Chronic Lymphoid Leukemia Patients Research Articles

Circular RNA as a Biomarker for Diagnosis, Prognosis and Therapeutic Target in Acute and Chronic Lymphoid Leukemia.

Circular RNAs (circRNAs) are single-stranded RNAs that have received much attention in recent years. CircRNAs lack a 5' head and a 3' poly-A tail. The structure of this type of RNAs make them resistant to digestion by exonucleases. CircRNAs are expressed in different cells and have various functions. The function of circRNAs is done by sponging miRNAs, changing gene expression, and protein production. The expression of circRNAs changes in different types of cancers, which causes changes in cell growth, proliferation, differentiation, and apoptosis. Changes in the expression of circRNAs can cause the invasion and progression of tumors. Studies have shown that changes in the expression of circRNAs can be seen in acute lymphoid leukemia (ALL) and chronic lymphoid leukemia (CLL). The conducted studies aim to identify circRNAs whose expression has changed in these leukemias and their more precise function so that these circRNAs can be identified as biomarkers, prediction of patient prognosis, and treatment targets for ALL and CLL patients. In this study, we review the studies conducted on the role and function of circRNAs in ALL and CLL patients. The results of the studies show that there is a possibility of using circRNAs as biomarkers in the identification and treatment of patients in the future.

Determination of Some miRNA Expression Levels in Chronic Lymphoid Leukemia Patients

Chronic lymphoid leukemia (CLL) is a type of cancer that occurs as a result of the accumulation of morphologically small, mature-looking lymphocytes. CLL is the most common type of leukemia in Western countries, where it accounts for 30% of all leukemias. Accounts for only 10% of all leukemias in Asian populations. MicroRNAs (miRNAs) are non-protein-coding single-stranded RNA molecules approximately 18-25 nucleotides long, forming a class of endogenous small RNAs. Research has shown that microRNAs can function as oncogenes or tumor suppressors in CLL. Although the expression levels of microRNA 133a and microRNA 452 have been determined in many cancers, including lung, prostate and colon cancer, expression levels in CLL patients have not been determined. Therefore, in our study, the expression levels of miRNA 133a and miRNA 452 in CLL patients will be calculated quantitatively using the Real-Time PCR method. As for the method steps, in the first stage, whole blood samples will be taken. miRNA will be isolated from the whole blood samples taken, cDNA will be synthesized from the miRNA samples, and finally, expression levels will be determined with the Real-Time PCR method using miRNA 133a, miRNA452 specific primers and U6 primer as the reference gene. The data obtained will be analyzed and interpreted with the SPSS package program. This study will be conducted to determine whether these two miRNAs can guide early diagnosis and diagnosis in CLL patients and to provide preliminary information to clinicians and contribute to the literature on this subject.

S2623 Chronic Lymphoid Leukemia With Extensive Liver Infiltration in the Absence of Leukemic Transformation

Introduction: Chronic lymphoid leukemia (CLL) is the most common hematologic cancer diagnosed in the elderly. Its association with liver dysfunction secondary to leukemic infiltration ranges from mild elevations in liver enzymes (LE) to acute liver failure. Severe hepatic infiltration is typically associated with acute leukemic transformation. Here we report an atypical manifestation of CLL with rising LE and white blood cell (WBC) count mimicking leukemic transformation. Case Description/Methods: 62-year-old male with history of CLL not on therapy presented to clinic for abnormal LE and rising leukocytosis. Patient reported fatigued, jaundice, right upper quadrant pain and 25lb weight loss over five months attributed to dietary modifications. Patient reported taking glimepiride, atorvastatin and herbal supplements (GNC50+ multivitamin, fertility and prostate multivitamin) that were stopped five months prior to encounter due to concerns of drug-induced liver injury (DILI). Labs were significant for absolute lymphocytosis >53,000/uL, AST 156U/L, ALT 336U/L, total bilirubin 4.4 mg/dl, direct bilirubin 3.57mg/dl and gamma-glutamyl transferase of 94U/L from previously normal LE. Additional work up was negative for viral and autoimmune etiologies. NASH Fibrosure was consistent with F4 Fibrosis. Computer tomography (CT) of abdomen did not demonstrate fatty changes but did show significant abdominal lymphadenopathy. Patient was hospitalized for rapidly rising WBC and LE. Abdominal ultrasound doppler showed patent liver vasculature. Transjugular liver biopsy was negative for acute leukemic transformation, but did identify extensive, >60% sinusoidal infiltration by CLL with hepatic venous pressure gradient (HVPG) of 11 mmHG attributed to periportal and peripancreatic adenopathy observed on CT imaging (Imaging A-D). Patient was initiated on Venetoclax and Obinutuzumab as an outpatient. LE peaked two months after chemotherapy initiation. Liver synthetic function remained normal (Table 1). Discussion: After ruling out DILI with failed improvement upon discontinuation of suspected agents and liver biopsy, extensive CLL extranodal liver involvement was attributed to have caused liver dysfunction and subsequent portal hypertension. Severe pattern of involvement is rare without Richter Transformation, which can be seen in 8% of CLL patients. Due to severe pattern of liver infiltration, worsening LE, portal hypertension, and hepatosplenomegaly it was ultimately decided to start patient on chemotherapy.Figure 1.: Image A:CT abdomen and pelvis with IV contrast depicting an enlarged 33.4x42.9mm periportal lymph node Image B: CT abdomen and pelvis with IV contrast depicting 11.7x18.3mm pancreatic lymph node Image C: : H&E (400x) section demonstrates an atypical lymphoid follicle surrounding bile ductules, with adjacent liver parenchyma. Characteristic immunohistochemical staining of the neoplastic cells shows positivity for CD20 (dim), CD5, CD23, and is negative for CD3 Image D: ZAP 70 (400x) staining (brown) of neoplastic cells has been associated with a poor prognosis.Table 1.: Patient laboratory information from date of diagnosis to most recent outpatient follow up. Footnote: *Represents laboratory data for time of hospital admission for elevated liver enzyme work up **Represents the laboratory data prior to initiation of chemotherapy ***Represents peak liver enzyme abnormality ****Represents most recent outpatient follow up laboratory data.

