CircBCAR3 sponges miR-27a-3p and mediates ferroptosis in human B-prolymphocytic leukaemia cells via SLC7A11.

B-lymphocytic leukaemia is one of the most commonly diagnosed blood malignancies, and our knowledge of B-prolymphocytic leukaemia remained barely comprehensive. CircRNAs and miRNAs were identified of important regulatory roles in tumours. This study focused on the possibly existing interaction of circBCAR3 and miR-27a-3p, and downstream molecules thereafter in B-prolymphocytic leukaemia cells. CircBCAR3 and miR-27a-3p expression was evaluated in JVM-2 cell line and normal lymphocytes. Dual-luciferase luminescence assay was conducted for validation of circBCAR3 and miR-27a-3p interaction, as well as western blot and flow cytometry for evaluation and validation of their association with SLC7A11, reactive-oxygen species and Fe2+ regarding ferroptosis. CircBCAR3 were upregulated in JVM-2 cells and were reversely correlated with the expression of miR-27a-3p. CircBCAR3 targeted at miR-27a-3p and was consequently associated with SLC7A11 expression positively, inhibiting ferroptosis and peroxidative damage in JVM-2 cells. This study identified a circBCAR3-miR-27a-3p-SLC7A11 axis regulating ferroptosis and peroxidation of B-prolymphocytic leukaemia cells which might be a key mechanism facilitating the survival of tumour cells. However, further validation based on more diverse cell lines and animal models might be required.

Long-Smoldering T-prolymphocytic Leukemia: A Case Report and a Review of the Literature.

T-prolymphocytic leukemia (T-PLL) is a rare malignancy of mature T-cells with distinct clinical, cytomorphological, and molecular genetic features. The disease typically presents at an advanced stage, with marked leukocytosis, B symptoms, hepatosplenomegaly, and bone marrow failure. It usually follows an aggressive course from presentation, and the prognosis is often considered dismal; the median overall survival is less than one year with conventional chemotherapy. This case report describes a patient with T-PLL who, after an unusually protracted inactive phase, ultimately progressed to a highly invasive, organ-involving disease. After initial treatments failed, a novel treatment approach resulted in a significant response.

Chronic lymphocytic leukemia and prolymphocytic leukemia with MYC translocations: a subgroup with an aggressive disease course.

Translocations involving MYC are rare in chronic lymphocytic leukemia (CLL), and up to now, their prognostic significance remains unclear. We report the characteristics of 21 patients with CLL and nine patients with prolymphocytic leukemia (PLL), diagnosed in multiple centers (n = 13), which showed an MYC translocation demonstrated by fluorescence in situ hybridization. The prevalence was estimated to be <1%. Advanced age and male predominance were observed. Morphological analysis frequently revealed the presence of prolymphocytes. A typical "CLL-immunophenotype" was found in four of nine cases with PLL. Moreover, CD5 and CD23 were frequently expressed in PLL. The latter findings are atypical for PLL and may suggest transformation or progression of an underlying CLL. MYC translocations were frequently observed with concomitant adverse cytogenetic markers, such as del(11q) (n = 8/30) and/or del(17p)/monosomy 17 (n = 7/30). In addition, the presence of unbalanced translocations (n = 24 in 13/30 cases) and complex karyotype (n = 16/30) were frequent in cases with MYC translocations. Altogether, del(17p)/monosomy 17, del(11q), and/or complex karyotype were observed in 22 of 30 patients. Survival outcome was poor: the median time to treatment was only 5 months, and overall survival (OS) from clinical diagnosis and from genetic detection was 71 and 19 months, respectively. In conclusion, CLL/PLL with MYC translocations is a rare entity, which seems to be associated with adverse prognostic features and unfavorable outcome.

