Course Of Chronic Lymphocytic Leukemia Research Articles

Nasolacrimal duct obstruction caused by lymphoproliferative infiltration in the course of chronic lymphocytic leukemia.

Background:Endoscopic dacryocystorhinostomy (DCR) is the standard treatment of nasolacrimal duct obstruction. Only in rare cases, blockage may be caused by malignant tumors and even more exceptionally by lymphatic neoplasms so that biopsies are not routinely taken for diagnostic purposes.Methods:A computerized retrieval system was used for this retrospective study to identify all patients with histologically documented lymphoproliferative infiltration in the lacrimal drainage system from 2001 to 2009.Results:In four of 191 patients (2.1%), infiltration of the nasolacrimal sac mucosa with a small lymphocytic lymphoma (SLL)/chronic lymphatic leukemia (CLL) was found. Patients who develop symptoms like epiphora within the course of known CLL are highly suspicious for lymphoproliferative infiltration of the lacrimal drainage associated lymphoid tissue.Conclusion:A proactive approach with ophthalmologic consultation and DCR should be followed in these patients to avoid dacryocystitis.

Autoimmune Hemolytic Anemia and Immune Mediated Thrombocytopenia in the Phase III RESONATETM Study of Ibrutinib Vs Ofatumumab in Relapsed/Refractory Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma, Including a Case Report

Autoimmune Hemolytic Anemia and Immune Mediated Thrombocytopenia in the Phase III RESONATETM Study of Ibrutinib Vs Ofatumumab in Relapsed/Refractory Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma, Including a Case Report

Other Cancers in Long-Term Survivors of Chronic Lymphocytic Leukemia: Incidence and Prognostic Relevance

Introduction. The clinical course of chronic lymphocytic leukemia (CLL) is mostly indolent. About one third of the patients are managed with lifelong watch-and-wait (WW) and those who receive therapy often achieve a durable remission. As a result, the majority of patients with CLL will live with their disease for long periods of time, and be exposed to several complications, including the occurrence of other cancers (OC). Patients with CLL may have an increased incidence in OC. Published reports indicate an incidence of 3-27%, mostly in treated patients, however, very little is known on OC in patients with CLL not requiring therapy. Furthermore, observation time in published studies is limited to <5 years, and the incidence of OC in patients followed for longer than 10 years is unknown. We, therefore, studied the incidence and prognostic impact of OC in treatment-naïve patients with CLL followed for ≥10 years.Methods. We reviewed our database and identified all patients with CLL untreated at the time of referral. We selected long-term survivors (LTS), defined as patients with a follow-up ≥10 years, and analyzed the incidence and prognostic impact of OC in this population. Non-melanoma skin cancers were excluded since these were diagnosed and treated promptly in virtually all cases and felt not to have prognostic impact. Standardized incidence ratios (SIR) were calculated for OC occurring after the diagnosis of CLL that were reportable to the Surveillance, Epidemiology and End Results program.The estimated overall survival (OS) according to the presence of OC was plotted considering OC as a time-dependent covariate.Results. We identified 797 LTS of CLL seen at our institution between 1957 and 2003. Median age was 56 years (24-88). 57% of patients were males. Median follow-up for the entire population is 154 months (120-485). We recorded 383 OC in 286 (36%) patients. 76/286 (26%) patients had >1 OC (62 had 2 OC, 10 had 3, 2 had 4, 1 had 5 and 1 had 6).The firstOC preceded or was diagnosed concomitantly with CLL in 100 patients (35%), while in the remaining 186 (65%) it occurred later during the course of the disease. 570 patients (71%) required treatment for CLL. Median time to treatment was 18 months (0-454). In treated patients, the cumulative frequency of OC was 205/570 (36%) and in WW patients 81/227 (36%). The SIR for all OC was 1.2 (p = .034). Males and patients younger than 60 years had a significantly higher incidence of OC (SIR 1.31 and 1.27, respectively). Among OC types, secondary leukemia, melanoma and head and neck cancers had the highest observed-to-expected ratio. Surprisingly, lung, digestive tract, and bladder cancer had a lower-than-expected incidence (table). 474 patients (59%) are alive. 222/570 (39%) treated patients and 101/227 (44%) WW patients have died. The median OS was longer in patients without OC (279 months) vs. those with OC (189 months). Independent predictors of shorter survival in multivariate analysis included higher creatinine, the presence of OC, and older age.Discussion. This is the first study to address the incidence of OC in LTS of CLL, including WW patients. In our population, the frequency of OC is similar in treated and WW patients. Although the incidence of OC in LTS of CLL is higher compared to matched general population, the incidence of lung, digestive and bladder cancer is lower than expected. Reasons of this finding remain to be identified.The occurrence of OC is an independent predictor of shorter survival, thus constituting a relevant competing risk of mortality in LTS of CLL.VariableObservedExpectedPerson-yearsSIR (O/E)95% CI for O/EP -valueOverall148123.34109561.201.01 – 1.400.034Male9673.458851.311.06 – 1.580.013Female5249.9350711.040.78 – 1.360.67Age ≥60 years6054.3334161.100.84 – 1.420.44Age <60 years8869.0275401.271.02 – 1.570.027OC typeProstate2825.92118091.080.72 – 1.560.64Lung2029.08119420.690.42 – 1.060.04Breast1918.60118551.020.62 – 1.590.96Melanoma164.23119263.782.16 – 6.140.00Leukemia154.27120093.511.96 – 5.790.00Non-Hodgkin lymphoma66.38119960.940.34 – 2.051.00Digestive1640.4119370.400.23 – 0.640.00Colon819.42119720.410.18 – 0.810.006Pancreas24.83120240.410.05 – 1.490.18Rectal38.69120110.340.07 – 1.000.05Bladder311.18119930.270.05 – 0.780.009Multiple Myeloma21.98120121.010.12 – 3.641.00Lip30.021201515031.00 – 438.50.00Salivary gland20.031202666.668.00 – 240.060.00 DisclosuresNo relevant conflicts of interest to declare.

