B-cell Prolymphocytic Leukemia Research Articles

MLT/MALT1 involving translocations and amplifications are important mechanisms in the pathogenesis of non-Hodgkin’s lymphoma

6582 Background: The t(11;18)(q21;q21) and the t(14;18)(q32;q21) involving the MLT/MALT1 gene are the main structural abnormalities in extranodal marginal zone lymphoma (MZL). In addition, amplification of MLT/MALT1 has been proposed as a pathogenetic mechanism in NHL. Amplifications in 18q21 frequently involve the BCL2 gene, which lies about 5Mb telomeric to MLT/MALT1. However, a recent study described amplification of MLT/MALT1 without BCL2 coamplification, suggesting that MLT/MALT1 and BCL2 are independent targets of amplification in NHL. Methods: In order to screen for translocations and amplifications of MLT/MALT1, we have analyzed 214 NHL with a novel FISH assay using probes flanking the MLT/MALT1 gene (PACs 117B5 and 59N7). The assay was applied to 91 MALT lymphomas, 19 splenic MZL, 17 nodal MZL, 17 follicular lymphomas (FL), 8 mantle cell lymphomas (MCL), 15 chronic lymphocytic leukemias (CLL), 3 B-cell prolymphocytic leukemias (PLL), 9 Burkitt’s lymphomas (BL), 30 diffuse large B-cell lymphomas (DLBCL), 5 T-cell NHL, and 10 cell lines (7 BL, 1 transformed MCL, 1 transformed FL, and 1 MZL). Results: In 20 MALT lymphomas (22% of MALT lymphomas) a translocation involving MLT/MALT1 was detected. FISH analyses with API2 and IGH specific probes revealed the t(11;18) in 13 cases and the t(14;18) in 7 cases. Amplification of MLT/MALT1 was observed in 2 DLBCL, the MZL cell line SSK41, and the BL cell line NAMALWA. Further FISH analyses showed a concomitant amplification of BCL2 in the 2 DLBCL but in SSK41 and NAMALWA an amplification of MLT/MALT1 without BCL2 involvement was found. Trisomy or polysomy of the chromosomal region 18q21 was found in 13 MZL, 9 DLBCL, 8 FL, 1 MCL, 1 CLL, and the transformed MCL cell line Granta-519. In addition, a heterozygous deletion of MLT/MALT1 was detected in two MALT lymphomas of the stomach. Conclusions: 1) MLT/MALT1 associated translocations occur exclusively in extranodal MALT lymphomas and represent either the t(11;18) or the t(14;18); 2) true amplifications of MLT/MALT1 occur in some aggressive NHL and some transformed cell lines; and 3) trisomy or polysomy of 18q21 are seen in different subtypes of B-cell NHL and cell lines. No significant financial relationships to disclose.

Allogeneic peripheral stem-cell transplantation with reduced-intensity conditioning regimen in refractory primary B-cell prolymphocytic leukemia: a long-term follow-up

Allogeneic peripheral stem-cell transplantation with reduced-intensity conditioning regimen in refractory primary B-cell prolymphocytic leukemia: a long-term follow-up

Prolymphocytic leukemia

Prolymphocytic leukemia is a rare chronic lymphoproliferative disorder that includes two subtypes, B cell and T cell, each with its own distinct clinical, laboratory and pathological features. T-cell prolymphocytic leukemia has an aggressive course with short median survival and poor response to chemotherapy. With the use of the purine analogue pentostatin more than half of patients will have a major response and a minority will have a complete remission, usually lasting months. With the introduction of alemtuzumab, most patients who progressed despite treatment with pentostatin had a major response with a complete remission rate higher than that obtained with pentostatin when used as a first line. Unfortunately, progression still follows shortly. We recommend alemtuzumab as initial therapy and offer stem cell transplant (SCT) to selected young, healthy patients who respond. Although B-cell prolymphocytic leukemia is also a progressive disease, some patients can achieve a prolonged progression-free-survival with fludarabine. Patients presenting with massive splenomegaly may be effectively palliated with splenic irradiation or splenectomy. Rituximab is a promising agent and further investigations are warranted to better define its role in treatment of this disorder.

