Assessment of fludarabine plus cyclophosphamide for patients with chronic lymphocytic leukaemia (the LRF CLL4 Trial): a randomised controlled trial.

BACKGROUND: Previous studies of patients with chronic lymphocytic leukaemia reported high response rates to fludarabine combined with cyclophosphamide. We aimed to establish whether this treatment combination provided greater survival benefit than did chlorambucil or fludarabine. METHODS: 777 patients with chronic lymphocytic leukaemia requiring treatment were randomly assigned to fludarabine (n=194) or fludarabine plus cyclophosphamide (196) for six courses, or chlorambucil (387) for 12 courses. The primary endpoint was overall survival, with secondary endpoints of response rates, progression-free survival, toxic effects, and quality of life. Analysis was by intention to treat. This study is registered as an International Standard Randomised Controlled Trial, number NCT 58585610. FINDINGS: There was no significant difference in overall survival between patients given fludarabine plus cyclophosphamide, fludarabine, or chlorambucil. Complete and overall response rates were better with fludarabine plus cyclophosphamide than with fludarabine (complete response rate 38%vs 15%, respectively; overall response rate 94%vs 80%, respectively; p<0.0001 for both comparisons), which were in turn better than with chlorambucil (complete response rate 7%, overall response rate 72%; p=0.006 and 0.04, respectively). Progression-free survival at 5 years was significantly better with fludarabine plus cyclophosphamide (36%) than with fludarabine (10%) or chlorambucil (10%; p<0.00005). Fludarabine plus cyclophosphamide was the best combination for all ages, including patients older than 70 years, and in prognostic groups defined by immunoglobulin heavy chain gene (V(H)) mutation status and cytogenetics, which were tested in 533 and 579 cases, respectively. Patients had more neutropenia and days in hospital with fludarabine plus cyclophosphamide, or fludarabine, than with chlorambucil. There was less haemolytic anaemia with fludarabine plus cyclophosphamide (5%) than with fludarabine (11%) or chlorambucil (12%). Quality of life was better for responders, but preliminary analyses showed no significant difference between treatments. A meta-analysis of these data and those of two published phase III trials showed a consistent benefit for the fludarabine plus cyclophosphamide regimen in terms of progression-free survival. INTERPRETATION: Fludarabine plus cyclophosphamide should now become the standard treatment for chronic lymphocytic leukaemia and the basis for new protocols that incorporate monoclonal antibodies.

Cytogenetic abnormalities additional to t(11;14) correlate with clinical features in leukaemic presentation of mantle cell lymphoma, and may influence prognosis: a study of 60 cases by FISH.

Mantle cell lymphoma (MCL), characterised by t(11;14)(q13;q32), has a poor prognosis. Many cases have additional cytogenetic abnormalities, and often have a complex karyotype. Fluorescence in situ hybridisation (FISH) was used to study 60 cases with leukaemic presentation of MCL, to determine the frequency, clinical correlations and prognostic impact of a panel of molecular cytogenetic abnormalities: 17p13 (TP53 locus), 13q14, 12 p11.1-q11 (centromere), 6q21 and 11q23. CD38 expression, of prognostic value in chronic lymphocytic leukaemia (CLL), was also studied, and correlations with clinical and cytogenetic abnormalities sought. Eighty per cent of cases had at least one abnormality in addition to t(11;14). Deletions at 17p13 (TP53) and 13q14 were most frequent and involved the majority of the leukaemic clone. Cases with TP53 deletion were more likely to have splenomegaly and marked leucocytosis (>30 x 10(9)/l), and less likely to have lymphadenopathy than those without deletion. Deletions at 11q23 and 6q21 were associated with extranodal disease. 13q14 and 11q23 deletions showed a trend towards worse prognosis by univariate analysis. In multivariate analysis, deletions at 13q14 and 6q21 were independent predictors of poor outcome. Deletion at 17p13 did not show prognostic impact in this series. CD38, positive in two-thirds of cases, was associated with male gender and nodal disease but not with any cytogenetic abnormality, or with survival.

IgVH genes mutation and usage, ZAP-70 and CD38 expression provide new insights on B-cell prolymphocytic leukemia (B-PLL).

