Prognostic Factor In Chronic Lymphocytic Leukemia Research Articles

V(D)J Recombination Excision Circles of B- and T-cells as Prognostic Marker in B-Cell Chronic Lymphocytic Leukemia

Background & Aims. T-cell receptor excision circles (TREC) and к-deleting recombination excision circles (KREC) are extrachromosomal DNA segments generated during V(D)J recombination process that characterize the diversity of the antigen repertoire of T- and B-cells. The aim of our study is to identify the prognostic value of the excision circles in the chronic lymphocytic leukemia (CLL) setting. Methods. The excision circles' levels were assessed by means of real time PCR in 109 patients with high-risk CLL and 16 matched healthy individuals. Results. KREC levels were significantly (p < 0.001) lower in CLL patients vs. the reference group. TREC levels were lower in groups with unmutated status of immunoglobulin heavy chain variable region genes (p < 0.05) and 11q deletions (p < 0.1). Moreover, the KREC levels were higher in NOTCH1 mutation carriers than in noncarriers (p < 0.05). The comparison of treatment outcomes demonstrated a correlation between a high TREC level and achievement of complete remission. The prognostic value of the bio-marker was confirmed by ROC-analysis: AUC<sub>TREC</sub> = 0.713 (p = 0.001) Conclusion. Association between excision circles' le' and clinical/laboratory CLL prognostic factors, as well complete remission achievement, makes possible the plementation of the test for early prediction of the treatm outcome.

Lipids and Their Effects in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is an incurable common B-cell malignancy with a spectrum of clinical outcomes. Over the past decade, our increasing understanding of the drivers of CLL progression has led to the development and use of novel therapeutics. For example, B-cell receptor (BCR) signaling was shown to be overactive in CLL, and subsequently the kinase inhibitors ibrutinib (BTK inhibitor) and idelalisib (PI3K delta inhibitor) were found to have clinical efficacy in this malignancy (Byrd et al., 2013, Furman et al., 2014). Despite these new treatments, CLL remains incurable and there remains a need to identify new therapeutic targets.

Skin Cancers Among Chronic Lymphocytic Leukemia (CLL) Patients - the Effect of UV Radiation and CLL Clinical Characteristics

Background: It is well established that the incidence of skin cancers in patients with CLL are significantly elevated compared to age- and sex- matched controls. However, little is known about the characteristics of CLL patients who develop skin cancer. Herein, we evaluate the associations of CLL clinical and prognostic characteristics, along with UV radiation exposure, with risk of first skin cancer following CLL diagnosis.Methods: Newly diagnosed CLL patients from Minnesota, Iowa, and Wisconsin were enrolled in the Mayo Clinic case-control study from 2002-2015 and systematically followed in the Iowa/Mayo Lymphoma SPORE. Clinical and prognostic CLL data were obtained from the Mayo Clinic CLL database, and skin cancer clinical data were abstracted from medical records using a standard protocol. The CLL international prognostic index (CLL-IPI) was computed using a weighted average of five independent CLL prognostic factors (IGHV mutational status, serum b2-microglobulin, Rai stage, age, and FISH 17p deletion/TP53 status). Self-reported history of midday sun exposure at various ages (birth to age 12; 13 to 21 years; 22 to 40 years, and 41+ years) was obtained from a risk factor questionnaire. For each age, we asked the extent of mid-day sun as: practically no exposure (under 3 hours per week), little exposure (4-7 hours per week), moderate exposure (8 to 14 hours per week) and extensive exposure (15+ hours per week). Midday sun exposure was modeled as an ordinal covariate. To evaluate associations with risk of skin cancer following CLL diagnosis, we calculated time from date of CLL diagnosis to date of first skin cancer, death, or last known follow-up. We used Cox regression analysis to estimate hazard ratios (HRs) and 95% CIs. CLL treatment was considered a time-dependent covariate.Results: Among 846 CLL patients enrolled, the median age at diagnosis was 63 years (range 28-91), 68% were male, 7% had Rai stage III-IV at diagnosis. Based on the CLL-IPI, 42% were categorized as low risk, 33% as intermediate risk, and 25% as high or very-high risk. 109 CLL cases (13%) had one or more reported skin cancers at or prior to CLL diagnosis. Melanoma was observed in 19 (2%) cases and non-melanoma was in 90 (11%) cases. At a median follow-up of 7 years from CLL diagnosis, 165 patients (20%) had one or more skin cancers after CLL diagnosis. Among these patients, 49 had skin cancer before CLL diagnosis. The most frequent skin cancer was squamous cell carcinoma (59%), followed by basal cell (31%), melanoma (5%), and Merkel cell (1%). 552 (65%) of the 846 patients returned a questionnaire. Significant associations of clinical and prognostic characteristics with risk of first skin cancer were observed for age (HR=1.35 per 10 year increase, 95% CI=1.17-1.56, P<0.001), male sex (HR=1.38, 95% CI 0.98-1.96, p=0.07), prior history of skin cancer (HR=4.19, 95% CI=2.98-5.88, P<0.001), and CLL-IPI (HR=1.26, 95% CI= 1.03-1.54, P=0.026, after adjusting for age, sex, and prior skin cancer). Of note, the risk of first skin cancer in those CLL patients categorized as very high via CLL-IPI had 2.28 fold risk (95% CI 1.02-5.11). Midday sun exposures for each of the ages considered showed no evidence of association with risk of first skin cancer (all P>0.05). 50 CLL patients were treated prior to first skin cancer following CLL diagnosis; we observed no evidence of association between treatment and risk of first skin cancer (HR=1.44, 95% CI= 0.93-1.92, P=0.12).Conclusion: CLL patients who are at an increased risk of skin cancer following CLL diagnosis are those who either have had a prior history of skin cancer or a more aggressive CLL disease at diagnosis, according to CLL IPI. Routine skin cancer screening is currently recommended for CLL patients. Our data suggest that more frequent screening would be particularly important among patients with aggressive CLL and who have a prior history of skin cancer. Unexpectedly, we found no evidence of association of skin cancer risk with UV radiation following CLL diagnosis or with CLL treatment. Further investigation is needed to determine whether other factors increase the risk of skin cancer following CLL diagnosis. DisclosuresShanafelt:Genentech: Research Funding; GlaxoSmithkKine: Research Funding; Janssen: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding.