Clinicopathologic study of 369 B-cell non-Hodgkin lymphoma cases, with reference to the 2001 World Health Organization classification of lymphoid neoplasms

To describe the relative frequency, morphologic features, immunophenotype and clinical data of different types of B-cell non-Hodgkin lymphoma (B-NHL) and to evaluate the practical application of the 2001 World Health Organization (WHO) classification of lymphoid neoplasms. 369 documented cases of B-NHL were further subtyped according to the 2001 WHO classification of lymphoid neoplasms, on the basis of hematoxylin and eosin staining, immunohistochemistry and in-situ hybridization techniques. Amongst the 369 cases of B-NHL studied, 353 cases could be further classified into 11 subtypes. Diffuse large B-cell lymphoma, extranodal marginal zone lymphoma and follicular lymphoma were the commonest subtypes, accounting for 51.2% (189 cases), 14.9% (55 cases) and 10.6% (39 cases) of all cases respectively. Tumors in lymph nodes were seen in 158 cases (42.8%) and in extra node in 211 cases (57.2%). B-cell prolymphocytic leukemia and hairy cell leukemia were not identified. When comparing the diagnosis based on morphologic examination alone with the diagnosis based on both morphology and immunophenotype, there was a 80% concordance rate. Immunohistochemical study was helpful in reaching the correct diagnosis in many cases and could improve the overall diagnostic accuracy by about 20%. Amongst cases of B-NHL, diffuse large B-cell lymphoma is the commonest subtype, followed by MALToma and follicular lymphoma. While morphologic examination forms the basis for lymphoma diagnosis, immunohistochemical study also plays an important role in further subtyping. A combination of both modalities are sufficient for arriving at an accurate diagnosis in most cases of B-NHL, in keeping with the recommendation of the 2001 WHO classification of lymphoid neoplasms.

Detection of CD5 in B-cell chronic lymphoproliferative diseases by flow cytometry: a strong expression in B-cell chronic lymphocytic leukemia

PURPOSE: CD5 is a T cell marker, aberrantly express in B cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL). Other chronic B cell malignancies including hairy cell leukemia (HCL) and B cell prolymphocytic leukemia (B-PLL) are CD5 negative or express this antigen in a weak way. In this study, CD5 expression was investigated in leukemic cells from 42 patients with chronic B cell lymphoproliferative disease. METHODS: We studied the CD5 expression in leukemic cells from 42 patients with chronic B-cell malignancies by flow cytometry. Demographic features such as age, sex and clinical date were also analyzed. RESULTS: There were 22 males and 20 females. The immunophenotyping showed that 35 cases were B-CLL, 3 B-PLL and HCL and one patient was MCL. CD5 expression was present in all B-CLL and MCL. Low expression of CD5 was observed in one patient with B-PLL and negative in all cases of HCL. CONCLUSION: Our date demonstrated that CD5 expression can help distinguish among B-CLL from HCL and B-PLL, but is similar expressed in MCL.

B- Prolymphocytic Leukemia Shows Heterogeneous IgVH Mutational Status and Expression of ZAP-70 and CD38.