Computational gene expression analysis reveals distinct molecular subgroups of T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is a rare blood cancer with poor prognosis. Overexpression of the proto-oncogene TCL1A and missense mutations of the tumor suppressor ATM are putative main drivers of T-PLL development, but so far only little is known about the existence of T-PLL gene expression subtypes. We performed an in-depth computational reanalysis of 68 gene expression profiles of one of the largest currently existing T-PLL patient cohorts. Hierarchical clustering combined with bootstrapping revealed three robust T-PLL gene expression subgroups. Additional comparative analyses revealed similarities and differences of these subgroups at the level of individual genes, signaling and metabolic pathways, and associated gene regulatory networks. Differences were mainly reflected at the transcriptomic level, whereas gene copy number profiles of the three subgroups were much more similar to each other, except for few characteristic differences like duplications of parts of the chromosomes 7, 8, 14, and 22. At the network level, most of the 41 predicted potential major regulators showed subgroup-specific expression levels that differed at least in comparison to one other subgroup. Functional annotations suggest that these regulators contribute to differences between the subgroups by altering processes like immune responses, angiogenesis, cellular respiration, cell proliferation, apoptosis, or migration. Most of these regulators are known from other cancers and several of them have been reported in relation to leukemia (e.g. AHSP, CXCL8, CXCR2, ELANE, FFAR2, G0S2, GIMAP2, IL1RN, LCN2, MBTD1, PPP1R15A). The existence of the three revealed T-PLL subgroups was further validated by a classification of T-PLL patients from two other smaller cohorts. Overall, our study contributes to an improved stratification of T-PLL and the observed subgroup-specific molecular characteristics could help to develop urgently needed targeted treatment strategies.

Molecular characterization of Novel ATM fusions in chronic lymphocytic leukemia and T-cell prolymphocytic leukemia.

ATM deletions and/or mutations are recurrent in lymphoid neoplasms while rearrangements are rare. In this study, we used mate pair sequencing (MPseq) technology to characterize two novel ATM rearrangements in one patient with chronic lymphocytic leukemia (CLL) and one patient with T-prolymphocytic leukemia (T-PLL). Both patients showed chromosome 11q22 aberrations encompassing ATM by conventional karyotype and fluorescence in situ hybridization: isolated t(11;13)(q22;q14) in CLL and a complex karyotype with apparent 11q deletion and unbalanced der(14)t(11;14)(q22;p11.2) in T-PLL. MPseq identified ATM-LINC00371 fusion in CLL and ATM-USP28 in T-PLL, both of which led to ATM inactivation, confirmed by loss of immunohistochemical protein expression. Next-generation sequencing mutation analysis detected concurrent ATM mutation(s) CLL patient, while T-PLL lacked ATM mutation. ATM rearrangements, not apparently detectable using standard laboratory technologies, represent another mechanism of loss-of-function. Recent high-throughput technologies such as MPseq can uncover novel pathogenic gene fusions and resolve complex chromosomal rearrangements in hematologic malignancies.

Clinical efficacy of immunochemotherapy with fludarabine, epirubicin and rituximab in the treatment for chronic lymphocytic leukaemia and prolymphocytic leukaemia.

Despite some considerable progress in the therapy for chronic lymphocytic leukaemia (CLL) owing to fludarabine-based regimens and rituximab, no curative treatment is available so far. We conducted an explorative phase II study in patients with CLL, prolymphocytic leukaemia (PLL) and leukaemic lymphoplasmacytic lymphoma (LL) with the combination of fludarabine, epirubicin and rituximab (FER) to improve the complete remission (CR) rate and progression-free survival (PFS). Fludarabine 25 mg/m(2) was administered i.v. on days 1-5 and epirubicin 25 mg/m(2) i.v. on days 4 and 5, and rituximab was added at a dose of 375 mg/m(2) i.v. day 1 in the first cycle and at a dose of 500 mg/m(2) in all consecutive cycles. Patients exhibiting responsive disease after FER were eligible to receive maintenance therapy of up to 12 cycles of rituximab 375 mg/m(2) bimonthly. Forty-four patients (38 CLL, 4 PLL and 2 LL) with a median age of 65 yrs (43-84 yrs) were evaluable. Seventeen patients with CLL had stage Binet C, 14 Binet B and seven symptomatic or rapid progressive stage Binet A. Cytogenetic features showed normal karyotype in nine cases, an isolated deletion (del) 13q in 12 patients, trisomy 12 in 7, del 11 in two and del 17p in 4. Half of the patients (48%) had mutated IgVH genes. Treatment with FER achieved an overall response rate of 95%, including 63% CRs and 32% PRs. Haematological toxicity was considerable. After a median follow-up period of 34 months (range: 8-84 months), median PFS was 61 months and overall survival was yet not reached. All patients with PLL and LL achieved CR. The data support the high efficacy of the combination of rituximab with chemotherapy (FE) and are suggestive of possible benefit with rituximab maintenance therapy for PFS and DFS.