Increased IL-10/IL-17 ratio is aggravated along with the prognosis of patients with chronic lymphocytic leukemia

ObjectiveThis study is to investigate the association between the Treg/Th17 cells and prognosis of chronic lymphocytic leukemia (CLL). MethodsTotally 50 CLL patients and 20 Health controls were included in this study. Regulatory T (Treg) cells and the cell subset secreting IL-17 (Th17) in peripheral blood were detected with flow cytometry. Serum levels of IL-10 and IL-17 were determined with ELISA, and expression of Foxp3 and RORγt was assessed with quantitative real-time PCR. ResultsTreg and Th17 cell proportions in peripheral blood in the CLL patients were significantly higher than control. Serum levels of IL-10 and IL-17, and expression of Foxp3 and RORγt, were significantly increased in the CLL patients. Ratios of Treg/Th17 and IL-10/IL-17 were significantly elevated in the CLL patients. Compared with those before treatment, Treg/Th17 and IL-10/IL-17 ratios were declined in the CLL patients in remission. Compared with the non-remission group, Treg cells were significantly decreased, while Th17 cells were significantly increased, resulting in decreased Treg/Th17 ratio, in the remission group. Moreover, the serum IL-10 level was significantly decreased, while the serum IL-17 level was significantly increased, resulting in declined IL-10/IL-17 ratio, in the remission group. Correlation analysis showed that, Treg and Th17 cell counts were significantly associated with CD38 and ZAP-70 expression in the CLL patients. Moreover, the IL-10/IL-17 ratio was also significantly associated with CLL prognostic factors. ConclusionAltered Treg/Th17 and IL-10/IL-17 ratios in CLL would be aggravated along with the disease progression, which might be used as indicators for the disease prognosis.

Low expression of CD200 predicts shorter time-to-treatment in chronic lymphocytic leukemia.

CD200, formerly known as OX-2, is a type I glycoprotein that is expressed on a variety of cell types. CD200 has been shown to be overexpressed in chronic lymphocytic leukemia (CLL). Although previous studies have confirmed the diagnostic value of CD200 in differentiating CLL from to other B-cell chronic lymphoproliferative disorders especially mantle cell lymphoma, whether CD200 has prognostic significance in CLL remains to be determined. We evaluated the mean fluorescence intensity (MFI) of CD200 in 307 consecutive, untreated patients with CLL in our center using flow cytometry. Using a CD200 MFI cutoff of 189.5, these cases could be divided into two groups. Patients with lower CD200 MFI (< 189.5) had a significantly shorter time-to-treatment (TTT) than those with higher CD200 MFI (≥ 189.5) (median TTT: 2 months vs 28 months, p = 0.0008). However, the effect of CD200 MFI on overall survival was not significant (CD200 MFI < 189.5: undefined vs CD200 MFI ≥ 189.5: undefined, P = 0.2379). In subgroup analysis, CD200 MFI retained its prognostic value in patients with favourable characteristics such as Binet stage A disease, mutated IGHV status, normal TP53 or negative CD38 expression. In conclusion, our study identified CD200 MFI as a potential prognostic factor in CLL.