Significance of Frequencies, Compositions, and/or Antileukemic Activity of (DC-stimulated) Invariant NKT, NK and CIK Cells on the Outcome of Patients With AML, ALL and CLL

Invariant natural killer T (iNKT)/natural killer (NK)/cytokine-induced killer (CIK) cells are important for immune surveillance. (I) Novel combinations of antibody 6B11 (targeting the Vα24-Jα18-invariant T-cell receptor) with CD4/CD8/CD1d/Vα24 for iNKT subset detection and "T/NK cell-like"-iNKT subsets were defined. Compared with healthy peripheral blood mononuclear cells (MNC) (significantly) lower proportions of iNKT cells (6B11/6B11CD3/6B11CD161), NK cells (CD3CD56/CD3CD161), and CIK cells (CD3CD56/CD3CD161) were found in peripheral blood MNC from acute myeloid (AML)/acute myeloid, lymphoid (ALL)/chronic lymphoid leukemia (CLL) patients in acute disease stages. Subtyping of iNKT cells revealed (significantly) higher proportions of CD3 T cells and CD161 NK cells in AML/ALL/CLL expressing 6B11 compared with healthy MNC. Prognostic evaluations showed higher proportions of iNKT/NK/CIK cells in favorable AML subgroups (younger age, primary, no extramedullary disease, achievement/maintenance of complete remission) or adult ALL and CLL patients. (II) iNKT/NK/CIK cell frequencies increased after (vs. before) mixed lymphocyte cultures of T-cell-enriched immune reactive cells stimulated with MNC/whole blood with or without pretreatment with "cocktails" (dendritic cells generating methods/kits inducing blasts' conversion to leukemia-derived dendritic cells from AML patients). Individual "cocktails" leading to "highest" iNKT cell frequencies could be defined. Antileukemic blast lytic activity correlated significantly with frequencies of iNKT/NK/CIK cells. In summary healthy MNC show significantly more iNKT/NK/CIK cells compared with AML/ALL/CLL MNC, a shift in the iNKT cell composition is seen in healthy versus leukemic samples and iNKT/NK/CIK cell-proportions in AML/ALL/CLL MNC samples correlate with prognosis. "Cocktail"-treated AML blasts lead to higher iNKT/NK/CIK cell frequencies and samples with antileukemic activity show significantly higher frequencies of iNKT/NK/CIK cells. Proportions of iNKT/NK/CIK cells should regularly be evaluated in AML/ALL/CLL diagnosis panels for quantitative/prognostic estimation of individual patients' antileukemic potential and their role in dendritic cells/leukemia-derived dendritic cells triggered immune surveillance.

Therapeutic CD94/NKG2A blockade improves natural killer cell dysfunction in chronic lymphocytic leukemia

ABSTRACTNatural killer (NK)-cell count is predictive of chronic lymphoid leukemia (CLL) disease progression and their dysfunction is well documented, but the etiology of this is currently lacking. CLL cells have been shown to over-express HLA-E, the natural ligand for NKG2A expressed on NK-cells that generates a distinct negative signal relative to direct NK-cell cytotoxicity in other disease models. Utilizing a novel anti-NKG2A monoclonal blocking antibody (mAb), monalizumab, we explored the in vitro preclinical activity of targeting the NKG2A receptor, and the NKG2A/HLA-E interaction as a mechanism of tumor evasion in CLL patients. Our work confirmed overexpression of HLA-E on CLL B-cells and demonstrated NKG2A expression on CD56+/16+ NK-cells from CLL patients. We also demonstrate that blocking NKG2A on CLL NK-cells was sufficient to restore direct cytotoxicity ability of NK-cells against HLA-E-expressing targets without impacting NK-cell mediated antibody-dependent cellular cytotoxicity. Additionally, we proved the specificity of monalizumab in blocking NKG2A through Fc-blocking mechanisms. This paper provides justification for the potential clinical utility of monalizumab in the treatment of patients with CLL.

