B-cell Chronic Lymphocytic Leukemia Research Articles

CXCL13 plasma levels function as a biomarker for disease activity in patients with chronic lymphocytic leukemia.

The chemoattractant CXCL13 organizes the cellular architecture of B-cell follicles and germinal centers. During adaptive immune responses, CXCL13 plasma concentrations transiently increase and function as a biomarker for normal germinal center activity. Chronic lymphocytic leukemia (CLL) cells express high levels of CXCR5, the receptor for CXCL13, and proliferate in pseudofollicles within secondary lymphoid organs (SLO). Given the morphologic and functional similarities between normal and CLL B-cell expansion in SLO, we hypothesized that CXCL13 plasma concentrations would correlate with CLL disease activity and progression. We analyzed CXCL13 plasma concentrations in 400 CLL patients and correlated the findings with other prognostic markers, time to treatment (TTT), CCL3 and CCL4 plasma concentrations, and in vivo CLL cell proliferation. We found that CXCL13 plasma concentrations were higher in CLL patients with active and advanced stage disease, resulting in a significantly shorter TTT. Accordingly, high CXCL13 levels correlated with other markers of disease activity and CCL3 levels. Higher CLL cell birth rates in vivo also associated with higher CXCL13 plasma concentrations. Interestingly, elevated CXCL13 plasma levels normalized during ibrutinib therapy, and increased in ibrutinib resistance patients. Collectively, these studies emphasize the importance of CXCL13 in crosstalk between CLL cells and the SLO microenvironment.

Polyclonal B-cell lymphocytosis in English bulldogs.

BackgroundEnglish bulldogs disproportionally develop an expansion of small B‐cells, which has been interpreted as B‐cell chronic lymphocytic leukemia (BCLL). However, clonality testing in these cases has often not been supportive of neoplasia.HypothesisEnglish bulldogs have a syndrome of nonneoplastic B‐cell expansion.AnimalsEighty‐four English bulldogs with small‐sized CD21+ B‐cell lymphocytosis in the blood as determined by flow cytometry.MethodsThis is a retrospective study. We characterized this syndrome by assessing B‐cell clonality, clinical presentation, flow cytometric features, and immunoglobulin gammopathy patterns. We identified 84 cases with CD21+ lymphocytosis among 195 English bulldogs with blood samples submitted to the Colorado State University‐Clinical Immunology laboratory for immunophenotyping between 2010 and 2019. Flow cytometry features were compared to normal B‐cells and BCLL cases. PCR for antigen receptor rearrangements (PARR) by multiple immunoglobulin primers was performed to assess B‐cell clonality. A subset of cases with gammopathy were examined by protein electrophoresis, immunofixation, and immunoglobulin subclass ELISA quantification.ResultsSeventy percent (58/83) of cases had polyclonal or restricted polyclonal immunoglobulin gene rearrangements, suggesting nonmalignant B‐cell expansion. The median age of all dogs in the study was 6.8 years and 74% were male. The median (range) lymphocyte count was 22 400/μL (2000‐384 400/μL) and B‐cells had low expression of class II MHC and CD25. Splenomegaly or splenic masses were detected in 57% (26/46) of cases and lymphadenopathy in 11% (7/61). Seventy‐one percent (52/73) of cases had hyperglobulinemia and 77% (23/30) with globulin characterization had IgA ± IgM polyclonal or restricted polyclonal gammopathy patterns.Conclusions and Clinical ImportancePolyclonal B‐cell lymphocytosis in English bulldogs is characterized by low B‐cell class II MHC and CD25 expression, splenomegaly and hyperglobulinemia consisting of increased IgA ± IgM. We hypothesize that this syndrome has a genetic basis.

EFFECT OF HLA-DR ANTIBODY CONCENTRATION ON ESTIMATION OF THE NUMBER OF CD14+HLA-DRLOW /–-CELLS IN THE BLOOD OF PATIENTS WITH B-CELL chronic lymphocytic leukemia

Introduction. Immunoregulatory functions of myeloid-derived suppressor cells have been extensively studied over the recent decades. Additionally, myeloid-derived suppressor cells have been investigated as a prognostic factor.Objective. To optimize the measurement of monocytic myeloid-derived suppressor cells in the peripheral blood of patients with B-cell chronic lymphocytic leukemia.Materials and methods. The number of myeloid-derived suppressor cells with CD14+HLA-DRlow/–-phenotype was determined in the peripheral blood of patients with B-cell chronic lymphocytic leukemia and healthy donors by flow cytometry. The measurement was carried out at two points, which differed in the concentration of anti-HLA-DR antibodies − 15 and 4 μl.Results. The median amount of myeloid-derived suppressor cells in the peripheral blood of B-cell chronic lymphocytic leukemia patients with 15 μl of anti-HLA-DR antibody was 1.9 %, and with 4 μl of antibody concentration – 7 %. Healthy donors had that median of 0.15 % with 15 μl of antibody and 0.3 % with 4 μl concentration.Conclusion. The number of CD14+HLA-DRlow/–-cells in the blood of patients with B-cell chronic lymphocytic leukemia is sensitive to the concentration of the HLA-DR antibody.Compliance with patient rights and principles of bioethics. The study protocol No 3 of 13.02.2020 was approved by the biomedical ethics committee of Research Institute of Experimental Diagnostics and Therapy of Tumors N.N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of the Russian Federation. All patients gave written informed consent to participate in the study.