Cancer related fibroblasts (CAFs) control killer activity of natural killer cell through the poliovirus receptor (PVR: CD155) and have an important role for tumor expansion

Rituximab (RTX, anti-CD20 antibody) combined with chemotherapy is currently standard treatment for chronic lymphocytic leukemia (CLL). Serum level of IL-8 is a prognostic factor for CLL that correlates with disease stage. We investigated whether endogenous IL-8 affects RTX or Obinutuzumab (GA101) B-leukemic depletion mediated by natural killers (NK). Using whole peripheral blood lymphocytes from untreated CLL patients, RTX, but most significantly GA101, were effective in B-cell depletion and NK activation. IL-8 inhibition completely inhibited B-cell depletion by RTX and reduced GA101-induced B-cell depletion. Altogether results underline IL-8 as an endogenous NK co-activator and confirm GA101 therapeutic potential for CLL treatment.

Predicting Prognosis in Chronic Lymphocytic Leukemia in the Contemporary Era.

Next-generation sequencing has identified new genetic markers that have altered prognosis for patients with chronic lymphocytic leukemia (CLL) at diagnosis. Understanding the significance of these prognostic indicators and recognizing their potential impact on treatment selection and patients' outcomes is critical for clinicians and investigators. To review novel prognostic factors at CLL diagnosis that have shown an impact on the prognosis and outcomes of this disease. Literature from January 2004 through December 2014 was searched in PubMed, Cochrane Central Register of Controlled Trials, and Scopus to identify English-language, peer-reviewed articles on clinical and prognostic factors for CLL (TP53, ATM, NOTCH1, SF3B1, BIRC3, and MYD88). Reference lists were subsequently reviewed for additional articles. A total of 450 articles was identified, and 48 articles meeting inclusion criteria were reviewed. Among prognostic markers reviewed, chromosomal aberrations have been validated and are currently used clinically to predict prognosis. Patients with 17p13.1 deletion have poor response to chemoimmunotherapy and are treated differently, with some undergoing allogeneic transplantation in first remission. CD38 and ZAP-70 status of malignant cells and unmutated immunoglobulin variable heavy chain gene have similarly been validated to predict adverse prognosis, but their implications on treatment selection have not been proven. The presence of TP53 and ATM mutations predicts worse prognosis, which has been corroborated in various studies. Patients with TP53 mutations have lower responses to chemoimmunotherapy. Furthermore, patients with TP53 and ATM mutations have inferior progression-free survival and overall survival, independent of other factors. Patients carrying the NOTCH1 and SF3B1 mutations have worse prognosis; patients with the NOTCH1 mutation have lower response rates to standard chemoimmunotherapy. The impact of BIRC3 on prognosis and survival requires further confirmation. The heterogeneous clinical course of CLL is likely explained by underlying molecular prognostic factors. Moving forward, analyzing these factors at diagnosis is recommended for better prognostication.

A novel diagnostic biosensor for distinguishing immunoglobulin mutated and unmutated types of chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), the immunoglobulin heavy-chain variable (IgVH) region may be mutated (Ig-mutated CLL) or unmutated (Ig-unmutated CLL); and the presence or absence of mutations in this region of CLL cells distinguishes two clinically distinct forms. It is important for physicians to distinguish between patients with Ig-unmutated CLL, where typically have more indolent disease with median survivals close to 25 years, and Ig-mutated CLL, where have more aggressive disease with median survivals around eight years. In this work, a biosensor capable of diagnosis and distinguishing between these two types of CLL was reported. The biosensor was fabricated by modifying a gold electrode with gold nanoparticles (AuNPS) followed by coating of ZAP70 oligonucleotide probe on the surface to detect specific sequence of ZAP70 gene. ZAP70 could predict the IgVH mutation status and is a good marker for differentiating Ig-mutated and Ig-unmutated CLL and serve as prognostic marker. First, we focused on achieving hybridization between probe and its complementary sequence. Hybridization between probe and target was determined with electrochemical impedance spectroscopy (EIS). Then, our efforts turned to optimize the conditions for the detection of any point mutation and also to maximize the selectivity. Under optimal conditions, the biosensor has a good calibration range between 2.0 × 10(-14) and 1.0 × 10(-9)molL(-1), with ZAP70 DNA sequence detection limit of 4.0 × 10(-15)molL(-1). We successfully detect hybridization first in synthetic samples, and ultimately in blood samples from patients. Experimental results illustrated that the nanostructured biosensor clearly discriminates between mutated and non-mutated CLL and predict the IgVH mutation status, which it has been considered as the single most informative stage independent prognostic factor in CLL.

Proteins interaction network and modeling of IGVH mutational status in chronic lymphocytic leukemia.