Direct Coombs Test Positivity in B-Chronic Lymphoid Leukemia: a Marker of Advanced Clinical Disease.

Chronic lymphoid leukemia (CLL) is a malignant hematopoietic disorder, the most common of all adult leukemias with a distinctive immunophenotype. It is well established that CLL patients can have autoimmune complications, amongst them autoimmune hemolytic anemia as the most frequent. This study was carried out to determine the frequency of direct Coombs Test positivity in CLL patients and its possible correlation with Rai staging, hematological parameters and biochemical markers. This descriptive cross sectional study was carried at Liaquat National Hospital from January 2011 to June 2013. Sixty untreated patients with B- chronic lymphoid leukemia were enrolled. Complete blood count, direct Coombs test, serum urea, creatinine, uric acid and LDH levels were determined. Data were compiled and analyzed using SPSS version 21. Out of 60 patients, 42(70%) were males and 18(30%) were females. Mean age was 59±9.2 years. Male to female ratio was 2.1: 1. The frequency of direct antiglobulin test (DAT) positivity was found to be 23.3%. The monospecific IgG was positive in 11 patients (18.3%); C3d positivity was evident in 1 patient (1.6%) and 2 patients (3.3%) had dual IgG and C3d positivity. The mean hemoglobin was 10.8±2.4gm/ dl. Significantly low mean hemoglobin of 8.3±3.0 gm/dl was seen in Coombs positive patients compared with negative patients having a mean hemoglobin level of 11.7±1.6 gm/dl (P<0.001). DAT positivity also demonstrated a positive association with advanced Rai stage III disease (P<0.01). No associations were noted with age, gender and biochemical markers. Direct Coombs test positivity in CLL in our patients, unlike in Western studies, appears relatively high, indicating significant autoimmune hemolytic anemia and advanced Rai stage in our setting. DAT positivity can be considered as a surrogative marker for advanced clinical disease.

Development of a High-Throughput Screening Assay with Nurse-like Cell-Based Microenviroment in Chronic Lymphoid Leukemia Cells.

Background and Objectives: B-cell chronic lymphoid leukemia (CLL) is a lymphoproliferative disorder where specific microenvironment between B-cells and nurse-like Cells (NLC) seem to be involved in disease progression providing cell survival, proliferation and drug resistance. Consequently, functional screening platforms that can assess drug candidates within this microenvironment are needed. Our aim is to show the ability of the Exvitech® automated flow cytometry platform to screen agents that interfere with the microenvironmentxs protective scenario such as ibrutinib or idelalisib or standard CLL drugs such as fludarabine or prednisolone. This approach will allow us to select candidates in an in vitro assay and could personalize the treatment according the response to the drugs.Patients and methods: We have adapted Jan Burger’s published assay1. Peripheral blood mononuclear cells (PBMCs) from not previously treated CLL patients were isolated by density gradient centrifugation over Ficoll-Pacque and were used fresh (N=14) or cryopreserved (N=6). B-cells were assessed to be >90% viable by flow cytometry. NLC co-cultures were stablished by suspending PBMCs from CLL patients in complete RPMI medium with 10% FBS to a concentration of 2x107/ml. Cells were incubated for 14 days in 96-well plates and presence of NLC was confirmed by microscopy. After that, viability was investigated in B-cells treated with 8 concentrations of ibrutinib, idelalisib, fludarabine and prednisolone after 72h of incubation with annexin-V and the appropriate CLL flow cytometry markers. Drug response was evaluated as a depletion survival index of the B-cell population relative to the average of the control wells with NLC but without drug.Results: As expected, depletion of B-cells cultured without NLCs were significant greater than with NLC after the 72h incubation supporting the assay where NLCs protect B-CLL cells from in vitro spontaneous apoptosis. In a similar way, viability of fresh samples with NLC was higher than the corresponding frozen samples (84% vs 25%), though both could be used. Our results show a lower pharmacological median potency, measured as a higher EC50, when we work with NLC versus without NLC for ibrutinib (10µM vs 4µM), idelalisib (17µM vs 0.4µM) and prednisolone (3.5µM vs 1.5µM). However, the effect of fludarabine seems to be independent of the presence of the NLC in the cell culture (7µM vs 6µM). This is consistent with the protective role of microenvironment; more pronounced for ibrutinib and idelalisib. Interestingly with NLC, for each drug there is a significant interpatient variability (Figure 1); each line correspond to a different patient, reflecting the possibility that patients might be more sensitive or resistant to a certain drug in this particular scenario. There is a higher degree of patient sample stratification for idelalisib, fludarabine or prednisolone, where there are still an important % of B-cells alive for some patients after drug exposure at high concentration, supporting the notion of drug resistance. Synergism between some of these drugs was evaluated in 3 samples, with some samples being more synergistic than other, requiring a larger number of samples.Conclusions: Cellular and molecular interactions between B-cells and the microenvironment represented here with the NLC, have become an attractive target for CLL therapy. Because novel drugs such as ibrutinib or idelalisib are transforming CLL therapy targeting the microenvironment, novel technologies that could predict its effect are necessary. Here we have adapted a Nurse-Like Cell assay mimicking the microenvironment published by Burger1 to our ExviTech platform. The automated platform enables scaling of the data points acquired with this assay supporting characterization of drug activity by pharmacological dose response curves, as well as exploring synergistic interactions. As showed in the results and illustrated in Figure 1, there is a interpatient variability of the pharmacological profile for the studied drugs, if clinically validated, could help guiding a personalized treatment selection; measuring the drug activity inside this particular microenvironment responsible of drug resistance.1.- Burger JA et al. Blood. 2000 Oct 15;96(8):2655-63. [Display omitted] DisclosuresPrimo:Vivia Biotech: Employment. Martinez:Vivia Biotech: Membership on an entity’s Board of Directors or advisory committees. Gorrochategui:Vivia Biotech: Employment. Espinosa:Vivia Biotech: Employment. Arroyo:Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Employment. Hernandez:Vivia Biotech: Employment.