Fluorescent in situ hybridization in patients with chronic lymphocytic leukemia with autoimmune hemolytic anemia

Background. Gene aberrations are an important prognostic criterion for the course of B-cell chronic lymphocytic leukemia (B-CLL) and response to treatment, which includes not only immunochemotherapy, but also concomitant infusion therapy for the prevention and correction of complications.
 Objective. To investigate the presence of prognostic cytogenetic changes in patients with B-CLL with autoimmune hemolytic anemia (AIGA). To analyze the course of the disease and the direct effect of treatment in patients with cytogenetic changes of different nature.
 Materials and methods. Cytogenetic studies were performed by fluorescent in situ hybridization (FISH) on the interphase nuclei of peripheral blood lymphocytes in 11 patients with B-CLL with AIGA. Probes to the ATM genes (gene localized in region 11q23) and TP53 (gene localized in region 17p13) were used in the work, the deletions of which have prognostic value in B-CLL. All patients received treatment.
 Results. Among 11 patients with AIGA, signals to both genes were detected in nuclei 4. No deletions were detected. In the cells of the other 7 patients, the absence of a single signal to the ATM gene was detected, indicating the presence of a deletion of del(11)(q23). In recent patients, an unfavorable course of B-CLL disease was observed without response to treatment. Deletions of the TP53 gene in patients of the studied group were not detected.
 Conclusions. FISH study in patients with B-CLL with AIGA revealed the presence of important and prognostically unfavorable chromosomal rearrangement of the ATM gene in 63 % of patients.

Berberine affects mitochondrial activity and cell growth of leukemic cells from chronic lymphocytic leukemia patients

B-cell chronic lymphocytic leukemia (CLL) results from accumulation of leukemic cells that are subject to iterative re-activation cycles and clonal expansion in lymphoid tissues. The effects of the well-tolerated alkaloid Berberine (BRB), used for treating metabolic disorders, were studied on ex-vivo leukemic cells activated in vitro by microenvironment stimuli. BRB decreased expression of survival/proliferation-associated molecules (e.g. Mcl-1/Bcl-xL) and inhibited stimulation-induced cell cycle entry, irrespective of TP53 alterations or chromosomal abnormalities. CLL cells rely on oxidative phosphorylation for their bioenergetics, particularly during the activation process. In this context, BRB triggered mitochondrial dysfunction and aberrant cellular energetic metabolism. Decreased ATP production and NADH recycling, associated with mitochondrial uncoupling, were not compensated by increased lactic fermentation. Antioxidant defenses were affected and could not correct the altered intracellular redox homeostasis. The data thus indicated that the cytotoxic/cytostatic action of BRB at 10–30 μM might be mediated, at least in part, by BRB-induced impairment of oxidative phosphorylation and the associated increment of oxidative damage, with consequent inhibition of cell activation and eventual cell death. Bioenergetics and cell survival were instead unaffected in normal B lymphocytes at the same BRB concentrations. Interestingly, BRB lowered the apoptotic threshold of ABT-199/Venetoclax, a promising BH3-mimetic whose cytotoxic activity is counteracted by high Mcl-1/Bcl-xL expression and increased mitochondrial oxidative phosphorylation. Our results indicate that, while CLL cells are in the process of building their survival and cycling armamentarium, the presence of BRB affects this process.

Development of Anti-CD32b Antibodies with Enhanced Fc Function for the Treatment of B and Plasma Cell Malignancies

