Blastic Plasmacytoid Dendritic Cell Neoplasm: A Single-Center Experience. Clinical Characterization, Mutational Landscape, and Clinical Outcome of Patients Undergoing Hematopoietic Stem Cell Transplantation Intensive Therapy.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematodermic neoplasm usually involving the skin. In this retrospective case series, 10 cases of BPDCN were identified, 90% of which had skin involvement and exhibited predominantly violaceous nodules and/or bruise-like plaques. Skin lesions showed diffuse or nodular dermal-based infiltrates of intermediate sized blasts with a grenz zone. Tumor immunophenotyping was CD4(+), CD56(+), CD123(+) and CD303(+). The most frequently mutated genes according to targeted next-generation sequencing were TET2 (3/7) and NRAS (2/7). Multiagent chemotherapy (CT) was administered as first-line therapy, and a total of 5 patients underwent allogenic hematopoietic stem cell transplantation (allo-HSCT). Better outcomes were observed in younger patients and those treated with acute lymphoblastic leukemia (ALL)-like CT followed by allo-HSCT. This study shows the clinical range of cutaneous lesions of BPDCN. Despite the absence of a gold standard therapy, patients treated with myeloablative intensive regimens and allo-HSCT seems to have a more favorable prognosis.

Clinicobiological features and prognostic impact of diffuse large B-cell lymphoma component in the outcome of patients with previously untreated follicular lymphoma.

BACKGROUND: The co-existence at diagnosis of follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) components (FL/DLBCL) has been considered a transformed lymphoma and accordingly treated although clinicobiological information on these patients is scarce. The aim of this study was to analyze the initial features and outcome of FL/DLBCL patients in the rituximab era. PATIENTS AND METHODS: All patients consecutively diagnosed at a single institution with FL/DLBCL (n = 40), as well as those with pure FL (n = 328) or de novo DLBCL (n = 510) as controls. RESULTS: The proportion of the DLBCL component was highly variable (median 50%). In 29 FL/DLBCL cases analyzed, the cell of origin was GCB in 86%, ABC in 10% and unclassifiable in 4%. NOTCH1-2 was mutated in 10% of these cases. The proportion of DLBCL component did not impact on overall survival (OS). Regarding initial characteristics, patients with FL/DLBCL were closer to FL in terms of primary nodal origin, good performance status and advanced stage, whereas the other features were intermediate between FL and DLBCL. FL/DLBCL patients were treated as DLBCL with no further intensification. Complete response and primary refractory rates were 65% and 20%, respectively, with these figures being similar to DLBCL and worse than FL. Progression-free survival and OS were intermediate between FL and DLBCL (5-year OS: 85%, 73% and 63% for FL, FL/DLBCL and DLBCL, respectively). FL/DLBCL histology did not reach independent prognostic value for OS in the multivariate analyses. CONCLUSIONS: The outcome of FL/DLBCL patients is not worse than that of de novo DLBCL. These cases should be treated with immunochemotherapy as DLBCL, but intensification with ASCT may not be necessary. The biological insights of FL/DLBCL warrants further genetic and molecular studies.

Human organic cation transporter 1 (hOCT1) as a mediator of bendamustine uptake and cytotoxicity in chronic lymphocytic leukemia (CLL) cells.

Bendamustine is used in the treatment of chronic lymphocytic leukemia (CLL). Routes for bendamustine entry into target cells are unknown. This study aimed at identifying transporter proteins implicated in bendamustine uptake. Our results showed that hOCT1 is a bendamustine transporter, as bendamustine could cis-inhibit the uptake of a canonical hOCT1 substrate, with a Ki in the micromolar range, consistent with the EC50 values of the cytotoxicity triggered by this drug in HEK293 cells expressing hOCT1. hOCT1 polymorphic variants determining impaired bendamustine-transporter interaction, consistently reduced bendamustine cytotoxicity in HEK293 cells stably expressing them. Exome genotyping of the SLC22A1 gene, encoding hOCT1, was undertaken in a cohort of 241 CLL patients. Ex vivo cytotoxicity to bendamustine was measured in a subset of cases and shown to correlate with SLC22A1 polymorphic variants. In conclusion, hOCT1 is a suitable bendamustine transporter, thereby contributing to its cytotoxic effect depending upon the hOCT1 genetic variants expressed.