Insulin Growth Factor 1 Receptor Expression Is Associated with NOTCH1 Mutation, Trisomy 12 and Aggressive Clinical Course in Chronic Lymphocytic Leukemia

Insulin growth factor 1 receptor (IGF1R) is emerging as an important gene in many solid and hematological cancers and its over expression has been reported to be associated with aggressive disease and pharmacologic resistance. Specifically, the IGF1R-IGF1-2 interaction was recently described to be involved in the constitutive activation of many important cell signaling such as NOTCH1 and PI3K/Akt pathways that play a key role in many solid and hematological cancers. In this study we performed a clinical and biological investigations about the role of IGF1R expression in a large and representative prospective series of chronic lymphocytic leukemia (CLL) in Binet A clinical stage enrolled in observation O-CLL1 protocol (clinicaltrial.gov identifier NCT00917540). Total RNA extraction, preparation of DNA single-stranded sense target, and hybridization to gene expression profiling arrays were carried out according to manufacturer’s protocols in 217 CLL patients enrolled in the multicentre O-CLL1 protocol. Gene expression data has been deposited in the National Centre for Biotechnology Information’s Gene Expression Omnibus database http://www.ncbi.nlm.mih.gov/geo and are accessible through series accession number GSE51529. High IGF1R expression was significantly associated with IGHV unmutated (IGHV-UM) status (p<0.0001), high ZAP-70 and CD38 expression (p<0.0001) and unfavorable cytogenetic deletion [i.e. del(11)(q23) and del(17)(p13)], particularly with del(11)(q23) (p=0.03). Cases with del(13)(q14) as single lesion were characterized by the lowest IGF1R gene levels. On the contrary, among the most common cytogenetic aberrations, trisomy 12 showed stronger IGF1R expression compared with the other patients (p<0.0001) and this association was independent from IGHV mutational status. Patients with stereotyped HCDR3 sequences showed a greater IGF1R expression compared to not stereotyped HCDR3 (p=0.001) even if this can be related to the high frequency of IGHV-UM among stereotyped HCDR patients. Interestingly, subset #4 patients, who are known to exhibit an indolent clinical course and distinct biological profile, were also characterized by lower IGF1R expression compared to other M-IGHV and UM-IGHV patients. NOTCH1 c.7541_7542delCT mutation was investigated by next generation sequencing Roche 454 technology in 199 (92%) patients (Lionetti et al, BJH 2014). Globally, median depth of coverage was 1510x, ranging from 605 to 2842. Mutant allele frequency estimated by NGS ranged from 0.02% to 75% of total reads per sample. The presence of NOTCH1 mutation was confirmed by ASO-PCR and Sanger sequencing in all patients with allele burden higher > 0.7% (31; 15.5%) and 7% (19; 9.5%) respectively. We considered as mutated only the 31 patients in whom the presence of the dinucleotide deletion was confirmed by ARMS-PCR. Patients carrying NOTCH1 mutation were characterized by a greater IGF1R expression compared with wild type cases (p=0.002). In addition high IGF1R expression was not significantly different comparing patients with low and high NOTCH1 mutation burden. In order to avoid the bias represented by the strong association between NOTCH1 and trisomy 12, we compared the IGF1R expression between NOTCH1 mutated and wild type cases excluding trisomy 12, and confirmed the previous association (p=0.004). IGF1R expression represented a strong clinical prognostic factor in our CLL cohort: by Kaplan-Maier analysis we observed a significant time to first treatment stratification in all CLLs, in all IGHV-UM and in all IGHV-M patients (p<0.0001). Furthermore, IGF1R retained its significance in multivariate analysis with most important clinical and molecular prognostic factors (CD38 expression, unfavorable FISH and IGHV mutational status). Overall, our study shows the importance of IGF1R expression in CLL and its strong association with specific clinical and biological features, confirming the interest for the study of this gene as a potential prognostic factor and its possible role as a therapeutic target in a specific group of CLL patients carrying trisomy 12 and NOTCH1 mutations. DisclosuresNo relevant conflicts of interest to declare.

EBV- DNA Viral Load in Peripheral Blood Mononuclear Cells of Patients with Chronic Lymphocytic Leukemia Is Associated with Unfavorable Prognosis

Background: Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus that infects more than 90% of the world population. Following primary infection in the oropharynx, EBV remains latently present in B cells. Asymptomatic EBV reactivations periodically occur in oral mucosa-associated lymphoid tissues in healthy carriers. B cells infected by EBV are suspected to be involved in the etiology of various human lymphoma. Previously, EBV reactivation has been diagnosed by serologic IgG, IgM, IgA profiles that suggested virus replication. However, serologic responses, in immunocompromised subjects, like patients with chronic lymphocytic leukemia (CLL), do not necessarily indicate ongoing replicative activity. CLL is the most frequent form of leukemia in adults in western countries. Despite by definition chronic character, CLL is characterized by marked heterogeneity. Potential involvement of EBV in the pathogenesis of CLL is still to be explain. Latent EBV infection in healthy carriers is controlled by a cell-mediated immune response that is impaired in CLL patients and might result in poor control of EBV infection. Since EBV may activate B cells, stimulate their proliferation and inhibit their apoptosis, we hypothesize that it could contribute to unfavorable clinical course of CLL and may be one of reason of its heterogeneity.Objective: The aim of this study was to determine whether EBV-DNA load in the peripheral blood mononuclear cells (PBMCs) of CLL patients may influence heterogeneity in clinical course of the disease. We analyzed the association between EBV-DNA load in PBMC and stage of the disease, adverse prognostic factors (including CD38 and ZAP-70 expression and cytogenetic abnormalities) and clinical outcome.Material and methods: Peripheral blood samples were obtained from 115 untreated CLL patients and 20 healthy individuals. Real-time PCR was used to quantitate EBV-DNA copy number in PBMCs (viral load). As the sensitivity of the system amounts to 10 copies per µl, all the samples with the EBV-DNA copy number below this detection threshold were considered EBV-negative. The immunophenotype of peripheral blood lymphocytes was analyzed by flow cytometer.Results: Detectable EBV-DNA load in PBMCs was found in 62 out of 115 CLL patients (53.91%). The median EBV-DNA copy number in these group was 184 per 1 µg DNA (range: 12.79-8957.69). The EBV-DNA copy number per 1 µg DNA was significantly higher in patients who required the treatment (p<0.0001) than in untreated ones, in patients with lymphocyte count doubling time <12 months than in than patients with lymphocyte count doubling time >12 months (p<0.0001), in patients with hepatomegaly (p=0.023) and, splenomegaly (p=0.005) than in patients without organomegaly. It was also significantly higher in patients with CD38 (p<0.0001) and ZAP-70 expression (p=0.008) comparing to the patients CD38- and ZAP-70-, respectively, as well as in the patients with del (11q22.3) as comparing to the patients without this cytogenetic abnormality. Furthermore, the EBV-DNA copy number per 1 µg DNA showed significant positive correlation with the concentrations of LDH (r=0.531, p=0.00001) and beta-2-microglobulin (r=0.425, p=0.00057). The presence of more than 12.79 copies of EBV DNA in 1 µg DNA isolated from PBMCs was associated with reduced time to first treatment (p=0.00001), and lymphocyte count doubling-free survival (p<0.000001).Conclusions: We proved for the first time, that higher of EBV-DNA copy number in PBMCs of patients with CLL is associated with adverse prognostic factors as well as more progressive clinical course of the disease expressed by reduced time to first treatment. These results suggest that higher of EBV-DNA copy number in peripheral blood mononuclear cells blood predicts poor clinical outcome of CLL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical Impact of Telomere Shortening in Normal and Leukemia Cells in Chronic Lymphocytic Leukemia

