Early Chronic Lymphocytic Leukemia Research Articles

Whole Genome Classification of Pre-Malignant B-Cells Disorder of the Oxplored Study

Background. Chronic lymphocytic leukaemia (CLL) is always preceded by preclinical clonal B-cell disorders such as monoclonal B-cell lymphocytosis (MBL). Pre-malignant clonal B-cells are identifiable in asymptomatic individuals, a minority of whom will progress to symptomatic CLL. While some people remain at pre-cancerous or asymptomatic early stage CLL, others experience clinically aggressive disease. There is currently no method to effectively stratify individuals at higher risk of developing symptomatic CLL. Despite recent advances in treatment options available, the disease remains incurable. Therefore, we need to expand our knowledge and improve current classification methods. Here, we propose a molecular classifier using whole genome sequencing (WGS). This classifier was obtained by performing genomic characterization in pre-malignancy / early stages of the disease and comparing the genomic subgroups with those of symptomatic CLL. Methods. Individuals with newly diagnosed monoclonal B-cell lymphocytosis and early asymptomatic chronic lymphocytic leukaemia were enrolled in the OXPLORED clinical trial (Oxford Pre-cancerous Lymphoproliferative Disorders: Analysis and Interception study). We performed WGS in 400 samples from 200 individuals (sorted CD19+ B-cells as tumour, and matched salivary DNA as germline) to detect somatic alterations. We derived immunoglobulin heavy chain variable (IGHV) mutational status, stereotype and an additional 186 genomic features as previously described (Robbe*, Ridout* et al. Nature Genetics 2022) including known and recently discovered candidate drivers of CLL, recurrent structural variants, mutational signatures, genomic complexity measures, mutational burden, and pathway alterations. All the data was compiled into a matrix to extract meaningful sets of features to cluster patients' genomes using non-negative matrix factorization (NMF). Genomic findings were compared to the largest CLL whole genome cohort of symptomatic patients requiring frontline treatment (n=443). Results. The IGHV mutational status was found unmutated in 31% of individuals and hypermutated in 69% (excluding missing data for 6 genomes). Next, we considered mutations and CNAs in 58 common CLL drivers and 34 regions with recurrent CNAs. Although numbers were lower than in frontline CLL, most individuals presented at least one driver (86%) or more (57%) (by comparison, the CLL frontline cohort presented at least one CLL driver in 98% of patients). Genomic complexity, defined as 4 or more CNAs was detected in 12%, and complex genome defined as the presence of both CN gains and CN losses in 22%, indicating that newly diagnosed monoclonal B-cell lymphocytosis and early asymptomatic CLL present a relatively high degree of genomic complexity similar to what was observed in frontline CLL. The most common driver was del13q (60%), including as a sole driver in a third of these genomes. Other recurrent alterations were IGLL5 mutations (20%), trisomy 12 (9%), and mutations in MYD88 (8%), CREBBP (5%), NOTCH1 (5%), and SPEN (5%). Noncoding elements were also found mutated, including 30 promoters (including BIRC3 in 7%), five UTRs and eight enhancers, including BCL6 and PAX5 enhancers in 18% and 3.4% of individuals, respectively. When comparing this cohort with CLL genomes, we found that alterations in the main cancer pathways were depleted in OXPLORED, including known drivers such as ATM, NOTCH1, SF3B1. Finally, moving away from single-alteration grouping, we clustered the 643 genomes (443 CLL + 200 OXPLORED) using 186 genomic features by applying NMF. Importantly, we found distinct genomic profiles in the early disease cohort, including a subgroup like symptomatic CLL at frontline and another distinct subgroup, hinting towards identifying a genomic subgroup at higher risk of progressing to CLL. Conclusion: The comprehensive characterisation of genomic events that determine progression to malignancy are essential to understand the mechanisms of disease progression. Our study opens new horizons towards identifying individuals at higher risk and might pinpoint to individuals who need early intervention with curative intent. Therefore, we propose to analyse newly generated whole-genome sequencing (WGS) data to establish genomic subgroups in early CLL / pre-malignancy stages.

Platelet-derived microparticles provoke chronic lymphocytic leukemia malignancy through metabolic reprogramming.

It is well established that inflammation and platelets promote multiple processes of cancer malignancy. Recently, platelets have received attention for their role in carcinogenesis through the production of microvesicles or platelet-derived microparticles (PMPs), which transfer their biological content to cancer cells. We have previously characterized a new subpopulation of these microparticles (termed mito-microparticles), which package functional mitochondria. The potential of mitochondria transfer to cancer cells is particularly impactful as many aspects of mitochondrial biology (i.e., cell growth, apoptosis inhibition, and drug resistance) coincide with cancer hallmarks and disease progression. These metabolic aspects are particularly notable in chronic lymphocytic leukemia (CLL), which is characterized by a relentless accumulation of proliferating, immunologically dysfunctional, mature B-lymphocytes that fail to undergo apoptosis. The present study aimed to investigate the role of PMPs on CLL metabolic plasticity leading to cancer cell phenotypic changes. CLL cell lines were co-incubated with different concentrations of human PMPs, and their impact on cell proliferation, mitochondrial DNA copy number, OCR level, ATP production, and ROS content was evaluated. Essential genes involved in metabolic-reprogramming were identified using the bioinformatics tools, examined between patients with early and advanced CLL stages, and then validated in PMP-recipient CLLs. Finally, the impact of the induced metabolic reprogramming on CLLs' growth, survival, mobility, and invasiveness was tested against anti-cancer drugs Cytarabine, Venetoclax, and Plumbagin. The data demonstrated the potency of PMPs in inducing tumoral growth and invasiveness in CLLs through mitochondrial internalization and OXPHOS stimulation which was in line with metabolic shift reported in CLL patients from early to advanced stages. This metabolic rewiring also improved CLL cells' resistance to Cytarabine, Venetoclax, and Plumbagin chemo drugs. Altogether, these findings depict a new platelet-mediated pathway of cancer pathogenesis. We also highlight the impact of PMPs in CLL metabolic reprogramming and disease progression.