BackgroundChronic lymphocytic leukemia (CLL) is an incurable malignancy of mature B-lymphocytes, characterized as being a heterogeneous disease with variable clinical manifestation and survival. Mutational statuses of rearranged immunoglobulin heavy chain variable (IGVH) genes has been consider one of the most important prognostic factors in CLL, but despite of its proven value to predict the course of the disease, the regulatory programs and biological mechanisms responsible for the differences in clinical behavior are poorly understood.MethodsIn this study, (i) we performed differential gene expression analysis between the IGVH statuses using multiple and independent CLL cohorts in microarrays platforms, based on this information, (ii) we constructed a simplified protein-protein interaction (PPI) network and (iii) investigated its structure and critical genes. This provided the basis to (iv) develop a Boolean model, (v) infer biological regulatory mechanism and (vi) performed perturbation simulations in order to analyze the network in dynamic state.ResultsThe result of topological analysis and the Boolean model showed that the transcriptional relationships of IGVH mutational status were determined by specific regulatory proteins (PTEN, FOS, EGR1, TNF, TGFBR3, IFGR2 and LPL). The dynamics of the network was controlled by attractors whose genes were involved in multiple and diverse signaling pathways, which may suggest a variety of mechanisms related with progression occurring over time in the disease. The overexpression of FOS and TNF fixed the fate of the system as they can activate important genes implicated in the regulation of process of adhesion, apoptosis, immune response, cell proliferation and other signaling pathways related with cancer.ConclusionThe differences in prognosis prediction of the IGVH mutational status are related with several regulatory hubs that determine the dynamic of the system.Electronic supplementary materialThe online version of this article (doi:10.1186/s12976-015-0008-z) contains supplementary material, which is available to authorized users.

Second Myeloid Malignancies in a Large Cohort of Patients With Chronic Lymphocytic Leukemia: A Single Institution Experience.

Patients with chronic lymphocytic leukemia (CLL) have a 2- to 5-fold risk of developing a second malignancy compared with the general population. The incidence of myeloid malignancies, such as acute myeloid leukemia and myelodysplastic syndrome, is increased in CLL and has been linked to previous therapy. In this study, we aim at describing characteristics and determining risk factors for developing second myeloid disorders (SMDs) in patients with CLL. From a total of 1269 patients diagnosed with CLL during the study time period, 30 (2.4%) were found to have an SMD. The majority of SMDs were myelodysplastic syndrome or acute myeloid leukemia (76.7%). The median time from diagnosis of CLL to diagnosis of SMD was 4.47 years. Most patients who developed an SMD had received treatment for their CLL (86%). The median time from treatment of CLL to diagnosis of SMD was 4.19 years. The overall survival of patients with CLL with no second malignancy was significantly longer than those with an SMD (11.9 vs. 7.1 years, P = .001). There was no association between developing SMD and age, gender, expression of CD38, expression of ZAP-70, and unmutated immunoglobulin heavy chain gene status. The risk of developing SMD in our cohort was higher in patients who received fludarabine- or alkylator-based therapy. Our analysis is one of the largest series showing that patients receiving fludarabine or alkylator-based therapies for CLL have a higher risk of developing SMD. Our study also confirms previous reports that prognostic factors in CLL do not increase the risk for development of SMD. Clinicians should understand the leukemogenicity of fludarabine or alkylator-based treatments when considering treatments for patients with CLL.

Opposite Prognostic Significance of Cellular and Serum Circulating MicroRNA-150 in Patients with Chronic Lymphocytic Leukemia.

MicroRNAs (or miRs) play a crucial role in chronic lymphocytic leukemia (CLL) physiopathology and prognosis. In addition, circulating microRNAs in body fluids have been proposed as new biomarkers. We investigated the expression of matched cellular and serum circulating microRNA-150 by quantitative real-time PCR (qPCR) from purified CD19(+) cells or from CLL serums obtained at diagnosis in a cohort of 273/252 CLL patients with a median follow-up of 78 months (range 7-380) and correlated it to other biological or clinical parameters. We showed that miR-150 was significantly overexpressed in CLL cells/serums compared with healthy subjects (P < 0.0001). Among CLL patients, a low cellular miR-150 expression level was associated with tumor burden, disease aggressiveness and poor prognostic factors. In contrast, a high level of serum miR-150 was associated with tumor burden markers and some markers of poor prognosis. Similarly, cellular and serum miR-150 also predicted treatment-free survival (TFS) and overall survival (OS) in an opposite manner: patients with low cellular/serum miR-150 levels have median TFS of 40/111 months compared with high-level patients who have a median TFS of 122/60 months (P < 0.0001/P = 0.0066). Similar results were observed for OS. We also found that cellular and serum miR-150 levels vary in an opposite manner during disease progression and that cellular miR-150 could be regulated by its release into the extracellular space. Cellular and serum levels of miR-150 are associated with opposite clinical prognoses and could be used to molecularly monitor disease evolution as a new prognostic factor in CLL.