Essential thrombocythemia in young individuals: frequency and risk factors for vascular events and evolution to myelofibrosis in 126 patients.

The frequency of vascular events and evolution to myelofibrosis (MF) in young individuals with essential thrombocythemia (ET) is not well known. The incidence and predisposing factors to such complications was studied in 126 subjects diagnosed with ET at a median age of 31 years (range: 5-40). Overall survival and probability of survival free of thrombosis, bleeding and MF were analyzed by the Kaplan-Meier method and the presence of the Janus Kinase 2 (JAK2) V617F mutation correlated with the appearance of such complications. The JAK2 mutation (present in 43% of patients) was associated with higher hemoglobin (Hb) (P<0.001) and lower platelets at diagnosis. With a median follow-up of 10 years (range: 4-25), 31 thrombotic events were registered (incidence rate: 2.2 thromboses/100 patients/year). When compared with the general population, young ET patients showed a significant increase in stroke (odds ratio 50, 95% CI: 21.5-115) and venous thromboses (odds ratio 5.3, 95% CI: 3.9-10.6). Thrombosis-free survival was 84% at 10 years, with tobacco use being associated with higher risk of thrombosis. Actuarial freedom from evolution to MF was 97% at 10 years. In conclusion, young ET patients have thrombotic events, especially stroke and venous thrombosis, more frequently than generally considered, whereas they rarely transform to MF.

Outcome of patients with acute promyelocytic leukemia failing to front-line treatment with all-trans retinoic acid and anthracycline-based chemotherapy (PETHEMA protocols LPA96 and LPA99): benefit of an early intervention.

To determine prognosis of acute promyelocytic leukemia (APL) failing to front-line therapy with all-trans retinoic acid (ATRA) and anthracyclines, outcome of 52 patients (32 M/20 F; age: 37, 3-72) included in PETHEMA trials LPA96 and LPA99 who presented with either molecular failure (MOLrel, n=16) or hematological relapse (HEMrel, n=36) was analyzed. Salvage therapy consisted of ATRA and high-dose ara-C-based chemotherapy (HDAC) in most cases (83%), followed by stem-cell transplantation (autologous, 18; allogeneic, 10; syngeneic, 1). Fourteen patients with MOLrel (88%) achieved second molecular complete response (molCR), whereas 81% HEMrel patients responded to second-line treatment, with 58% molCR. After median follow-up of 45 months, four MOLrel and 18 HEMrel patients, respectively, experienced a second relapse. Outcome after MOLrel compared favorably to HEMrel, with longer survival (5-year survival: 64+/-14 vs 24+/-8%, P=0.01) and lower relapse risk (5-year relapse risk: 30+/-13 vs 64+/-9%; P=0.044). Additionally, age

International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia.

The eradication of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) predicts for improved outcome. However, the wide variety of MRD techniques makes it difficult to interpret and compare different clinical trials. Our aim was to develop a standardized flow cytometric CLL-MRD assay and compare it to real-time quantitative allele-specific oligonucleotide (RQ-ASO) Immunoglobulin heavy chain gene (IgH) polymerase chain reaction (PCR). Analysis of 728 paired blood and marrow samples demonstrated high concordance (87%) for patients off-therapy. Blood analysis was equally or more sensitive than marrow in 92% of samples but marrow analysis was necessary to detect MRD within 3 months of alemtuzumab therapy. Assessment of 50 CLL-specific antibody combinations identified three (CD5/CD19 with CD20/CD38, CD81/CD22 and CD79b/CD43) with low inter-laboratory variation and false-detection rates. Experienced operators demonstrated an accuracy of 95.7% (specificity 98.8%, sensitivity 91.1%) in 141 samples with 0.01-0.1% CLL. There was close correlation and 95% concordance with RQ-ASO IgH-PCR for detection of CLL above 0.01%. The proposed flow cytometry approach is applicable to all sample types and therapeutic regimes, and sufficiently rapid and sensitive to guide therapy to an MRD-negativity in real time. These techniques may be used as a tool for assessing response and comparing the efficacy of different therapeutic approaches.

The BET bromodomain inhibitor CPI203 overcomes resistance to ABT-199 (venetoclax) by downregulation of BFL-1/A1 in in vitro and in vivo models of MYC+/BCL2+ double hit lymphoma.