Germline CHEK2 and ATM Variants in Myeloid and Other Hematopoietic Malignancies

An intact DNA damage response is crucial to preventing cancer development, including in myeloid and lymphoid malignancies. Deficiencies in the homologous recombination (HR) pathway can lead to defective DNA damage responses, and this can occur through inherited germline mutations in HR pathway genes, such as CHEK2 and ATM. We now understand that germline mutations can be identified frequently (~ 5-10%) in patients with myeloid and lymphoid malignancies, and among the most common of these are CHEK2 and ATM. We review the role that deleterious germline CHEK2 and ATM variants play in the development of hematopoietic malignancies, and how this influences clinical practice, including cancer screening, hematopoietic stem cell transplantation, and therapy choice. In recent large cohorts of patients diagnosed with myeloid or lymphoid malignancies, deleterious germline loss of function variants in CHEK2 and ATM are among the most common identified. Germline CHEK2 variants predispose to a range of myeloid malignancies, most prominently myeloproliferative neoplasms and myelodysplastic syndromes (odds ratio range: 2.1-12.3), and chronic lymphocytic leukemia (odds ratio 14.83). Deleterious germline ATM variants have been shown to predispose to chronic lymphocytic leukemia (odds ratio range: 1.7-10.1), although additional studies are needed to demonstrate the risk they confer for myeloid malignancies. Early studies suggest there may also be associations between deleterious germline CHEK2 and ATM variants and development of clonal hematopoiesis. Identifying CHEK2 and ATM variants is crucial for the optimal management of patients and families affected by hematopoietic malignancies. OPENING CLINICAL CASE: "A 45year-old woman presents to your clinic with a history of triple-negative breast cancer diagnosed five years ago, treated with surgery, radiation, and chemotherapy. About six months ago, she developed cervical lymphadenopathy, and a biopsy demonstrated small lymphocytic leukemia. Peripheral blood shows a small population of lymphocytes with a chronic lymphocytic leukemia immunophenotype, and FISH demonstrates a complex karyotype: gain of one to two copies of IGH and FGFR3; gain of two copies of CDKN2C at 1p32.3; gain of two copies of CKS1B at 1q21; tetrasomy for chromosome 3; trisomy and tetrasomy for chromosome 7; tetrasomy for chromosome 9; tetrasomy for chromosome 12; gain of one to twocopies of ATM at 11q22.3; deletion of chromosome 13 deletion positive; gain of one to two copies of TP53 at 17p13.1). Given her history of two cancers, you arrange for germline genetic testing using DNA from cultured skin fibroblasts, which demonstrates pathogenic variants in ATM [c.1898 + 2T > G] and CHEK2 [p.T367Metfs]. Her family history is significant for multiple cancers. (Fig.1)." Fig. 1 Representative pedigree from a patient with germline pathogenic ATM and CHEK2 variants who was affected by early onset breast cancer and chronic lymphocytic leukemia. Arrow indicates proband. Colors indicate cancer type/disease: purple, breast cancer; blue, lymphoma; brown, melanoma; yellow, colon cancer; and green, autoimmune disease.

The therapy for early relapsed chronic lymphocytic leukemia: the development of the problem
 The results of the project with the discussion concerning the possibility of optimization approaches for the therapy

The Russian Oncohematology Society with the support of AbbVie completed the project that allows Russian hematologists to discuss the possibilities of optimization approaches for the therapy for early relapsed chronic lymphocytic leukemia, during 2021. The consolidated opinion of the specialists for several clinical situations was formulated at the final meeting.

Comparison of four prognostic scoring system in patients with early asymptomatic chronic lymphocytic leukemia patients

目的比较四种预后评分系统预测Binet A期中国慢性淋巴细胞白血病（CLL）患者诊断到治疗时间（TTFT）的预后评估价值。方法回顾性分析南京医科大学第一附属医院（浦口慢淋中心）2009年6月至2020年1月诊断的110例Binet A期CLL患者的基线临床资料，采用无症状早期CLL国际预后评分（IPS-E）、CLL国际预后指数评分（CLL-IPI）、CLL1预后评分模型（CLL1-PM）与巴塞罗那预后评分（Barcelona-Brno）模型对患者进行危险度分层和预后评估。结果110例Binet A期CLL患者中位年龄58（25～84）岁，中位随访时间35（4～189）个月，其中57例（51.8％）患者因病情进展达到治疗指征启动治疗。对患者年龄、Rai分期、淋巴细胞绝对计数（ALC）、淋巴结大小、淋巴细胞倍增时间（LDT）、β2-微球蛋白、IGHV突变状态、TP53缺失和（或）突变、11q缺失等9个因素进行Log-rank检验，其中RaiⅠ～Ⅲ期、ALC>15×109/L、淋巴结≥1 cm、β2-微球蛋白>3.5 mg/L、IGHV无突变、TP53缺失和（或）突变、11q缺失是影响TTFT的独立危险因素。使用一致性指数（C-index）和赤池信息准则（AIC）对上述模型进行预后评估效能评价，其中CLL1-PM C-index＝0.736，AIC＝777；CLL-IPI C-index＝0.722，AIC＝933；IPS-E C-index＝0.683，AIC＝1004；Barcelona-Brno C-index＝0.663，AIC＝986。结论四种预后评分模型均具有预测TTFT的效能。IPS-E因纳入指标的临床可及性高，价格较低，可作为指导临床检测的理想工具。对于完善FISH及二代测序检查的患者，使用CLL-IPI或CLL1-PM可进行更为全面的预后评价。