Biallelic mutations in the ATM gene in T-prolymphocytic leukemia.

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by cerebellar ataxia, oculocutaneous telangiectasia, immune deficiency, genome instability and predisposition to malignancies, particularly T-cell neoplasms. The responsible gene, designated ataxia-telangiectasia mutated (ATM), was recently identified by positional cloning in the chromosomal region 11q22.3-23.1 (ref. 4, 5) ATM is 150 kb in length, consists of 66 exons and encodes a nuclear phosphoprotein of approximately 350 kDa (ref. 4-9). Although ATM is considered to be a tumorigenic factor in several human cancers, it has not yet been found mutated in tumors of non-AT patients. Given the marked predisposition of AT patients to develop neoplasms of the T-cell lineage, we analyzed a series of T-cell leukemias (T-prolymphocytic leukemia, or T-PLL) in non-AT patients in search of genomic changes associated with the development of this disease. Among the recurrent aberrations identified, deletion of the chromosome arm 11q was very frequent. Subsequent molecular cytogenetic analyses allowed us to define a small commonly deleted segment at 11q22.3-23.1 in 15 of 24 T-PLLs studied. Since this critical region contained ATM, we further analyzed the remaining copy of the gene in six cases showing deletions affecting one ATM allele. In all six cases, mutations of the second ATM allele were identified, leading to the absence, premature truncation or alteration of the ATM gene product. Thus, our study demonstrates disruption of both ATM alleles by deletion or point mutation in T-PLL, suggesting that ATM functions as a tumor-suppressor gene in tumors of non-AT individuals.

An Unusual Case of Prolymphocytic Leukemia Transformation in a Patient With Chronic Lymphocytic Leukemia.

B-cell prolymphocytic leukemia (B-PLL) is a rare leukemia characterized by rapidly increasing leukocytosis with splenomegaly and lymphadenopathy. Treatment strategies are largely based on studies of chronic lymphocytic leukemia (CLL). Antibodies against the cell surface protein CD20 are considered to be first-line therapy. A 76-year-old male with known CLL presented 2 weeks after starting chemoimmunotherapy for newly refractory CLL after failing ibrutinib therapy. White blood cell count was elevated at 226.7 × 103/µL. Fluorescent in situ hybridization analysis of a bone marrow specimen showed new development of complex cytogenetics. Flow cytometry revealed B cells appearing slightly dimmer on CD45 and brighter on CD20 compared with typical B-CLL suggestive of less mature lymphocyte forms. The patient was diagnosed with B-PLL and started on obinutuzumab and venetoclax with rapid normalization of white blood cells. This case recapitulates the challenges in diagnosing and treating B-PLL. Ibrutinib resistance is a growing area of study with several proposed mechanisms of acquired resistance. The pathogenesis of B-PLL is not completely understood, although mutations in MYC are presumed to play a role.

Biological and clinical characterization of recurrent 14q deletions in CLL and other mature B-cell neoplasms.

