Regulatory T-cell modulation by green tea in chronic lymphocytic leukemia.

Regulatory T cells (Tregs) are considered to be key immunomodulatory cells of the immune system and are increased in chronic lymphocytic leukemia (CLL). Rai stage 0 identifies patients with early stage CLL for which there is no effective intervention at the present time and a "wait and see" policy is usually adopted. Some biological and clinical studies have reported that green tea constituents, such as epigallocatechin-gallate (EGCG), have antitumor effects on hematologic malignancies including CLL. We report data on a clinical trial in which green tea extracts were given orally to 12 patients with stage 0 CLL and 12 healthy subjects. Ten patients and 10 controls completed the 6-month scheduled therapy. Two patients and 2 controls stopped therapy within 1 month because of tachycardia and epigastralgia. Eight out 10 evaluable patients (80 percent) showed a reduction of lymphocytosis and absolute number of circulating Tregs, as well. One patient (10 percent) had a stabilization of lymphocytosis and a reduction of Tregs, and 1 patient (10 percent) showed an increase of both lymphocytosis and Tregs. Only the non-responding patient progressed after 5 months from the end of green tea administration and chemotherapy was given. Interestingly, both IL-10 and TGF-beta serum levels declined throughout the green tea intake period, in both patients and controls. These data seem to indicate that green tea is able to modulate circulating Tregs in CLL patients with early stage of the disease. This can result in the control of lymphocytosis as well as in the prevention of disease progression.

Frontline chemotherapy with bortezomib-containing combinations improves response rate and survival in primary plasma cell leukemia: a retrospective study from GIMEMA Multiple Myeloma Working Party.

BACKGROUND: The best therapeutic approach for primary plasma cell leukemia (PPCL) remains unknown so far. In very limited studies, the poor clinical outcome of this aggressive variant of multiple myeloma seemed to be ameliorated by the use of the proteasome inhibitor bortezomib. Aiming to provide more consolidated data, this multicenter retrospective survey focused on unselected and previously untreated PPCL patients who had received bortezomib as frontline therapy. PATIENTS AND METHODS: Twenty-nine patients with PPCL were collected. Bortezomib was given at standard doses and schedules, in various combinations with dexamethasone, thalidomide, doxorubicin, melphalan, prednisone, vincristine, and cyclophosphamide. RESULTS: An overall response rate of 79% was observed, with 38% of at least very good partial remission. Grade 3-4 hematological, neurological, infectious, and renal toxic effects occurred in 20%, 21%, 16%, and 4% of patients, respectively. After a median follow-up of 24 months, 16 patients were alive (55%), 12 of whom were in remission phase and 4 relapsed. The best long-term results were achieved in patients who received stem-cell transplantation after bortezomib induction. CONCLUSION: Bortezomib, used as initial therapy, is able to increase the percentage and the quality of responses in PPCL patients, producing a significant improvement of survival.

Combination treatment of flag with non-pegylated liposomal doxorubicin (MYOCET(TM)) in elderly patients with acute myeloid leukemia: a single center experience.

The incidence of acute myeloid leukemia (AML) increases with age, but results of intensive chemotherapy in elderly patients are disappointing. Non-pegylated liposomal formulations of doxorubicin (Myocet™) have been developed with the aim of reducing systemic and cardiac toxicity especially in the elderly. We evaluated the efficacy and toxicity profiles of fludarabine, cytarabine and granulocyte colony-stimulating factor (FLAG) regimen given in association with Myocet™ in 35 patients with AML, median age 69 years (range 61-83 years). Nineteen (54.3%) had newly-diagnosed AML, twelve (34.3%) patients had secondary AML (ten with Myelodisplastic Syndrome, two with Primary Myelofibrosis) and 4 (11.4%) patients had had a late relapse (>12 months) of AML. Complete remission (CR) and partial remission (PR) were obtained in twenty-two (63%) and 3 (8.5%) patients, respectively. Seven (20%) patients showed a resistant disease. There were 3 early deaths (8.5%). Six patients (17%) experienced severe cardiovascular toxicity. The median overall survival (OS) was 12 months (range 1-52 months) with a median disease-free survival (DFS) of 20 months (range 1-48 months). One-year and two-year DFS were 78.9% and 26.7%, respectively. This study demonstrates that in elderly patients with AML, FLAG-Myocet combination shows promising efficacy response with acceptable toxicity, enabling most patients to receive further treatments, including transplantation procedures.