High Prevalence of Multiple B Cell Clones in Patients with Chronic Lymphocytic Leukemia

INTRODUCTIONStudies suggest that the first clonal events in chronic lymphocytic leukemia (CLL) may be acquired at hematopoietic stem cell stage. Consistently, it has been reported that CD34+cells from CLL patients engrafted in immunodeficient mice give rise to clonal B cell lymphoproliferation while CD34+ cells from healthy donors did not (Kikushige, Cancer Cell 2011). Furthermore, in rare patients with early CLL or monoclonal B cell lymphocytosis two clonal populations can be demonstrated. We hypothesized that immunofixation electrophoresis could serve as a sensitive tool to assess the prevalence of a second, clonally distinct, lymphoproliferative event in CLL patients. METHODSWe studied 262 treatment-naïve patients with a diagnosis of chronic lymphocytic leukemia (CLL) enrolled in our natural history study (NCT00923507). All patients had diagnostic peripheral blood flow cytometry testing (Laboratory of Pathology, NCI) and serum immunofixation electrophoresis (Department of Laboratory Medicine, Clinical Center, NIH) done. RESULTSOf the 262 patients, 148 (56.49%) were male and 114 (43.51%) were female. The median age of the study population was 65 years. Amongst the 262 patients, 243 (92.74%) were Caucasians, 8 (3.05%) were African Americans, 3 (1.14%) were Asians and 3 (1.14%) were multiracial. The race was undisclosed for 5 patients. Two of the 262 CLL patients had another B-cell malignancy on diagnostic workup, Waldenstrom's macroglobulinemia (Lymphoplasmacytic Lymphoma) and Hairy Cell Leukemia. Nine (3.44%) patients had two distinct clones of CLL by flow cytometry. In 4 both kappa and lambda clones were present. In 5, the two clones expressed the same light chains but had a distinct immunophenotype resulting in bi-phenotypic clones. By serum IFE, 62 (23.66%) of 262 patients had monoclonal gammopathy; in 44 (16.8%) patients one monoclonal band was detected, while 18 (6.9%) patients had 2 or more bands. Among patients with one monoclonal band 25 were kappa and 19 lambda light chain. In 19 of 25 patients with kappa clonal CLL by flow cytometry, the monoclonal band on IFE was kappa (concordant flow and IFE): IgM Kappa (in 8), IgG Kappa (10), and IgA Kappa (1); and in 6 patients with kappa clonal CLL the monoclonal band was lambda (discordant flow and IFE): IgM Lambda (in 2), and IgG Lambda (4). In 15 of 19 patients with lambda clonal CLL, flow cytometry and IFE were concordant: IgM Lambda (in 6), IgG Lambda (6), IgA Lambda (1), free lambda light chain (2), and discordant in 4: IgM Kappa (in 2), IgG Kappa (2). Amongst the 18 patients with 2 or more bands on IFE, only 5 patients had concordant flow cytometry and IFE, while 13 had discordant findings. In summary, using IFE we detected a second clonal event in 23 (8.8%) of 262 CLL patients. Considering the 11 patients with two distinct clones by flow cytometry, 34 (13%) of 262 treatment-naive CLL patients had at least one additional clonal B cell process. In addition, 18 patients with concordant flow and IFE findings had IgG or IgA type gammopathy, consistent with a concurrent plasma cell dyscrasia of the same light chain restriction as the CLL clone. CONCLUSIONIn 13% of CLL patients we found evidence for a second B cell lymphoproliferative process. The prevalence of biclonal CLL by flow cytometry at 3.4% is comparable to findings by others, reporting between 0.7% and 3.4%. In contrast, the prevalence of monoclonal gammopathy (23.7%) in our patients (median age 65) was much higher than reported in the general population: 3.2 % at age 50 and above, and 5.3% at age 70 and above (Kyle, NEJM 2006). Importantly, in 8.8% of all patients the monoclonal gammopathy was discordant with the CLL clone. These data support the existence of a genetic event in an early progenitor cell that predisposes to the development of clonal B cell expansions.This research was supported by the Intramural Research Program of NHLBI and NCI. DisclosuresWiestner:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding.

A Randomized Phase 2 Study Comparing Acalabrutinib with or without Obinutuzumab in the Treatment of Early Stage High Risk Patients with Chronic Lymphocytic Leukemia (CLL) or Small Lymphocytic Lymphoma (SLL)

A Randomized Phase 2 Study Comparing Acalabrutinib with or without Obinutuzumab in the Treatment of Early Stage High Risk Patients with Chronic Lymphocytic Leukemia (CLL) or Small Lymphocytic Lymphoma (SLL)

Association of gene mutations with time-to-first treatment in 384 treatment-naive chronic lymphocytic leukaemia patients.