Introduction: In chronic lymphocytic leukemia (CLL), short telomere length in the leukemia cells predicts poor prognosis. However, it is not known whether telomere length in normal tissues also predicts patient outcome and can improve the prognostic value of CLL telomere length. Prognosis in CLL is heterogeneous with the primary cause of death being cancer and infections, thought related to immunosuppression. The elderly, males and those with comorbidities have a particularly poor relative survival. Whether this reflects differences in the biology of CLL in these patient groups, or to extrinsic factors such as increased immunosuppression or fragility, is unknown. In normal individuals, the telomeres in somatic cells (ie, buccal cells (BC)) shorten with aging and prior comorbidities; short telomeres being predictive of early mortality related to infections, cancer and cardiovascular disease. In the present study, we have evaluated BC telomere length in patients with CLL, to determine if this is predictive of survival, and can enhance the predictive value of leukemia cell telomere length.Methods: The Manitoba CLL tumor bank contains 235 samples from newly diagnosed CLL patients between 2007-2011; with a median follow-up of 2 years. One-quarter of these patients required chemotherapy and one-tenth have died. Genomic DNA was extracted from purified CLL cells and buccal cells (BC) collected at diagnosis. Telomere length was established by multiplex quantitative real-time PCR. Telomere/standard (t/s) ratio was calculated using the beta-globulin gene as the standard. Statistical analysis was performed using Statistical Analysis Software (SAS) and Prism software.Results: The median adjusted telomere length was much shorter in CLL cells than in BCs being 0.53 and 2.01, respectively. In BCs, telomere length significantly shortened with increasing age (OR 1.04, CI 95% (1.00-1.078)) and at this short follow-up time, correlation with second malignancies was approaching significance (p=0.06). However, BC telomere length was not reflective of the number of comorbidities or survival. In contrast, telomere length in CLL cells was independent of age (p=0.44) and sex (p=0.75) confirming that these factors did not influence the cellular biology of the disease. Telomere length also correlated with other biological markers with short telomeres correlating with unmutated IgHV status (p<0.0001), Zap-70 positivity (p=0.05), and CD38 positivity (p=0.003). These patients with short telomeres also had clinical markers of poor prognosis including short lymphocyte doubling time (p=0.004), higher Rai stage (p=0.02) and an earlier time to treatment (p<0.0001). The prognostic value of CLL telomere length was not enhanced by the addition of BC telomere length.Conclusions: These results demonstrate that BC telomere length in CLL patients shorten with age, and short telomeres may predict subsequent second malignancies. Telomere length in CLL cells correlates with biological and clinical markers of aggressive disease, but it does not explain the poor prognosis seen in the elderly and male patients. While BC telomere length does not seem to influence the initial clinical course of CLL, ongoing studies are evaluating whether it is predictive of the long-term risk of infections and second malignancies. In addition, whether BC telomere length in CLL is shorter than in the normal population and whether shortening correlates with specific comorbidities is presently being determined. DisclosuresNo relevant conflicts of interest to declare.

A Rare Case of Chronic Lymphocytic Leukemia (CLL), Transforming to Two Different Types of Aggressive Lymphoma during the Course of the Disease.