Circulating regulatory T cells in "clinical" monoclonal B-cell lymphocytosis.

Regulatory T-cells (Tregs) constitute a small subset of cells involved in antitumour immunity and are generally increased in patients with chronic lymphocytic leukemia (CLL). No data is available on Tregs in monoclonal B-cell lymphocytosis (MBL), a disease entity characterized by less than 5000/microL circulating clonal B-cells in absence of other features of lymphoproliferative disorders. We used multicolour flow cytometry to evaluate the number of circulating Tregs in 56 patients with "clinical" MBL, 74 patients with previously untreated CLL and 40 healthy subjects. MBL patients showed a lower absolute number of Tregs, compared to CLL patients, but slightly higher than controls. Moreover, the absolute cell number of Tregs directly correlated both with more advanced Rai/Binet clinical stages and peripheral blood B-cell lymphocytosis. Of note, the absolute number of Tregs was found lower in MBL patients than in CLL patients staged as 0/A Rai/Binet. The study showed that Treg increase gradually from normal subjects to "clinical" MBL patients and are significantly higher in CLL patients as compared to MBL patients. Moreover, a significant direct relationship was found between higher Treg values and a higher tumor burden expressed by B-lymphocytosis or more advanced clinical stages. In light of this data, MBL seems to be a preliminary phase preceding CLL. The progressive increase of Treg numbers might contribute both to the clinical evolution of MBL to overt CLL and to CLL progression.

Successful treatment of Schnitzler's syndrome with anakinra after failure of rituximab trial.

We report a 50-year-old man who presented with a 5-year history of an intermittent widespread pruritic urticarioid rash and fever, fatigue, arthralgia and a monoclonal immunoglobulin-M paraprotein. The patient was initially treated with antihistamines and corticosteroids without the disappearance of symptoms. A skin biopsy from the urticarial rash on the thorax was performed, revealing dermal mononuclear and polymorphonuclear cell infiltrate and normal epidermis. A diagnosis of Schnitzler's syndrome (SS), a rare disorder in which the simultaneous occurrence of monoclonal gammopathy and chronic urticaria is usually observed, was made. After an unsuccessful trial with rituximab at a dosage of 375 mg/sqm weekly for 4 consecutive weeks, the patient was treated with anakinra, an inhibitor of interleukin-1alpha that is thought to be involved in the pathogenesis of the disease, at a dose of 100 mg daily given subcutaneously. He showed a prompt response to the drug and he is still well and symptom-free after 12 months of follow-up. On the basis of both this experience and the review of the literature we conclude that anakinra may be a promising option for the treatment of SS. However, these results need to be confirmed on a larger number of patients.

Chronic lymphocytic leukemia-associated pure red cell aplasia.

Pure red cell aplasia (PRCA) is a well-known marrow failure which may be acquired or constitutional/congenital, as the Diamond-Blackfan syndrome. Acquired PRCA may show as a primary hematological disorder or secondary to an associated disease, infection or drug. PRCA rarely complicates chronic lymphocytic leukemia (CLL), may occur anytime in the course of the disease and, in this context, it is a minority of total PRCA. Anemia due to PRCA in CLL patients must be carefully evaluated and differentiated from other causes (autoimmune hemolytic anemia, neoplastic lymphocyte infiltration of bone marrow, chemotherapy) that require a different therapeutic approach. PRCA is thought to be an immunologically mediated disorder, but there is no uniformity in the setting of the management. Immunosuppressive therapy is frequently given, such as steroids and cyclosporin-A. Recently, anecdotal cases have been published on the effectiveness of monoclonal antibodies rituximab and alemtuzumab.