The sole inhibitory Fcγ receptor CD32b (FcγRIIb) is expressed throughout B and plasma cell development and on their malignant counterparts. CD32b expression on malignant B cells is known to provide a mechanism of resistance to rituximab that can be ameliorated with a CD32b-blocking antibody. CD32b, therefore, represents an attractive tumor antigen for targeting with a monoclonal antibody (mAb). To this end, two anti-CD32b mAbs, NVS32b1 and NVS32b2, were developed. Their complementarity-determining regions (CDR) bind the CD32b Fc binding domain with high specificity and affinity while the Fc region is afucosylated to enhance activation of FcγRIIIa on immune effector cells. The NVS32b mAbs selectively target CD32b+ malignant cells and healthy B cells but not myeloid cells. They mediate potent killing of opsonized CD32b+ cells via antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP) as well as complement-dependent cytotoxicity (CDC). In addition, NVS32b CDRs block the CD32b Fc-binding domain, thereby minimizing CD32b-mediated resistance to therapeutic mAbs including rituximab, obinutuzumab, and daratumumab. NVS32b mAbs demonstrate robust antitumor activity against CD32b+ xenografts in vivo and immunomodulatory activity including recruitment of macrophages to the tumor and enhancement of dendritic cell maturation in response to immune complexes. Finally, the activity of NVS32b mAbs on CD32b+ primary malignant B and plasma cells was confirmed using samples from patients with B-cell chronic lymphocytic leukemia (CLL) and multiple myeloma. The findings indicate the promising potential of NVS32b mAbs as a single agent or in combination with other mAb therapeutics for patients with CD32b+ malignant cells.

S1675 Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma Presenting as Chronic Diarrhea: An Unusual Presentation of a Common Lymphoproliferative Disorder

INTRODUCTION: B-cell chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the United States. It has a variable presentation with most patients having asymptomatic lymphocytosis. CLL can present with extramedullary involvement. Most commonly this is in the form of skin or central nervous system involvement, though rarely it can present with gastrointestinal involvement. CASE DESCRIPTION/METHODS: We are highlighting a case of a 70-year-old male who presented with complaints of diarrhea for the last 4 months. He was having 4–5 bowel movement a day ranging from semi-solid to watery stools. He denied any history of fevers, abdominal pain, vomiting, weight loss, night sweats, skin rashes, travel history, or exposure to anyone with similar symptoms. On physical examination, his abdomen was soft, non-tender, and without apparent hepatomegaly or splenomegaly. The rest of the system examinations were unremarkable. The initial laboratory evaluation was unremarkable. After failing conservative treatment, a colonoscopy was performed which showed diffuse mucosal nodularities (Image 1 and 2). Histopathology of the biopsied tissue revealed multiple aggregates for small mature lymphocytes in the submucosa (Image 3). Immunohistochemical staining revealed a diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma. CT scan revealed extensive bilateral axillary, hilar, abdominal, and pelvic lymphadenopathy. He subsequently underwent bone marrow biopsy histopathology and flow cytometric immunophenotyping which were consistent with CLL. Oncology started him on chemotherapy and he went into remission from his CLL. On 3- and 6-month follow-ups, the patient was asymptomatic. DISCUSSION: A review of the literature revealed a total of n = 4 prior reported cases where the initial presentation of CLL/SLL was diarrhea. In the existing case reports with similar presentation a mean age of presentation was n = 74 years, ages ranging from 65 to 81 years. They had fecal urgency, abdominal pain, and weight loss as associated symptoms. Only one patient had history of CLL for which he had undergone treatment and was thought to be in remission. Imaging with CT with contrast in all cases revealed abdominal and pelvic lymphadenopathy. Histopathology of the colonic biopsy in all cases revealed lymphocytic infiltration. This patient was treated successfully with chemotherapy and his diarrhea improved. This is a differential to keep in mind in patients with chronic diarrhea, once the more common causes have been ruled out.Figure 1.: Image 1: Colonoscopy showing submucosal nodularity.Figure 2.: Image 2: Colonoscopy showing submucosal nodularity.Figure 3.: Image 3: Hematoxylin and eosin stain showing lymphoid aggregate with normal colon mucosa.

Realization of the rescue effect at the cytogenetic level due to the interaction between intact normal and irradiated malignant human blood lymphocytes

The effect of intact blood lymphocytes from conditionally healthy persons on the genome stability in blood lymphocytes of patients with primary diagnosis of B-cell chronic lymphocytic leukemia (CLL) exposed in vitro to γ-quanta 137Cs at the G0 stage of the cell cycle in a dose of 0.5 Gy was investigated. For the study, the own model system of co-cultivation of blood lymphocytes from persons with different genders that permit to study va rious manifestations of the bystander response phenomenon at the cytogenetic level was used. The decrease in the radiation-induced total frequency of chromosome aberrations in irradiated cells of patients with CLL (from 12.88 till 9.56 per 100 metaphases, p < 0.01) due to a reduction in the level of chromatid-type aberrations (from 5.35 till 2.83 per 100 cells, p < 0.001), which are considered as markers of the chromosomal instability, was established. The frequencies of unstable cytogenetic markers of a radiation exposure (dicentric and ring chromosomes) remained unchanged (p> 0.05). The obtained data indicate that the result of the interaction between irradiated in vitro blood cells of patients with B-cell CLL (target cells) with intact blood lymphocytes of relatively healthy individuals (bystander cells) is a decrease of the chromosome instability in target cells, which is similar to the radiation-induced rescue effect type one.