This study correlated somatic mutation results and known prognostic factors with time-to-first treatment (TTFT) in 384 treatment-naïve (TN) chronic lymphocytic leukaemia (CLL) patients to help determine disease-specific drivers of early untreated CLL. CLL DNA from either peripheral blood or bone marrow underwent next generation targeted sequencing with a 29-gene panel. Gene mutation data and concurrent clinical characteristics, such as Rai/Binet stage, fluorescence in situ hybridisation (FISH), ZAP70/CD38, karyotype and IGHV mutation, status were analysed in univariable and multivariable analyses to identify associations with TTFT. TTFT was defined as time from diagnosis to initial treatment. In univariable analyses, mutated ATM (P<0·001), NOTCH1 (P<0·001) and SF3B1 (P=0·002) as well as unmutated IGHV (P<0·001), del(11q) (P<0·001) and trisomy 12 (P<0·001) by hierarchal FISH and advanced Rai (P=0·05) and Binet (P<0·001) stages were associated with shorter TTFT. Importantly, del(17p), mutated TP53 and complex karyotype were not associated with shorter TTFT. In a reduced multivariable analysis, mutated ATM (P<0·001) and unmutated IGHV status (P<0·001) remained significant, showing their importance in early leukaemogenesis. High-risk prognostic markers such as del(17p), mutated TP53 and complex karyotype, were not correlated with TTFT, suggesting that these abnormalities have limited roles in early disease progression but are more important in relapsed CLL.

Role of long non-coding RNAs in disease progression of early stage unmutated chronic lymphocytic leukemia.

Predicting disease progression in chronic lymphocytic leukemia (CLL) remains challenging particularly in patients with Rai Stage 0/I disease that have an unmutated immunoglobulin heavy chain variable region (UM IGHV). Even though patients with UM IGHV have a poor prognosis and generally require earlier treatment, not all UM IGHV patients experience more rapid disease progression with some remaining treatment free for many years. This observation suggests biologic characteristics other than known prognostic factors influence disease progression. Alterations in long non-coding RNA (lncRNA) expression levels have been implicated in diagnosis and prognosis of various cancers, however, their role in disease progression of early Rai stage UM CLL is unknown. Here we use microarray analysis to compare lncRNA and mRNA profiles of Rai 0/I UM IGHV patients who progressed in <2 years relative to patients who had not progressed for >5 years. Over 1,300 lncRNAs and 940 mRNAs were differentially expressed (fold change ≥ 2.0; p-value ≤ 0.05). Of interest, the differentially expressed lncRNAs T204050, NR_002947, and uc.436+, have known associated genes that have been linked to CLL. Thus, our study reveals differentially expressed lncRNAs in progressive early stage CLL requiring therapy versus indolent early Rai stage UM CLL. These lncRNAs have the potential to impact relevant biological processes and pathways that influence clinical outcome in CLL.

Functional Activation of Osteoclast Commitment in Chronic Lymphocytic Leukaemia: a Possible Role for RANK/RANKL Pathway

Skeletal erosion has been found to represent an independent prognostic indicator in patients with advanced stages of chronic lymphocytic leukaemia (CLL). Whether this phenomenon also occurs in early CLL phases and its underlying mechanisms have yet to be fully elucidated. In this study, we prospectively enrolled 36 consecutive treatment-naïve patients to analyse skeletal structure and bone marrow distribution using a computational approach to PET/CT images. This evaluation was combined with the analysis of RANK/RANKL loop activation in the leukemic clone, given recent reports on its role in CLL progression. Bone erosion was particularly evident in long bone shafts, progressively increased from Binet stage A to Binet stage C, and was correlated with both local expansion of metabolically active bone marrow documented by FDG uptake and with the number of RANKL + cells present in the circulating blood. In immune-deficient NOD/Shi-scid, γcnull (NSG) mice, administration of CLL cells caused an appreciable compact bone erosion that was prevented by Denosumab. CLL cell proliferation in vitro correlated with RANK expression and was impaired by Denosumab-mediated disruption of the RANK/RANKL loop. This study suggests an interaction between CLL cells and stromal elements able to simultaneously impair bone structure and increase proliferating potential of leukemic clone.

Clinical and serological autoimmune complications in chronic lymphocytic leukemia.

Autoimmune disorders often develop during the course of chronic lymphocytic leukemia (CLL). The aim of our study was to investigate the incidence of autoimmune complications (AIC) and serological autoantibodies, and to assess the relationship of these to patient characteristics. We prospectively collected screenings of AIC and serological markers from atotal of 192patients. AIC was observed in 18(9.4%) patients. Autoimmune hemolytic anemia (AIHA) was observed in 8patients. Autoimmune thrombocytopenia (AITP) was observed in 3patients. Other various types of AIC were observed in the remaining 7patients. Serological autoantibodies were positive in 17.2% of patients with CLL. The mean age of patients with AIC was higher than the control group (p= 0.036). Patients with AIC were mostly in advanced disease stage (p= 0.004), and they had received more first-line treatments than the control group (p= 0.003). Patients with AIC had ahigher mean age and more advanced disease stage than patients with positive serological autoantibodies (p= 0.020 and p= 0.009; respectively). In addition, patients with AIC had also received more first-line treatment than the patients with positive serological autoantibodies (p= 0.015). Hematologic AIC was associated with older age, advanced disease stage, and treatment. Conversely, non-hematological AIC and serological autoantibodies are generally observed in early stages. Our study has established acoexistence of CLL and autoimmune complications. Hematologists are usually familiar with AIHA and AITP, but less so with non-hematologic AIC. The latter complications should be carefully searched for, particularly in patients with early CLL.