B-cell prolymphocytic leukemia (B-PLL) is a rare lymphoid leukemia with an aggressive course. Diagnosis is based on morphology (>55% peripheral blood prolymphocytes), immunophenotype and histology when available, as there is no cytogenetic hallmark for this disease. There are scanty data regarding the mutational status of the variable region of immunoglobulin heavy chain (IgVH) genes and ZAP-70 expression in B-PLL. Davi et al ( Blood 1996; 88:970–6) showed that 9 of 11 B-PLL cases had mutated IgVH genes (<98% homology) with a preferential use of the V3-23 and V4-34 genes, accounting for more than half of the B-PLL repertoire.We analyzed the mutational status of IgVH genes and ZAP-70 expression in 16 B-PLL cases and correlated the findings with clinical and biological features such as CD38 expression, chromosome abnormalities detected by FISH and overall survival. There were 7 females and 9 males, with a median age of 71 years (range 42–91). All except one were untreated at the time of the study. The diagnosis was established by peripheral blood morphology and immunophenotype (CLL score ≤ 3) in all cases and bone marrow and spleen histology in 6. Mantle cell lymphoma was excluded by the absence of t(11;14) by FISH in 12 out of 12 cases tested, including 4 which were CD5 positive. IgVH mutational status was performed by PCR (cut off >98% homology) and ZAP-70 expression was evaluated by 4-colour flow cytometry. Seven of 13 cases (54%) in which IgVH mutational status was evaluable, showed unmutated IgVH genes (99.7–100% homology) and 6 (46%) mutated IgVH genes (90.1–97.4% homology). V3-23 and V4-34 genes were used in one third of the cases. ZAP-70 was expressed (≥ 20% CD19+ cells) in 7 of 10 evaluable cases and CD38 (≥ 30% of CD19+ cells) in 7 of 11 cases. FISH analysis showed delp53 in 6/13 (46%) and del(13)(q14) in 3/11 (27%) cases; trisomy 12 was absent in 7 cases tested. Correlation between IgVH status and other features is shown in the table. There was a prevalence of delp53 (83%) among the unmutated group, while ZAP-70 expression did not correlate with IgVH mutational status. Median overall survival was 41 months. Six patients (4 mutated, 2 unmutated) are alive at 50 months (range 8-112) from diagnosis, 9 (5 unmutated and 2 mutated) died at 17 months (range 0–55) and 1 was lost to follow-up at 4 months. The number of patients is too small to conclude whether IgVH mutational status and/or ZAP-70 expression have a significant impact on survival. In summary, B-PLL is heterogeneous with respect to IgVH mutational status as in most chronic B-cell disorders, with equal representation of unmutated and mutated cases. P53 deletion is more common in the unmutated subgroup and most of the cases express ZAP-70 and CD38.Laboratory findings according to mutational statusZAP (>/=20%)CD38 (>/=30%)Del p53Del (13)(q14)Trisomy 12Unmutated (7)4/51/45/62/50/3Mutated (6)2/34/51/50/40/4

The role of allogeneic transplantation in non‐Hodgkin's lymphoma

The evolution of combination chemotherapy regimens, combined with improvements in supportive care, has incrementally improved survival outcomes for patients with non-Hodgkin's lymphomas (NHL). Although 40-60% of younger patients with diffuse large cell lymphoma can now expect to be cured, significant numbers will either fail to achieve a remission or relapse after attaining a remission. In addition, certain histological subtypes are associated with particularly poor prognoses with combination chemotherapy alone (e.g. mantle cell lymphoma, B-cell prolymphocytic leukaemia). Relatively few of these patients can achieve long-term responses. Other NHL subtypes, whilst associated with more favourable prognoses in terms of overall survival, are rarely, if ever, cured (e.g. most low grade NHL including follicular lymphoma, chronic lymphocytic leukaemia and small lymphocytic lymphoma). For these reasons dose escalation and allogeneic transplantation have been investigated as potential ways of improving outcome, although this has mainly been in the setting of advanced disease. Any possible benefits have frequently been out-weighed by procedural morbidity and mortality. The parallel development of transplantation approaches that limit procedural toxicity along with advances in supportive care require that the role of allogeneic haematopoietic stem cell transplantation in the management of lymphoma be re-evaluated.