High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, mostly known as double-hit lymphoma (DHL), is a rare entity characterized by morphologic and molecular features between Burkitt lymphoma and the clinically manageable diffuse large B-cell lymphoma (DLBCL). DHL patients usually undergo a rapidly progressing clinical course associated with resistance to standard chemo-immunotherapy. As a consequence, the prognosis of this entity is particularly poor with a median overall survival inferior to 1 year. ABT-199 (venetoclax) is a potent and selective small-molecule antagonist of BCL-2 recently approved for the treatment of a specific subtype of lymphoid neoplasm. In this study, we demonstrate that single-agent ABT-199 efficiently displaces BAX from BCL-2 complexes but fails to maintain a significant antitumor activity over time in most MYC+/BCL2+DHL cell lines and primary cultures, as well as in a xenograft mouse model of the disease. We further identify the accumulation of the BCL2-like protein BFL-1 to be a major mechanism involved in acquired resistance to ABT-199. Noteworthy, this phenomenon can be counteracted by the BET bromodomain inhibitor CPI203, since gene expression profiling identifies BCL2A1, the BFL-1 coding gene, as one of the top apoptosis-related gene modulated by this compound. Upon CPI203 treatment, simultaneous downregulation of MYC and BFL-1 further overcomes resistance to ABT-199 both in vitro and in vivo, engaging synergistic caspase-mediated apoptosis in DHL cultures and tumor xenografts. Together, these findings highlight the relevance of BFL-1 in DH lymphoma-associated drug resistance and support the combined use of a BCL-2 antagonist and a BET inhibitor as a promising therapeutic strategy for patients with aggressive DHL.

Clinical impact of the subclonal architecture and mutational complexity in chronic lymphocytic leukemia.

Genome studies of chronic lymphocytic leukemia (CLL) have revealed the remarkable subclonal heterogeneity of the tumors, but the clinical implications of this phenomenon are not well known. We assessed the mutational status of 28 CLL driver genes by deep-targeted next-generation sequencing and copy number alterations (CNA) in 406 previously untreated patients and 48 sequential samples. We detected small subclonal mutations (0.6-25% of cells) in nearly all genes (26/28), and they were the sole alteration in 22% of the mutated cases. CNA tended to be acquired early in the evolution of the disease and remained stable, whereas the mutational heterogeneity increased in a subset of tumors. The prognostic impact of different genes was related to the size of the mutated clone. Combining mutations and CNA, we observed that the accumulation of driver alterations (mutational complexity) gradually shortened the time to first treatment independently of the clonal architecture, IGHV status and Binet stage. Conversely, the overall survival was associated with the increasing subclonal diversity of the tumors but it was related to the age of patients, IGHV and TP53 status of the tumors. In conclusion, our study reveals that both the mutational complexity and subclonal diversity influence the evolution of CLL.

Coding sequence and intron-exon junctions of the c-myb gene are intact in the chronic phase and blast crisis stages of chronic myeloid leukemia patients.

The c-myb gene encodes a transcription factor required for proliferation, differentiation and survival of normal and leukemic hematopoietic cells. c-Myb has a longer half-life in BCR/ABL-expressing than in normal cells, a feature which depends, in part, on PI-3K/Akt-dependent regulation of proteins interacting with the leucine zipper/negative regulatory region of c-Myb. Thus, we asked whether the stability of c-Myb in leukemic cells might be enhanced by mutations interfering with its degradation. We analyzed the c-myb gene in 133 chronic myeloid leukemia (CML) patients in chronic phase and/or blast crisis by denaturing-high performance liquid chromatography (D-HPLC) and sequence analysis of PCR products corresponding to the entire coding sequence and each exon-intron boundary. No mutations were found. We found four single nucleotide polymorphisms (SNPs) and identified an alternatively spliced transcript lacking exon 5, but SNPs frequency and expression of the alternatively spliced transcript were identical in normal and CML cells. Thus, the enhanced stability of c-Myb in CML blast crisis cells and perhaps in other types of leukemia is not caused by a genetic mechanism.

Activity of the novel BCR kinase inhibitor IQS019 in preclinical models of B-cell non-Hodgkin lymphoma.