A rare case of chronic lymphocytic leukemia (CLL), Transforming to two different types of aggressive lymphoma during the course of the disease.BACKGROUNDPatients with chronic lymphocytic leukemia may have disease transformation to non-Hodgkin’s lymphoma (DLBL)or prolymphocytic leukemia (PLL)To date there have been no cases in the english literatures reporting a CLL patient presenting with transformation to both of these different types of more aggressive diseases during the course of their disease. We describe a rare case of CLL who developed transformation to PLL and DLBL, also known as Richter’s transformation(RT) respectively during the course of disease.CASEA 53 year old woman with no significant prior medical history who initially presented with leukocytosis of 14500 and cervical lymphadenopathy on physical examination. Excisional biopsy and microscopic examination confirmed the diagnosis of chronic lymphocytic leukemia with microscopic features of monotonous population of small mature-appearing lymphocytes. Immunohistochemical studies are positive for CD5 and CD23, CD38 and ZAP 70. Concurrent review of peripheral blood smear was showed leukocytosis with relative lymphocytosis and smudge cells. A computed tomography scan of the neck, chest, abdomen and pelvis performed and was remarkable for cervical lymphadenopathy and she was subsequently staged as RAI I of CLL/SLL.The patient was asymptomatic and was therefore observed expectantly for 10 months. Then due to progression of lymphocytosis and extensive cervical lymphadenopathy, she began treatment with chemotherapy consisting of Rituxan, Cytoxan and Fludarabine. After 18 months of being in remission after treatment she again developed cervical lymphadenopathy and night sweats.Exisional biopsy of left cervical lymph node performd and flow cytometric analysis of the lymph node was positive for CD-20, PAX-5, and BCL-2 . Also 97% of these B cells expressed CD38, which is associated with a more aggressive course in CLL, and was supportive for prolymphocytic transformation of CLL. She has been initiated on chemotherapy including bendamustine and Rituxan for a total of four cycles.Patient had a good response to treatment for nine months until she palpated a lymph node in left submandibular area and subsequent excisional lymh node biopsy showed recurrence of the disease with prolymphocytic transformation. The patient began to receive chemotherapy with (R-CHOP) regimen after confirmation of recurrence. A month after completion of chemotherapy, patient had developed increasing lymphadenopathy in neck.Microscopic exam of the biopsy showed foci of CLL/SLL cells and Some binucleated large cells with Immunohistochemical stains, positive for CD20 and weak positivity for CD5 and negativity for CD23, CD10 and BCL6. Immunohistochemical stain for KI-67 shows 70-80% proliferation rate in large cells and less than 10% proliferation rate in background CLL cells which indicates Richter’s transformation of CLL/SLL to diffuse large B-cell lymphoma with an activated B-cell phenotype, which is the most common histologic variant of Richter transformation.CONCLUSIONChronic lymphocytic leukemia(CLL) is an indolent B-cell non-Hodgkin’s lymphoma and is the mostcommon leukemia in the western world with a heterogenous and mostly prolonged disease course, that may transform into higher grade lymphoma. Transformation of CLL to a more aggressive lymphoma occurs in about 5-10% of patients, and is generally accociated with acceleration of the disease and poor clinical prognosis. The transformation involves an increased number of Prolymphocytic cells, termed PLL transformation or the development of DLBCL which also reffered to as Richter’s transformation. DisclosuresNo relevant conflicts of interest to declare.

Short Response Duration to Initial Treatment of Autoimmune Cytopenias in Chronic Lymphocytic Leukemia

Introduction: The incidence of chronic lymphocytic leukemia (CLL) is believed to be low in India. Furthermore, there is no information on the incidence and outcome of autoimmune cytopenias. The incidence of autoimmune cytopenias is 5-10% at any point of the course of CLL. This diagnosis can have an important impact on therapeutic decisions. The prognostic significance of these autoimmune cytopenias in CLL continues to be a topic of debate.Methods: This was a retrospective study done at a tertiary level referral center in North India. Patient records of all CLL patients visiting the hematology clinic for the period 1994-2014 (20 years) were analyzed. Diagnosis of autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia (ITP) and pure red cell aplasia (PRCA) was made according to standard criteria. The initial treatment options were categorized as prednisone, chlorambucil and observation. Responses were categorized as overall response rate (ORR) including CR+PR+SD and progressive disease (PD). Time to progression (TTP) was also calculated for each of the treatments and autoimmune cytopenias.Result: Out of a total of 412 CLL patients registered and following up at our clinic, a total of 62 patients (15.0%) developed autoimmune cytopenias. AIHA was seen in 21 patients (5.09%), ITP in 19 patients (4.6%) and PRCA in 22 (5.3%) patients. Autoimmune cytopenia at diagnosis was present in 44 and at follow-up in 18 patients. The median age of the patients was 63 years (SD±10.4). The median hemoglobin of patients with AIHA and PRCA was 70g/l (SD±28) and 60g/L (SD±16) respectively and median platelet count of patients with ITP was 81x109/L (SD±78.3). The best response to steroids was seen in patients with AIHA (ORR=71.4%) and ITP (ORR=80%), while only 40% PRCA patients show response to steroids. The response to chlorambucil was best for ITP (71.4%) followed by PRCA (33.3%) and AIHA (28.5%). The median time to progression of ITP, AIHA and PRCA patients on the above treatment options was 12, 6 and 0 months respectively (p<0.05).Table 1Number of patients as per the autoimmune cytopenia and treatment givenAIHAITPPRCATreatmentTotalORRPDTotalORRPDTotalORRPDPrednisone1410410821046Chlorambucil7257521248None000200000Conclusions: Recent studies have shown contrasting results of autoimmune cytopenias in CLL with most studies showing no impact on overall survival and a few studies showing negative impact. While most studies have concentrated on the survival impact, there is paucity of data on the initial treatment options for these cytopenias and their follow-up. Our study shows that steroids which are the first line in autoimmune conditions are effective only in 70-80% of AIHA and ITP patients, while oral chemotherapy with chlorambucil is effective only in 70% ITP patients and a third of AIHA and PRCA patients. Most other patients have progressive disease. Even those who show initial responses to treatment ultimately progress with time to progression shortest for PRCA patients followed by AIHA and ITP. In conclusion initial treatment of autoimmune cytopenias may atmost delay starting standard therapy for CLL by a few months. DisclosuresNo relevant conflicts of interest to declare.

IgM peak independently predicts treatment-free survival in chronic lymphocytic leukemia and correlates with accumulation of adverse oncogenetic events.

We examined the significance of IgM peaks in chronic lymphocytic leukemia (CLL), including its association with newly reported MYD88, BIRC3, NOTCH1 and SF3B1 mutations. A total of 27, 25, 41 and 57 patients with monoclonal IgM or IgG peaks (IgM and IgG groups), hypogammaglobulinemia (Hypo-γ group) and normal immunoglobulin serum levels (normal-γ group) were, respectively, included. IgM peaks were mainly associated with Binet stage C and the del(17p). Biased usage of IGHV3-48 was shared by both IgM and IgG groups. IGHV3-74 and IGHV4-39 gene rearrangements were specific for IgM and IgG peaks, respectively. SF3B1, NOTCH1, MYD88 and BIRC3 mutation frequencies were 12%, 4%, 2% and 2%, respectively, being over-represented in IgM, IgG and Hypo-γ groups for SF3B1, and being equal between normal-γ and IgM groups for MYD88. Overall, 76%, 87%, 49% and 42% of cases from IgM, IgG, Hypo-γ and normal-γ groups had at least one intermediate or poor prognosis genetic marker, respectively. By multivariate analysis, IgM peaks were associated with shorter treatment-free survival independently from any other univariate poor prognosis biological parameters, including IgG peaks, Hypo-γ, IGHV status, SF3B1 mutations, cytogenetics and lymphocytosis. Therefore, as with IgG peaks, IgM peaks aggravated the natural course of CLL, with increased accumulation of adverse genetic events.