Translocation (8;14)(q24;q32) as the sole cytogenetic abnormality in B-cell prolymphocytic leukemia

B-cell prolymphocytic leukemia is a relatively rare lymphoproliferative disorder. No specific cytogenetic abnormality has yet been associated with it. The most common translocation reported in patients with this disease is t(11;14)(q13;q32). We describe the case of a patient with B-cell prolymphocytic leukemia and a hitherto unreported genetic translocation (8;14)(q24;q32) as the sole genetic abnormality, classically seen in patients with B-cell acute lymphoblastic leukemia/Burkitt lymphoma. This patient presented with an asymptomatic leukocytosis and splenomegaly. Her marrow showed lymphoid hyperplasia, with immunophenotyping consistent with prolymphocytic leukemia; however, t(8;14)(q24;q32) was the only cytogenetic aberration with both standard karyotyping and fluorescence in situ hybridization analysis.

Level of CD 20-expression and Efficacy of Rituximab Treatment in Patients with Resistant or Relapsing B-cell Prolymphocytic Leukemia and B-cell Chronic Lymphocytic Leukemia

Rituximab (IDEC C2B8) is a chimeric human-mouse monoclonal anti-CD20 antibody that has been proven to be effective for the treatment of patients with CD20 positive leukemia and lymphoma. The level of CD20-expression in patients with B-cell chronic lymphocytic leukemia (B-CLL) is low in comparison to other B-cell lymphomas and normal B-cells. Previous experience with rituximab treatment in small series of patients with B-CLL suggest that it is less effective in B-CLL than in follicular lymphomas. We analyzed the correlation between CD20-expression level and efficacy of rituximab treatment in eight patients with refractory or relapsed B-CLL and two patients with B-cell prolymphocytic leukemia (B-PLL). We could not identify any correlation between CD20-expression and efficacy of rituximab treatment.

Mature B-cell leukemias with more than 55% prolymphocytes. A heterogeneous group that includes an unusual variant of mantle cell lymphoma.

We studied 20 cases of mature B-cell leukemia with more than 55% prolymphocytes in peripheral blood or bone marrow, fulfilling the French-American-British criteria for B-cell prolymphocytic leukemia (PLL). Cases segregated into 3 groups: de novo PLL, 6; PLL occurring in patients with a previous well-established diagnosis of chronic lymphocytic leukemia (PLL-HxCLL), 10; and t(11;14)(q13;q32)-positive neoplasms, 4. All cases expressed monotypic immunoglobulin light chain, and most were positive for CD5. All t(11;14)-positive neoplasms were CD23- and uniquely positive for cyclin D1. Cytogenetic abnormalities were present in 19; in all 19, the karyotype was complex, indicating clonal evolution and genomic instability. The most frequent cytogenetic abnormality in de novo PLL involved chromosome 7 in 4 cases. Trisomy 12 or add(12p) was present in 4 cases of PLL-HxCLL. We conclude that mature B-cell leukemias with more than 55% prolymphocytes are a heterogeneous group that includes t(11;14)-positive neoplasms, which we suggest are best classified as mantle cell lymphoma. We also suggest that prolymphocytic morphologic features are a common end-stage of transformation for several B-cell neoplasms.

B-Cell Prolymphocytic Leukemia

B-cell prolymphocytic leukemia (B-PLL) is a rare lymphoproliferative disorder with clinical, morphologic, and immunophenotypic features that overlap with other mature B-cell leukemias and lymphomas. Patients with B-PLL typically have marked splenomegaly and leukocytosis, have no significant lymphadenopathy, and have a clinically aggressive course and poor prognosis. In peripheral blood (PB) and bone marrow (BM) aspirate smears, numerous large prolymphocytes with a round to oval nucleus and a single large nucleolus are observed. The French-American-British group recommends requiring that >55% prolymphocytes be present in the PB (or BM) to establish the diagnosis of B-PLL. In BM biopsy sections, B-PLL extensively replaces the medullary space in a pattern that is diffuse, interstitial, or both. Immunophenotypic studies have shown that B-PLL patients express monotypic immunoglobulin light chain (of bright intensity) and pan-B-cell antigens, and variably express the CD5 antigen. Cytogenetic studies have reported several chromosomal abnormalities, which are usually complex. The differential diagnosis of B-PLL is reviewed.