Verbena officinalis essential oil and its component citral as apoptotic-inducing agent in chronic lymphocytic leukemia.

We evaluated the pro-apoptotic activity of Verbena officinalis essential oil and of its main component citral, on lymphocytes collected from normal blood donors and patients with chronic lymphocytic leukemia (CLL). The number of apoptotic cells was greater in CLL patients than in healthy subjects at all different times of incubation (4, 8 and 24 hours) for samples treated with Verbena officinalis essential oil (A) and citral (B) as well vs controls at different concentrations (0.1% and 0.01%). The greater pro-apoptotic ability was shown by both essential oil of Verbena officinalis and citral at lower concentrations (after 4 h A 0.1%: 17.8% vs 37.1%; A 0.01%: 15.8% vs 52%; B 0.1%: 18.4% vs 46.4%; B 0.01%: 15.8% vs 54.2%; after 8 h A 0.1%: 23% vs 38%; A 0.01%: 22.2% vs 55%; B 0.1%: 32% vs 42.2%; B 0.01%: 22% vs 54.3%; after 24 h A 0.1%: 5% vs 20.7%; A 0.01%: 25.8% vs 47.2%; B 0.1%: 18.4% vs 46.4%; B 0.01%: 15.8% vs 54.2%). Patients carrying deletion 17p13 (p53 mutation) showed a reduced ability to undergo apoptosis with respect to patients with other genomic aberrations or normal karyotype. The proapoptotic activity of Verbena officinalis essential oil and citral is thought to be due to a direct procaspase 3 activation. These data further support evidence that indicate natural compounds as a possible lead structure to develop new therapeutic agents.

Epigenetic Changes Induced by Green Tea Catechins a re Associated with Prostate Cancer.

Prostate cancer is one of the most difficult cancers to treat especially when it becomes hormone resistant such as castrate resistant prostate cancer (CRPC) and subsequent metastatic CRPC. Apart from the genetic alterations in prostate cancer, epigenetic modifications also play an important role in the development and neoplastic progression of this disease. These include DNA methylation, histone modifications, and non-coding microRNAs. miRNAs are a novel class of small endogenous single-stranded non-coding RNAs of 19-25 nucleotides in length that typically silence gene expression. Considering the reversibility of epigenetic alterations in early carcinogenesis process, reversion (correction) of these modifications by green tea catechins could be a promising strategy for cancer chemoprevention and therapy. Recent evidence suggests that green tea catechins such as epigallocatechin gallate (EGCG) not only act as epigenetic modulators but can also modify miRNA expression and their target mRNAs, consistently contributing to the inhibition of prostate carcinogenesis. Various studies also indicate that several green tea polyphenols (GTPs) exert synergistic effects with other cancer chemotherapeutic agents. Therefore, the use of appropriate combinations of green tea catechins with the existing chemotherapeutics will lead to a reduction in side effects without decreasing the chemotherapeutic effects. This review will summarize the key results from recent studies detailing the effects of green tea catechins such as EGCG on epigenetic alterations and miRNA expression in prostate cancer.

NOTCH1-mutated chronic lymphocytic leukemia cells are characterized by a MYC-related overexpression of nucleophosmin 1 and ribosome-associated components.

In chronic lymphocytic leukemia (CLL), the mechanisms controlling cell growth and proliferation in the presence of NOTCH1 mutations remain largely unexplored. By performing a gene expression profile of NOTCH1-mutated (NOTCH1-mut) versus NOTCH1 wild-type CLL, we identified a gene signature of NOTCH1-mut CLL characterized by the upregulation of genes related to ribosome biogenesis, such as nucleophosmin 1 (NPM1) and ribosomal proteins (RNPs). Activation of NOTCH1 signaling by ethylenediaminetetraacetic acid or by coculture with JAGGED1-expressing stromal cells increased NPM1 expression, and inhibition of NOTCH1 signaling by either NOTCH1-specific small interfering RNA (siRNA) or γ-secretase inhibitor reduced NPM1 expression. Bioinformatic analyses and in vitro activation/inhibition of NOTCH1 signaling suggested a role of MYC as a mediator of NOTCH1 effects over NPM1 and RNP expression in NOTCH1-mut CLL. Chromatin immunoprecipitation experiments performed on NOTCH1 intracellular domain (NICD)-transfected CLL-like cells showed the direct binding of NOTCH1 to the MYC promoter, and transfection with MYC-specific siRNA reduced NPM1 expression. In turn, NPM1 determined a proliferation advantage of CLL-like cells, as demonstrated by NPM1-specific siRNA transfection. In conclusion, NOTCH1 mutations in CLL are associated with the overexpression of MYC and MYC-related genes involved in protein biosynthesis including NPM1, which are allegedly responsible for cell growth and/or proliferation advantages of NOTCH1-mut CLL.