Development and characterization of a physiologically relevant model of lymphocyte migration in chronic lymphocytic leukemia

AbstractThere is growing evidence that lymphocyte trafficking contributes to the clinical course of chronic lymphocytic leukemia (CLL), but to date, only static in vitro cultures have been used to study these phenomena. To address this lack of data, we have developed a dynamic in vitro model in which CLL cells experience shear forces equivalent to those in capillary beds and are made to flow through capillary-like hollow fibers lined with endothelial cells. CLL cells treated in this way increased their expression of CD62L and CXCR4 (both P < .0001) and of CD49d and CD5 (both P = .003) directly as a result of the shear force. Furthermore, CLL cells migrated through the endothelium into the “extravascular” space (mean migration, 1.37% ± 2.14%; n = 21). Migrated CLL cells had significantly higher expression of CD49d (P = .02), matrix metallopeptidase-9 (P = .004), CD38 (P = .009), CD80 (P = .04), and CD69 (P = .04) compared with CLL cells that remained in the circulation. The degree of migration observed strongly correlated with CD49d expression (r2, 0.47; P = .01), and treatment with the CD49d-blocking antibody natalizumab resulted in significantly decreased migration (P = .01). Taken together, our data provide evidence for a novel, dynamic, and tractable in vitro model of lymphocyte migration and confirm that CD49d is a critical regulator of this process in CLL.

Incorporating Targeted Agents Into Future Therapy of Chronic Lymphocytic Leukemia

Treatment of chronic lymphocytic leukemia (CLL) is currently undergoing profound changes. Several monoclonal antibodies (ofatumumab and obinutuzumab), and the first agent targeting essential signaling cascades in CLL (ibrutinib) have recently been approved. Inhibiting the B-cell receptor pathway seems of particular importance in effective targeted therapies of CLL. Here, inhibition of Bruton's tyrosine kinase and phosphatidylinositol 3-kinase delta currently offer the most promising targeted approaches. The clinical course of CLL presents with an impressive heterogeneity. During recent years, the combined use of clinical, biologic, and genetic parameters has allowed characterization of at least three categories of patients(1): (1) patients with a very mild onset and course; (2) patients with an intermediate prognosis; and (3) patients with an aggressive course of high-risk leukemia. With this background, it becomes increasingly challenging to select the right treatment strategy for each patient. In the present article, we summarize the current therapeutic tools and their combination partner drugs. Moreover, we offer a perspective on how to integrate the novel targeted agents for CLL therapy into sequential treatment approaches.

Hepatitis B and Hepatitis C Viral Infections in Patients with Chronic Lymphocytic Leukemia

Whether chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections contribute to the pathogenesis and⁄or course of chronic lymphocytic leukemia is unclear. To document the prevalences of HBV and HCV infections in chronic lymphocytic leukemia patients, and to determine whether infected patients experience more aggressive disease than those without infection. Patient sera were screened for antibodies to HBV core antigen and HCV (anti-HCV) using ELISA; both sera and peripheral blood lymphocytes were further tested (regardless of antibody results) for HBV-DNA and HCV-RNA using real-time polymerase chain reaction. Prognostic markers for chronic lymphocytic leukemia included Rai stage, IgVH mutational status, β2-microglobulin levels, Zap-70 and CD38 status. Fourteen of 222 (6.3%) chronic lymphocytic leukemia patients and two of 72 (2.8%) healthy controls tested positive for previous or ongoing HBV infection (OR 2.4 [95% CI 0.5 to 7.7]; P=0.25) while four of 222 (1.8%) chronic lymphocytic leukemia patients and one of 72 (1.4%) controls tested positive for HCV markers (OR 1.3 [95% CI 0.2 to 6.4]; P=0.81). The levels and distribution of the various indicators of aggressive chronic lymphocytic leukemia disease were similar among HBV- and HCV-infected and uninfected patients. Survival times were also similar. Occult HBV and HCV infection (HBV-DNA or HCV-RNA positive in the absence of diagnostic serological markers) were uncommon in chronic lymphocytic leukemia patients (0.5% and 1.8%, respectively). The results of the present study do not support the hypothesis that HBV or HCV infections play an important role in the pathogenesis or course of chronic lymphocytic leukemia.

Kronik Lenfositik Lösemili Hastaların Kemik İliği Biyopsi Örneklerinde IgVH Mutasyon Durumu ile ZAP70 ve CD38'in İmmünohistokimyasal Olarak Analizinin Prognostik Önemi

Objective: Chronic lymphocytic leukemia (CLL) is one of the most common leukemia type among adults in the industrialized countries. Due to the nature of CLL, it is important to recognize patients with a more rapid course of disease. The goal of our study was to study ZAP70 and CD38 antibodies along with immunglobulin heavy chain variable region (IgVH) mutation status, which have been associated with rapid progression and aggressive clinical course in CLL, and to correlate the expression of these molecules with patterns of bone marrow infiltration. Material and Methods: We included 84 bone marrow biopsy samples into the study to determine ZAP70 and CD38 status using immunohistochemistry. Expression patterns for both antibodies were then correlated with the bone marrow infiltration patterns. We also analyzed IgVH mutations in 20 patients using DNA obtained from paraffin-embedded formalin-fixed bone marrow biopsies. These findings were then correlated with immunohistochemical results. Results: We identified a positive correlation between the expression patterns of ZAP70 and CD38, factors that were previously identified as poor prognostic factors (p<0.001). However, there was no correlation between these two markers and IgVH mutation status (p=1.000 and p=0.931). In addition, we showed a statistically significant positive correlation with ZAP70 immunostaining, and the necessity for an early intervention (p=0.046). ZAP70 and CD38 expressions were statistically significantly correlated with the diffuse pattern infiltration of bone marrow (p<0.001 and p<0.001, respectively). Conclusion: Despite small number of our patients, the findings of our study suggested that ZAP70 and CD38 expression patterns as well as IgVH mutation status might be helpful to determine the course of the disease, and the risk of progression. Particularly ZAP70 immunopositive patients appear to have a faster disease progression, and may require earlier intervention and a closer follow up.

