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.

[Acute myeloid leukemia with the morphological characteristics of prolymphocytic leukemia].

To investigate the clinical, cellular morphology, immunophenotype, and cytogenetic characteristics of acute myeloid leukemia (AML) which are very similar to the morphological characteristics of prolymphocytic leukemia (PLL), the morphological features of bone marrow cells from patient were observed by light microscope, the immunophenotypes were detected by flow cytometry, the karyotypes were analyzed by conventional cytogenetic method, the hybridization signals were determined by fluorescence in situ hybridization. The results indicated that the clinical features were in accordance with acute leukemia and the immunophenotyping results showed malignant cells originated from myeloid lineage, while the cytomorphology analysis showed that the blastic cells were more like the lymphoid lineage. Trisomy 8 was found in the patient by cytogenetic study, the patient did not show good response to chemotherapy. In conclusion, acute leukemia has high heterogenicity, which could be defined as AML, but more like lymphocytic origination by morphological study. Immunophenotyping analysis could contribute to the final diagnosis of malignant cells.

Typhlitis as a complication of alemtuzumab therapy.

Alemtuzumab is a humanized monoclonal antibody directed against lymphocytes through the CD-52 receptor, an antigen being found on > 95% of peripheral blood lymphocytes and monocytes, and to a smaller extent on granulocytes. It is an effective immunotherapeutic agent in patients with malignancies such as non-Hodgkin lymphoma, B cell chronic lymphocytic leukemia and T cell pro- lymphocytic leukemia. Adverse side effects are increasingly recognized in patients receiving alemtuzumab, mainly including fever, rigors, nausea/vomiting, skin rash; other severe alemtuzumab-related reactions have also been described, such as lymphopenia and neutropenia leading to both opportunistic (e.g. cytomegalovirus) and non-opportunistic infections. Digestive complications have more rarely been described, i.e.: gastroenteritis and peritonitis. We recently observed a case of particular interest as the patient with T cell prolymphocytic leukaemia treated with alemtuzumab, exhibited symptomatic reactivation of CMV infection and developed subsequently typhlitis.

Cryptococcal immune reconstitution inflammatory syndrome following alemtuzumab therapy.

Alemtuzumab is a lymphocyte ablative agent that may cause susceptibility to severe opportunistic infections similar to those seen in AIDS. Pathogen-specific immune reconstitution syndromes can complicate antiretroviral therapy and immune recovery in HIV-infected patients. We present the first reported case of immune reconstitution syndrome associated with T lymphocyte recovery after alemtuzumab therapy.

Production of vascular endothelial growth factor in T-cell prolymphocytic leukemia.

We describe a 79-year-old man who had massive pleural effusion and a proliferation of prolymphocytic leukemia cells in the peripheral blood, bone marrow, and pleural effusion fluid. Immunophenotyping of leukemia cells revealed either CD3+CD4+CD8-CD25+ or CD3+CD4+CD8+CD25+. The antibody against human T-cell lymphotropic virus type I was negative. A diagnosis of T-PLL was made. The level of VEGF in the plasma or pleural effusion fluid was very high. Moreover, polymerase chain reaction analysis demonstrated an expression of VEGF mRNA in the leukemia cells, indicating a production of VEGF from leukemia cells and its involvement in the pathogenesis of T-PLL.

Powerful strategy for polymerase chain reaction-based clonality assessment in T-cell malignancies Report of the BIOMED-2 Concerted Action BHM4 CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal T-cell receptor (TCR) and immunoglobulin (Ig) gene rearrangements is an important diagnostic tool in mature T-cell neoplasms. However, lack of standardized primers and PCR protocols has hampered comparability of data in previous clonality studies. To obtain reference values for Ig/TCR rearrangement patterns, 19 European laboratories investigated 188 T-cell malignancies belonging to five World Health Organization-defined entities. The TCR/Ig spectrum of each sample was analyzed in duplicate in two different laboratories using the standardized BIOMED-2 PCR multiplex tubes accompanied by international pathology panel review. TCR clonality was detected in 99% (143/145) of all definite cases of T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, peripheral T-cell lymphoma (unspecified) and angioimmunoblastic T-cell lymphoma (AILT), whereas nine of 43 anaplastic large cell lymphomas did not show clonal TCR rearrangements. Combined use of TCRB and TCRG genes revealed two or more clonal signals in 95% of all TCR clonal cases. Ig clonality was mostly restricted to AILT. Our study indicates that the BIOMED-2 multiplex PCR tubes provide a powerful strategy for clonality assessment in T-cell malignancies assisting the firm diagnosis of T-cell neoplasms. The detected TCR gene rearrangements can also be used as PCR targets for monitoring of minimal residual disease.