14q-deletions have been repeatedly described in mature B-cell neoplasms, but not yet characterized in a larger cohort. Based on chromosome banding analysis, the present study identified 47 del(14q) cases in 3054 mature B-cell neoplasms (1·5%) (chronic lymphocytic leukaemia [CLL]: 1·9%; CLL/prolymphocytic leukaemia [PL]: 9·0%; others: 0·2%). Interphase fluorescence in situ hybridization was performed with probes for 14q22.1, 14q24.1, 14q32.33, and IGH@ (14q32.3). The del(14q) had heterogeneous size but showed a breakpoint cluster at the centromeric site in 14q24.1 (62% of cases). At the telomeric side, the most frequent breakpoint was within the IGH@ locus (14q32.3) between IGH@ 3'-flanking and IGHV (IgVH) probes (45%). In 16 cases (34%), breakpoints occurred within 14q24.1 and 14q32.3. Eighty-one percent of del(14q) cases showed 1-3 additional cytogenetic alterations (in 45%, +12), and 56% were IGHV-unmutated. In all cases (16/16) with breakpoints in 14q24.1 and 14q32.3, a B-CLL immunophenotype was found. Clinical follow-up in 32 del(14q) patients was compared to 383 CLL and CLL/PL patients without del(14q). While 3-year-overall survival did not differ significantly, time to treatment was significantly shorter in the del(14q) cohort (21·0 months vs. 80·1 months, P = 0·015). In conclusion, the del(14q) is a rare recurrent alteration in diverse mature B-cell neoplasms, shows variable size but distinct clustering of breakpoints, and is associated with short time to treatment.

Proto-oncogene TCL1: more than just a coactivator for Akt.

Serine threonine kinase Akt, also called PKB (protein kinase B), plays a central role in regulating intracellular survival. Deregulation of this Akt signaling pathway underlies various human neoplastic diseases. Recently, the proto-oncogene TCL1 (T cell leukemia 1), with a previously unknown physiological function, was shown to interact with the Akt pleckstrin homology domain, enhancing Akt kinase activity; hence, it functions as an Akt kinase coactivator. In contrast to pathological conditions in which the TCL1 gene is highly activated in various human neoplasmic diseases, the physiological expression of TCL1 is tightly limited to early developmental cells as well as various developmental stages of immune cells. The NBRE (nerve growth factor-responsive element) of the proximal TCL1 promoter sequences can regulate the restricted physiological expression of TCL1 in a negative feedback mechanism. Further, based on the NMR structural studies of Akt-TCL1 protein complexes, an inhibitory peptide, "Akt-in," consisting of the betaA strand of TCL1, has been identified and has therapeutic potential. This review article summarizes and discusses recent advances in the understanding of TCL1-Akt functional interaction in order to clarify the biological action of the proto-oncogene TCL1 family and the development avenues for a suppressive drug specific for Akt, a core intracellular survival regulator.

Combined single nucleotide polymorphism-based genomic mapping and global gene expression profiling identifies novel chromosomal imbalances, mechanisms and candidate genes important in the pathogenesis of T-cell prolymphocytic leukemia with inv(14)(q11q32).

T-cell prolymphocytic leukemia (T-PLL) is a rare aggressive lymphoma derived from mature T cells, which is, in most cases, characterized by the presence of an inv(14)(q11q32)/t(14;14)(q11;q32) and a characteristic pattern of secondary chromosomal aberrations. DNA microarray technology was employed to compare the transcriptomes of eight immunomagnetically purified CD3+ normal donor-derived peripheral blood cell samples, with five highly purified inv(14)/t(14;14)-positive T-PLL blood samples. Between the two experimental groups, 734 genes were identified as differentially expressed, including functionally important genes involved in lymphomagenesis, cell cycle regulation, apoptosis and DNA repair. Notably, the differentially expressed genes were found to be significantly enriched in genomic regions affected by recurrent chromosomal imbalances. Upregulated genes clustered on chromosome arms 6p and 8q, and downregulated genes on 6q, 8p, 10p, 11q and 18p. High-resolution copy-number determination using single nucleotide polymorphism chip technology in 12 inv(14)/t(14;14)-positive T-PLL including those analyzed for gene expression, refined chromosomal breakpoints as well as regions of imbalances. In conclusion, combined transcriptional and molecular cytogenetic profiling identified novel specific chromosomal loci and genes that are likely to be involved in disease progression and suggests a gene dosage effect as a pathogenic mechanism in 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.

Complex karyotype including chromosomal translocation (8;14) (q24;q32) in one case with B-cell prolymphocytic leukemia.