B-prolymphocytic leukemia (B-PLL) is a rare disease with poor prognosis. To further characterize the biological features of this disease, we analyzed immunoglobulin heavy chain (IgVH) mutations, ZAP-70 and CD38 in 19 cases with de novo B-PLL. Immunoglobulin heavy chain genes analysis showed an unmutated pattern (>98% homology to germ line) in 9/17 cases (53%), with 100% homology in eight. In the remaining, it ranged from 90 to 97.4%, with three cases slightly mutated (98-95%) and five heavily mutated (<95%). All B-PLL utilized members of VH3 (11/17) and VH4 (6/17) families, with V3-23, V4-59 and V4-34 gene accounting for more than half of them, regardless of mutational status. ZAP-70, assessed by flow cytometry, ranged from 1 to 91% cells, being > or =20% in 57% of cases. CD38 ranged from 1 to 99% (median 21%). There was no correlation between IgVH status and ZAP-70 or CD38 expression, but male gender and del(17p) were more common in the unmutated group. Neither IgVH mutations, CD38 expression nor del(17p) influenced patients' outcome. Unexpectedly, ZAP-70+ B-PLL patients survived longer (40 months) than ZAP-70- B-PLL (8 months). B-PLL appears biologically heterogeneous regarding IgVH mutations, ZAP-70 and CD38 expression, showing a pattern distinct from that of other lymphoproliferative disorders.

The leukemic presentation of mantle-cell lymphoma: disease features and prognostic factors in 58 patients.

Mantle-cell lymphoma (MCL) is a B-cell malignancy with distinct molecular genetics and pathological features. Peripheral blood involvement has been reported with variable frequency, but information on the natural history of cases presenting with leukemia is lacking. This study aimed to determine the clinical and prognostic features of such cases. We studied clinical features, tumor characteristics, prognostic factors and outcome in 58 patients with leukemic presentation of MCL. Diagnosis was based on morphology, immunophenotype, presence of t(11;14), histology and cyclin D1 expression. The median age was 62 years and male:female 2.4:1. Presenting features included splenomegaly (74%), lymphadenopathy (45%), hepatomegaly (17%) and, in a minority, gastro-intestinal involvement or involvement of Waldeyer's ring; 10% had lymphocytosis alone. Six patients developed central nervous system disease. Median lymphocyte count was 58 x 10(9)/l, 55% had anemia and 17% had thrombocytopenia. Morphology of peripheral blood showed small-cell MCL in 15% of cases, typical MCL in 46% and blastoid MCL in 39%. Immunological markers showed a typical phenotype (CD5+ CD23 -) in 68%, and atypical phenotypes, CD5- CD23- in 17% or CD5+ CD23+ in 15%. CLL scores were 0, 1 or 2 in 96%. Median overall survival was 36 months. Good response to first-line treatment (P = 0.0008) and splenomegaly (P = 0.03) were favorable prognostic factors, while other features including morphology and CD38 expression had no impact on survival or treatment response. This analysis demonstrates that except for splenomegaly, survival of MCL patients presenting with leukemia is not significantly influenced by clinical or tumor characteristics. Splenectomy is a useful treatment option in this group of patients.

High remission rate in T-cell prolymphocytic leukemia with CAMPATH-1H.

T-cell prolymphocytic leukemia (T-PLL) is a chemotherapy-resistant malignancy with a median survival of 7.5 months. Preliminary results indicated a high remission induction rate with the human CD52 antibody, CAMPATH-1H. This study reports results in 39 patients with T-PLL treated with CAMPATH-1H between March 1993 and May 2000. All but 2 patients had received prior therapy with a variety of agents, including 30 with pentostatin; none achieved complete remission (CR). CAMPATH-1H (30 mg) was administered intravenously 3 times weekly until maximal response. The overall response rate was 76% with 60% CR and 16% partial remission (PR). These responses were durable with a median disease-free interval of 7 months (range, 4-45 months). Survival was significantly prolonged in patients achieving CR compared to PR or no response (NR), including one patient who survived 54 months. Nine patients remain alive up to 29 months after completing therapy. Seven patients received high-dose therapy with autologous stem cell support, 3 of whom remain alive in CR 5, 7, and 15 months after autograft. Stem cell harvests in these patients were uncontaminated with T-PLL cells as demonstrated by dual-color flow cytometry and polymerase chain reaction. Four patients had allogeneic stem cell transplants, 3 from siblings and 1 from a matched unrelated donor. Two had nonmyeloablative conditioning. Three are alive in CR up to 24 months after allograft. The conclusion is that CAMPATH-1H is an effective therapy in T-PLL, producing remissions in more than two thirds of patients. The use of stem cell transplantation to consolidate responses merits further study.

Deletions of D13S25, D13S319 and RB-1 mapping to 13q14.3 in T-cell prolymphocytic leukaemia.