Cutaneous manifestations of B-cell chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a malignant lymphoproliferative disease characterized by the accumulation of immature monoclonal B lymphocytes in blood cells, bone marrow, spleen and lymph nodes. This is the most common type of leukemia among the Caucasoid race. When CLL skin lesions occur in about 25% of patients, they are extremely diverse. These lesions can be divided into specific, including infiltration of the skin by leukemic cells and the skin form of Richter's syndrome, secondary skin tumors, nonspecific lesions and associated skin diseases.Leukemic infiltration of the skin in patients with leukemia is called specific skin lesions (SSL). Many authors associate the unfavorable prognosis with the transformation of CLL with specific infiltration of the skin into Richter syndrome, as well as the appearance of SSL before the diagnosis of CLL. The risk of developing various cancer pathologies in patients with CLL is three times higher than in healthy people identical in sex and age. It was found that the risk of skin cancer in these patients is eight times higher than in the healthy population. The most common secondary skin tumors in CLL are basal-cell carcinoma, squamous-cell carcinoma, melanoma, and Merkel tumor.Nonspecific skin changes are extremely diverse and occur in patients with CLL in 30-50% of cases. The most common secondary changes in the skin in CLL are those of infectious nature. There are also increased reactions to insect bites, generalized itching, exfoliative erythroderma, nodular erythema, paraneoplastic pemphigoid, bullous pemphigoid, drug eruption. Concomitant dermatoses in these patients are more severe and often torpid to the previously conducted therapy. There is no doubt that together with the clarification of the etiology and pathogenesis of CLL, particular issues related to the study of clinical and morphological changes in individual organs and systems, in particular the skin, formed at various stages of the development of this disease should be studied in detail. This can not only expand and clarify our understanding of this pathology, but also can help to clarify the essence of the disease.

CLL-155: Differential Diagnosis of Aplastic Anaemia in Chronic Lymphocytic Leukaemia

A 73-year-old lady presented in September of 2019 with pancytopenia on a background of 17p and 13q deleted chronic lymphocytic leukaemia (CLL) on ibrutinib. The diagnosis of CLL was made in 2008 and no treatment was required until she developed progressive cytopenias in January of 2019, with a lymphocyte count of 193.8 ×109/L. Fluorescence in-situ hybridisation (FISH) demonstrated 17p and 13q deletions and she was commenced on ibrutinib. She developed atrial fibrillation but otherwise tolerated the therapy well. She represented with significant fatigue and bleeding in September of 2019 and her blood count revealed a haemoglobin (Hb) of 8.2 g/dL, a platelet count of 6 ×109/L, a lymphocyte count of 32 ×109/L, a neutrophil count of 0.24 ×109/L and a reticulocyte count of References [1] Zonder JA, Keating M, Schiffer CA. Chronic Lymphocytic Leukemia presenting in association with Aplastic Anemia. Am. J. Hematol. 2002 71:323–327. [2] Castiglioni MG, Scatena P, Pandolfo C et al. Rituximab Therapy of Severe Aplastic Anemia Induced by Fludarabine and Cyclophosphamide in a Patient Affected by B-cell Chronic Lymphocytic Leukemia. 2006 Sep;47(9):1985-6. [3] Singal R, Winfield DA, Greaves M. Bone marrow aplasia in B cell chronic lymphocytic leukaemia: Successful treatment with antithymocyte globulin. J Clin Pathol 1991; 44:954-956. [4] Vitale C, Ahn IE, Sivina M et al. Autoimmune cytopenias in patients with chronic lymphocytic leukemia treated with ibrutinib. Haematologica 2016; 101:e257.

BDA-366, a putative Bcl-2 BH4 domain antagonist, induces apoptosis independently of Bcl-2 in a variety of cancer cell models

Several cancer cell types, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) upregulate antiapoptotic Bcl-2 to cope with oncogenic stress. BH3 mimetics targeting Bcl-2’s hydrophobic cleft have been developed, including venetoclax as a promising anticancer precision medicine for treating CLL patients. Recently, BDA-366 was identified as a small molecule BH4-domain antagonist that could kill lung cancer and multiple myeloma cells. BDA-366 was proposed to switch Bcl-2 from an antiapoptotic into a proapoptotic protein, thereby activating Bax and inducing apoptosis. Here, we scrutinized the therapeutic potential and mechanism of action of BDA-366 in CLL and DLBCL. Although BDA-366 displayed selective toxicity against both cell types, the BDA-366-induced cell death did not correlate with Bcl-2-protein levels and also occurred in the absence of Bcl-2. Moreover, although BDA-366 provoked Bax activation, it did neither directly activate Bax nor switch Bcl-2 into a Bax-activating protein in in vitro Bax/liposome assays. Instead, in primary CLL cells and DLBCL cell lines, BDA-366 inhibited the activity of the PI3K/AKT pathway, resulted in Bcl-2 dephosphorylation and reduced Mcl-1-protein levels without affecting the levels of Bcl-2 or Bcl-xL. Hence, our work challenges the current view that BDA-366 is a BH4-domain antagonist of Bcl-2 that turns Bcl-2 into a pro-apoptotic protein. Rather, our results indicate that other mechanisms beyond switching Bcl-2 conformation underlie BDA-366’s cell-death properties that may implicate Mcl-1 downregulation and/or Bcl-2 dephosphorylation.