Prognostic relevance of angiopoietin-2, fibroblast growth factor-2 and endoglin mRNA expressions in chronic lymphocytic leukemia.

Elevated levels of circulating angiogenic cytokines and increased expression of genes encoding angiogenic factors have been reported in recent years in patients with chronic lymphocytic leukemia (CLL) but data regarding prognostic and predictive significance are still limited. Therefore, in the present study based upon our prior pilot results, we measured mRNA expressions of angiopoietin-2 (Ang-2), fibroblast growth factor-2 (FGF-2) and endoglin (CD105) by reverse transcription quantitative PCR in purified CD19+ cells from 70 untreated CLL patients (median age, 63 years; males, 64%; Rai III/IV stages, 29 %; unmutated IgVH genes, 60 %) and evaluated their possible association with established prognostic factors and clinical course of the disease. Higher expression of Ang-2 was significantly associated with unmutated IgVH genes (n = 55, p = 0.003). Higher CD105 expression was significantly associated with unmutated IgVH genes (n = 55, p < 0.001), high CD38 expression (n = 66, p = 0.022), high ZAP-70 expression (n = 66, p = 0.010), Rai stage I-IV (n = 70, p < 0.001), progressive clinical course of CLL (n = 70, p = 0.001) and shorter time to treatment (n = 70; p < 0.001). Expression of FGF-2 was not significantly associated with any of the prognostic markers. These results indicate that elevated expression of Ang-2 and in particular CD105 by CLL cells is associated with unfavorable prognostic features and clinical outcome; thus, both cytokines appear to play an important role in biology and progression of CLL and warrant further investigation.

Clonal evolution in CLL patients as detected by FISH versus chromosome banding analysis, and its clinical significance

The acquisition of new aberrations during the course of chronic lymphocytic leukemia (CLL) named clonal evolution (CE) is usually detected by one of the two methods: chromosome banding analysis (CBA) and interphase fluorescence in situ hybridization (I-FISH). The purpose of this study was to compare the usefulness of FISH and CBA for detecting CE and to evaluate its influence on clinical outcome. FISH and CBA were performed at two time points: baseline and follow-up. Thirty-eight previously untreated patients with CLL were included in this study. CBA and I-FISH revealed CE in 15 (39.5%) and 10 (26.3%) patients, respectively. High-risk CE was detected in six cases by CBA and in five cases by I-FISH. In four cases with CE-dependent 17p abnormalities detected by CBA, metaphase FISH was needed for the confirmation of 17p13.1 deletion. Time from first-line to second-line treatment (TTST) and overall survival (OS) did not differ between patients with and without CE, irrespective of the CE-detecting method used. However, shorter OS (P=0.043) and TTST (P=0.006) were observed for the patients with potentially relevant CE (rCE) detected by CBA, in which acquired aberrations were present in at least 20% of undivided cells and/or changed baseline karyotype to abnormal or complex and were not resulting from 13q deletion. Our results suggest that some, but not all, CE-dependent aberrations detected by CBA influence clinical outcome. Moreover, I-FISH, which was aimed at detecting aberrations of prognostic significance, was found to be more precise than CBA in their detection, especially TP53 deletion.

Richter's transformation in CLL—a distinct lymphoma

A landmark study has delineated and identified the genetic pathways that drive the natural course of chronic lymphocytic leukaemia (CLL) B cells into Richter’s transformation. CLL-related Richter’s transformation is a distinct lymphoma, and poses various questions about B-cell ontogeny and relevance of B cell receptor signalling inhibitors.

Old DAT / New Data : Positive Direct Antiglobulin Test Identifies a Specific Subgroup Of CLL Patients

Autoimmune cytopenias represent a well-known complication of chronic lymphocytic leukemia (CLL). The most frequent one is autoimmune haemolytic anemia (AIHA), occurring in about 5% of the patients during the course of the disease. The prognostic significance of autoimmune cytopenias remains uncertain and controversial. In previous studies, a positive direct antiglobulin test (DAT) was found in up to 35% of CLL cases, however only a minority of the patients would develop AIHA at any time of the disease. The aim of this monocentric study was to explore the clinical and biological characteristics and the outcome of CLL patients showing a positive direct antiglobulin test at any time during the course of the disease. We reviewed all CLL patients seen at our institution who had at least one positive DAT between January 2007 and May 2013.Fifty-four patients were found, representing about 10% of our CLL cohort. The sex ratio was 1,8 (35M/19F). Median age at diagnosis was 66,2 years (range 44,7 to 87,3). According to the Binet classification, 41 (76 %) patients were in stage A, 7 in stage B, and 6 in stage C. Study of usual prognostic parameters evidenced that these patients represented a very high risk group: 82% of cases harbored unmutated IGHV, and CD38 was expressed in 60% cases. Cytogenetic alterations were studied by FISH analysis: 11q deletion, trisomy 12 and 17p deletion were found in 12,5%, 22,9% and 14,3% cases respectively.In this cohort, with a median follow up of 85 months, 41 (76 %) patients required treatment. The first line of treatment was initiated for CLL progression in 33 cases, for symptomatic AIHA along with CLL progression in 5, and for symptomatic AIHA (without CLL progression) in 3. The median time to first treatment was very short (16 months), and the median overall survival of this cohort was 84 months. In 22 cases, the DAT was positive from the time of diagnosis, while in 34 patients it became positive later during the course of CLL. Only 19 patients (35%) developed symptomatic AIHA. DAT specificity was IgG, IgG+ C or C for respectively 36, 10 and 8 patients. Interestingly, there was no impact of the time of first positive DAT during the course of the disease (at diagnosis or later). Moreover, occurrence of a symptomatic AIHA had no impact on treatment free survival or overall survival when compared with cases with positive DAT only.Among these cases, 41 were in stage A at diagnosis and up to 78% harbored unmutated IGHV (as compared with 30 % in our institutional stage cohort). In order to evaluate the impact of a positive DAT on a homogenous population, we focused on the 31 IGHV unmutated stage A cases and compared them with our cohort of IGHV unmutated stage A with consistently negative DAT. We found a significant prevalence of VH1-69 and VH 3-21 use (43 %) and a high percentage belonging to a stereotyped subset (34%). The majority of the cases (19/28 tested) were responders to in vitro B-cell receptor stimulation by anti IgM. In this population, time to first treatment (26 months) was slightly shorter but median overall survival (55 months) was significantly reduced highlighting the poor response to treatment.In conclusion, occurrence of a positive DAT at any time of the course of CLL is associated with poor outcome and has the same adverse prognostic impact as the onset of a symptomatic AIHA. Moreover, study of B-Cell receptor (BCR stimulation by anti-IgM, CDR3 stereotypy) show that these patients may represent a pathophysiological distinct subgroup in which antigen stimulation is likely to play a major role. Disclosures:No relevant conflicts of interest to declare.