T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is a rare aggressive post-thymic malignancy with poor response to conventional treatment and short survival. It can readily be distinguished from other T-cell leukemias on the basis of the distinctive morphology, immunophenotype, and cytogenetics. Consistent chromosomal translocations involving the T-cell receptor gene and one of two protooncogenes (TCL-1 and MTCP-1) are seen in the majority of cases and are likely to be involved in the pathogenesis of the disorder. The CD52 antigen is expressed at high density on the malignant T-cells and therapy with alemtuzumab, a humanized IgG1 antibody that targets this antigen, has produced promising results. In relapsed/refractory patients overall and complete response rates have been seen in up to 76% and 60%, respectively. In previously untreated patients, complete remission rates of 100% have been reported. These responses are durable and translate into improved survival for responders. However, relapse is inevitable and strategies using both autologous and allogeneic stem cell transplantation are currently being explored. Additional clinical trials are investigating the use of alemtuzumabin combinations with chemotherapy, either concurrent or sequential. In the future we hope to have a betterunderstanding of how best to integrate these therapeutic approaches to further prolong survival for patients with T-PLL.

Prolymphocytoid transformation of follicular lymphoma with coexpression of CD5 and CD10.

Histologic transformation of follicular lymphoma is usually to a diffuse large B-cell lymphoma. We present a rare example of a histologic transformation of follicular lymphoma manifested by prolymphocytoid morphology and an unusual immunophenotype characterized by coexpression of CD5 and CD10. The transformed prolymphocytoid lymphoma was positive for CD5 and CD10 antigens by both flow cytometry and immunohistochemistry. The case also expressed bcl-2 and bcl-6 proteins, and exhibited t(14;18), consistent with derivation from a pre-existing follicular lymphoma. Polymerase chain reaction analysis of the immunoglobulin kappa light chain genes derived from the follicular lymphoma and prolymphocytoid lymphoma showed identical rearranged bands, suggesting clonal identity of the two neoplasms. The basis for coexpression of CD5 and CD10 remains unclear. Because the preceding low-grade follicular lymphoma was positive only for CD10 and did not express CD5, CD5 expression appears to be an acquired phenomenon accompanying the process of histologic transformation in this particular case. Prolymphocytoid transformation, similar to other histologic forms of transformation of follicular lymphoma, appears to accompany clinical progression of disease.

[Allogeneic bone marrow transplantation for chemotherapy-resistant T-prolymphocytic leukemia].

A 34-year-old female was referred to our hospital for the evaluation of atypical lymphocytosis. Leukocyte count at diagnosis was 17,900/microl with 58% atypical lymphocytes having a convoluted nucleus and prominent nucleoli. Because the leukocyte count increased to 43,600/microl, the patient was treated with 2'deoxycoformycin followed by CHOP combination chemotherapy. However, both treatments failed to achieve remission. We planned an allogeneic bone marrow transplantation from an HLA-matched unrelated donor. The patient was treated with Ara-C and etoposide before conditioning to decrease the high leukemia burden. After administration of total body irradiation (12 Gy in six fractions) and cyclophosphamide (total dose of 120 mg/kg) unmanipulated marrow cells were infused. Under prevention of GVHD by CsA and short-term MTX, leukocyte engraft was prompt at day 16, and acute GVHD grade II was observed. Because 9.4% of residual recipient type T-cells was seen with STR analysis on day 22, we decreased the dose of Cs'A. After the occurrence of mild acute GVHD, the residual T-cell number decreased. The patient is still in complete remission for up to 22 months after BMT. We conclude that allogeneic SCT is effective for the treatment of T-PLL.

CD3-negative, CD20-positive T-cell prolymphocytic leukemia: case report and review of the literature.