CLL-132: Mutational Status of IGVH in Correlation with Genetic Abnormalities of Patients with Chronic Lymphocytic Leukemia in Macedonia - A Single-Centre Experience

Context B-cell chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. One of the most important molecular predictors, the immunoglobulin VH gene mutational status, divides CLL into two prognostic groups, where unmutated U-CLL is associated with remarkably worse prognosis than mutated M-CLL. In many studies, multiple recurring chromosomal aberrations appear to be effective prognostic markers in CLL. Objective The aim of the study was first-time evaluation of rearrangement of IG genes and genetic abnormalities in Macedonian CLL patients. Design Prospective study of Macedonian patients with CLL at early and advanced stage of disease in a period of time from January 2011 to January 2019. The median follow-up was 48 months (1-96 months). Setting Mutational status and configuration of IGHV-IGHD-IGHJ rearrangements in 90 treatment-naive CLL patients were analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing methodology. We used multiplex ligation-dependent probe amplification (MLPA) for detection of most common genetic abnormalities. Interventions We introduced prognostic markers like cytogenetic aberrations, and mutational status of immunoglobulin heavy chain in prognostic stratification of Macedonian patients with CLL. Results Our evaluation has shown that 54.7% patients belonged to the U-CLL subset, whereas 45.3% belonged to the M-CLL subset. The most frequently expressed IGHV subgroup was IGHV3 (40%), followed by IGHV4 (29%), IGHV1(27%), and IGHV5 (3%). The most frequent subgroup with 8% were IGHV1-69*13 and IGHV4-34*0. In the IGHD and IGHJ sets, most frequently expressed genes were IGHD3 (59.7%) and IGHJ6(53.1%), respectively. We found that 80% of IGHV1, 65.6% of IGHD3, and 83.3% of IGHJ6 genes were U-CLL (p=0.0005). Frequency of genetic aberrations by MLPA were 13q14 deletion (20%) followed by 11q deletion (14%), then trisomy 12 (13%) and 17p deletion (6.6%), multiple mutations (6.7%), and NOTCH mutation (5.3%). Conclusions Our evaluation of mutational status on IGVH, IGDH, and IGJH genes in Macedonian CLL patients resulted with data that are consubstantial to those from the Mediterranean area. Novel mutations discovered in this study must be validated through large cohort studies and may offer clues to the mechanisms underlying the difference in CLL survival. In the future, therapeutic strategies targeting these genes should be evaluated and considered.

Combining T-cell-based immunotherapy with venetoclax elicits synergistic cytotoxicity to B-cell lines in vitro.

Alterations of B-cell lymphoma 2 (BCL-2) family proteins contribute to the survival of B-cell malignancies. Recently, venetoclax, a BCL-2 inhibitor, was approved for B-cell chronic lymphocytic leukemia therapy and is being investigated in clinical trials for a variety of hematologic cell malignancies. Furthermore, combination therapy with other molecularly targeted drugs was reported to be more effective than monotherapy. However, combining venetoclax with immunotherapy based on T-cells has not been tested. Because both venetoclax and granzyme B activate the mitochondrial apoptosis pathway by targeting different BCL-2 family molecules, it is possible that combinations of venetoclax with immunotherapy will be effective treatments. We examined the effect of combining venetoclax with immunotherapy using an in vitro model system involving cytomegalovirus (CMV) pp65 antigen-specific cytotoxic T-cells (CMV-CTLs) as the effector cells and CMVpp65 antigen-expressing B-cell lines as the target cells. Cytotoxicity of CMV-CTLs to the target B-cell lines was enhanced by venetoclax with combination index values of 0.47-0.83. This suggests that venetoclax synergizes with T-cell-based immunotherapy to affect B-cell malignancies. Interestingly, venetoclax synergized not only with antigen-specific cytotoxicity but also with nonspecific cytotoxicity. Importantly, CMV-CTLs could be expanded in the presence of venetoclax at the maximum concentration (5 μM) that induced apoptosis in resting CMV-CTLs. B-cell lymphoma-extra large (BCL-xL) expression in CMV-CTLs increased transiently after activation by CMVpp65-transfected B-cell lines, indicating that the expression of BCL-xL was important for the effectiveness of combination treatment with venetoclax. These findings suggest that T-cell-based immunotherapy combined with venetoclax is effective against B-cell malignancies.