Activation-Induced Release of the TNF-Family Member BAFF By NK Cells Facilitates Resistance of Chronic Lymphoid Leukemia Cells to Direct and Rituximab-Induced NK Lysis

NK cells are cytotoxic lymphocytes that play an important role in the immunosurveillance of leukemia and, due to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab. Available data indicate that the ability of NK cells to mediate ADCC is compromised in Chronic Lymphoid Leukemia (CLL), but the underlying mechanisms are still unclear. The TNF family member B cell activating factor (BAFF) was described to be aberrantly produced in mature B cell malignancies and contributes to disease pathophysiology e.g. by acting as a growth and survival signal for CLL cells. Here we report that NK cells express and release BAFF, and NK cell activation, notably including FcγRIIIa stimulation by Rituximab, results in increased secretion of BAFF (but not its close relative APRIL). Expression on the cell surface was neither detectable in resting nor in activated state. NK cell-derived BAFF enhanced the metabolic activity of primary CLL cells and protected CLL cells from chemotherapy-induced cell death. Moreover, exposure to BAFF profoundly diminished direct and Rituximab-induced lysis of primary CLL cells by allogeneic and autologous NK cells, while NK activation and degranulation per se remained unaffected. Notably, sensitivity of CLL cells to both chemotherapeutic treatment as well as direct lysis and Rituximab-induced ADCC of NK cells could be restored by the anti-BAFF antibody Belimumab (Benlysta®), which is approved for the treatment of systemic lupus erythematosus. Thus, our data provide evidence for the involvement of BAFF in the resistance of CLL cells to chemotherapy. Moreover, our results offer a functional explanation for the reportedly compromised ability of NK cells to combat lymphoid as compared to myeloid leukemias as well as their impaired ability to mediate ADCC upon Rituximab treatment in CLL patients. Our findings point to a possible benefit of combinatory approaches employing Rituximab and Belimumab for chemo-immunochemotherapy of B cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

METABOLIC STATE OF BLOOD LYMPHOCYTES IN CHRONIC MYELOID LEUKEMIA AND CHRONIC LYMPHOID LEUKEMIA

Abstract. We examined metabolic status of blood lymphocytes in forty-four patients with chronic myeloid leukemia (CML), and in fifty-seven patients with chronic lymphoid leukemia (CLL). The most pronounced changes of enzyme activities are found in blood lymphocytes of CML and CLL patients during terminal stage of the disease. All the patients with chronic leukemia at both stages showed a decreased intensity of metabolic processes in blood lymphocytes. In addition, CML patients at both clinical stages exhibited a decrease in antioxidant cell protection. The more pronounced biochemical disturbances in CML patients at progression stage concerned carbohydrate metabolism, whereas the changes at terminal stage of disease affected both carbohydrate and fat metabolism. The most marked biochemical alterations in CLL at progression stage are registered for carbohydrate and protein metabolism, and carbohydrate, fat and protein metabolism at terminal stage of disease. The revealed changes showed indicate to more severe clinical course in the patients with CML. These changes and differences depict some aspects of immune pathogenesis in development and progression of CML and CLL.

Bimodal Induction of NK Cell Reactivity Against Acute Myeloid (AML) and Chronic Lymphoid Leukemia (CLL) by Fc-Engineered GITR-Fc Fusion Proteins.