Role of Lncrnas in Early Stage Immunoglobulin Heavy Chain Variable Region (IGHV) Unmutated CLL Disease Progression

BACKGROUND: Chronic lymphocytic leukemia (CLL) remains a heterogeneous disease despite significant advances in disease classification based upon cellular and molecular markers. Rai stage, immunoglobulin heavy chain variable region mutation status (IGHV), cytogenetic abnormalities, beta 2 microglobulin levels, expression of ZAP-70, CD38 and CD49d provide useful guides alone or in prognostic models for progression risk assessment but variability in clinical outcome in early Rai stage (Rai 0/I) unmutated (UM) IGHV patients persists. This variability in clinical outcome suggests that there are yet-to-be defined biologic/genetic factors that contribute to disease heterogeneity. Long non-coding RNAs (lncRNAs) display a range of diverse activities including regulation of gene transcription, post translational regulation and epigenetic regulation and represent an intriguing prospect for influencing CLL disease progression. LncRNAs have been implicated in diagnosis, prognosis and therapy of various cancers however their role in influencing disease progression in early Rai stage UM CLL has not yet been studied.Methods: To investigate a role for lncRNAs in UM Rai 0/I CLL patients we employed the Arraystar Human lncRNA Microarray v4.0. Using blood samples collected within 3 months of diagnosis, two cohorts of UM Rai 0/I patients were compared: one defined as progressive disease with time to first treatment (TTT) ≤ 2 years after diagnosis (n=34) and one as indolent disease with TTT ≥5 years after diagnosis (n=29). Differentially expressed lncRNAs were identified using Agilent GeneSpring GX Software v12.1. To be considered for further analysis, the difference in lncRNA expression had to have a fold change (FC) ≥ 2.0, a t-test p-value ≤ 0.05 and a false discovery rate (FDR) ≤ 0.05.Results: Over 1100 lncRNAs were found to be differentially expressed between the progressive and indolent UM CLL cohorts. Greater than 150 of these lncRNAs have known associated genes and are well annotated and validated. Among the differentially expressed lncRNAs, several are of particular interest and potentially relevant to CLL disease biology. For example, the lncRNA AL833181 was overexpressed in the progressive group. The associated gene for AL833181 is BCL11A and encodes a zinc finger protein that interacts with BCL6 and may itself serve as a proto-oncogene. Translocations involving BCL11A have been shown to identify a very aggressive subset of CLL (Am J Pathology, 2009). Therefore it is possible this lncRNA may be playing a role in deregulated expression of BCL11A.Within the indolent disease group, CTD-2566J3.1 was overexpressed >10-fold as compared to the progressive disease group. Its associated gene RAD51B is essential for DNA repair, suggesting the possibility that CTD-2566J3 may protect CLL cells from further DNA damage and disease progression. Also overexpressed in the indolent disease group were 2 lncRNAs that are associated with genes that encode proteins involved in the ubiquitin-proteasome system. USP2-AS1 lncRNA is associated with the USP2 gene and encodes an ubiquitin-specific protease required for TNF-alpha induced NF-kB signaling and is a specific deubiquitinase for cyclin D1, a known proto-oncogene. Furthermore, USP2-AS1 may also be linked to MYC. RP11-522I20.3 is associated with the QBLN1gene which encodes a protein that mediates the proteasomal degradation of misfolded or accumulated proteins. Thus, the associated lncRNAs may be acting as post-transcriptional gene silencers.Although CLL is not viewed as an "invasive" cancer, there were 5 lncRNAs overexpressed in the indolent group that are associated with Ankyrin genes which play a role in cell motility, activation and proliferation along with the lncRNA RP11-477D19 that is associated with theTIAM2 gene. TIAM2is a Rac guanine nucleotide exchange factor that can promote invasion and motility of cells. The respective lncRNAs may be down-regulating these genes and prohibiting malignant cell expansion.Conclusions: Our study reveals that there are indeed specific lncRNAs that are expressed at different levels in progressive versus indolent early Rai stage UM CLL and have the potential to impact a number of relevant biological processes and pathways. While these data are preliminary, the lncRNA AL833181 and its associated gene BCL11A may prove to be a potential marker and therapeutic target for aggressive disease. DisclosuresShanafelt:Pharmacyclics: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; GlaxoSmithkKine: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Hospira: Research Funding.

Transcriptomic analysis of monocytes and macrophages derived from CLL patients which display differing abilities to respond to therapeutic antibody immune complexes.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia. While therapeutic antibodies show clinical activity in CLL patients, resistance inevitably develops resulting in treatment failure. Identifying mechanisms of antibody resistance and methods to reduce resistance would be valuable in managing CLL. Monocyte derived cells (MDCs), also known as nurse like cells (NLCs) in CLL [1], [2], are known to be crucial components of the CLL microenvironment network and following “maturation” in in vitro culture systems are able to provide support for the survival of the malignant B cells from CLL patients. In addition to their protective role, MDCs are key effector cells in mediating responses to therapeutic antibody therapies [3]. We have determined that macrophages from patients with early stable CLL are able to elicit superior cytotoxic response to therapeutic antibodies than macrophages derived from patients with progressive CLL. We have exploited this unique finding to gain insight into antibody resistance. Thus, we have profiled monocytes on day 0 and MDCs on day 7 from antibody sensitive and antibody resistant CLL patients (GEO accession number GEO: GSE71409). We show that there are no significant differences in transcriptomes from the monocytes or MDCs derived from sensitive or resistant patient samples. However, we show that MDCs acquire an M2-like macrophage transcriptomic signature following 7 days culture regardless of whether they were derived from sensitive or resistant patient samples.