NOTCH1 mutations associate with low CD20 level in chronic lymphocytic leukemia: evidence for a NOTCH1 mutation-driven epigenetic dysregulation.

In chronic lymphocytic leukemia (CLL), NOTCH1 mutations have been associated with clinical resistance to the anti-CD20 rituximab, although the mechanisms behind this peculiar behavior remain to be clarified. In a wide CLL series (n=692), we demonstrated that CLL cells from NOTCH1-mutated cases (87/692) were characterized by lower CD20 expression and lower relative lysis induced by anti-CD20 exposure in vitro. Consistently, CD20 expression by CLL cells was upregulated in vitro by γ-secretase inhibitors or NOTCH1-specific small interfering RNA and the stable transfection of a mutated (c.7541-7542delCT) NOTCH1 intracellular domain (NICD-mut) into CLL-like cells resulted in a strong downregulation of both CD20 protein and transcript. By using these NICD-mut transfectants, we investigated protein interactions of RBPJ, a transcription factor acting either as activator or repressor of NOTCH1 pathway when respectively bound to NICD or histone deacetylases (HDACs). Compared with controls, NICD-mut transfectants had RBPJ preferentially complexed to NICD and showed higher levels of HDACs interacting with the promoter of the CD20 gene. Finally, treatment with the HDAC inhibitor valproic acid upregulated CD20 in both NICD-mut transfectants and primary CLL cells. In conclusion, NOTCH1 mutations are associated with low CD20 levels in CLL and are responsible for a dysregulation of HDAC-mediated epigenetic repression of CD20 expression.

Should we continue to study high-dose chemotherapy in metastatic breast cancer patients? A critical review of the published data.

Data from eight randomised trials on high-dose chemotherapy (HDC) for metastatic breast cancer (MBC) have been published, but only seven studies are evaluable after the Bezwoda trial was discredited. Moreover, overall survival (OS) has been evaluated in only four out of seven studies since three had a crossover design. OS was similar for the HDC and standard-dose chemotherapy (SDC) group in the four evaluable trials, while disease-free survival (DFS) was improved in the HDC group in six of the seven trials. The delay in relapse for patients with metastatic disease represents an important clinical outcome; furthermore, since none of the reported studies randomised more than 220 patients, their statistical power may have been too limited to detect meaningful survival differences. Finally, preliminary experiences have shown that HDC seems to be the ideal platform upon which to build novel therapies. In conclusion, HDC remains an important field of clinical research for breast cancer patients with stage IV disease and, from the studies reported in this article, there is some evidence for offering this therapeutic modality to selected patients who are interested in a medically aggressive approach.

Effective autologous peripheral blood stem cell transplantation in plasma cell leukemia followed by T-large granular lymphocyte expansion: a case report.

We report a case of de novo plasma cell leukemia, resistant to standard VMD (vincristine, mitoxantrone, dexamethasone) and CVP (cyclophosphamide, vincristine and prednisone) protocols, treated with a chemotherapy intensification regimen (high-dose cyclophosphamide, modified EDAP, Dexa-BEAM) and peripheral blood stem cell transplantation, performed using fractionated total body irradiation and high dose melphalan. The patient is currently alive and well, in very good partial remission 12 months after transplant and 22 months after diagnosis, disclosing a significant proportion of bone marrow and peripheral blood CD3+, CD8+, CD57+, HLA-Dr+ large granular lymphocytes with cytotoxic activity against neoplastic plasma cells.