AbstractAbstract 2143NK cells play an important role in anti-tumor immunity and largely contribute to the efficacy of therapeutic strategies like allogenic stem cell transplantation in AML and application of Rituximab that induces antibody-dependent cellular cytotoxicity (ADCC) in CLL. Recently, we demonstrated that the TNF family member GITR ligand (GITRL) is expressed on leukemia cells in a high proportion of AML and CLL patients and impairs direct and Rituximab-induced reactivity of NK cells which constitutively express its counterpart GITR (e.g., Buechele et al., Leukemia 2012). Here we developed a strategy to reinforce NK anti-leukemia reactivity by combining disruption of NK-inhibitory GITR-GITRL interaction with induction of ADCC against the GITRL-expressing leukemia cells using GITR-Ig fusion proteins with modified Fc moieties. Fc parts were engineered by amino acid exchange as previously described (Lazar et al., PNAS 2006; Armour et al., Eur. J. Immunol. 1999). Compared to wild type GITR-Ig (GITR-Fc-WT), our mutants (S239D/I332E and E233P/L234V/L235A/deltaG236/A327G/A330S) displayed highly enhanced (GITR-Fc-ADCC) and abrogated (GITR-Fc-KO) affinity to the Fc(gamma)RIIIa receptor (CD16) expressed on NK cells, respectively. In functional analyses of NK cells and primary leukemia cells, GITR-Fc-KO, which does not induce ADCC, already increased NK reactivity due to disruption of GITR-GITRL interaction. Treatment with GITR-Fc-WT further enhanced NK reactivity due to modest induction of ADCC, while GITR-Fc-ADCC induced highly increased NK-mediated target cell lysis, degranulation and cytokine production in a target-antigen dependent manner. With CLL cells, combined treatment with GITR-Fc-ADCC fusion protein and Rituximab caused additive effects, resulting in significantly enhanced NK cell ADCC. Notably, the effects of our fusion proteins were observed both in an allogenic setting and when employing NK cells of patients with autologous leukemia cells as targets. Our results demonstrate that Fc-engineered GITR-Fc-ADCC fusion protein may combine both neutralization of the NK-inhibitoryeffects of GITR-GITRL interaction and targeting GITRL-expressing malignant cells for NK anti-tumor reactivity and thus constitute an attractive immunotherapeutic means for the treatment of AML and CLL. Disclosures:No relevant conflicts of interest to declare.

Characterization of Peripheral B Cells Expressing CLL-Associated Receptor Tyrosine Kinase ROR1 in Healthy Donors

AbstractAbstract 1787Recent studies have described an extensive expression of the receptor tyrosine kinase-like orphan receptor 1 (ROR1) on the surface of malignant B cells in patients suffering from chronic lymphoid leukemia (CLL). ROR1 expression has also been detected in ovarian cancer, renal cancer, melanoma, and lung adenocarcinoma, suggesting a general role of ROR1 in cancer genesis and/or maintenance. However, low levels of ROR1 mRNA or protein expression have also been found on undifferentiated embryonic stem cells, adipose tissue and on early stage of B-cells in bone marrow, but not in major adult tissues and peripheral B cells.In contrast, our results describe for the fist time expression of ROR1 on rare B cells in peripheral blood of healthy donors. Using magnetic cell sorting techniques we were able to enrich ROR1 positive B cells from PBMCs of healthy donors. The average frequency of ROR-1+ B cells in PBMCs ranged from 1×10−3 to 1×10−4. Interestingly, ROR1+ B cells revealed a heterogeneous phenotype and could further be classified into a CD19+/CD27+/CD38− and a CD19+/CD27−/CD38+ subpopulation as assessed by flow cytometry. Remarkably, in contrast to the CD27+ subpopulation the CD38+ subpopulation showed higher levels of CD5 and CD23 expression, which are both markers known to be upregulated on B cells of CLL patients. Both populations do not show elevated expression of transcription factor ZAP70 and Bcl-2 but expressed higher level of Bcl-6 compared to CD19 B cells. Bcl-6 is known to be required for pre-B cell self-renewal but has also been described to play an essential role in resistance of leukemic cells to therapeutic tyrosine kinase inhibitors. Moreover ROR1+ B cells revealed an immature and non-activated sate as evidenced by the expression of IgD and IgM, and the lack of IgG and CD69. Further functional examination and immunophenotyping of these rare cells are under investigation.We have identified rare B cells expressing the tumor-associated receptor tyrosine kinase ROR1, which are heterogenic in CD27 and CD38 expression and show an immature phenotype. We are currently assessing a potential relationsphip ROR1+ peripheral B cells and ROR1 expressing B cells in malignancies like CLL, mantel cell (MCL) and marginal zone lymphoma (MZL). Disclosures:Milleck:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec GmbH: Employment. Jähn:Miltenyi Biotec GmbH: Employment.

Investigation of thrombopoietin (TPO) function in primary chronic lymphocytic leukemia cells.