Exclusion of SERUM Thymidine Kinase Does NOT Reduce the Predictive VALUE of a Comprehensive Prognostic INDEX in Chonic Lymphocytic Leukemia. Results of an External Validation on a Prospective Cohort of Patients with EARLY Disease

▪A comprehensive prognostic index that includes clinical (i.e., age, sex, ECOG performance status), serum (i.e., B2-microglobulin, thymidine kinase) and molecular (i.e., IGVH mutational status, del 17p, del 11q) markers with independent prognostic value and combines them into a single risk score was recently developed by the German CLL Study Group (GCLLSG).We sought to externally validate this novel score in an independent, community-based cohort consisting of 338 patients with early chronic lymphocytic leukemia (CLL) who were registered on a prospective basis during 2006 to 2010 on an observational Italian database (O-CLL1 protocol; clinicaltrial.gov identifier NCT00917540).Because serum thymidine kinase was not available a slightly modified version of the German model based on 6 instead of 7 prognostic variables was used.According to clinical caracteristics of our patients (i.e., all with Binet stage A disease), a point score higher than 5, necessary for inclusion in the high-risk group, was found only in 2 cases. By German prognostic index, 62.9% of patients were scored as having low-risk CLL (score 0-2) whereas 37.1% had intermediate-risk CLL (score 3-5). This stratification translated into a significant difference in the time to first treatment (TTFT). In other words, the higher the score risk the shorter the TTFT [HR = 4.21; 95% C.I. (2.71-6.53) P<0.0001; (Fig 1)]. Interestingly, the prognostic index category remained a predictor of TTFT also when the analysis was restricted to 262 patients with Rai stage 0 disease [HR= 3.12; 95% C.I. (1.84-5.31);P <0.0001]The model proposed in 2007 by investigators at MD Anderson Cancer Center (MDACC) and barely based on traditional clinical parameters (i.e., age, gender, Rai substage, absolute lymphocyte count, number of involved nodal groups and B2-microglobulin) worked in predicting the TTFT [HR = 2,73; 95% C.I. (1.79-4.17); P<0.0001] in our patient cohort. In comparison to the MDACC score the German model provided similar results in terms of sensitivity (German score, 67%; MDACC score, 58.2; P = 0.28). However, both specificity (74% vs 65.4%; P=0.05) and accuracy (72.1% vs 63.5%; P=0.02) were significantly better with the German score.In order to investigate which prognostic model (i.e. German or MDACC score) provided a more accurate prediction of TTFT, a C-statistic analysis, considered a measure of concordance between observed and predicted time-dependent events, was performed. Interestingly, the C-statistic of the German score was 0.71 (range, 0.60-0.82), a value higher than c-statistic observed with MDACC score (c=0.65; range,0.53-0.78).In conclusion, we demonstrated that the German score works in predicting the TTFT of patients with early CLL also when a modified version which does not include serum thymidine kinase is utilized. These changes affected only partially the predictive value of model because C-statistic remained above 0.70, which is considered a necessary threshold of utility at the individual patient level. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tracing the roots of cancer evolution.

By comparing the genomes of progenitor cells and mature cells of lymphoid and myeloid lineages in patients with chronic lymphocytic leukemia (CLL), Damm and colleagues confirmed that CLL originates from preleukemic CD34(+) progenitor cells and identified early CLL mutations that are associated with these progenitor cells. Moreover, they discovered that deregulation of B-cell receptor signaling may be one of the hallmarks of CLL, particularly in tumors with EGR2 mutations.

Changes in the incidence, pattern of presentation and clinical outcome of early chronic lymphocytic leukemia patients using the 2008 International Workshop on CLL guidelines

Objectives: Before 2008, diagnosis of chronic lymphocytic leukemia (CLL) relied on 1996 National Cancer Institute Working Group criteria which required an absolute lymphocyte count of 5.0 × 109/l or higher. The up-dated 2008 International Workshop on CLL (IWCLL) guidelines recommend using the B-cell count as a basis for the diagnosis of CLL and indicate a B-cell threshold of 5.0 × 109/l. The objective of this study was to assess the effects of these changes on the pattern of presentation and clinical outcome of early CLL. Methods: For this purpose we analysed 414 patients with previously untreated, Binet stage A CLL diagnosed between January 2006 and December 2010 and registered prospectively at a national database. Results: After patients were reclassified according to updated 2008 IWCLL guidelines, 130 Rai stage 0 CLL patients were reclassifed as clinical monoclonal B-cell lymphocytosis, resulting in a 31.4% decrease (from 414 to 284) in the diagnosis of early stage CLL. Moreover, a shift towards a more advanced Rai clinical stage under 2008 IWCLL criteria was observed. Finally, the estimated 3-year time to first treatment decreased from 77.2 to 69.9% according to whether 1996 National Cancer Institute Working Group criteria or 2008 IWCLL criteria were used. Conclusion: In conclusion, the 2008 IWCLL guidelines modify the distribution of Rai stage at diagnosis and shorten the time to first treatment in early CLL patients. We expect that these changes might be considered in the interpretation of epidemiologic studies in CLL and in designing clinical trials of early therapeutic intervention in patients with asymptomatic CLL.

The clinical significance of patients' sex in chronic lymphocytic leukemia.