Chronic lymphoproliferative disorders: an integrated point of view for the differential diagnosis.

Morphology is regarded as the principle basis for the identification of lymphoid neoplasms. Sometimes, however, it fails to discriminate among several chronic lymphoproliferative disorders (CLDs). Improved immunophenotyping has resulted in a better characterization of a number of variants of these diseases, some of which may benefit from different therapeutic approaches. In particular, the proposal of scoring systems using a panel of monoclonal antibodies (MoAbs) has represented a critical step in this field. In fact, to date, some MoAbs (CD5, CD23, FMC7, CD22, CD79b, and surface immunoglobulin density) are able to distinguish among several entities, thus allowing for a correct diagnosis in the majority of cases. However, there is still a small percentage of patients where the combined diagnostic approach (morphology and immunophenotyping) should be further refined by other techniques, such as cytogenetic and molecular characterization. Here numerous questions are raised indicating the need to more accurately differentiate the disease entities under discussion and better understand some of their clinical manifestations.

Minimally differentiated acute myeloid leukemia (AML M0): clinico-biological findings of 29 cases.

Twenty-nine cases of minimally differentiated acute myeloid leukemia or AML M0 identified among 441 AML diagnosed in the last 12 years are reported. In all cases, flow cytometric analysis using a large panel of monoclonal antibodies and cytogenetic and molecular studies (IgH, TcRbeta, BCR/ABL, AML1/ETO and CBFB-MYH11 rearrangements) were performed. Of the 29 patients, 27 were treated with intensive chemotherapy based on GIMEMA protocols. We noted a greater incidence of older (over 60 years) and male patients (52% and 65%, respectively). CD33, CD13, CD7 and TdT were expressed in 79.3%, 82.7%, 58.6% and 42.8% of cases, respectively. Antigenic MPO was present in 17 of 22 cases (77.3%). Most cases expressed CD34 (93.1%), HLA-DR (93.1%), CD117 (80%) and CD45RA (87%). CD45RO and CD90 were consistently negative. In all cases, we observed an up-expression of bcl-2 and a down-expression of CD95 with an inverse trend between the two markers (r -5253; p 0.03). Karyotypic abnormalities were demonstrated in 53.6% of cases. Of these, 6 involved chromosomes 5, 7 and 8, t(9;22), confirmed by the BCR/ABL transcript, was detected in one case. Rearrangements of the TcRbeta and IgH chains were observed in 3 and 2 cases, respectively. No AML1/ETO and CBFB-MYH11 transcripts were found. Twelve out of 27 patients (44%) achieved a complete remission (CR) (in 2 cases after rescue therapy). Seven early (range 1-9 months) and one late (32 months) relapses were observed. Five patients are alive, but only the 4 who underwent bone marrow transplantation are in persistent first CR. In conclusion, AML M0 is a subtype of AML antigenically well detectable, endowed with many adverse parameters (older age, TdT and CD34 expression, resistance to apoptosis, unfavorable cytogenetic abnormalities) and poor prognosis. A very aggressive consolidation treatment can be useful to improve the outcome.

Are "early" and "late" T-acute lymphoblastic leukemias different diseases? A single center study of 34 patients.