Frequent deletions at 11q23 and 13q14 in B cell prolymphocytic leukemia (B-PLL).

Deletions of the long arm of chromosomes 11 and 13 are the most frequent structural chromosome aberrations in various types of lymphoproliferative disorders. However, these regions have not been studied so far in B cell prolymphocytic leukemia (B-PLL). We have investigated the incidence of 13q deletions in 18 B-PLL cases by fluorescence in situ hybridization (FISH), using molecular probes for the RB1 and D13S25 loci. Chromosome 11q deletions were evaluated by FISH using the yeast artificial chromosome (YAC) clone 755b11 from the chromosome 11q22.3-q23.1 region, which has been previously shown to be deleted in 20% of cases of chronic lymphocytic leukemia. Chromosome 11q23 deletions were found in 7/18 (39%) cases of B-PLL. Monoallelic loss of RB1, D13S25 and BRCA2 was present in 10/18 (55%), 6/18 (33%) and 3/18 (16%) of the cases, respectively. All the cases with D13S25 and BRCA2 deletion showed RB1 loss. Deletions of 13q14 and 11q23 are frequent chromosome aberrations in B-PLL and, in contrast to CLL, there is a preferential loss of RB1 with respect to the D13S25 locus suggesting that allelic loss of the RB1 gene may play a role in the pathogenesis of B-PLL.

Expression of the CD11c Antigen in B-Cell Chronic Lymphoproliferative Disorders

This study analyzed the expression of the β2 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.

Pentostatin in T-cell malignancies – a phase II trial of the EORTC

Within this phase II EORTC trial, we have investigated the safety and efficacy of pentostatin in lymphoid malignancies. We have previously reported the results in T- and B-cell prolymphocytic leukemia, B-cell chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia. This report focuses on the outcome in T-cell malignancies: T-CLL, Sézary syndrome (Sézary), mycosis fungoides (MF) and T-zone lymphoma (TZL). Of the 92 patients with these diagnoses enrolled, 76 were evaluable for response and toxicity, i.e., 25 of 28 with T-CLL, 21 of 26 with Sézary, 22 of 26 with MF, and 8 of 12 with TZL. All patients had progressive and advanced disease. Pentostatin was administered at a dosage of 4 mg/m2 every week for the first 3 weeks, then 4 mg/m2 every 14 days for another 6 weeks, followed by maintenance therapy of 4 mg/m2 monthly for a maximum of 6 months. Response rates (complete and partial responses) in patients with Sézary (n = 22) or MF (n = 21) were 33% and 23%, respectively, and in patients with T-CLL (n = 21) or TZL (n = 8) 8% and 25%, respectively. Sixteen (21%) patients died during the first ten weeks of treatment: twelve of progressive disease, two of infectious complications with progressive disease, one of myocard infarction and one of renal failure related to administration of i.v. contrast. Major toxicity (grade 3-4) included infection in 11% of patients, nausea/vomiting in 4%, diarrhea in 3%. Hematologic toxicity was mild to non-existent. We conclude that pentostatin is active in Sézary and MF but showed marginal activity in T-CLL or TZL. Toxicities are mild to moderate at the dose schedule administered. Due to its relatively specific lympholytic effect and its favorable toxicity spectrum, pentostatin might be especially useful for the palliative treatment of T-cell malignancies.

FISH analysis for BCL-1 rearrangements and trisomy 12 helps the diagnosis of atypical B cell leukaemias.

