Background Chronic Lymphocytic Leukemia Research Articles

SF3B1 Mutations Associate with Low CD20 Expression in CLL: Another NOTCH1-Dependent Mechanism?

BackgroundChronic lymphocytic leukemia (CLL) is characterized by a CD20 expression lower than other lymphoproliferative disorders, hampering treatment efficacy of anti-CD20 antibodies. Although most mechanisms behind this phenomenon are still obscure, we demonstrated that mutations of NOTCH1 associate with a lower CD20 expression, ascribed to a NOTCH1 mutation-driven repression of CD20 transcription by hystone deacetylases (Pozzo et al. Leukemia 2016). This may explain the association between NOTCH1 mutations and clinical resistance to anti-CD20 immunotherapy in CLL patients treated with FCR (Stilgenbauer et al. Blood 2014).SF3B1 mutations affect ~10% CLL cases at diagnosis; a recent characterization of their functional meaning highlighted transcriptome-wide alterations of splicing patterns affecting several pathways, including the NOTCH1 pathway (Wang et al. Cancer Cell 2016). In this context, one of the most aberrantly-spliced genes is DVL2, a key component of the Wnt pathway and negative regulator of the NOTCH1 pathway.AimTo investigate CD20 expression in SF3B1-mutated CLL and the correlation with a putative secondary activation of the NOTCH1 pathway through splicing alterations of the NOTCH1 negative regulator DVL2.MethodsMutational status of NOTCH1 and SF3B1 was evaluated by next generation sequencing. Mutations with VAF-variant allele frequency below 2% were discarded. CD20 expression was evaluated by flow cytometry on the CD19+5+ population. Percentage of low-CD20 expressing (CD20dim) population was defined as %P1-(100-%P1), P1 being a linear gate spanning from zero to CD20 mode. Transcript levels of MS4A1 (encoding for CD20 protein) and spliced DVL2 were evaluated by QRT-PCR in cases with at least 75% of CD19+5+ cells. Statistical analyses were performed using Mann-Whitney rank-test.ResultsIn a cohort of 537 unselected CLL cases, 93 cases (17.3%) carried NOTCH1 mutations: 62 delCT, 20 other truncating, 11 3′UTR (VAF range 3-84%, mean 32%; NOTCH1-mut); 46 cases carried SF3B1 mutations (8.5%, VAF range 5-53%, mean 32%; SF3B1-mut), the hotspot surrounding K700 being the most frequent (50%) followed by the hotspot surrounding G742 (30%); 7 cases (1,3%) carried both NOTCH1 and SF3B1 mutations. As trisomy 12 CLL is characterized by increased expression of CD20 and mutation of SF3B1 co-occurred in only 5/121 cases, the trisomy 12 subset was excluded from further analyses. Confirming previous findings, NOTCH1-mut cases were characterized by a lower CD20 mean fluorescence intensity (MFI) compared to WT cases (p=0.0154). In agreement with the hypothesis of a more active NOTCH1 pathway, CD20 expression was found diminished also in those cases carrying SF3B1 mutations, compared to WT cases (p=0.0059).Since CD20 expression is highly variable between cases, spanning a 2/3-log range of fluorescence intensity, we evaluated the percentage of the CD20dim population, this quantity being independent from the actual MFI. This analysis confirmed that CLL cases with NOTCH1 or SF3B1 mutations show a greater proportion of CD20dim cells (NOTCH1-mut p<0.001, SF3B1-mut p<0.001, NOTCH1+SF3B1-mut p=0.0266; Figure 1a). Consistently, CD20 transcript expression was lower in both NOTCH1-mut (p=0.0021) and SF3B1-mut (p=0.0197) cases.Presence of the alternatively spliced isoform of DVL2 (altDVL2) was identified only in those CLL cases with SF3B1 mutations (p<0.001), including the few cases bearing also NOTCH1 mutations (6 cases; Figure 1b). We performed cell sorting experiments on clonal SF3B1-mut cases (VAF>35%) to isolate CD20high and CD20low populations. MS4A1 expression (higher in the CD20high subset) inversely correlated with altDVL2 expression (Figure 1c).As DVL2 can act as negative regulator of NOTCH1, we followed the hypothesis that loss of WT DVL2 due to alternative splicing may result in a more active NOTCH1 pathway. To simulate loss of DVL2 in the SF3B1-wild type CLL-like MEC1 cell line, cells were trasfected with siRNA for DVL2 itself and screened for CD20 expression. After 24 hours from transfection, CD20 expression was found downregulated at both protein (p=0.0062; Figure 1d) and transcript (p=0.0129) level.ConclusionsIn addition to NOTCH1-mut cases, also CLL cases bearing SF3B1 mutations are characterized by a lower CD20 expression, allegedly through a more active NOTCH1 pathway, potentially resulting in clinical resistance to anti-CD20 monoclonal antibodies. [Display omitted] DisclosuresZaja:Amgen: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria; Sandoz: Honoraria; Abbvie: Honoraria.