The relationship between oxidative stress and cytogenetic abnormalities in B-cell chronic lymphocytic leukemia

BackgroundProspective data on the value of oxidative stress in the pathogenesis of B-CLL are limited, and data on the relationship between oxidative stress and the presence of cytogenetic abnormalities (CA) in this pathology are almost absent. In the present study, we evaluated the serum levels of oxidative stress biomarkers [conjugated dienes (CD), malondialdehyde (MDA), and nitrite levels] and antioxidant biomarkers [ceruloplasmin (CP) level and glutathione peroxidase (GPx) activity] in B-CLL and investigated the relationship between these biomarkers and the presence of CA. Material and methodsA total of 64 B-CLL patients were grouped with respect to the Rai stages of the disease, then to the mutated/unmutated status of IGHV genes as well as to the presence/absence of CA. The type and frequency of CA in the B-CLL cells were determined by fluorescence in situ hybridization. Control group included 30 healthy volunteers. The CD, MDA, and nitrite levels as well as the GPx activity were evaluated spectrophotometrically; the CP level was evaluated turbidimetrically. ResultsCompared to control, the B-CLL patients had increased CD, MDA, and nitrite levels as well as increased CP level and decreased GPx activity, which was observed at all Rai stages of the disease. CA were identified in 47 patients: del(13q14.3) as a single CA occurred in 18 patients, del(11q22.3) in 5 patients, del(17p13.1) in 4 patients, tri 12 in 5 patients, and multiple CA occurred in 15 patients. Compared to patients without CA, isolated del(17p13.1) was associated with higher CD and MDA levels while multiple CA with elevated CD levels only. The nitrite and CP levels and the activity of GPx in patients with CA were close to those in patients with normal FISH. The odds of harboring CA increased by a factor of 1.88 (p = 0.004) for every one unit increase in serum CD level (μmol/L), as assessed by binomial logistic regression. ConclusionThe results indicate that B-CLL patients experience increased oxidative stress and the relative deficiency of the antioxidant defense system. Increased CD level was independently associated with greater likelihood of harboring CA.

Mediating effect of soluble B-cell activation immune markers on the association between anthropometric and lifestyle factors and lymphoma development

Sustained B-cell activation is an important mechanism contributing to B-cell lymphoma (BCL). We aimed to validate four previously reported B-cell activation markers predictive of BCL risk (sCD23, sCD27, sCD30, and CXCL13) and to examine their possible mediating effects on the association between anthropometric and lifestyle factors and major BCL subtypes. Pre-diagnostic serum levels were measured for 517 BCL cases and 525 controls in a nested case–control study. The odds ratios of BCL were 6.2 in the highest versus lowest quartile for sCD23, 2.6 for sCD30, 4.2 for sCD27, and 2.6 for CXCL13. Higher levels of all markers were associated with increased risk of chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). Following mutual adjustment for the other immune markers, sCD23 remained associated with all subtypes and CXCL13 with FL and DLBCL. The associations of sCD23 with CLL and DLBCL and CXCL13 with DLBCL persisted among cases sampled > 9 years before diagnosis. sCD23 showed a good predictive ability (area under the curve = 0.80) for CLL, in particular among older, male participants. sCD23 and CXCL13 showed a mediating effect between body mass index (positive) and DLBCL risk, while CXCL13 contributed to the association between physical activity (inverse) and DLBCL. Our data suggest a role of B-cell activation in BCL development and a mediating role of the immune system for lifestyle factors.

Abstract 6466: Serum immunoglobulins are an independent prognostic marker of time to first therapy in newly diagnosed chronic lymphocytic leukemia (CLL) and monoclonal B-cell lymphocytosis (MBL)