6613 Background: Immune thrombocytopenia purpura (ITP) is associated with poor prognosis of chronic lymphoid leukemia (CLL). TPO mimetics have been approved for the treatment of chronic ITP and have been suggested for the use in CLL-related ITP. Before testing this hypothesis in the clinic, the hypothesized role of TPO function in CLL cells was investigated. TPO mimetics stimulate platelet production via activation of the TPO receptor, c-Mpl, thus we analyzed c-Mpl cell surface expression and functional response to TPO in primary CLL cells in samples collected from early and late stage CLL patients. Methods: Peripheral blood was collected from CLL patients and mononuclear cells isolated by Ficoll separation. CLL cells were analyzed by flow cytometry and gated based upon viability and CD5+/CD19+ expression. c-Mpl expression was measured using a tested and validated novel c-Mpl-specific monoclonal antibody by flow cytometry. CLL samples were stimulated with TPO (10 ng/mL for 10 min) and induction of pSTAT5 was measured by intracellular flow cytometry. Platelets (CD41a+) provided internal controls for c-Mpl expression and response to TPO and phorbol myristate acetate was used as a control stimulation to demonstrate that CLL cells were responsive. Results: Analysis of expression and function of c-Mpl was performed on CLL samples (n=89) including 14 patients with thrombocytopenia. While robust c-Mpl expression was observed consistently in platelets from normal and CLL patients (normal: 31.90 ± 2.83, CLL: 26.76 ± 3.31; mean ± SEM), no significant expression of c-Mpl was observed on CD5+/CD19+ CLL cells (1.06 ± 0.01; mean ± SEM). No activation of downstream signaling (pSTAT5) was detected in CLL cells in any of the samples stimulated with TPO (normal: 0.90 ± 0.007, CLL: 1.04 ± 0.02; mean ± SEM), while robust pSTAT5 was detected in platelets (normal: 10.22 ± 0.29, CLL: 6.21 ± 0.23; mean ± SEM). Conclusions: A robust/sensitive platform was developed to profile biologically relevant c-Mpl expression and function in platelets. Using this platform we demonstrate a lack of c-Mpl expression and functional response to TPO challenge in CLL cells suggesting that CLL cells are unlikely to be activated in patients treated with TPO mimetics.

Characterization of Treg Subpopulations in Chronic Lymphocytic Leukemia Using 15 Color Flow Cytometry.

Abstract 1644Poster Board I-670CD4+ regulatory T cells (Treg) act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens. Tregs have been shown to be increased in the peripheral circulation of patients with various types of cancer including CLL and have been postulated to play a role in inhibition of anti-tumor immune responses. 15 color staining was performed on mononuclear cells isolated from peripheral blood from 33 healthy controls and 12 patients with chronic lymphoid leukemia (CLL). Treg cells were defined as viable CD45+ CD3+ CD8- CD4+ CD25+ Forkhead box P3+ (FoxP3)+ mononuclear cells. Among the Treg population we defined 4 sub-populations; Naïve Treg (CD45-RA+ CD27+ CD197+), Effector Treg (CD45-RA- CD27- CD197-), Central memory Treg (CD45-RA- CD27+ CD197+) and Effector memory Treg (CD45-RA- CD27+ CD197-). We report level of expression of activation markers CD39, HLA-DR and CD38 and IL-7 receptor CD127 on Treg subsets as defined above. The percentage of Treg was increased significantly in CLL patients compared with healthy controls 12.8±1.3% vs 5.7±0.3% (p<0.0001). In healthy controls, the majority of Treg are CD45-RA- (80.2±2.9 %) whereas in CLL patients, we observed Treg cells in both CD45-RA- and CD45-RA+ populations (62.6±4.9% and 40.4±5% respectively). Inside the CD45-RA+ populations, we showed that there was a significant higher proportion of naïve Treg among CLL patients compare to healthy controls ((83.8±5% and 41.8±5% respectively (p<0.0001)). Among the CD45-RA- populations, there was no significant difference in the proportion of each memory subtypes. We observed a different pattern of activation among the Treg in CLL patients, naïve Treg expressed more CD39 and HLA-DR compare to healthy controls (2x to 3x fold increase, p<0.0001), but less CD38. In central memory subpopulation, CD39 and CD127 expressions were increased in Treg isolated from CLL patients (p=0.04 and p=0.01 respectively), whereas CD38 was decreased (p=0.01). In effector memory population, HLA-DR expression was lower in CLL patients compared to healthy control (3±0.9% and 9.8±0.8% respectively, p<0.0001). Finally, in the effector subpopulation, HLA-DR expression was also decreased in Treg from CLL patients (p<0.0001). In summary, in these studies, we showed proportions of naïve and memory Treg as well as a different activation pattern of Treg cells present in CLL patients compared to healthy controls. The assessment of complex patterns of sub-phenotype expression in diseases vis a vis healthy individuals should expand our ability to understand the intricacies of both adaptive and innate cellular immune responses and their dysregulation in cancer patients. DisclosuresNo relevant conflicts of interest to declare.