Detecting Clonal Evolution in CLL Patients Treated with Targeted Therapies Versus Chemotherapy Utilizing Cytogenetic Microarray Analysis

BackgroundChronic lymphocytic leukemia (CLL) is a common B-cell lymphoproliferative neoplasm. Although not required for the diagnosis of CLL, important prognostic information is obtained with fluorescence in-situ hybridization (FISH) testing for chromosomal aberrations including del(11q), trisomy 12, del(13q), and del(17p). Abnormalities involving TP53, the ATM gene, and cases constituting complex cytogenetics (3 or more abnormalities), among others, have been shown to impact patient survival and response to traditional chemoimmunotherapy (CIT). Cytogenetic microarray analysis (CMA) is a laboratory technique used to detect unbalanced chromosomal abnormalities. CMA allows for comparison of hundreds of discrete loci within a chromosome and has been validated in CLL, with greater sensitivity in detecting genomic alterations compared to FISH. The acquisition of new genomic alterations is termed “clonal evolution” and has been shown to have a detrimental effect on survival in patients with CLL (Shanafelt TD, et al. J Clin Oncol. 2006). CIT results in a higher proportion of complete responses (CR) and eradication of CLL clones compared to targeted therapies. While targeted therapies may successfully suppress CLL clones, a CR is rare with single-agent treatment (Byrd JC, et al. Blood. 2015). CMA may be a valuable tool in monitoring clonal evolution throughout a patient's disease course and may have a role in evaluation of patients on suppressive therapies due to the relatively high risk of clonal emergence.MethodsThis was a single-center retrospective study. Patients with pathologically-confirmed CLL were analyzed utilizing data obtained from the electronic medical record. Data was extracted from the CMA reports and clinic documentation. To be included in the study, at least two serial CMA evaluations were required, separated by at least four weeks. In the interval between CMA results, subsequent systemic treatment was recorded, if any. Treatments were categorized as ibrutinib/idelalisib (“I”), any cytotoxic chemotherapy (“C”), and no treatment/observation (“N”). Change in the number of abnormalities detected by CMA was calculated for each interval and evaluated as both continuous and categorical (increase/no change/decrease). Patients with over three abnormalities were deemed to have complex disease.Genomic DNA of bone marrow or peripheral blood was extracted using Qiagen's Gentra Puregene kit. Chromosome microarray assay was performed using ThermoFisher Affymetrix CytoScan HD according to the manufacturer's protocol. Copy number and allele analysis was performed using Affymetrix Chromosome Analysis Suite using the default settings.ResultsThere were 34 patients who met inclusion criteria, each with 2-4 CMA results, for a total of 81 CMA results. Forty-seven treatment intervals were analyzed. The median age was 61 years, and 64% of patients were male. Based on the first CMA result, the median number of detected abnormalities was 2 and mean was 2.6; 13 patients (38%) had complex disease. Complexity was not associated with a clinically significant change in the number of chromosomal abnormalities regardless of what treatment was received, if any (p=0.14).Overall, 18% of patients (6/34) had a decrease in the number of abnormalities detected by CMA, while 21% (7/34) had an increase. Analyzing the data by treatment group, the mean change in number of abnormalities was -0.5, -0.6 and +0.3, in groups I, C and N, respectively (p=0.26) (Figure 1). Among the patients in group I, 23% (3/13) had a decrease in the number of abnormalities, while 23% demonstrated an increase. For those in group C, 25% (2/8) and 25% experienced increase and decrease, respectively. For patients in group N, 14% (3/21) had an increase and 0% had a decrease in CMA abnormalities. Among the two patients who experienced Richter's transformation, the complexity of their chromosomal abnormalities increased over time and preceded clinical emergence of transformation.DiscussionCMA can sensitively identify the emergence or clearance of CLL clones and help detect clonal evolution throughout periods of treatment or observation. Though a relatively small number of patients had serial CMA data for evaluation, these data suggest that CMA should be prospectively analyzed in patients with CLL and may be used as a dynamic clinical tool to potentially modify treatments in patients with sub-clinical evidence of treatment failure. DisclosuresBarta:Janssen: Membership on an entity's Board of Directors or advisory committees; Merck, Takeda, Celgene, Seattle Genetics, Bayer: Research Funding.