We report a case of a 64-year-old white female patient, who presented with symptomatic anemia (Hgb: 6.8g/dl), thrombocytopenia (platelets: 94,000/mcl) and leukocytosis (WBC: 156,000/mcl). Peripheral blood smear revealed markedly increased white blood cell count with predominance of atypical lymphoid cells of intermediate size, moderately dense chromatin, and prominent large single nucleoli. Bone marrow aspirate smear showed predominance (78%) of atypical lymphoid cells morphologically identical to those seen in the peripheral blood. The bone marrow core biopsy was hypercellular and packed with prominent infiltrate of prolymphocytes. Immunophenotypic analysis revealed a population of monoclonal cells (75% of all -erythroid cells) characterized by CD45+, CD19+, CD20+, CD5+, HLA-DR+, CD10-, CD23+/-, CD38+ and FMC7-. The abnormal cells were restricted to kappa light chain immunoglobulin with low intensity. Cytogenetic study showed an abnormal clone of eight cells with the following karyotype: 45,X,-X,add(8)(p11.2),t(8;14)(q24;q32),add(20)ql3[8]/46,XX[12]. The relative rarity of B-PLL and the heterogeneity of clinical and laboratory parameters make it difficult to define the natural history and prognosis in all cases. The optimal treatment for B-PLL is still unknown and to date there are no reports of chromosomal abnormalities as a prognostic factor. The patient was treated with six cycles of cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP). Complete remission was achieved according to the criteria defined by National Cancer Institute Working Group for CLL.

Mature T-cell leukemias including T-prolymphocytic leukemia, adult T-cell leukemia/lymphoma, and Sézary syndrome.

The 2005 Society for Hematopathology/European Association for Haematopathology Workshop Session 1 was devoted to case presentations with discussions of 3 types of mature T-cell leukemias--T-cell prolymphocytic leukemia, adult T-cell leukemia/lymphoma, and Sézary syndrome. These 3 disorders are clonal proliferations of postthymic alphabeta T cells that are often characterized by systemic manifestations and a leukemic blood picture. The application of clinical, morphologic, immunophenotypic, and genetic studies to the assessment and characterization of these 3 disorders is presented, along with specific diagnostic recommendations and differential diagnostic considerations.

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.

Prolymphocytic leukemias.

T and B subtypes of prolymphocytic leukemias (PLLs) are rare, highly aggressive lymphoid malignancies with characteristic morphologic, immunophenotypical, cytogenetic, and molecular features. Recent studies have highlighted the role of specific oncogenes such as TCL1, MTCP-1, and ATM in the case of T-cell and p53 mutations in the case of B-cell PLLs. Despite the advances in the understanding of the biology of these conditions, prognosis for these patients remains poor with short survival and no curative treatment. The advent of monoclonal antibody therapy has improved treatment options for this group. In particular, the use of Campath-1H, in T-PLL has more than doubled median survival. The role of allogeneic transplant with nonmyeloablative conditioning needs to be explored further.

T-cell prolymphocytic leukemia: an aggressive T cell malignancy with frequent cutaneous tropism.