CC-122 Has Robust Anti-Proliferative Activity in a Primary Chronic Lymphocytic Leukemia (CLL) Co-Culture Model and Is Superior to Lenalidomide

Background: The Ikaros family zinc finger transcription factor, Aiolos, is upregulated in more than 80% of CLL patients with data suggesting that it may be an important driver of CLL survival and hence a bona fide target for therapeutic intervention. To date, transcription factors such as Aiolos have not been amenable to drug therapy. We and others have shown that the IMiDs™ immunomodulatory drugs such as Lenalidomide (LEN) bind Cereblon (CRBN), a substrate receptor of the cullin ring E3 ubiquitin ligase complex (CRL4CRBN) and promote ubiquitination and subsequent proteosomal degradation of lymphoid transcription factors Ikaros and Aiolos. Such insights have been critical for the design and development of next generation IMiD analogues. CC-122 is a novel non-phthalimide, first-in-class, PPM™ pleiotropic pathway modifier drug with anti-proliferative, anti-angiogenic, and immunomodulatory properties. CC-122 specifically targets CRBN and promotes degradation of Aiolos and Ikaros. We have now investigated the effects of LEN and CC-122 on CLL proliferation, and Aiolos and Ikaros degradation, in a cohort of primary CLL cases using an in vitro co-culture system based on CD154 expressing mouse fibroblasts and exogenous IL-21 that recapitulates the in vivo CLL microenvironment.Results: Forty CLL primary patient PBMC samples, selected to represent diverse prognostic subgroups, were cultured in media supplemented with LEN, CC-122, or control and assessed in a BrdU incorporation assay. Among these analyzed samples, four distinct ‘response’ subsets were identified. In subset 1 (n=10, 25% of cases), we found that CC-122 is superior to LEN in inhibiting proliferation (mean IC50: LEN 5.7µM; CC-122 1.2µM). Subset 2 (n=23; 57% of cases) was sensitive to CC-122, but unresponsive to LEN. In these cases, CC-122 inhibited proliferation by at least 50% at a concentration of ≤3µM despite the failure of LEN (IC50: >10 µM) to inhibit proliferation, highlighting a qualitative difference between the two agents. In subset 3, the anti-proliferative activities of CC-122 and LEN were comparable (n=2, 5% of cases). Finally, both CC-122 and LEN were inactive (up to 10µM) in a 4th subset (n=5; 12% of cases). We subsequently explored associations between CC-122 activity and known CLL prognostic risk factors in these samples. In 5 out of 6 cases with 17p deletion CC-122 inhibited proliferation (IC50: <0.3-0.8 mM). Similarly, in 4 out 6 cases harbouring an 11q- defect CC-122 was active (IC50: 0.8-3 µM). In general, the efficacy of either drug at inhibiting CLL cell proliferation did not segregate with established prognostic variables (e.g. IgVH mutation status, cytogenetic abnormalities, etc.), or previous treatment exposure (e.g. chlorambucil, fludarabine, alemtuzumab, etc.). Preliminary investigations of the CLL cells demonstrate wide variation in baseline Ikaros, Aiolos, and CRBN levels between individual cases as measured by either flow cytometry and/or western blot. We show that treatment of CLL cells with CC-122 or LEN results in variable degradation of Ikaros (LEN 0-100%; CC-122 55-100%) and Aiolos (LEN 72-100%; CC-122 97-100%) compared to untreated controls. Of note, CC-122 was consistently superior to LEN in promoting Aiolos and Ikaros degradation. In contrast, levels of CRBN, the molecular target of CC-122 and LEN, were unaffected by either compound. The predictive value of baseline Aiolos, Ikaros and CRBN expression, as well as their degradation, by CC-122 in relation to cellular response is currently being assessed.Conclusions: Taken together, these data demonstrate that CC-122 has robust anti-proliferative activity in primary CLL cells through a unique mechanism of selective degradation of Aiolos and Ikaros transcription factors. CC-122 is superior to LEN and is active across several prognostic backgrounds. Furthermore, we show significant activity of CC-122 even in cases that are non-responsive to LEN suggesting that CC-122 targets a novel pathway in CLL. These data suggest that further evaluations of CC-122 in pre-clinical and clinical studies, as a single agent or in combination with established or novel compounds (such as Btk and PI3Kd inhibitors), are warranted. DisclosuresBlocksidge:Celgene: Research Funding. Glenn:Celgene: Research Funding. Gandhi:Celgene Corp: Employment, Equity Ownership. Klippel:Celgene: Employment, Equity Ownership. Pourdehnad:Celgene: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership. Kalakonda:Celgene: Research Funding.

Clinical relevance of vascular endothelial growth factor type A (VEGFA) and VEGF receptor type 2 (VEGFR2) gene polymorphism in chronic lymphocytic leukemia.