We examined the prognostic influence of gender in chronic lymphocytic leukemia. Data from four randomized trials (involving 1821 patients) and three registration studies of stage-A disease (involving 1299 patients) were analyzed. Overall survival at 10 years was better for women than men in all trials (27% versus 15%; P=0.0001) and in the registration series (55% versus 43%; P<0.0001). More women than men in the trials were Binet stage A-progressive (26% versus 15%), but gender was an independent predictor of survival in multivariate analysis of clinical variables (P<0.0001). Women responded better to treatment (overall response 83%) than men (71%; P<0.0001), within each stage and age group, although fewer women than men received the full treatment dose (79% versus 85%; P=0.01). Women were more likely than men to experience toxicity (85% versus 78%, P=0.01), particularly gastro-intestinal toxicity (57% versus 42%, P<0.0001). Laboratory markers in the LRF CLL4 trial showed a significantly lower incidence in women than men of unmutated IGHV genes, raised beta-2 microglobulin, CD38 and Zap-70 positivity and TP53 deletions/mutations and/or 11q deletions. We also highlight the higher male:female ratios in randomized trials versus studies of early chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis. Chronic lymphocytic leukemia in women runs a more benign clinical course than in men. Gender was also an independent predictor of response, suggesting that pharmacokinetic differences between the sexes and a possible effect of estrogens may contribute to the better outcome. Understanding the reasons for the different outcome by gender may improve patients' management. (LRF CLL4 controlled-trials.com identifier: ISRCTN58585610).

Alterations Of The Chemokine Microenvironment In Chronic Lymphocytic Leukemia

BackgroundIn chronic lymphocytic leukemia (CLL), proliferation of the leukemic cell clone occurs in the bone marrow and lymphatic tissues rather than peripheral blood. In this microenvironment, CLL cells interact with accessory cells, such as T-cells and CD68+ nurselike cells (NLCs). In addition, cytokines and chemokines secreted by leukemic cells, stromal cells and T-cells are essential in forming the disease-specific microenvironment. However, the exact biological role of cytokines and chemokines needs to be defined. MethodsIn order to address this issue, we measured serum levels of 20 chemokines and cytokines in a prospective cohort of 159 previously untreated Binet stage A patients (pts) (GCLLSG CLL1 trial), 50 pts with advanced CLL in need of first line treatment (GCLLSG CLL8 trial) and 27 healthy individuals. For the study cohorts, serum samples had been centrally collected at study entry and stored at -80°C. Sera were analyzed on a Luminex-based multiplex platform, allowing simultaneous screening of multiple serum parameters. ResultsSerum levels of 13 chemokines (EGF, MCP-1, MIP-1alpha, MIP-1beta, sIl2Ralpha, VEGF, MCP-2, MCP-4, SDF-1, 6CKine, CTACK, TRAIL, SCF) differed significantly between healthy controls and CLL patients (p<0.05), indicating that the expression of these cytokines may potentially impact pathways common to CLL and the disease specific microenvironment. In addition, 7 chemokine serum levels were significantly higher in advanced stage CLL compared to patients at primary diagnosis, indicating a pronounced shift of the chemokine homeostasis upon disease progression. (Table)TableMedian chemokine serum levels in healthy individuals and patients with early (CLL1 cohort) and advanced (CLL8 cohort) CLLMedian serum level (pg/ml)Healthy controlCLL1CLL8MIP-1beta116266196p<0.001sIl2Ralpha215042255p<0.001TNFalpha124080p=0.008Il-10319p=0.001IL-1655203425p=0.0136CKine319307764p<0.001SCF107955p=0.001We identified a strong correlation of inflammatory cytokines (TRAIL, TNFalpha; p=0.039), growth factors (EGF, VEGF, TPO, SCF; p<0.001) and chemokines involved in B- and T-cell migration and homing (MIP-1alpha, MIP-1beta, 6CKine, Il-16; p<0.05). These coordinately regulated cytokines may reflect pathways important to the pathobiology of CLL. Hierarchical cluster analyses revealed distinct chemokine expression patterns in healthy individuals, early CLL and advanced stage disease. Finally, sIl2Ralpha has been previously confirmed as an independent prognostic factor in early stage CLL ConclusionDue to the comparison of 27 healthy individuals and 209 CLL pts we were able to demonstrate that the chemokine homeostasis is significantly altered in CLL compared to healthy individuals. Chemokines involved in B- and T-cell migration and homing and recruitment, inflammation and growth factors are coordinately overexpressed, pointing towards pathways crucial to the formation of a disease specific microenvironment. These pathways might serve as potential targets for future therapeutic strategies. Disclosures:No relevant conflicts of interest to declare.

Early Versus Deferred Treatment With Combined Fludarabine, Cyclophosphamide and Rituximab (FCR) Improves Event-Free Survival In Patients With High-Risk Binet Stage A Chronic Lymphocytic Leukemia – First Results Of a Randomized German-French Cooperative Phase III Trial