We report a case of CD3-negative, CD20-positive T-cell prolymphocytic leukemia (T-PLL). The leukemic cells were of medium-to-large size, mature-looking, and did not have cytoplasmic granules. The leukemic cells were negative for surface CD3, CD2, and CD7 and strongly positive for CD20. T-cell lineage markers such as CD4, CD5, and cytoplasmic CD3 were also positive. A monoclonal rearrangement of the T-cell receptor (TCR) beta chain gene was detected. CD3-negative T-PLL has been reported often, but CD20-positive T-PLL has not. We reviewed seven cases of CD20-positive immature and mature T-cell leukemias, including the present case. Three were immature T-cell leukemias (acute lymphoblastic leukemia), and four were mature T-cell leukemias (granular lymphocytic leukemia, small lymphocytic lymphoma/chronic lymphocytic leukemia, adult T-cell leukemia, and the present case). Splenomegaly was a common feature. However, our case alone had "bright" CD20 expression on the leukemic cells. This is the first report of CD20(+) T-PLL.

CD20 levels determine the in vitro susceptibility to rituximab and complement of B-cell chronic lymphocytic leukemia: further regulation by CD55 and CD59.

Complement-dependent cytotoxicity is thought to be an important mechanism of action of the anti-CD20 monoclonal antibody rituximab. This study investigates the sensitivity of freshly isolated cells obtained from 33 patients with B-cell chronic lymphocytic leukemia (B-CLL), 5 patients with prolymphocytic leukemia (PLL), and 6 patients with mantle cell lymphoma (MCL) to be lysed by rituximab and complement in vitro. The results showed that in B-CLL and PLL, the levels of CD20, measured by standard immunofluorescence or using calibrated beads, correlated linearly with the lytic response (coefficient greater than or equal to 0.9; P <.0001). Furthermore, the correlation remained highly significant when the 6 patients with MCL were included in the analysis (coefficient 0.91; P <.0001), which suggests that CD20 levels primarily determine lysis regardless of diagnostic group. The role of the complement inhibitors CD46, CD55, and CD59 was also investigated. All B-CLL and PLL cells expressed these molecules, but at different levels. CD46 was relatively weak on all samples (mean fluorescence intensity less than 100), whereas CD55 and CD59 showed variability of expression (mean fluorescence intensity 20-1200 and 20-250, respectively). Although CD55 and CD59 levels did not permit prediction of complement susceptibility, the functional block of these inhibitors demonstrated that they play an important role in regulating complement-dependent cytotoxicity. Thus, lysis of poorly responding B-CLL samples was increased 5- to 6-fold after blocking both CD55 and CD59, whereas that of high responders was essentially complete in the presence of a single blocking antibody. These data demonstrate that CD20, CD55, and CD59 are important factors determining the in vitro response to rituximab and complement and indicate potential strategies to improve the clinical response to this biologic therapy.

Typical and atypical chronic lymphocytic leukemia differ clinically and immunophenotypically.

We compared the features of 17 cases of atypical chronic lymphocytic leukemia (aCLL) with those of a clinical control group of 24 cases of CLL. Quantitative flow cytometric data, available for 12 cases, were compared with an immunophenotypic control group of 58 cases using a relative fluorescence indexfor CD5, CD23, CD79b, and surface immunoglobulin light chain (sIg). Compared with the clinical control group, patients with aCLL had a higher mean WBC count and a lower platelet count. Patients with aCLL had a significantly higher probability of disease progression. Compared with an immunophenotypic control group of 58 CLL cases, 12 cases of aCLL demonstrated significantly higher expression of CD23. There was no significant difference in expression of sIg, CD79b, or CD5 between the groups. CD38 expression was noted in only 1 (9%) of 11 tested cases; 2 (18%) of 11 cases had trisomy 12. aCLL can be distinguished from typical CLL morphologically, clinically, and immunophenotypically. Atypical morphologic features in CLL seem to be a marker of aggressive clinical behavior.

Immunophenotype changes and loss of CD52 expression in two patients with relapsed T-cell prolymphocytic leukaemia.

T-cell prolymphocytic leukaemia (T-PLL) is an aggressive disease often resistant to conventional chemotherapy. Long lasting remissions with the monoclonal antibody CAMPATH-1H (anti-CD52) have been documented. We describe two unusual T-PLL patients treated successfully first with CAMPATH-1H in whom, at the time of relapse, the cells underwent a phenotypic switch with loss of CD52 expression. In one of them, cytogenetic analysis demonstrated the same chromosome abnormalities in the cells at diagnosis and relapse. The reasons for the immunophenotypic changes are unknown but it is likely that loss of CD52 antigen expression contributed to the resistance to CAMPATH-1H in one of the patients when re-treated.