Vascular endothelial growth factor type A (VEGFA) is a key regulator of angiogenesis and vascular permeability. Chronic lymphocytic leukemia (CLL) cells are able to secrete VEGFA and express VEGFA receptors, thus it can be hypothesized that VEGFA-mediated signaling influences CLL clone survival. In this case-control study we verified whether inherited differences in activities of VEGFA and its main receptor VEGFR2 impact predisposition to CLL or the course of the disease. Four functional single nucleotide polymorphisms (SNPs) including two SNPs in VEGFA gene, namely rs2010963 (+405G>C) and rs3025039 (+936C>T) and two SNPs in VEGFR2 gene including rs7667298 (-271G>A) and rs1870377 (+1719A>T) were genotyped using PCR-based assays in 223 Caucasian CLL patients and 150 matched controls. Regarding VEGF rs2010963 SNP, we observed an association between CLL and allele C distribution with an OR of 1.52 (95% CI, 1.002-2.312), p=0.04. The distribution of other genotypes and alleles was similar in CLL and control groups. No genotype or allele was significantly associated with important prognostic factors in CLL including clinical stage, IgVH mutational status, ZAP-70 expression and FISH cytogenetic abnormalities. In conclusion, the results of our study indicate that genetic polymorphisms in VEGFA mediated pathway may influence the susceptibility to CLL.

Overexpressed BAG3 is a potential therapeutic target in chronic lymphocytic leukemia

Bcl-2-associated athanogene 3 (BAG3), a member of BAG family, is shown to sustain cell survival and underlie resistance to chemotherapy in human neoplastic cells. We aimed to determine the exact role and underlying mechanisms of BAG3 in human chronic lymphocytic leukemia (CLL). One hundred human CLL samples and 20 normal B-cell samples from healthy controls were collected. We measured the BAG3 expression in these cells and explored its relationship with known prognostic factors for CLL. The roles of BAG3 in cell apoptosis and migration were evaluated by small interfering RNA-mediated knockdown of BAG3 in primary CLL cells. We showed that BAG3 expression level was increased in CLL cells compared with normal B cells. Moreover, BAG3 expression was particularly upregulated in CD38 positive, unmutated immunoglobulin heavy-chain patients and those with lymphadenopathy and/or splenomegaly. Importantly, patients with increased BAG3 expression level have poor overall survival in subgroups with positive ZAP-70 or those without any "p53 abnormality". In addition, knocking down of BAG3 expression resulted in increased apoptotic ratio and decreased migration in primary CLL cells. Our data indicate that BAG3 is a marker of poor prognostic in specific subgroups of CLL patients and may be a potential therapeutic target for this disease.

New insights into biology, prognostic factors, and current therapeutic strategies in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by the clonal proliferation and accumulation of mature B lymphocytes. CLL cells show an antiapoptotic profile, suggesting the important role of apoptosis inhibition in the disease development. However, there is some population of proliferating CLL cells, which may also play a role in progression of the disease. There are several newer, biological prognostic factors in CLL. Currently, cytogenetic abnormalities with different prognostic values seem to be the most biologically relevant. During the last decades, the treatment of CLL has been significantly changed. Different strategies such as monotherapy with chlorambucil and purine nucleoside analogues (PNA) used alone or in combination with cyclophosphamide have been introduced. Most recently, immunochemotherapy with anti-CD20 monoclonal antibody, rituximab, combined with fludarabine and cyclophosphamide, became a gold standard of first-line treatment in eligible CLL patients. Currently, new treatment strategies including new monoclonal antibodies, bendamustine, lenalidomide, or inhibitors of several cell signaling pathways are under clinical studies in resistant/relapsed CLL patients. Moreover, allogeneic stem cell transplantation has to be considered, especially in younger high risk patients, for example, those who are resistant to PNA or those with 17p deletion. In this paper, we present the most important recent advances in CLL biology and treatment.

Endogenous IL-8 acts as a CD16 co-activator for natural killer-mediated anti-CD20 B cell depletion in chronic lymphocytic leukemia

Rituximab (RTX, anti-CD20 antibody) combined with chemotherapy is currently standard treatment for chronic lymphocytic leukemia (CLL). Serum level of IL-8 is a prognostic factor for CLL that correlates with disease stage. We investigated whether endogenous IL-8 affects RTX or Obinutuzumab (GA101) B-leukemic depletion mediated by natural killers (NK). Using whole peripheral blood lymphocytes from untreated CLL patients, RTX, but most significantly GA101, were effective in B-cell depletion and NK activation. IL-8 inhibition completely inhibited B-cell depletion by RTX and reduced GA101-induced B-cell depletion. Altogether results underline IL-8 as an endogenous NK co-activator and confirm GA101 therapeutic potential for CLL treatment.