Colon Cancer Secondary to Hematologic Disease

Purpose: The incidence of secondary malignancies in hematologic patients is known to be higher than it is in other patients. However, the characteristics of secondary malignancy and surveillance have not yet been established for colorectal cancer in leukemic patients. Methods: From 1995 to 2007, 6,030 patients who were diagnosed with acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphoid leukemia (CLL), and multiple myeloma (MM) were enrolled in this study. Among them, 9 patients were diagnosed with colorectal cancer at St. Mary's Hospital and were analyzed retrospectively. Results: Three of the 2,570 patients with AML, 1 of the 1,158 patients with CML, 2 of the 83 patients with CLL, 2 of the 422 patients with MM, and none of the 1,797 patients with ALL were found to have colorectal cancer. There were no operative mortalities, but 2 patients refused to have surgery. The ratio of observed to expected subsequent colorectal cancer in CLL was higher than it was in the other groups, indicating that the relative risk of colorectal cancer is higher in patients with CLL. Conclusion: Compared to the Surveillance, Epidemiology and End-Result (SEER) program at the National Cancer Institute (NCI) in the United State, we have the same high relatively risk in CLL patients. Careful attention should be paid to the possibility of colorectal cancer in CLL patients.

Expression Profiles in Chronic Lymphoid Leukemia by Hematochip Analysis.

Aim: The gene expression analysis system is a good tool to identify biomarkers related to prognosis or response predictors of therapy in haematological malignancies. Low density microarrays are an useful technology to obtain genetic expression profiles in different neoplastic disorders. Recently we have designed an oligonucleotide biochip (Hematochip, Alvarez et al, Clin Chem 2007; 53:259) useful for study peripheral blood in hematological malignancies. Chronic Lymphoid Leukemia (CLL) is a frequent clonal lymphocyte disorder characterized by clinical diversity and that have known prognosis markers but a more accurate subclassification could be required. Our objective was to define CLL subgroups with differential genetic profile expression. Patients andMethods: Analytical, prospective study in 49 consecutive CLL patients diagnosed since January 2003 to December 2004 in the Hematology Department of Miguel Servet University Hospital. Zaragoza. Spain. At diagnosis demographic, clinical stage, analytical, immunophenotype expression, genetic aberrations and mutational status of IgVH were collected at electronically data base. Simultaneously, peripheral blood from 20 healthy donors and all CLL patients were collected in PAXgene tubes (PreAnalytix). cDNA was generated from total RNA and double-strand cDNA was used as a template to generate biotinylated cRNA by in vitro transcription using the MEGA-script T7 High Yield Transcription kit (Ambion). Fragmented cRNA was denatured and hybridized using a Ventana Discovery Station (Ventana Medical Systems). Arrays were stained with Cy3-conjugated streptavidin (Amersham Biosciences) and scanned using a ScanArray 4000 scanner (Perkin-Elmer). The data were treated and analyzed by GeneSpring clusters method. The expression profiles were compared and stratified with clinical stability or progression, gender, ZAP 70 expression, associated neoplasia, time free of therapy and response or not to Fludarabine.Results: The Hematochip has identified 9 probes obtained with filtered data and statistical significance (p<0.01), related to stable or progressive disease. The results had been validated by RT-PCR.Comments: the study of genetic profiles by Hematochip could be a useful analysis to predict the stability or progression of CLL patients at diagnosis. Further studies analyzing more cases will be necessary in order to validate these results. This work has been partially sponsorized by grants: Mutua Madrilena del Automovil, Fundacion para el Estudio de la Hematologia y Hemoterapia de Aragon and I+CS.

P163 Sequential and comparative Zap-70 and CD38 analysis in chronic lymphoid leukemia patients

P163 Sequential and comparative Zap-70 and CD38 analysis in chronic lymphoid leukemia patients

Effect of chlorambucil on bone mineral density in the course of chronic lymphoid leukemia.

The purpose of this work was to study the effects of chronic lymphoid leukemia (CLL) and its treatments on bone mineral density (BMD). Lumbar and femoral BMD was measured by X-ray absorptiometry in 50 (32 M, 18 F, median age 65, range age: 47-87 yr) CLL patients. In order to gauge the respective effects of CLL and corticoids on bone mass, 31 CLL patients under treatment were compared with 31 controls on cortisone. Nineteen untreated patients with CLL were compared with controls devoid of osteopenia risk factor. There was no significant difference regarding lumbar and femoral BMD between the untreated patients with CLL and the healthy controls. An increase in lumbar and femoral BMD was noted in the treated CLL group compared with the controls on cortisone (lum BMD: 1.018 vs. 0.861 g/cm2, p=6.10(-4); fem BMD: 0.773 vs. 0.699 g/cm2, p=0.037). This increase was observed only in patients who had received chlorambucil (lum BMD: 1.066 vs. 0.861 g/cm2, p=0.10(-4); fem BMD: 0.806 vs. 0.699 g/cm2, p=4.10(-3)), whereas there was no difference between the CLL patients treated without chlorambucil and the controls on cortisone. Multiple linear regression analysis confirmed the marked effect of chlorambucil (r=0.3715, p<10(-3)) on BMD increase in the course of CLL.

