PLL) and antibodies against HTLV-I and -II have been reported in individuals with large granular lymphocytic (LGL) leukemia. We examined 36 patients with T-PLL and 28 with LGL leukemia for evidence of HTLV-I and -II. Polymerase chain reaction (PCR) was performed on DNA from fresh peripheral blood mononuclear cells (PBMCs) and PBMCs after short-term culture (STC) using primers against all parts of the HTLV-I genome (LTR, gag, env, pol, tax/rex) and against HTLV-II pol and gag. Reverse transcriptase (RT) activity was measured on supernatants from STCs using a sensitive PCR-based technique. No HTLV-I or -II sequences were found by PCR nor RT activity detected in the 64 cases. Our findings do not provide evidence of HTLV-I or -II infection in T-PLL and LGL leukemia patients from an HTLV-I nonendemic area. Previous positive reports on these disorders may represent technical artefacts, detection of endogenous HTLV-like sequences or reflect patients from endemic areas and a variable etiology of T cell diseases.

Engraftment of chronic prolymphocytic and T cell leukemia in SCID mice.

Engraftment of human acute leukemia cells in immunocompromised (SCID) mice has resulted in in vivo models for exploration of human tumor biology. Attempts at engraftment of chronic leukemia cells have been generally unsuccessful. We have engrafted cells from three human chronic leukemias in SCID mice. Cell populations were from two patients with chronic lymphocytic leukemia (CLL) and either increased proolymphocytes (CLL-Pro; patient 1), or prolymphocytic transformation (PLL; patient 2) and from a third patient with newly diagnosed T cell CLL. Both fresh and cryopreserved cells were used and were injected intravenously, intraperitoneally, or both, after conditioning with cyclophosphamide. In addition, cells derived from a mouse spleen engrafted with human leukemia were passaged into another mouse. The animals were observed daily for signs of disease or appearance of tumors and sacrificed when terminally ill. At intervals blood samples were obtained and analyzed for the presence of human cells or DNA. Human leukemic cells were demonstrated by polymerase chain reaction (PCR) analysis of the human DQalpha gene or positive staining for human leukocyte common antigen (LCA). The presence of Epstein-Barr virus (EBV)-positive cells was also investigated by PCR analysis. Disseminated tumors developed in most mice inoculated with cells from the first patient, and this was associated with shortened survival times. The methods of administration, use of fresh or frozen samples, or the size of the inoculum had no effect on the development of leukemia. Survival of the mouse receiving passaged cells was similar to mice inoculated with fresh cells. Extensive histologic, immunophenotypic, and DNA studies were performed on organs from mice engrafting with cells from patient 1. PCR analysis for EBV sequences was negative in the mice engrafting from all three cases. The successful engraftment of human CLL-Pro PLL and T cell CLL in SCID mice, and the reproducibility of this effect using frozen cells, will provide a model for exploration of disease biology and for investigations of new drugs or combinations that may be useful in the treatment of CLL.