Prior Treatment with Chemotherapy Is Associated with Poor Outcomes of High Risk Skin Cancers in Patients with Chronic Lymphocytic Leukemia

Abstract 3920The increased incidence of skin cancer in patients with chronic lymphocytic leukemia (CLL) has been well-documented; however, the outcomes for CLL patients with high risk skin cancers are not as well-characterized. Moreover, little is known about the association between outcomes of high risk skin cancers and CLL prognostic factors or exposure to prior chemotherapy.We identified 225 patients at our institutions over the last 20 years with concurrent CLL and skin cancer, and we retrospectively examined outcomes for the 139/225 (61.8%) who had high risk skin cancer as defined by a diagnosis of squamous cell carcinoma (SCC), melanoma, or Merkel cell carcinoma (MCC). Poor skin cancer outcome (defined by local recurrence, nodal metastasis, and disease specific death) was determined by review of patient medical records and verified by pathology reports. Associations between various risk factors and poor skin cancer outcomes were evaluated via chi-square statistics (STATA 12.0 College Station, TX).The study group included a representative CLL population, with 98/139 (70%) males and a median age at diagnosis of 65 years (range 35–84), with a median follow-up of 120 months (range 2–410). Over the course of the study, 76 CLL patients remained alive (55%), 16 patients died due to CLL (12%), and 42 patients died from other causes (30%), including 18 (13%) from skin cancer. Median age at time of skin cancer diagnosis was 72 years (range 44–98), and median follow-up time after skin cancer diagnosis was 41 months (range 2–182). Skin cancer diagnoses included SCC in 122 patients (81%), melanoma in 22 patients (13%), and MCC in 8 patients (6%). Sixty-three (45%) patients had more than one skin cancer diagnosed. In the 122 patients with SCC, there were a total of 353 tumors, 297 of which occurred in males (84%). Half of the SCCs were in the head and neck (n=175, 49.6%). Fifty-four SCCs (15%) were greater than 2 cm in diameter. Rates of nodal metastases and death due to SCC were 12% and 6%, respectively. In the 22 patients with melanoma, 14 occurred in men (63.6%), with the largest number of tumors again located on the head and neck (n=8, 36.4%). Breslow thickness was greater than 1 mm in 12 patients (54.5%). Local recurrence and nodal metastases each occurred in 5 patients (22.7%). The five patients (22.7%) who died due to melanoma were the same patients with nodal metastases. In the 8 patients with MCC, 5 patients (62.5%) were male, and there was no location predominance. Five tumors (62.5%) were greater than 2 cm at time of diagnosis, local recurrence occurred in 1 patient (12.5%), and 5 patients (62.5%) had nodal metastases, with 4 of these 5 dying from MCC. Interestingly, prior treatment with any CLL chemotherapy was significantly associated with poorer skin cancer outcome, with increased rates of local recurrence, metastatic disease, or death due to skin cancer (p=0.001). We also explored whether high risk CLL prognostic factors were associated with poorer skin cancer outcomes. Eleven out of 18 patients tested (61%) had unmutated IGHV, 9/69 patients tested (13%) had del(17p) or del(11q) cytogenetics, and 71 patients did not have prognostic marker data available. In this limited prognostic marker data set, neither unmutated IGHV, poor risk cytogenetics, nor advanced Rai stage was associated with skin-cancer specific outcomes.Overall, the majority of the skin cancers in our cohort occurred in males, and the most common site of disease was the head and neck, highlighting an area in need of especially close surveillance. Relative to the historical experience of high-risk skin cancer patients in the general population, we found skin cancers in our CLL population to be more aggressive. For example, 2 of the skin-cancer related deaths were in CLL patients with stage I melanoma, which is unusual in the general population. Additionally, rates of nodal metastases and death in our SCC group were 12% and 6%, respectively, compared to rates of 4% and 2% in most studies of SCC in the general population. Strikingly, the risk of death from skin cancer was equivalent to the risk of death from CLL. The poor skin cancer outcomes in patients in our study were driven by the group who had received prior chemotherapy. Outcomes were not influenced by high risk CLL prognostic markers, although this latter analysis was limited by incomplete data. Disclosures:No relevant conflicts of interest to declare.