Abstract Immune dysfunction is a hallmark of CLL/MBL; ~25% individuals with newly diagnosed CLL have hypogammaglobulinemia at diagnosis. In this study of newly diagnosed CLL/MBL, we sought to a) investigate the association between serum immunoglobulins and specific mutations identified by next generation sequencing, and b) determine if serum immunoglobulin levels predict TFT, independent of the CLL-International Prognostic Index (CLL-IPI) and tumor mutational load (TML). We used the Mayo Clinic CLL Resource to identify newly diagnosed CLL/MBL individuals consented within 2 years of diagnosis. Baseline clinical characteristics and the CLL-IPI score was ascertained on all individuals. DNA was extracted from peripheral blood CD5+/CD19+ clonal B-cells; and the entire coding regions of 60 somatically recurring mutated CLL driver genes were enriched using a customized Agilent SureSelect gene panel and sequenced in Illumina Hiseq4000. Serum immunoglobulins were quantitated by radial immunodiffusion using Immunoplates. The relationship between serum immunoglobulins was assessed: a) with individual gene mutations (mutated vs. not); and b) using the TML score (using a value of 0, 1, and 2 or greater [2+] mutated genes). The impact of serum immunoglobulin levels on TFT was analyzed for all individuals. Of 356 individuals identified, 80 (22%) had MBL, median age was 63 years (28-87), 261 (73%) were male, and the CLL-IPI distribution was: low risk (n=149, 42%), intermediate risk (n=112, 32%), and high/very high risk (n=94, 26%). We observed 43%, 39%, and 18% individuals with 0, 1, or 2+ mutated genes, respectively. The most commonly mutated genes were NOTCH1 (13%), SF3B1 (13%), TP53 (8%), ATM (8%), BIRC3 (7%), and NFKBIE (7%). Using standard cutoffs, 135 (38%) individuals had low serum IgG, 73 (21%) had low serum IgA, and 154 (44%) had low serum IgM at diagnosis. There were no differences in individual gene mutation frequencies between individuals with low and normal serum IgG. In contrast, individuals with low serum IgA were more likely to have mutations in NOTCH1 (25% vs 10%) and ATM (14% vs 7%) compared to those with normal IgA, and individuals with low serum IgM were more likely to have mutations in NOTCH1 (19% vs 9%) and BIRC3 (11% vs 4%) compared to those with normal IgM (all p-values <0.05). There were no significant differences in individuals with low serum IgG based on TML scores of 0, 1, and 2+ (31%, 35%, 40%, respectively, p=0.28). However, individuals with low serum IgA and low serum IgM were more likely to have a higher TML (8%, 20%, 36% for IgA, p<0.0001; and 35%, 45%, 54% for IgM, p<0.0001, for TML scores of 0, 1, and 2+, respectively). After a median follow-up of 6.9 years, 157 individuals required CLL therapy. After adjusting for CLL-IPI and TML, low serum IgG, low serum IgA, and low serum IgM (hazard ratio [HR]: 1.8, 2.3, and 2.4, respectively, all p-values <0.05) were independently associated with a shorter TFT. Our study shows that individuals with CLL/MBL who harbor mutations in NOTCH1, ATM and BIRC3 are more likely to have low serum IgA and IgM at diagnosis. Serum IgA and IgM (but not serum IgG) levels decrease with an increase in the TML score. Finally, low serum immunoglobulins can predict TFT, independent of the CLL-IPI and TML, which has important implications for risk stratification in newly diagnosed CLL/MBL. Citation Format: Sameer A. Parikh, Cristine Allmer, Geffen Kleinstern, Nicholas J. Boddicker, Shulan Tian, Cecilia B. De Campos, Laura A. Bruins, Daniel R. o"brien, Brian F. Kabat, Kari G. Rabe, Aaron D. Norman, Huihuang Yan, Xing Li, Timothy G. Call, Wei Ding, Jose F. Leis, Saad J. Kenderian, Celine M. Vachon, James R. Cerhan, Neil E. Kay, Susan L. Slager, Esteban Braggio. Serum immunoglobulins are an independent prognostic marker of time to first therapy in newly diagnosed chronic lymphocytic leukemia (CLL) and monoclonal B-cell lymphocytosis (MBL) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6466.

Abstract 73: Development of APG-3526 as a novel and highly efficacious MCL-1 inhibitor

Abstract Background MCL-1 is an important anti-death BCL-2 family protein and plays a key role in blocking apoptosis in cancer cells. MCL-1 gene is located in one of the most frequently amplified locus in various hematologic malignancies and solid tumors, including prostate, lung, pancreatic, breast, ovarian and cervical cancers, as well as melanoma, B-cell chronic lymphocytic leukemia (B-CLL), acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL)); moreover, MCL-1 overexpression is implicated as a resistance factor for multiple therapies including widely prescribed microtubule-targeted agents for breast cancers. Therefore, MCL-1 is an attractive therapeutic target for the treatment of cancers. In this study, we discovered the lead preclinical compound APG-3526, from its chemical synthesis optimization to potent antiproliferative and antitumor activity using multiple in vitro and in vivo xenograft models, respectively. Methods and Experiments APG-3526 was chemically synthesized by using one convergent multistep synthesis in gram scale. The binding affinity of APG-3526 was determined by a FP assay using a fluorescein labelled peptide (FAM-Bid) which binds to the MCL-1 protein leading to an increased anisotropy measured in milli-polarization (mP) values. The effect of APG-3526 on cell viability was determined using CCK-8 assay and its antitumor activities were tested in NCI-H929 and OPM-2 multiple myeloma (MM) xenograft SCID mice model (dosed i.v. at 50 mg/kg and p.o. at 25 mg/kg). Results Our biochemical study has demonstrated that APG-3526 binds to MCL-1 with a nanomolar affinity (IC50 = 7 nM). It is highly potent in inhibition of cell growth in MCL-1-dependent multiple myeloma cell lines (NCI-H929 cells: IC50 = 14 nM; OPM-2 cells: IC50 = 62 nM). Administration of APG-3526 (p.o. at 25 mg/kg, QDx14D, or i.v. at 50 mg/kg, once) achieved complete tumor regression in both NCI-929 and OPM-2 multiple myeloma xenograft models. The pharmacodynamics (PD) study using tumor samples further revealed caspase 3 activation and PARP cleavage triggered by APG-3526. MCL-1 complex MSD assay confirmed that APG-3526 disrupted MCL-1:BIM binding thus freeing BIM to initiate the apoptotic cascade. In conclusion, we have developed a novel and highly potent MCL-1 inhibitor, APG-3526, which displays clinically relevant pharmacokinetic properties and elicits potent antiproliferative and antitumor activities via disrupting MCL-1 complex and triggering caspase activation, especially in MCL-1 driven MM models. These results support APG-3526 as a promising MCL-1inhibitor for further clinical development. Citation Format: Jianyong Chen, Chengzhe Wu, Lingling Jiao, Leilei Zhao, Yunlong Zhou, Dongbo Li, Guozhi Tang, Shoulai Gu, Jing Deng, Guangfeng Wang, Douglas D. Fang, Shaomeng Wang, Dajun Yang, Yifan Zhai. Development of APG-3526 as a novel and highly efficacious MCL-1 inhibitor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 73.