BACKGROUND: Pharmacological inhibition of B cell receptor (BCR) signaling has recently emerged as an effective approach in a wide range of B lymphoid neoplasms. However, despite promising clinical activity of the first Bruton's kinase (Btk) and spleen tyrosine kinase (Syk) inhibitors, a small fraction of patients tend to develop progressive disease after initial response to these agents. METHODS: We evaluated the antitumor activity of IQS019, a new BCR kinase inhibitor with increased affinity for Btk, Syk, and Lck/Yes novel tyrosine kinase (Lyn), in a set of 34 B lymphoid cell lines and primary cultures, including samples with acquired resistance to the first-in-class Btk inhibitor ibrutinib. Safety and efficacy of the compound were then evaluated in two xenograft mouse models of B cell lymphoma. RESULTS: IQS019 simultaneously engaged a rapid and dose-dependent de-phosphorylation of both constitutive and IgM-activated Syk, Lyn, and Btk, leading to impaired cell proliferation, reduced CXCL12-dependent cell migration, and induction of caspase-dependent apoptosis. Accordingly, B cell lymphoma-bearing mice receiving IQS019 presented a reduced tumor outgrowth characterized by a decreased mitotic index and a lower infiltration of malignant cells in the spleen, in tight correlation with downregulation of phospho-Syk, phospho-Lyn, and phospho-Btk. More interestingly, IQS019 showed improved efficacy in vitro and in vivo when compared to the first-in-class Btk inhibitor ibrutinib, and was active in cells with acquired resistance to this latest. CONCLUSIONS: These results define IQS019 as a potential drug candidate for a variety of B lymphoid neoplasms, including cases with acquired resistance to current BCR-targeting therapies.

Equilibrative nucleoside transporter-2 (hENT2) protein expression correlates with ex vivo sensitivity to fludarabine in chronic lymphocytic leukemia (CLL) cells.

Fludarabine is considered the treatment of choice for most patients with chronic lymphocytic leukemia (CLL). We have analyzed the role of plasma membrane transporters in nucleoside-derived drug bioavailability and action in CLL cells. Among the known plasma membrane transporters, we have previously observed a significant correlation between fludarabine uptake via ENT carriers and ex vivo sensitivity of CLL cells to fludarabine, although mRNA amounts of the equilibrative nucleoside transporters hENT1 and hENT2 do not show any predictive response to treatment. In this study, using polyclonal monospecific antibodies we have observed a significant correlation between the expression of hENT2 by Western blot and fludarabine uptake via hENT carriers and also with ex vivo sensitivity of CLL cells to fludarabine. These results suggest that the equilibrative nucleoside transporter hENT2 plays a role in fludarabine responsiveness in CLL patients.

Control of phosphofructokinase by fructose 2,6-bisphosphate in B-lymphocytes and B-chronic lymphocytic leukemia cells.

The levels of fructose 2,6-bisphosphate and glucose 1,6-bisphosphate and the activities of the key glycolytic enzymes have been studied in T- and B-lymphocytes, and in B-chronic lymphocytic leukemia cells (B-CLL). In both kinds of cells these two bisphosphorylated metabolites have been identified and are present at similar concentrations. Their phosphofructokinase, like that of other normal or tumoral cells, is sensitive to these activators. Fructose 2,6-bisphosphate is the most potent stimulator; it displays the properties of a positive effector. It greatly increases the affinity for fructose 6-phosphate and relieves the inhibition by adenosine triphosphate, without changing Vmax. This effect is also synergistic with adenosine monophosphate. Despite few differences in the activity of phosphofructokinase and in the content of its main effectors in B-lymphocytes and in B-CLL cells, the kinetic properties of the enzyme from B-CLL cells were different, the enzyme being more sensitive to fructose 2,6-bisphosphate (Ka 2 orders of magnitude lower) and to glucose 1,6-bisphosphate than the enzyme from normal lymphocytes. The results reported showing that phosphofructokinase from B-CLL lymphocytes is altered in regulatory properties and the observed changes, in comparison to phosphofructokinase from normal B-lymphocytes, fit well with the hypothesis that fructose 2,6-bisphosphate can also assume a regulatory role in these cancer cells characterized by proliferation and accumulation of relatively mature-appearing lymphocytes.

BMI-1 gene amplification and overexpression in hematological malignancies occur mainly in mantle cell lymphomas.