The Evolution and Impact of Subclonal Mutations in Chronic Lymphocytic Leukemia

Abstract 5The impact of clonal heterogeneity on cancer progression in chronic lymphocytic leukemia (CLL) is not well understood. We hypothesized that the evolutionary dynamics of subclonal mutations contribute to the variations in disease tempo and response to therapy that characterize CLL. We therefore carried out a large-scale analysis of subclonal and clonal point mutations and copy-number alterations in 149 CLLs, detected by whole exome sequencing (WES) and SNP arrays.We utilized a novel computational approach, which integrates purity and local ploidy information, to infer the cancer cell fraction (CCF) of each mutation from WES data, and to classify mutations as clonal or subclonal. Subclonal mutations were detected in 146/149 CLLs and were enriched with putative cancer driver events (P=0.001). Furthermore, higher numbers of subclonal mutations were associated with prior anti-leukemia therapy (P=0.017). Together, these results suggest that a strong extrinsic selection pressure, such as cytotoxic treatment, promotes the expansion of fitter subclones, driving them to above our detection threshold (CCF of ∼0.10).The order of mutation acquisition may be inferred from the aggregate frequencies at which driver events are clonal or subclonal, as clonal mutations represent earlier events and subclonal later events. Of the 149 samples, we found 3 drivers (MYD88, trisomy 12, and del(13q)) that were clonal in 80–100% of samples harboring these alterations –significantly higher than other driver events (q<0.1), suggesting that they arise earlier in typical CLL development. Other drivers (e.g., ATM, TP53 and SF3B1) were often observed at subclonal frequencies, indicating that they often arise later in leukemic development.We directly assessed the evolution of somatic mutations in 18 patients, in which data from two distant timepoints were available. Clonal evolution was observed in 11 of 18 patients (10 of 12 who received intervening treatment, but only 1 of 6 without intervening treatment, P=0.012) and confirmed that subclonal mutations (e.g., del(11q), SF3B1 and TP53) shifted towards clonality over time. Indeed, expanding subclonal mutations were enriched in putative drivers (P=0.021), suggesting that these mutations not only mark genetic evolution but also provide the fitness advantage driving it. Changes in the genetic composition of CLL cells with clonal evolution were associated with network level changes in gene expression.If treatment-associated genetic evolution leads to expansion of a fitter subclone, we would predict a shorter time to relapse in these individuals. Indeed, presence of a detectable subclonal driver mutation was associated with a shorter time to retreatment in these 18 samples (P=0.04), indicating that the presence of subclonal drivers adversely impacts clinical outcome. In the analysis of the full cohort of 149 samples, we observed that CLLs with subclonal driver mutations were associated with shorter times from diagnosis to first therapy (P=0.001) and between sample collection to treatment (P<0.001). Moreover, in the subset of 67 of 149 patients who were treated after sampling, presence of subclonal driver mutations evident in the pre-treatment sample was associated with earlier retreatment (P=0.003). Regression models adjusting for CLL prognostic factors (IGHV status, prior therapy and high risk cytogenetics) demonstrated that the presence of a subclonal driver was an independent risk factor for earlier retreatment (adjusted hazard ratio of 4.61 (CI 1.59–13.34), P=0.005). Thus, the detection of subclonal drivers(indicative of an active evolutionary process) is associated with shorter duration of remission.In conclusion, the analysis of clonal heterogeneity in CLL provides a glimpse into the past, present and future of a patient's disease. Through the cross-sectional analysis of 149 samples, we derived the number and genetic composition of clonal and subclonal mutations and thus uncovered footprints of the past history of CLL. Furthermore, we inferred a temporal order of genetic events implicated in CLL. Finally, our combined longitudinal and cross-sectional analyses revealed that knowledge of subclonal mutations anticipates the genetic composition of the future relapsing leukemia as well as the rapidity with which it will occur. These data challenge us to therapeutically address not only genetic targets but also their dynamic evolutionary landscape. Disclosures:No relevant conflicts of interest to declare.

Prognostic factors in CLL.

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease, as some patients progress rapidly toward the more advanced studies, whereas others survive for a long period without the need for treatment. This heterogeneity of clinical course was somehow unexplained until studies on the CLL cell features disclosed that the CLL clones were heterogeneous and were characterized by different phenotypic and genotypic features in the different patients. On the basis of these observations, it was determined in retrospective studies that clones characterized by unmutated IGHV genes, and/or CD38 and/or ZAP-70 expression conferred a more severe prognosis to the CLL patients. Here, we present data on prospective studies carried out on Binet A-stage patients, in whom the markers were determined at diagnosis and their predictive value was assessed in comparison with chromosomal abnormalities and gene expression or micro RNA profiles. In addition, hypothesis on the potential pathogenetic role of these markers will be presented.

Predicting survival in chronic lymphocytic leukemia

There is increasing interest in the use of prognostic markers that may predict survival and guide management in patients diagnosed with the early stages of chronic lymphocytic leukemia (CLL). Currently, the most important traditional prognostic factors include clinical staging, lymphocyte doubling time and β2-microglobulin/thymidine kinase; and the most important novel markers include karyotypic aberrations (typically assessed by FISH probes or CpG oligonucleotide karyotyping) and IgVH mutation status. Although each of these factors have individually shown significant correlations with survival, there is increasing appreciation that the most complete information may be obtained by using a combination of several factors in prognostic normograms or models. In this article, we review the current state-of-the-art with regards to CLL prognostic factors and discuss how they can be applied in the clinic.