T-cell receptor gammadelta T-cell leukemia with the morphology of T-cell prolymphocytic leukemia and a postthymic immunophenotype.

T-cell prolymphocytic leukemia (T-PLL) is a postthymic T-cell neoplasm with a characteristic morphology and heterogeneous immunophenotype. Most cases of T-PLL express membrane T-cell receptors (TCRs) of the alphabeta phenotype. We experienced a 30-year-old man suffering from TCRgammadelta T-cell leukemia with morphology compatible to T-PLL with a postthymic phenotype. He was admitted with skin eruption and pancytopenia. Peripheral blood and bone marrow were occupied with medium-sized lymphocytes, which had moderately condensed chromatin with a single nucleolus and sparse, nongranular basophilic cytoplasm. The immunophenotype was CD1a-, CD2-, CD3+, CD4-, CD5+, CD7+, CD8-, and terminal deoxynucleotidyl transferase negative. Hepatosplenomegaly was absent. He was diagnosed as having T-PLL and was treated with combination chemotherapy. Six months later the leukemic cell became chemoresistant. Although the patient showed transient improvement in response to pentostatin, he died 13 months after the diagnosis. To our knowledge, this is the first case of T-PLL with a TCRgammadelta phenotype.

Expression of the CD11c antigen in B-cell chronic lymphoproliferative disorders.

This study analyzed the expression of the beta2 integrin CD11c in 155 patients with well-characterized B-cell chronic lymphoproliferative disorders: 106 B-cell chronic lymphocytic leukemias (B-CLL), 21 hairy cell leukemias (HCL), 9 B-cell prolymphocytic leukemias (PLL) and 19 low grade non-Hodgkin's lymphomas (NHL) in leukemic phase. CD11c was expressed in 100% of patients with HCL and B-PLL, while in B-CLL and NHL it was expressed in only 49 and 57%, respectively. Furthermore, in B-CLL the expression of CD11c was found mainly in patients with early stage of disease. In addition, when the fluorescence intensity of CD11c, calculated by MFI, was evaluated, it proved significantly higher in HCL and B-PLL compared to the values recorded in B-CLL and NHL (325 and 387 vs 34 and 56, respectively) (p < 0.05). Our results demonstrate that the evaluation of CD11c, both in terms of overall positivity and of fluorescence intensity, represents an additional useful parameter for a more precise differential diagnosis within the spectrum of B-cell chronic lymphoproliferative disorders.

T-cell prolymphocytic leukaemia: does the expression of CD8+ phenotype justify the identification of a new subtype? Description of two cases and review of the literature.

T-cell chronic lymphocytic leukaemia (T-CLL) has recently been reclassified under the heading of T-cell prolymphocytic leukaemia (T-PLL) because of its unfavourable clinical course, independently of the morphologic features. This rare neoplasm usually shows CD4+/CD8- phenotype. Herein we report on two cases of T-PLL with CD8 expression that correspond to a possible variant of the disease first proposed by Hui et al. in 1987. These cases presented with malignant cells showing immunophenotypic features that can be easily identified and distinguished from other peripheral T-cell leukemias. However, the total number of cases studied is inadequate for defining a discrete clinico-pathologic entity with characteristic clinical features and cytogenetical abnormalities. An international collaboration in which tissue from similar cases is referred to a central pathologist for immunophenotyping and cytogenetical study, and clinical data are centrally compiled, may assist in defining this rare malady as a discrete clinico-pathologic entity.

B-cell prolymphocytic leukemia: a survey of 35 patients emphasizing heterogeneity, prognostic factors and evidence for a group with an indolent course.