Clinical and biological parameters were retrospectively reviewed in 34 cases of T-lineage acute lymphoblastic leukemia (T-ALL), classified as "early" (20 cases) or "late" (14 cases) subgroups, according to the degree of blast cell differentiation, assessed by immunophenotyping. In "early" T-ALL, age, co-expression of "immature" (CD34 and HLA-Dr) or myeloid (My+) antigens, proliferative activity (as evaluated by Ki67 monoclonal antibody), and expression of the "multidrug-resistance" (MDR) phenotype (as determined by C-219 monoclonal antibody) were significantly higher, while WBC count and expression of CDl0 were significantly lower, than in "late" T-ALL. Furthermore, although no statistically significant difference was found between the two groups, "late" T-ALL more frequently displayed a greater extramedullary tumor mass ("lymphoma-like" syndrome), LI FAB morphology and a normal karyotype. A single patient, with "late" T-ALL, also showed positivity for TCR gamma/delta chains, specific monoclonal antibodies. On the whole, 30 patients (88.2%) achieved complete remission: 16(80%) were "early" and 14(100%) "late" T-ALL. No statistical difference was found between the two groups with respect to disease free survival (42% vs 54% at six years), whereas median overall survival was significantly shorter in "early" T-ALL (23 months vs median not yet reached at six years for "late" T-ALL, p < 0.05). We conclude that "early" and "late" T-ALL show clinical and biological differences, that could perhaps justify differential therapeutic approaches.

CD38 expression correlates with adverse biological features and predicts poor clinical outcome in B-cell chronic lymphocytic leukemia.

CD38 identifies a surface molecule with multi-functional activity. Its prognostic importance in B-cell chronic lymphocytic leukemia (B-CLL) is currently under investigation in view of the fact that two different groups have recently indicated that CD38 expression could be an independent prognostic marker in B-CLL. We analyzed the clinico-biological features of 61 immunologically typical (CD5+CD23+) B-CLL patients stratified according to the CD38 expression. Twenty-two (36%) patients expressed CD38 in more than 30% of CD19-positive cells and were considered as CD38-positive B-CLL. Atypical morphology (p 0.02), peripheral blood lymphocytosis (p 0.01) and diffuse histopathologic bone marrow pattern (p 0.003) were findings found to be closely associated with CD38 expression. On the other hand, A and B Binet stages (p 0.02) and interstitial bone marrow involvement (p 0.005) were more represented in the CD38-negative B-CLL group. Trisomy 12 was detected more frequently in the CD38-positive B-CLL group, while 13q14 deletions mainly occurred in CD38-negative group (p 0.005). Finally, median survival of CD38-positive B-CLL patients was 90 months, while it was not reached at 180 months in CD38-negative patients. Taken together, our data strongly suggest that the evaluation of CD38 expression may identify two groups patients with B-CLL greatly differing in their clinico-biological features.

Morphologically typical and atypical B-cell chronic lymphocytic leukemias display a different pattern of surface antigenic density.

Recent evidences suggest that B-cell chronic lymphocytic leukemia (B-CLL) may have heterogeneous biological and clinical features. Immunological phenotype may be useful for distinguishing these different forms of disease. We used a quantitative flow cytometric approach to analyze the expression of several membrane molecules (CD19, CD20, CD22, CD23, CD11c, CD5, CD79b) commonly used to diagnose and characterize B-CLL in a choort of 84 consecutive B-CLL patients diagnosed according to morphological and immunological findings. We found that morphologically so-called "atypical" B-CLL displayed a significantly higher number of CD20 and CD22 molecules than typical forms. On the other hand, CD19 was found to be more expressed in typical B-CLL, although without reaching statistical significance. Finally, no difference was detected with respect to CD23, CD79b, CD11c and CD5 number of molecules/per cell between typical and atypical B-CLL. Other clinico-biological features, such as surface membrane immunoglobulin density, percentage of CD79b and FMC7 expression, peripheral blood lymphocytosis, trisomy 12 and advanced clinical stages were also found to be more frequent in atypical B-CLL. In conclusion, our data confirm the hypothesis that atypical B-CLL is a disease sustained by more mature B-cells, closely related but, at the same time, clearly distincted from neoplastic cells of typical B-CLL.

Treatment of "poor risk" acute myeloid leukemia with fludarabine, cytarabine and G-CSF (flag regimen): a single center study.