We have investigated the diagnostic value of fluorescence in situ hybridisation (FISH) to detect t(11;14) and trisomy 12 in 53 cases with a B cell leukaemia difficult to classify on clinical and laboratory grounds. These cases were initially diagnosed by morphology and immunophenotype and in 33 of them, on tissue histology, as follows: chronic lymphocytic leukaemia (CLL), 20, 18 of them with atypical features; B cell prolymphocytic leukaemia (B-PLL), two; mantle-cell lymphoma (MCL), 15; splenic lymphoma with villous lymphocytes (SLVL), five; lymphoplasmacytic lymphoma, six; follicular lymphoma, one and, four cases remained unclassifiable. FISH demonstrated BCL-1 rearrangement in the circulating cells from 15 cases classified as: MCL (10), atypical CLL (three) and B-PLL (two). A definitive diagnosis of MCL was made on review of the spleen histology in one out of the three atypical CLL with BCL-1 rearrangement. Trisomy 12 was detected in eight cases which included four atypical CLL, one typical CLL, two MCL and one unspecified B cell lymphoma by histology and morphology. One of the MCL had both trisomy 12 and BCL-1 rearrangement and the other was CD5+, CD23+ and had a CLL score of 3, suggesting the latter diagnosis. Our findings demonstrate that FISH analysis is useful to clarify the nature of the disease in patients presenting with a B cell leukaemia in which the diagnosis is difficult by conventional methods. FISH established with certainty the diagnosis of MCL by showing BCL-1 rearrangement in over two-thirds of cases in which this was suspected, including blastoid forms, and confirmed the diagnosis of most cases of atypical CLL.

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 × 109/1 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.

5′ Region and Exon 7 Mutations of the TP53 Gene in Two Cases of B-Cell Prolymphocytic Leukemia

We previously found that cases of typical B-chronic lymphocytic leukemia (CLL), atypical B-CLL with t(11;14) and mantle cell lymphomas characterized by rapid progression of the disease and resistance to therapy, had mutations of the TP53 gene. In this paper, abnormalities of the TP53 gene were investigated in two cases of prolymphocytic leukemia, one with t(11;14)(q13;q32), evolving from atypical CLL (patient 1), and one presenting as a de novo condition (patient 2). TP53 DNA was investigated by Southern blot and PCR-SSCP analysis, and TP53 expression was investigated by Northern blot analysis and immunocytochemistry. C-MYC and BCL-1/PRAD1 gene expression were also investigated. Restriction enzyme analysis of TP53 DNA in patient 1 showed alteration of fragments including exon I and intron I, and, in both patients, a specific loss of TP53 DNA. In patient 2, PCR direct sequencing showed in exon VII a 9 bp deletion including codons 252–254. In patient 1, TP53 RNA and protein were not found, indicating that the unusual 5′ rearrangement has affected TP53 gene expression. By contrast, patient 2 exhibited detectable TP53 RNA and protein. Detectable but weak BCL-1/PRAD1 RNA was present in both patients, whereas C-MYC RNA expression was clearly present only in case 1. The presence of TP53 hemizygous mutations in both patients suggests that TP53 abnormalities may be important in the pathogenesis of prolymphocytic leukemia (PLL), and may possibly account for the frequent resistance to therapy observed in this disease.

Cytogenetic Abnormalities in Three Patients with B-Cell Prolymphocytic Leukemia

We present the cytological features, conventional cytogenetics, and in situ hybridization (ISH) findings of three cases of B-cell prolymphocytic leukemia (B-PLL). The diagnosis was made according to the French-American-British (FAB) criteria. We considered a diagnosis of B-PLL when a predominance (>50%) of lymphoid cells with coarse chromatin but prominent central nucleoli and more abundant cytoplasm than typical chronic lymphocytic leukemia (CLL) cells were present. B-PLL express strong SIg, B-cell antigens, and reactivity with the monoclonal antibody FMC7. Chromosome analysis was carried out on lymphoid cells from peripheral blood and, in one patient, from lymph node. The phytohemagglutinin (PHA) mitogen was used. ISH was performed with two types of probes: the biotin-labeled chromosome 12-specific alpha satellite DNA probe to detect trisomy 12, and biotin-labeled libraries of whole chromosomes 1, 7, and 14. Clonal chromosome abnormalities were found in all three patients; in one, a complex karyotype was observed. The most frequent recurrent abnormality was trisomy 12. Our results suggest that PLL usually presents with cytogenetic abnormalities. The finding of translocation (11;14) is noteworthy; chromosomes 1 and 3 are also involved.