The BMI-1 gene is a putative oncogene belonging to the Polycomb group family that cooperates with c-myc in the generation of mouse lymphomas and seems to participate in cell cycle regulation and senescence by acting as a transcriptional repressor of the INK4a/ARF locus. The BMI-1 gene has been located on chromosome 10p13, a region involved in chromosomal translocations in infant leukemias, and amplified in occasional non-Hodgkin's lymphomas (NHLs) and solid tumors. To determine the possible alterations of this gene in human malignancies, we have examined 160 lymphoproliferative disorders, 13 myeloid leukemias, and 89 carcinomas by Southern blot analysis and detected BMI-1 gene amplification (3- to 7-fold) in 4 of 36 (11%) mantle cell lymphomas (MCLs) with no alterations in the INK4a/ARF locus. BMI-1 and p16INK4a mRNA and protein expression were also studied by real-time quantitative reverse transcription-PCR and Western blot, respectively, in a subset of NHLs. BMI-1 expression was significantly higher in chronic lymphocytic leukemia and MCL than in follicular lymphoma and large B cell lymphoma. The four tumors with gene amplification showed significantly higher mRNA levels than other MCLs and NHLs with the BMI-1 gene in germline configuration. Five additional MCLs also showed very high mRNA levels without gene amplification. A good correlation between BMI-1 mRNA levels and protein expression was observed in all types of lymphomas. No relationship was detected between BMI-1 and p16INK4a mRNA levels. These findings suggest that BMI-1 gene alterations in human neoplasms are uncommon, but they may contribute to the pathogenesis in a subset of malignant lymphomas, particularly of mantle cell type.

Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR(4.5) sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

Effect of TPA on fructose 2,6-bisphosphate levels and protein kinase C activity in B-chronic lymphocytic leukemia (B-CLL).

Normal B lymphocytes and peripheral mononuclear blood cells from B-chronic lymphocytic leukemia (B-CLL) patients were incubated in the presence of the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In normal B lymphocytes and lymphocytes from five patients with B-CLL, TPA stimulation increased lymphocyte fructose 2,6-bisphosphate (fructose 2,6-P2) content and activity of 6-phosphofructo 2-kinase (PFK-2), which is the enzyme that catalyzes the synthesis of fructose 2,6-P2. This effect was evident after 6 h and maximal after 12-24 h of TPA exposure. In three patients, lymphocytes seemed to be refractory to TPA stimulation in the conditions described here. Lymphocyte stimulation by TPA was associated with the translocation of protein kinase C (PKC) from the soluble to the particulate membrane fraction, except in B-CLL lymphocytes refractory to the TPA effect. These results give further support to the existence within B-CLL of subsets of cells which are refractory to the stimulation by TPA and demonstrate that the tumor promoter TPA induces important metabolic changes in lymphocytes of some patients with B-CLL.

Detection of circulating neoplastic cells by reverse-transcriptase polymerase chain reaction in malignant melanoma: association with clinical stage and prognosis.

PURPOSE: Circulating melanoma cells can be detected in peripheral blood by means of tyrosinase mRNA amplification by reverse-transcriptase polymerase chain reaction (RT-PCR). We conducted a prospective study to evaluate the clinical significance of the presence of circulating neoplastic cells in the blood of patients with malignant melanoma (MM). METHODS: A sensitive RT-PCR assay was used to detect tyrosinase mRNA in the peripheral blood of patients with stages I to IV melanoma. Healthy subjects or patients with other malignancies were used as negative controls. RESULTS: Ninety-one assessable patients were included in the study. There was a statistically significant association between RT-PCR positivity and clinical stage. Circulating melanoma cells were detected in 36% of patients with localized disease (stages I and II), in 45% of patients with regional nodal involvement (stage III), and in 94% of patients with metastatic disease (stage IV) (P < .001). In stage II-III patients who were RT-PCR-positive for mRNA tyrosinase in blood, the recurrence rate and disease-free survival were significantly worse than patients who were RT-PCR-negative. In multivariate analysis, RT-PCR was an independent prognostic factor for recurrence in patients with nonmetastatic disease (P = .002). CONCLUSION: The detection of circulating melanoma cells in peripheral blood by RT-PCR correlated with the clinical stage of patients with melanoma and was an independent prognostic factor for recurrence. Further studies are warranted to better assess the significance of this test in the evaluation of prognosis, early detection of relapse, and in monitoring the effectiveness of systemic therapy.

Phosphorylation of the MARCKS family of protein kinase C substrates in human B chronic lymphocytic leukemia cells.