We describe a single center experience of 41 consecutive patients with poor prognosis acute myeloid leukemia (AML) who received a single course of FLAG regimen consisting of Fludarabine 30 mg/m2/day plus Cytarabine 2 gr/m2/day (days 1-5) and G-CSF 5 mg/Kg/day (from day 0 to polymorphonuclear recovery) as salvage therapy. Eleven patients were primarily refractory to previous chemotherapy, 10 patients were in first relapse, 2 patients in second relapse and 7 patients in relapse after transplants. Eleven cases were defined as secondary AML (diagnosis of AML made after a preexisting diagnosis of myelodysplastic syndrome). The median age was 52.6 years (range 16-72); 29 patients were males and 12 females. Overall, 23 (56%) patients reached complete remission (CR), 3 patients died of infection (2) or hemorrhage (1) during induction, and 15 (36%) patients had resistant disease. The highest CR rates (80%) were obtained in relapsed cases; de novo and secondary AML registered 60% and 45% of CR rates, respectively. Patients achieving CR received a second FLAG course as consolidation and were submitted to an individualized program post-remission therapy, depending on the age and performance status. Hematological and non hematological toxicities were acceptable. In conclusion, our data confirm that FLAG is a an high effective treatment for poor prognosis AML and in young patients allows intensive post remissional therapy including allogeneic BMT.

Monoclonal B-cell lymphocytosis.

Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic hematologic condition defined by the presence of a small (<5 x 10(9)/L) clonal B-cell population in the peripheral blood in the absence of lymph-node enlargement, cytopenias or autoimmune diseases. It is found in approximately 3-12% of normal persons depending on the accuracy of analytical techniques applied. According to the immunophenotypic profile of clonal B-cells, the majority of MBL cases (75%) are classified as chronic lymphocytic leukemia (CLL)-like. This form may progress into CLL at a rate of 1-2% per year. It is thought that CLL is always preceded by MBL. The remaining MBL cases are defined as atypical CLL-like (CD5+/CD20(bright)) and CD5(-) MBL. The MBL clone size is quite heterogenous. Accordingly, two forms of MBL are identified: i) high-count, or 'clinical' MBL, in which an evidence of lymphocytosis (<5 x 10(9)/L clonal B-cells) is seen, and ii) a low-count MBL, in which a normal leukocyte count is found and that is identified only in population-screening studies. Both forms of MBL may carry the cytogenetic abnormalities that are the hallmark of CLL, including 13q-, 17p- and trisomy 12. Consistent with the indolent phenotype of this condition, genetic lesions, such as TP53, ATM, NOTCH1 and SF3B1 mutations, usually associated with high-risk CLL, are rarely seen. Overall, no prognostic indicator of evolution of MBL to overt CLL has been found at present time. However, taking into account this possibility, a clinical and lab monitoring (at least annually), is recommended.

Functional impact of NOTCH1 mutations in chronic lymphocytic leukemia.

The purpose of this study was to compare the expression and function of NOTCH1 in chronic lymphocytic leukemia (CLL) patients harboring a wild-type (WT) or mutated NOTCH1 gene. NOTCH1 mRNA and surface protein expression levels were independent of the NOTCH1 gene mutational status, consistent with the requirement for NOTCH1 signaling in this leukemia. However, compared with NOTCH1-WT CLL, mutated cases displayed biochemical and transcriptional evidence of an intense activation of the NOTCH1 pathway. In vivo, expression and activation of NOTCH1 was highest in CLL cells from the lymph nodes as confirmed by immunohistochemistry. In vitro, the NOTCH1 pathway was rapidly downregulated, suggesting that signaling relies upon micro-environmental interactions even in NOTCH1-mutated cases. Accordingly, co-culture of Jagged1(+) (the NOTCH1 ligand) nurse-like cells with autologous CLL cells sustained NOTCH1 activity over time and mediated CLL survival and resistance against pro-apoptotic stimuli, both abrogated when NOTCH1 signaling was pharmacologically switched off. Together, these results show that NOTCH1 mutations have stabilizing effects on the NOTCH1 pathway in CLL. Furthermore, micro-environmental interactions appear critical in activating the NOTCH1 pathway both in WT and mutated patients. Finally, NOTCH1 signals may create conditions that favor drug resistance, thus making NOTCH1 a potential molecular target in CLL.