BACKGROUND: T-cell prolymphocytic leukemia (T-PLL), formerly categorized as T-cell chronic lymphocytic leukemia, is a rare and aggressive hematologic malignancy. Although the skin is characteristically involved, it is not a well-recognized entity in the dermatologic literature. METHODS: Six cases of cutaneous T-PLL are presented from a clinical, light microscopic, and phenotypic perspective. RESULTS: The patient population comprised 2 women and 4 men, with a mean age of 69.8 years. The disease was associated in all with skin involvement with facial preference; edema, purpura, and lesional symmetry were characteristic. The skin biopsies demonstrated a largely non-epidermotropic angiocentric lymphocytic infiltrate with accompanying hemorrhage. The cells showed irregular- to reniform-shaped nuclei with small nucleoli and eosinophilic rims of cytoplasm. Phenotypic studies revealed three prevailing profiles: CD4 dominant in 4, CD8 dominant in one, and co-expression of CD4 and CD8 in one. CD3 loss was seen in one case. All expressed T-cell leukemia 1 (TCL-1) and CD7; cutaneous lymphocyte antigen expression was discernible in a dot-like perinuclear array. All cases tested excluding one expressed TCL-1 and CD52. In two cases tested, T-cell receptor beta rearrangements were observed. Cytogenetic studies demonstrated a paracentromeric chromosome 14 inversion. Polysomy 8 and MYC amplification was seen in one case, manifesting an aggressive clinical course. Four patients died from their disease within 18 months of diagnosis. LIMITATIONS: Cytogenetic MYC amplification, FISH, and TCR beta studies were conducted on each of 2 cases, respectively, due to limitations of tissue block samples and/or peripheral blood. cMYC translocation studies were conducted on 3 of the 6 cases, again due to limitations imposed by the tissue samples on the cases. The last case was recently diagnosed and, therefore, long-term follow-up is not possible. CONCLUSION: T-PLL is a distinctive post-thymic T-cell malignancy with frequent cutaneous tropism. A diagnosis is possible in almost all cases based on characteristic clinical, light microscopic, phenotypic, and cytogenetic features. While a chromosome 14 inversion is highly characteristic, additional inherent cytogenetic differences, such as trisomy 8 with CMYC over-amplification, may account for some case to case variation in clinical course.

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.

Ataxia-telangiectasia and T-cell leukemias: no evidence for somatic ATM mutation in sporadic T-ALL or for hypermethylation of the ATM-NPAT/E14 bidirectional promoter in T-PLL.

The ATM gene deficient in ataxia-telangiectasia, a recessive multisystem disease associated with a high risk of lymphomas and leukemias, was found previously to be inactivated in a rare sporadic malignancy, T-cell prolymphocytic leukemia (T-PLL), which is often associated with cytogenetic aberrations of chromosome 14. The ATM gene was shown to sustain frequent loss-of-function mutations in T-PLL tumor cells, consistent with functioning as a tumor suppressor gene in this leukemia. To investigate the possibility of nonmutational or nonrecombinational mechanisms of T-PLL development, we have used bisulfite genomic sequencing to analyze DNA methylation in the putative bidirectional promoter region of the closely linked ATM and NPAT/E14 genes within the CpG island at 11q22-q23. We show that this region is completely demethylated in lymphocytes expressing ATM; however, no extensive hypermethylation was found in 9 T-PLL tumor DNA samples without evidence of ATM/p53 mutations. Because acute T-cell lymphoblastic leukemias (T-ALL) were also observed in ataxia-telangiectasia patients and T-ALL tumor cells contain chromosome 14 abnormalities, 19 presentation samples of T-ALL patients were analyzed for ATM mutations. Although T-ALL patients exhibited rare nucleotide substitutions not previously found in ATM, all were identified in the germ-line, indicating constitutional polymorphisms, potentially confined to ethnic subpopulations. The absence of somatic nucleotide changes in ATM in T-ALL as compared with T-PLL suggests a distinct pattern of genetic events in the development of the two leukemias.

Alternative origin of p13MTCP1-encoding transcripts in mature T-cell proliferations with t(X;14) translocations.

The MTCP1 gene is involved in the t(X;14)(q28;q11) translocation associated with T-cell prolymphocytic leukemia and related conditions. This gene is unusual in that it codes for two distinct proteins: a small mitochondrial protein, p8MTCP1, and a putative oncogenic protein, p13MTCP1. Scarcity of material from t(X;14)-associated proliferations and very low levels of mRNA expression have so far prevented a thorough description of p13MTCP1-encoding transcripts. Here, we characterize two additional t(X;14) bearing leukemias allowing this analysis. In one case, with a breakpoint located 5' to the MTCP1 gene, the level of transcription of previously described p13MTCP1-encoding transcripts is enhanced. In the second case, with a breakpoint within the MTCP1 intron I, an alternative transcription initiation site is demonstrated in the tumor cells at 229 bp upstream to exon II. The identification of this internal promoter, together with the similarity between TCL1 and MTCP1 genomic structures, allow us to propose a model in which the duplication of an ancestral gene was followed by the insertion of one copy within the intron of a p8-encoding gene, accounting for the unusual feature of the MTCP1 gene.