▪ IntroductionPatients with asymptomatic early Rai or Binet stage chronic lymphocytic leukemia (CLL) do not benefit from mono-chemotherapy. Therefore, clinical observation without treatment (watch&wait; W&W) has been the gold standard for the management of these patients. Chemoimmunotherapy with FCR improves the outcome of patients with advanced CLL, but its efficacy in early stage disease has not been investigated. Several clinical and biological variables identify those patients who have a high risk of an aggressive disease course and who might benefit from early interventions. Consequently, this trial was conducted to test the value of FCR treatment in patients with early stage, high-risk CLL. MethodsThis report represents the endpoint and safety analysis of a randomized German-French cooperative phase III trial comparing the efficacy of early versus deferred FCR therapy in treatment-naïve Binet stage A CLL patients with a high risk of disease progression. Risk assessment was performed using 4 prognostic markers: Lymphocyte doubling time <12 months, serum thymidine kinase >10 U/L, an unmutated immunoglobulin heavy chain variable region gene (IGHV) status, and presence of unfavorable cytogenetics (del11q, del17p, trisomy 12) by fluorescence-in-situ hybridization. Presence of at least 2 versus less than 2 of these factors defined “high-risk” versus “low-risk” CLL. High-risk CLL patients were further randomized to receive either 6 cycles FCR (HR-FCR) or to be followed by a W&W strategy (HR-W&W). Patients with low-risk CLL were observed only (LR-W&W). ResultsBetween 2005 and 2010, a total of 824 patients was enrolled, 423 patients in 69 centers of the German CLL Study Group and 401 patients in 25 centers of the French Cooperative Group on CLL. The diagnosis of CLL needed to be established no longer than 12 months prior to enrollment and patients were required to present with previously untreated stage Binet A CLL at the time of inclusion. Overall, 800 patients (97.1%) were stratified, 201 of them categorized as high-risk CLL (25.1%). There was no significant difference between high-risk patients from the two study groups regarding common baseline characteristics (e.g., age, sex, comorbidity, immunophenotype) and the distribution of risk factors used for stratification.100 out of 201 high-risk patients were randomized to receive FCR therapy (HR-FCR), while 101 patients were allocated to the HR-W&W arm. 18 out of 100 patients (18%) withdrew consent for FCR therapy before treatment was started. 71 (86.6%) of 82 treated patients completed ≥4 cycles. The most common of 228 CTC grade III/IV adverse events reported within 12 months after treatment initiation were hematotoxicity (73.2% of patients) and infections (19.5% of patients). Three patients (3.7%) developed fatal CTC grade V infections (2 septic bacteremias, 1 of them with pulmonary aspergillosis; 1 encephalitis). Out of 79 patients available for response assessment until month 12 after treatment start, 76 showed a complete or partial remission (ORR 96.2%), 2 patients had stable disease (2.5%) and 1 patient had progressed (1.3%). After a median follow up of 46 months (range 0-88 months), HR-FCR patients demonstrated a significantly improved event-free survival (EFS) compared to HR-W&W patients (median EFS not reached versus 24.5 months, respectively, P<0.0001, Fig. 1). Overall survival was not significantly different between HR-FCR and HR-W&W with 181 high-risk patients (90%) being alive at last follow up. Both, HR-FCR and HR-W&W patients exhibited a significant shorter event-free and overall survival than LR-W&W patients, demonstrating an efficient prognostic segregation of patients by the risk assessment used for this trial (analysis based on the German LR-W&W cohort only, complete German-French LR-W&W data will be presented at the meeting). [Display omitted] ConclusionThis is the first randomized phase III trial investigating the efficacy of FCR chemoimmunotherapy in early stage CLL. So far, the study has revealed two major results: 1. A combination of clinical and biological factors can be used to identify early stage CLL patients who experience a rapid disease progression with unfavorable outcome, 2. FCR chemoimmunotherapy substantially improves event-free survival in early stage high-risk CLL. Disclosures:Langerbeins:Roche: travel grants Other. Cazin:roche: meeting invitation Other, Membership on an entity's Board of Directors or advisory committees; GSK: meeting invitation, meeting invitation Other, Membership on an entity's Board of Directors or advisory committees. Fischer:Mundipharma: Travel grants, Travel grants Other; Roche: Travel grants Other. Stilgenbauer:Roche: Consultancy, Research Funding, Travel grants Other; Mundipharma: Consultancy, Research Funding. Leblond:Roche : Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hallek:F. Hoffmann-La Roche: Consultancy, Honoraria, Research Funding.

The utility of two prognostic models for predicting time to first treatment in early chronic lymphocytic leukemia patients: Results of a comparative analysis

The use of both traditional and novel prognostic parameters combined in a statistical model for predicting patient clinical outcome has been recently proposed by both MD Anderson Cancer Center (MDACC) and German chronic lymphocytic leukemia (CLL) group. Using time to first treatment (TTFT) as end-point, we performed a comparative external validation of MDACC score versus a modified version of German score, which excluded thymidine kinase measurement, in a prospective, multicenter, community-based cohort consisting of 328 patients who had asymptomatic, early stage CLL. With both models a significant correlation between higher score and shorter TTFT could be found. As a matter of fact, patients with total point score ≥25 according to MDACC model (HR, 3.27; 95% CI, 2.07–5.18; P<0.0001) or ≥2 according to modified German model (HR, 2.02; 95% CI, 1.29–3.16; P=0.002) were more likely to receive therapy. Both models provided similar results in terms of sensitivity (MDACC score, 61.5%; modified German score, 57.7%; P=0.79), whereas specificity was significantly higher for MDACC score (72.1% versus 63%; P=0.02). The prognostic utility of either MDACC or modified German score was assessed by time-dependent Receiver Operating Characteristic (ROC) analysis. Results of this comparative analysis showed that after the 2nd year area under curve (AUC) for TTFT was higher than 0.60 for both models and kept unmodified this trend over the time. Results of this study suggest that in CLL both MDACC and modified German score group should be considered the benchmarking of comparison for any novel prognostic proposal having as endpoint TTFT in CLL and including both traditional and newer prognostic parameters.