Histidine decarboxylase in peripheral lymphocytes of healthy individuals and chronic lymphoid leukemia patients.

Histidine decarboxylase (HDC), the only enzyme capable of synthetizing histamine, has been found in many proliferating cells and tissues suggesting a role of histamine in cellular proliferation. In this study expression of HDC and the significance of histamine in the proliferation of peripheral lymphocytes of five healthy persons and six patients with chronic lymphoid leukemia (CLL) was examined. Expression of HDC mRNA and the protein was proved by reverse transcriptase polymerase chain reaction and by immunoblot, respectively. The role of histamine was studied in proliferation assays in the presence of irreversible inhibitor of the HDC (alpha-fluoromethylhistidine--aFMH) and also by competing for the intracellular binding sites of histamine using N,N-diethyl-2, 4-phenylmethyl-phenoxy-ethanamine-HCl (DPPE). By inhibiting the HDC enzyme activity by FMH and blocking the intracellular action of histamine by DPPE, a significant decrease in cell proliferation was observed in mitogen stimulated lymphocytes of healthy donors. In CLL patients the proliferation of leukemic lymphocytes was significantly inhibited by blocking the binding of histamine to intracellular binding sites by DPPE but not by FMH inhibiting only the de novo histamine formation. The observations suggest that HDC has functional relevance in lymphocytes, since mitogen induced lymphocyte proliferation of healthy donors is mainly enhanced by de novo synthesis and subsequent action of intracellular histamine. Alternatively, in constitutively proliferating chronic lymphoid leukemia cells we suggest that the preformed pool but not the de novo synthesized intracellular histamine interferes with cellular proliferation.

Inflammatory neuromuscular disorders associated with chronic lymphoid leukemia: Evidence for clonal B cells within muscle and nerve

It is frequently difficult to determine whether a neuromuscular disorder (NMD) related to a lymphoproliferative disease is neoplastic, paraneoplastic, or incidental. This may explain why neuromuscular complications of chronic lymphoid leukemia (CLL), a frequent disorder, have been rarely reported. We describe 7 patients with CLL and neuromuscular involvement in whom phenotypic and genotypic characterization of infiltrating lymphoid cells was carried out by immunocytochemistry and PCR-amplification of immunoglobulin heavy chain locus. One patient had massive neoplastic infiltration of muscle, and six presented with inflammatory-like NMDs (dermatomyositis: 2, polymyositis: 1, vasculitic mononeuropathy: 2; inflammatory demyelinating neuropathy: 1). Immunocytochemistry on nerve and muscle frozen sections showed a monotypic lymphoid cell population in 3 cases and failed in 4 cases. The PCR analysis of immunoglobulin heavy chain gene (FR3-FR4) rearrangement detected clonal B-cells in all biopsy specimens. There are arguments suggesting that incidental or paraneoplastic inflammatory NMDs may progress to neoplastic infiltration in CLL patients, as a result of the traffic of tumor B-cells from circulation to nerve and muscle tissues. This may question the traditional distinction between inflammatory and neoplastic NMDs in patients with lymphoid cell proliferations.

Enzyme-linked immunosorbent assay for human MRP-8/MRP-14 proteins and their quantitation in plasma of hematological patients

We developed a quantitative enzyme immunoassay for human MRP-8/MRP-14, neutrophil cytosolic proteins with calcium-binding and bacteriastatic properties. MRP-8/MRP-14 concentrations were measured in the plasma of 23 healthy volunteers and in 75 patients with hematological disorders. These proteins were detected in plasma of healthy blood donors with mean ± standard deviation 167 ± 114 ng/ml. MRP-8/MRP-14 plasma levels ranged from 435 to 13280 ng/ml in patients with chronic myeloid leukemia (CML), from 50 to 7570 ng/ml in chronic lymphoid leukemia (CLL), from 450 to 2790 ng/ml in polycythemia vera (PV), and were significantly higher than in healthy blood donors ( P < 0.01 for CML and P < 0.05 for others). In CML patients MRP-8/MRP-14 levels strongly correlated with total blood WBC count ( r = 0.82) and with neutrophilic granulocyte (NG) count ( r = 0.80). Correlation of these values in PV amounted to r = 0.70 and r = 0.46, respectively. In CLL patients MRP-8/MRP-14 levels strongly correlated with total WBC count ( r = 0.92), but not with the NG count. We suggest that MRP-8/MRP-14 quantitation may serve as a marker of neutrophil pool turnover in some hematological disorders.