Incubation of B chronic lymphocytic leukemia (B-CLL) cells with phorbol esters resulted in the phosphorylation of three Triton-soluble, heat-stable, acidic proteins with apparent M(r) of 80 KDa, 60 KDa and 43 KDa. The characteristics of the three proteins suggested that they could be related to the myristoylated, alanine-rich, C-kinase substrate (MARCKS). p80 was immunoprecipitated with an antibody against the N-terminal peptide of MARCKS. p43 co-migrated with mouse MRP/Mac-MARCKS (MARCKS-related protein). p60 is the most prominent substrate of protein kinase C in B-CLL cells.

Chronic myeloid leukemia of thrombocythemic onset: a CML subtype with distinct hematological and molecular features?

Among 84 consecutive patients with chronic phase Ph-positive chronic myeloid leukemia (CML) who were investigated for the hybrid BCR/ABL mRNA, in six cases (7%) the disease mimicked essential thrombocythemia (ET) at presentation, because of marked thrombocytosis (platelet counts ranging from 1003 x 10(9)/l to 2800 x 10(9)/l) and moderate leukocytosis (WBC counts from 10 x 10(9)/l to 19 x 10(9)/l). At initial examination, four of the six patients showed a few (4-9%) immature myeloid cells in the peripheral blood, and two had blood basophilia. All six patients later developed increasing leukocytosis, and two subsequently died from blast crisis and accelerated CML, respectively. In the overall series, 38 patients (45%) expressed the b2a2 type of BCR/ABL mRNA, and 46 (55%) either the b3a2 or both mRNAs. By contrast, only one of the six patients with CML of thrombocythemic onset expressed the b2a2 mRNA vs five with either b3a2 (n = 4) or both mRNA types (n = 1). The above results, in conjunction with similar data from a few previously published cases, suggest an association between the above form of CML and b3a2 type of BCR/ABL transcript.

Giant parallel tube arrays (PTAs), a new type of lymphocyte inclusion.

In a 56-year-old male patient, receiving chemotherapy after radical surgery for bladder carcinoma, an unusual type of cytoplasmic inclusion was discovered in about 30% of peripheral blood lymphocytes. This was a single, large (about 2 microns in diameter), round or ovoid body, darker than the nucleus and reddish-violet in May-Grünwald-Giemsa stain. The examination with transmission electron microscope demonstrated that such inclusions were made up of giant parallel tube arrays (PTAs). The absolute lymphocyte count was normal, but there was an expansion of CD3+, CD4-, CD8+, CD11b, TCR alpha beta lymphocytes. The lymphocytes bearing the inclusion were CD3+ and CD8+. DNA studies suggested an expansion of T-cell population with clonal rearrangement of TCR beta and TCR gamma. This case can be classified as an asymptomatic disorder of large granular lymphocytes, with unusual morphology. Giant PTAs should be taken into account in the differential diagnosis of lymphocyte cytoplasmic inclusions.

Nucleoside transporters in chronic lymphocytic leukaemia.

Nucleoside derivatives have important therapeutic activity in chronic lymphocytic leukaemia (CLL). Experimental evidence indicates that in CLL cells most of these drugs induce apoptosis ex vivo, suggesting that programmed cell death is the mechanism of their therapeutic action, relying upon previous uptake and metabolic activation. Although defective apoptosis and poor metabolism often cause resistance to treatment, differential uptake and/or export of nucleosides and nucleotides may significantly modulate intracellular drug bioavailability and, consequently, responsiveness to therapy. Two gene families, SLC28 and SLC29, encode transporter proteins responsible for concentrative and equilibrative nucleoside uptake (CNT and ENT, respectively). Furthermore, selected members of the expanding ATP-binding cassette (ABC) protein family have recently been identified as putative efflux pumps for the phosphorylated forms of these nucleoside-derived drugs, ABCC11 (MRP8) being a good candidate to modulate cell sensitivity to fluoropyrimidines. Sensitivity of CLL cells to fludarabine has also been recently correlated with ENT-type transport function, suggesting that, besides the integrity of apoptotic pathways and appropriate intracellular metabolism, transport across the plasma membrane is also a relevant event during CLL treatment. As long as nucleoside transporter expression in leukaemia cells is not constitutive, the possibility of regulating nucleoside transporter function by pharmacological means may also contribute to improve therapy.