Raman spectroscopy-based biomarker screening by studying the fingerprint characteristics of chronic lymphocytic leukemia and diffuse large B-cell lymphoma

Raman spectroscopy (RS) can provide fingerprint-type information on biochemical molecules. RS-based blood plasma analysis of solid tumors has been reported in recent years; however, there are no studies on the use of this analysis for detecting blood diseases. We studied the features of blood plasma in patients with diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL) by RS with the aim of developing a simple blood test for noninvasive DLBCL and CLL detection. We analyzed blood plasma from 33 DLBCL patients, 39 CLL patients and 30 healthy volunteers. Orthogonal partial least squares discriminant analysis (OPLS-DA) could build two clusters with almost no overlap between DLBCL/CLL and the controls. We used the prediction set to test the model built by OPLS-DA. For the CLL model, the sensitivity was 92.86%, and the specificity was 100%, whereas for the DLBCL model, the sensitivity was 80% and the specificity was 92.31%. We found Raman bands specific to both DLBCL and CLL patients in comparison with the healthy volunteers. Most importantly, we found that the combination of the 1445 cm−1 and 1655 cm−1 Raman shifts could discriminate DLBCL from CLL and even the other solid tumors reported to date. Further analysis of the assignments of 1655 cm−1 also gave us a clue to find potential important variables hemoglobin and serum albumin related with the CLL prognosis. Our exploratory study primarily demonstrated the great potential of developing RS blood plasma analysis as a novel clinical tool for the noninvasive detection of DLBCL and CLL.

Biomarkers in HCV-related mixed cryoglobulinemia patients withnon-Hodgkin lymphoma.

Chronic Hepatitis C virus (HCV) infection can cause severe extrahepatic manifestations, such as mixed cryoglobulins (MC), up to the development of B cell nonHodgkin's lymphoma (B-NHL). Mechanisms transforming of HCV infection into lymphoproliferative and/or autoimmune disorders are still poorly understood. In course of HCV infection, the sustained virus-driven antigenic stimulation may probably induce a B-cell clonal expansion. Measurements of serum free light chains (FLCs) levels, considered as a direct marker of B cell activity, are analyzed with increasing interest in clinical practice, for diagnosis, monitoring and follow-up of plasma cell dyscrasia. Syndecan-1 (CD138) is a transmembrane heparan sulfate proteoglycan expressed and actively shed by most myeloma cells. Membrane CD138 represents the major receptor protein for HCV attachment to the hepatocyte surface and high levels of circulating sCD138 levels are detected in patients at early stage of B-cell chronic lymphocytic leukemia. This study is aimed to evaluate sCD138 and FLC levels as diagnostic biomarkers of HCV-related MC with B-NHL. We enrolled 35 HCV-MC-NHL patients, characterized for the specific type of cryoglobulins, and 25 healthy blood donors (HBD) as negative control. Serum sCD138 levels were determined using ELISA kits specific for human sCD138. Serum FLCs were assessed by means of the turbidimetric assay. We found that serum levels of sCD138, as well as FLCs, were significantly higher in patients than in HBD (p<0.001). In agreement with the definition of HCV-driven lymphoproliferative disorders as the consequence of a multifactorial and multistep pathogenetic process, we suggest that sCD138 and FLCs could be considered putative independent markers of worsening progression of the disease.

Prognostic models for newly-diagnosed chronic lymphocytic leukaemia in adults: a systematic review and meta-analysis

Prognostic models for newly-diagnosed chronic lymphocytic leukaemia in adults: a systematic review and meta-analysis