Deletions of 13q14.3 are well known in several malignancies and are thought to be associated with tumour suppressor function. The RB-1 gene is a tumour suppressor gene, but other loci including D13S319 and D13S25 telomeric to this within 13q14.3 are deleted in B-cell chronic lymphocytic leukaemia (B-CLL), multiple myeloma and non-Hodgkin's lymphoma, with varying clinical significance. The fluorescence in situ hybridization screening of 22 patients with T-prolymphocytic leukaemia (T-PLL) for deletions of 13q14.3 revealed loss of D13S25 in 17 cases (mean 40% range 13-98%), with 11 patients having at least a 20% deletion. Mapping the deletions for the RB-1, D13S319,and D13S25 loci revealed D13S25 as the most frequently deleted marker. However, patients with only the D13S25 deletion had low percentages of cells with the deletion (12-13%), suggesting that loss of D13S25 on its own may not provide sufficient growth advantage. The use of the YAC 954c12, which maps immediately adjacent to D13S25, defined the telomeric border of the deletion in some of the cases. Inv(14)(q11q32) and t(14;14)(q11;q32) are characteristic of T-PLL, but are also observed in premalignant T-cell clones in patients with ataxia telangiectasia. Transition to overt leukaemia may result from loss of suppressor function. Thus, 13q14.3 deletions could contribute to the development of overt leukaemia in T-PLL, but the involvement of more than one gene in the region cannot be excluded.

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.

Unusual breakpoint distribution of 8p abnormalities in T-prolymphocytic leukemia: a study with YACS mapping to 8p11-p12.

Chromosome 8 abnormalities are seen in 80% of patients with T-cell prolymphocytic leukemia (T-PLL). The abnormalities described are idic(8)(p11),t(8;8)(p11;q12),+8, and 8p+ with the involvement of 8p. To localize 8p11-p12 breakpoints in T-PLL, metaphases from seven cases were karyotyped. Those with idic(8)(p11) and add(8)(p11) were probed with a panel of contiguous YACs derived from 8p11-p12 using fluorescence in situ hybridization (FISH). Analysis of FISH results showed that 8p11-p12 breakpoints cluster into two regions. The first region is telomeric to YAC 899e2, which contains the fibroblast growth factor receptor-1 gene (FGFR1) and appears to cluster within a 1.5-MB YAC 807a2. The second region is more centromeric with breakpoints on either side of YAC 806e9, flanked by YAC 940f10 distally and YAC 910d7 proximally, the latter containing the MOZ gene. These findings showed that a segment of 8p was still present in the isodicentric, but the pattern of clustering does not seem to correspond to a breakpoint affecting a single gene. The clustering regions are likely to be hot spots for recombination and result in idic(8)(p11) and 8p+. These changes point to the pathogenesis of T-PLL involving deletion of a gene sequence on 8p and/or gain of a copy of 8q.

T-cell prolymphocytic leukaemia: antigen receptor gene rearrangement and a novel mode of MTCP1 B1 activation.

T-cell prolymphocytic leukaemia (T-PLL) is a sporadic, mature T-cell disorder in which there is usually an aberrant T-cell receptor alpha (TCRA) rearrangement that activates the TCL1 or MTCP1-B1 oncogenes. As mutations of the Ataxia Telangiectasia (A-T) gene, ATM, are frequent in T-PLL and as ATM seems to act as a tumour suppressor through a mechanism involving V(D)J recombination, we examined V(D)J recombination in T-PLL. Using Southern blotting and the polymerase chain reaction, two of 60 TCRG coding joints were abnormal. In all cases, both TCRD alleles were deleted, IGH was germline, and patterns of TCRB and TCRA rearrangement were normal. However, in a case harbouring t(X;7)(q28;q35), we identified TCRB segment J beta 2.7 juxtaposed to MTCP1 exon 1. This is the first time that TCRB has been implicated in MTCP1 B1 activation. The structure of the breakpoint supports a model in which translocation activates a cryptic MTCP1 promoter. This analysis of V(D)J recombination is consistent with it being a variable that is independent of ATM in T-PLL.

p53 allele deletion and protein accumulation occurs in the absence of p53 gene mutation in T-prolymphocytic leukaemia and Sezary syndrome.