Characterization Of a Novel In Vitro Circulation System Designed To Model The Migration Of Primary CLL Cells Across The Vascular Endothelium

There is growing evidence that lymphocyte trafficking contributes to the clinical course of chronic lymphocytic leukemia (CLL). However, no in vitro models exist to quantify migration from the peripheral vasculature or establish the key molecular events that drive this process. We have therefore established and characterized a novel dynamic in vitro model in which CLL cells are placed under shear forces equivalent to those experienced in the capillary beds and are made to flow through capillary-like hollow fibers lined with endothelial cells (HUVECs). Electronic microscopy demonstrated that HUVEC cells subjected to a shear force of 10 dynes/cm2 flattened against the wall of the hollow fibers giving confluent coverage and allowing formation of tight junctions in our system. Shear force was subsequently reduced to levels found in capillary beds (5 dynes/cm2) and CLL cells were introduced into the hollow fibers (106 CLL cells/ml). CLL cells circulated under these conditions increased their expression of selectin CD62L (associated with lymphocyte homing), CXCR4 (SDF-1 receptor) (both P<0.0001), lymphocyte adhesion molecule CD49d and the regulator of B cell receptor signaling, CD5 (both P=0.003) when compared to static cultures. We then demonstrated that CLL cells could migrate though the HUVEC-lined hollow fibers into the ‘extravascular' space (EVS) and that this was an active process as 2mm fluorescent polystyrene beads were unable to move from the circulating compartment of the system to the EVS. CLL migration out of circulation into the EVS increased over time with a mean percentage migration of 1.37% ± 2.32% after 48h (n=22). Furthermore, CLL cells that migrated had significantly higher expression of CD49d (P=0.02), MMP-9 (breaks down extracellular matrix, P=0.004), the signaling molecule and activation marker CD38 (P=0.009), the co-stimulatory molecule CD80 (P=0.04) and the early activation marker CD69 (P=0.04) when compared with CLL cells that remained in the circulation. Similar results were also seen using HBMEC-lined hollow fibers suggesting that these effects were not HUVEC specific. Addition of an SDF-1 chemokine gradient from the EVS towards the circulating compartment resulted in significantly increased migration of CLL cells into the EVS after just 1h (P=0.04, n=8). Importantly, the degree of migration observed was strongly correlated with CD49d expression (r2=0.47, P=0.01) and in keeping with previously published data we showed a strong correlation between CD49, CD38 and MMP-9. Given these findings, we investigated the effects on migration of the CD49d blocking antibody, Natalizumab. This resulted in a significantly decreased migration (P=0.01) confirming that CD49d plays a critical role in CLL cell migration. Taken together, we have developed a dynamic in vitro model system of the peripheral vasculature. Using this unique model, we have identified some of the key pathophysiological events that drive CLL cell migration in contrast to normal cell migration and have shown the utility of our dynamic model to further understand the biology of CLL and as a novel potential drug testing platform. Disclosures:No relevant conflicts of interest to declare.

Systematic Mapping Of Drug and Pathway Sensitivity In Chronic Lymphocytic Leukemia Identifies Synthetic Lethal Interactions Of Mutant p53