We report a retrospective survey of 35 patients (18 males and 17 females) with B-Prolymphocytic leukemia (B-PLL) followed for a median of 63 months. Twelve patients fulfilled Galton's original clinical and hematological criteria, presented with prominent splenomegaly and hyperleukocytosis and showed rapid progression soon after diagnosis. Twelve cases with gradually increasing spleen size and prolymphocyte count had an indolent course. Seven of this group are alive 68 to 164 months after diagnosis, whereas five died from causes unrelated to PLL. Eleven patients who never developed impressive leukocytosis had a variable prognosis. In the group of 17 patients treated with chlorambucil and prednisone (CP) or cyclophosphamide, vincristine, prednisone (COP) 8 achieved a partial remission (PR) with a median response of 32 months. In the group of six cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) treated patients one achieved a complete remission and two PR (median response was maintained for 30 months). Three patients treated with 2CdA achieved good PR. Six patients remained untreated. Median survival was 65 months and the probability of overall survival for 3, 5, and 10 years was 63%, 56% and 35%, respectively. Anemia < 11 g/dl and lymphocytosis > 100 x 10(9)/l were predictors of shorter survival in this group of patients. Age over 70, gender, B-symptoms at presentation, spleen size, thrombocytopenia, low IgG and complement levels, presence of paraproteinemia and the pattern of bone marrow infiltrate were not significant. Our findings show that all B-PLL may not have such a poor prognosis as described in earlier reports. The existence of prior symptoms evolving gradually after years to obvious PLL and cases with mild prolymphocytosis could possibly lead to underdiagnosis of the entity. Identification and follow-up of such cases may suggest a different natural history, variable prognostic features and different survival curves for B-PLL patients. In the light of the above, we suggest that the therapeutic approach for B-PLL should always relate to the severity of the disease.

Indolent course as a relatively frequent presentation in T-prolymphocytic leukaemia. Groupe Français d'Hématologie Cellulaire.

T-prolymphocytic leukaemia (T-PLL) is a rare disorder with a poor outcome. Presentation features were studied in 78 T-PLL cases. Although 53 patients (group A) presented with typical progressive disease including rapidly increasing leucocytosis. 25 patients (group B) experienced an initial indolent clinical course with stable moderate leucocytosis. The morphology and antigenic profile of abnormal cells were similar in both groups, except for a lower incidence of CD45RO+ CD45RA- pattern in group B. A high incidence of inv(14)(q11;q32), t(14;14)(q11;q32) and i(8)(q10) chromosomal abnormalities were found in both groups. After an initial indolent phase (median 33 months; 6-103 months), 16 group B patients progressed to an aggressive stage with clinical and laboratory features similar to group A. Moreover, median survival after progression was short in both groups. In conclusion, T-PLL may start as an indolent disease similar to that reported in ataxia telangectasia. In this rare genetic disorder, some patients develop stable T-cell clones which progress toward T-PLL-like leukaemia. Moreover, ATM gene mutations have been reported in T-PLL. Thus, both diseases are likely to be closely related.

Levels of expression of CD19 and CD20 in chronic B cell leukaemias.

AIMS: To investigate whether the antigen levels of the B cell lineage markers CD19 and CD20 can distinguish between normal and neoplastic B cells or characterise distinct expression patterns among the chronic B cell leukaemias. METHODS: Peripheral blood cells from 70 patients with B cell disorders and 17 healthy donors were analysed by quantitative flow cytometry. Direct immunofluorescence staining was performed with phycoerythrin conjugated CD19 and CD20 monoclonal antibodies. Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity (MFI) values into number of antigen molecules/cell, expressed as antibody binding capacity (ABC). RESULTS: CD19 and CD20 ABC values in leukaemic B cells differed from those of normal blood B lymphocytes. The results identified distinct profiles of CD19 and CD20 expression in the various types of B cell leukaemias. In all leukaemias studied except hairy cell leukaemia (HCL), CD19 expression was significantly lower than the mean (SD) value in normal B cells (22 (7) x 10(3) molecules/cell), as follows: chronic lymphocytic leukaemia (CLL), 13 (7) x 10(3); B prolymphocytic leukaemia (B-PLL), 16 (9) x 10(3); splenic lymphoma with villous lymphocytes (SLVL), 15 (11) x 10(3); mantle cell lymphoma (MCL), 10 (7) x 10(3). In HCL there was strong CD19 expression (38 (16) x 10(3)). In contrast, the level of expression of membrane CD20 was higher than the mean (SD) value in normal B cells (94 (16) x 10(3) molecules/cell) in MCL (123 (51) x 10(3)); B-PLL (129 (47) x 10(3)); SLVL (167 (72) x 10(3)); and HCL (312 (110) x 10(3)); while it was significantly lower (65 (11) x 10(3)) in CLL compared with normal B cells and the other B cell leukaemias. CONCLUSIONS: Quantitative determination of CD19 and CD20 may provide useful diagnostic information for the study of B lymphoproliferative disorders.