In a series of 24 patients with chronic T-lymphoid disorders [13 T-prolymphocytic leukaemia (T-PLL) and 11 Sezary syndrome] we have studied (i) chromosome 17p abnormalities and p53 allele deletion by fluorescence in situ hybridization; (ii) mutation in the exons of the p53 gene by direct DNA sequencing; and (iii) p53 protein expression by immunocytochemistry and, in some cases, also by flow cytometry with DO-1, a monoclonal antibody to the p53 protein. The study revealed p53 deletion and accumulation of p53 protein in the absence of mutation in the exons that included the hot-spots and differs from that described in B-prolymphocytic leukaemia. Seven T-PLL and five Sezary syndrome patients had p53 overexpression, and five T-PLL and nine Sezary syndrome patients showed p53 deletion. Although the majority of cases with p53 accumulation had p53 deletion, the proportion of cells with the deletion did not correlate with the proportion of cells positive for p53 expression. Two cases of T-PLL showed strong p53 expression in the absence of p53 deletion, and one case of Sezary syndrome with p53 deletion in 97% of cells did not express p53. These findings suggest that a non-mutational mechanism exists for the accumulation of p53 protein in these T-cell disorders. The oncogenic effect of the accumulating wild-type protein has been reported in other malignancies. Whether haploidy resulting from p53 deletion contributes to this mechanism has yet to be determined. Alternatively, the frequent loss of the p53 gene could be associated with the deletion of an adjacent gene, which could be involved in the pathogenesis of these diseases.

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.

p53 gene deletion and trisomy 12 in hairy cell leukemia and its variant.

The deletion or mutation of the p53 tumour suppressor gene on chromosome 17p13 is known to be associated with aggressive disease in several B-cell malignancies. The present study describes the p53 gene status in 20 cases of hairy cell leukemia (HCL) and in 12 cases of its morphological variant (HCL-V) by fluorescence in situ hybridization (FISH). A high incidence of p53 deletion was found in both diseases (75-100% of cases). However, a significant difference was observed between the proportion of cells with p53 deletion in HCL-V cases (mean 31%) and HCL cases (mean 12%) P value < 0.01. The observed difference correlates with the well known tendency for transformation and poor response to therapy in HCL-V and seven cases of HCL-V with greater than 22% of cells with p53 deletion showed features of disease progression and transformation. Trisomy 12 was present in 8.5% of the cells in one case of HCL-V and in 6-8% of cells in three cases of HCL.

B cell prolymphocytic leukaemia (B-PLL) with complex karyotype and concurrent abnormalities of the p53 and c-MYC gene.

We report the cytogenetic, molecular and biological characterization of a case of B-PLL with a complex karyotype and concurrent abnormalities on the p53 and c-MYC genes. Conventional cytogenetics suggested that both 17q arms were translocated to chromosomes 1q and 14p, respectively, whereas both 17p arms were not identified. In addition, a Burkitt's-like variant translocation t(2;8) was found. Study of loss of heterozygosity at 17p13 and p53 direct sequencing demonstrated the presence of only one copy of the p53 gene. A 27 bp deletion in exon 8 that resulted in the expression of a p53 protein lacking nine amino acids from the DNA binding region was also found. To confirm the presence of one copy of the p53 gene and localize it, fluorescent in situ hybridization (FISH) studies using a p53 gene probe was performed. Only one signal of p53 was visualized. Moreover, the DAPI profile of the chromosome containing the hybridization spot for the p53 probe did correspond to the cytogenetic marker identified as der(14)t(14;17). Whole chromosome 14 paint, centromere-specific for chromosome 17 and p53 gene probes were cohybridized to the preparations. This demonstrated that the der(14) contained the 17 centromere and distally the p53 gene suggesting that the der(14) contained the short arm of chromosome 17 with the breakpoint occurring in the long arm. FISH studies confirmed the involvement of c-MYC and KAPPA in the t(2;8) translocation. To our knowledge, this is the first case of B-PLL with inactivation of the p53 gene by mutation together with a Burkitt's-like t(2;8) translocation involving the c-MYC gene. The cooperation of these genes may have conferred a growth advantage which was critical in the development of this aggressive form of B-PLL.

Abnormalities of chromosomes 8, 11, 14, and X in T-prolymphocytic leukemia studied by fluorescence in situ hybridization.