IntroductionCritical pathway alterations contribute to heterogeneous biology and clinical course in chronic lymphocytic leukemia (CLL). The role of particular mutations for drug sensitivity has been demonstrated for CLL and other entities. Here we systematically investigated heterogeneity of drug response and genetic lesions in primary CLL. The comprehensive identification of pathway dependencies will lead to a better understanding of hematologic malignancies and this may be exploited by development of rational-driven treatment approaches. MethodsPrimary CLL cells were characterized through an ex vivo high-throughput drug screening platform in 384-well format. The drug library consisted of 67 compounds (inhibitors targeting critical pathways in CLL and other cancers as well as compounds with known activity against CLL cells (including ABT-263, CAL-101, dasatinib, fludarabine, ibrutinib)) at different concentrations. Heat inactivated human serum was supplemented to mimic micro-environmental conditions. CLL samples represented untreated CLL, sensitive or refractory disease as well as major genetic subgroups. Cell viability was assessed by quantification of ATP (CellTiterGlo®) 48 and 72 hours after drug application. To understand heterogeneous pathway dependencies, drug sensitivity was regressed on genetic profiles. Genetic characterization was performed by FISH, targeted sequencing of recurrent aberrations (BRAF, MYD88, NOTCH1, SF3B1, TP53) as well as whole exome sequencing. ResultsWe report results from 97 primary CLL samples screened ex vivo. Validity of the approach was demonstrated by the response profiles of p53-interfering compounds: Nutlin-3a and fludarabine induced cell death in cells with wild-type (n=80) but considerably lessin CLL with mutated p53(n=17; Nutlin-3a 10µM: 51±18vs80±18;fludarabine 10µM: 40±36vs 67±23 [% viability of untreated control], both p<0.001). Furthermore inhibitors with related targets (e.g. drugs targeting the B-cell receptor (BCR) pathway: CAL-101, dasatinib, ibrutinib, R406) showed similar response profiles across patients, suggesting that prediction of drug effects can be based on functional and genetic properties of tumors. Patients can be divided into two major subgroups according to their sensitivity towards BCR and related kinase inhibition (e.g. Akt, Btk, Lyn, PI3K, Syk). A number of genotype-specific screening hits for single genetic aberrations were obtained. We identified compounds with preferential activity in groups with high risk aberrations (e.g. TP53 mutation, NOTCH1 mutation, 11q deletion; Fig. 1A) suggesting synthetic lethal interactions. Validation of the top hit with increased response of TP53-mutated CLL (compound A 10µM wt vs mutTP53: 58±31 vs 37±18 [% viability of untreated control]; Fig. 1A) confirmed genotype-specific cytotoxicity across concentration ranges (0.1-10µM; Fig 1B). Other compounds inhibiting the same target support our observation (compounds A2 and A3; Fig. 1B). The effect was reproducible in co-culture of CLL cells with HS-5 stroma cells (66±16 vs 32±13 [% viability of untreated control], p<0.05; Fig 1C). [Display omitted] ConclusionOur data provide a first comprehensive map of genetically determined drug sensitivity in CLL. The work offers a novel functional classification of CLL based on drug sensitivity and identifies synthetic lethal interaction for TP53 mutant CLL. Disclosures:No relevant conflicts of interest to declare.

Low Frequency Of IGHV4-39/IGHD6-13/IGHJ5 Rearrangement With Stereotyped HCDR3 (subset #8) In Russian and Ukrainian Chronic Lymphocytic Leukemia Patients

The leukemia cells of unrelated patients with chronic lymphocytic leukemia (CLL) display a restricted repertoire of immunoglobulin gene rearrangements and very similar structure of B-cell receptor (stereotyped BCR). Numerous different subsets of stereotyped BCRs were identified. Some associations between specific BCRs subsets, clinical course of CLL, and molecular abnormalities were found. Namely, short overall survival, accumulation of SF3B1 mutations in subset #2, high risk for Richter's transformation, trisomy 12, NOTCH1 mutations in subset #8, increased frequency of NOTCH1 mutations in subset #1 were revealed (Rossi et al., 2009, 2013; Streffoed et al., 2013).The aim of this study was to evaluate distribution of different stereotyped BCR subsets in Russian and Ukrainian CLL cohort with special attention on prognostic unfavorable subsets. MethodsImmunoglobulin heavy chain variable (IGHV) genes of 969 CLL patients (564 from Russia and 405 from Ukraine) were amplified according to BIOMED-2 rules, sequenced and analyzed with IMGT and IgBlast databases. Additionally, NOTCH1 c.7544_7545delCT mutations were analyzed in 301 patients (randomly collected and in the prognostic unfavorable subsets) by amplification refractory mutation system (ARMS) PCR as proposed by Rossi D. et al., 2012. In 19 cases NOTCH1 mutational status was confirmed by direct DNA Sanger sequencing. ResultsJoined Russian-Ukrainian CLL cohort was presented mainly by unmutated (UM) IGHV gene cases (64.2%). The distribution of major clusters of stereotyped BCR did not differ from those presented in the literature (comparison was made separately for cases with mutated (M) and UM IGHV genes, taking into account the differences in the mutational status of IGHV genes between our CLL cohort and CLL cohorts from European countries and USA), except for subset #8, which was found in one case only (0.16% of UM IGHV gene cases). At the same time, subset #8 (IGHV4-39/IGHD6-13/IGHJ5 rearrangement) belongs to the group of “major” subsets in CLL (Agathangelidis et al., 2012), and its frequency varies from 0.9% to 2% among cases with UM IGHV genes in the most of presented CLL cohorts (Murray et al., 2008; Messmer et al., 2009; Maura et al., 2011). Frequency of subset #2 had tendency to be lower among Russian CLL patients (1.4% of M CLL cases), but not among Ukrainian patients (4.5% of M CLL cases) comparing with European CLL cohorts.NOTCH1 c.7544_7545delCT was identified in 27 out of 301 patients (9.0%), which was in accordance with other CLL studies. Twenty five cases with NOTCH1 mutations had UM IGHV genes and 2 cases expressed M IGHV3-21 gene (both subset #2). As well, majority of NOTCH1 mutated cases (17/27, 63%) showed stereotyped BCRs, which belonged to the following subsets: #1 (2 of 12), #2 (2 of 7), #3 (1 of 6), #7 (2 of 10), #9 (2 of 10), #25 (1 of 2), #59 (1 of 3) (subset's designation according Murray et al., 2008), UA/ref2 (1 of 2), UA/ref11 (2 of 2) (subset's designation according Bilous et al., 2010), or having a homology with the CLL sequences of the public CLL database. No NOTCH1 mutations were found in 11 IGHV4-39-used cases, including one case from subset #8. In 10 of 27 (37.0%) NOTCH1 mutated cases IgHV1-69 gene was used, while among wild type NOTCH1 patients IgHV1-69 was presented in 65/274 (23.7%) cases (p=0.12). ConclusionThe distribution of the major subsets of stereotyped BCRs in CLL patients from Russia and Ukraine (eastern slavic countries) was similar to the western European countries and USA, except for low frequency of subset #8, associated with unfavorable clinical course. Our preliminary data suggested that NOTCH1 c.7544_7545delCT mutation is typical for UM CLL cases with stereotyped BCRs, but no clear association with specific subsets was found in the absence of subset #8. Disclosures:No relevant conflicts of interest to declare.