In Vivo Binding of Mouse IgG Via Polyreactive Surface IgM Abrogates Progressive Lymphocytosis in Prolymphocytic Leukemia

Surface IgM expressed by malignant CD5+ B-cells from patients with B-chronic lymphocytic leukemia (B-CLL) has previously been shown to bind mouse Ig in what appears to be an example of polyreactive antigen-binding activity. This report demonstrates the in vitro and in vivo binding of mouse Ig to the surface of malignant B-cells from a patient with B-cell prolymphocytic leukemia (B-PLL). In vitro studies showed that K121, a mouse monoclonal antibody, bound to the B-PLL cells via the same low-affinity binding interaction demonstrated to occur between mouse Ig and surface IgM expressed by B-CLL cells rather than in the conventional sense against a specific antigen via its antigen-binding site. With the view to using this phenomenon to target malignant B-cells, it was important to determine whether the low- affinity interaction also occurred in vivo. Infusions of K121 totalling 286 mg were administered to a B-PLL patient over 7 days. Binding of K121 to circulating B-PLL cells was demonstrable after the administration of 36 mg of antibody and was preceded by the appearance of free antibody in the serum. Throughout the period of the infusion, the rapid rise in the peripheral blood white cell count normally observed after leukopheresis was abrogated. However, the count rose markedly after cessation of the antibody infusion in parallel with a decrease in both free and cell-bound K121. There were no observable side effects and no host immune response to either species specific or idiotypic determinants on the mouse Ig was detected. The in vivo binding of mouse Ig together with the previous in vitro data suggest the potential for a novel targeting mechanism using a region of the mouse Ig molecule to target polyreactive Ig expressed by malignant cells in B-CLL and B-PLL.

P53 Abnormalities in B-Cell Prolymphocytic Leukemia

AbstractB-cell prolymphocytic leukemia (B-PLL) is an aggressive disorder of mature B cells with distinct clinical and pathologic features. To determine the incidence of abnormalities of p53, we analyzed 19 cases of B-PLL by DNA blot to assess loss of heterozygosity (LOH) at 17p13.3, by immunocytochemistry to assess p53 expression, and by direct DNA sequencing of polymerase chain reaction-amplified exons 5 to 9 of the p53 gene. LOH was detected in 10 of 19 (53%) cases, p53 expression was detected in 8 of 17 (47%), and p53 mutations were detected in 10 of 19 (53%) cases. The pattern of mutations was distinct from that observed in other B-cell malignancies. Six cases exhibited missense mutations; 4 were transversions and 2 were transitions. The G:C → A:T transition at cathepsin G dinucleotides commonly reported in p53 mutations in chronic lymphocytic leukemia (CLL) and other hematologic malignancies was observed in only 1 case of B-PLL. Three cases exhibited deletions (ranging from 3 to 35 bp in length) and one case exhibited a 2-bp insertion. In 1 case, a 27-bp deletion resulted in the expression of a p53 protein lacking 9 amino acids from the DNA binding region. All samples with p53 mutation showed loss of germline p53 sequences. However, 3 of 10 showed no LOH by Southern blot, indicating a localized deletion around the p53 locus at 17p13.1. Five of the 10 cases with p53 mutation exhibited detectable p53 expression, including 4 cases with p53missense mutation and 1 case with deletion. Two of 7 cases with no detectable mutation of p53 nevertheless overexpressed p53. Therefore, there was no correlation between protein expression and p53mutation in B-PLL. Our data indicate that the overall abnormalities of p53 occurred in 14 of 19 (75%) cases of B-PLL. The frequency of p53 mutation (53%) in B-PLL is the highest reported in B-cell malignancies and may be responsible for the frequent resistance to therapy of this disease. In addition, the pattern of p53 mutation was different from that observed in CLL and other hematologic malignancies and may indicate that a distinct pathogenic mechanism operates in B-PLL.