Twenty-one patients with T-prolymphocytic leukemia (T-PLL) were studied by FISH to characterize abnormalities of chromosomes 8, 11, 14, and X. A higher percentage of abnormalities of these chromosomes was detected by FISH than by cytogenetics. Seventy-one percent had inv(14) (q11q32)/t(14;14)(q11;q32). Four patients had abnormalities involving Xq28 (MTCP-1 locus) resulting from t(X;14)(q28;q11) or t(X;7)(q28;q35). These abnormalities have also been described in persistent expanding pre-malignant T-cell clones in patients with ataxia telangiectasia (AT). We have previously reported that in T-PLL and AT developing T-cell leukemia, the above abnormalities occur with additional abnormalities, mainly trisomy for 8q resulting predominantly from an i(8)(q10) and an increased expression of MYC. In this series, 81% of cases had chromosome 8 abnormalities including i(8)(q10)[43%]/t(8;8)(p12;q11)[14%], + 8[14%], and 8p + [14%]. The use of probes for MYC (8q24) and chromosome 8 centromere on metaphase chromosomes revealed that cases with i(8)(q10) were dicentric and t(8;8) monocentric. These abnormalities are not only associated with increase in dosage of 8q and the MYC gene, but also involved 8p. 8p is known to have several suppressor genes associated with solid tumors. Our findings suggest that the possible loss of a tumor suppressor gene plus the increased dosage of the q arm and/or the high expression of TCL-1/MTCP-1, which results from inv(14)/t(14;14), allows the malignant phenotype to emerge.

ATM is usually rearranged in T-cell prolymphocytic leukaemia.

T-prolymphocytic leukaemia (T-PLL) is a rare, sporadic leukaemia similar to a mature T-cell leukaemia seen in some patients with Ataxia Telangiectasia (A-T), a recessive multisystem disorder caused by mutations of the ATM gene at chromosome 11q23. ATM sequence mutations have been reported in 46% of T-PLL cases, but some cases also have karyotypic abnormalities at 11q, including 11q23. This led us to investigate the structure of the ATM locus in a panel of eight cases, two of which had 11q23 abnormalities. As expected, nucleotide changes were detected in some samples. Two remission samples were wild type. To test for structural lesions, DNA fibres were hybridized with a contig of four labelled cosmids spanning the ATM locus. In all samples there were structural lesions and in four samples both alleles were affected. This provides strong evidence for our suggestion that ATM acts as a tumour suppressor during T-PLL tumorigenesis. Some additional role for ATM during T-PLL tumorigenesis is possible since nucleotide changes were present in addition to structural lesions disrupting both alleles. The mechanism of inactivation appeared to be unusual because multiple structural lesions on one allele were often observed.

Sezary cell leukaemia: a distinct T cell disorder or a variant form of T prolymphocytic leukaemia?

We report the clinical, ultrastructural, immunophenotypic and virological features of nine cases of a rare type of mature T cell disorder formerly designated Sezary cell leukaemia. All patients presented with lymphocytosis ranging from 12.7 to 133 x 10(9)/l, bone marrow infiltration, splenomegaly and lymphadenopathy. Skin involvement was absent at presentation but developed as a terminal event in two patients, one of whom showed a pattern of dermal infiltration different from that characteristic of Sezary syndrome. Cells from eight cases bore a mature T cell phenotype and electronmicroscopy revealed lymphocytes with cerebriform nuclei resembling Sezary cells. All cases except one were HTLV-I negative. Patients were treated with various chemotherapy regimens but with poor outcome, the median survival being 13 months. Laboratory and clinical data suggest great similarity between Sezary cell leukaemia and T prolymphocytic leukaemia (T-PLL), namely coexpression of CD4 and CD8 (3/9 cases), identical chromosomal abnormalities in the three cases studied (isochromosome 8q plus inversion 14 or t(X;14)(q28;q11)) and a remarkable sensitivity to CAMPATH-1H (complete remission of 21 months' duration in one patient), suggesting that this entity could be considered a variant form of T-PLL. The alternative diagnosis of adult T cell leukaemia/lymphoma could not be excluded in one patient in whom positive HTLV-I serology was documented.

Two unbalanced translocations, t(12;22)(p13;q11) and t(12;?)(p13;?), in an aggressive chronic B-cell leukemia: TEL gene analysis using FISH.

The translocation t(12;22)(p13;q11) has been consistently described in myeloid malignancies and shown to result from a fusion between the TEL and MN1 genes. Previously described deletions of 12p in acute lymphoblastic leukemias have been recently shown to harbor undetected translocations involving the TEL gene at 12p13. We document a case of an aggressive chronic B-cell leukemia whose cells had trisomy 12 and two unbalanced translocations involving 12p13, including a t(12;22)(p13;q11) as shown by conventional cytogenetics and fluorescence in situ hybridization (FISH). The 12p13 breakpoint of the t(12;22)(p13;q11) was telomeric to the TEL gene, and the second unbalanced translocation with breakpoint 12p13 resulted in the deletion of TEL. This case demonstrates that TEL gene deletions may be relevant in cases of mature B-lymphoproliferative diseases.