Highly Comprehensive Genomic Testing for CLL: WGS, One Key to CLL Patient Stratification

BackgroundChronic Lymphocytic Leukemia (CLL) is characterised by a highly heterogeneous natural history and treatment response. Indeed, 50% of immunoglobulin heavy chain variable region (IgHV) hypermutated patients have an excellent progression free survival (PFS) after chemoimmunotherapy. Conversely, 25% of FCR treated patients relapse within 24 months (high risk CLL). Recent studies have shown that complex karyotype with or without TP53 disruption predicts for relapse after BCL2 therapy and BTK inhibitors. However, TP53 is the only marker for which routine testing is available. Overall, nearly 80% of patients relapsing after frontline FCR do not present a known poor risk genomic marker. Additional candidate genomic predictors of poor outcome including mutations in coding regions of NOTCH1, SF3B1 and RPS15, non-coding regions of NOTCH1 and enhancer regions of PAX5, telomere length, IgHV status, and DNA Damage Repair (DDR) germline mutations including TP53 and ATM have been reported in CLL. Further, the role of mutational signatures and regions of kataegis also merit additional investigation in progressive CLL. Evaluating all candidate predictors requires complex time consuming, multi-modality testing outside the scope of routine clinical diagnostic practice, however, in isolation, each has low predictive value. Here, we show preliminary data on a novel patient stratification method based on whole genome sequencing (WGS) data incorporating multiple genomic features in a single test.Patients and MethodsTumor (peripheral blood) and germline (saliva) samples were collected from 321 patients from 6 UK trials via the Genomics England CLL pilot: ARCTIC (n=61), AdMIRe (n=64), CLL 210 (n=30), CLEAR (n=12), RIAltO (n=88) and FLAIR (n=66). We performed WGS on the HiSeqX (Illumina). After read alignment, we detected somatic variants using Strelka 2.4.7 for small variants detection (SNV and InDels), Manta 0.28.0 for structural variant (SV) detection, and Canvas 1.3.1 for copy number variant (CNV) detection (Illumina). Non-coding regions were annotated with information from primary CLL, CLL cell lines and B-cell ENCODE databases. Mutational signatures and putative regions of kataegis were calculated based on Alexandrov et al. (Nature, 2013) and Lawrence et al. (Nature, 2013). Telomere lengths were assessed using Telomerecat. Data aggregation was performed using contingency tables combined with non-negative matrix factorization.ResultsMean coverage was 94.2X for tumor and 28.5X for germline samples. We found a median of 9172 SNPs/sample after filtering and 2348 indels/sample across 321 patients. High risk CLL was enriched for genomic complexity and poor prognostic mutations. The most frequently mutated genes were SF3B1 (17%), TP53 (13%), NOTCH1 (12%), IGLL5 (12%), and ATM (11%). Analysis of non-coding regions using DNA methylation markers, ATAC-seq and Hi-C revealed potential candidate regions associated with early relapse. Using CNA and SV data, we identified interesting patterns of genomic complexity and structural variants, including a trend towards enrichment of del8p in Relapse/Refractory and FCR non-responders. Additionally, we investigated mutation signatures and kataegis across coding and non-coding regions of the genome. We correlated exonic regions of DDR genes in germline data with clinical outcomes and extended this to genes mutated in both tumor and germline data, termed germline-tumor double-hits.We examined the relationship between the Alexandrov hypermutation signature, IgHV status (determined by % homology to the reference genome) and PFS, and combined mutational density at the Ig locus with mutation signature aiming to predict IgHV status.Finally, we produced a binary contingency matrix, using non-negative matrix factorization to cluster the samples. This method highlighted patient groups with shared genomic profiles.ConclusionWe present preliminary data on a patient stratification method derived from WGS of 321 paired germline and CLL trial samples. Our predictive signature includes driver gene mutations, CNAs, IgHV status, genomic complexity, telomere length, overall mutation burden and genes with germline-tumor double-hits. Our comprehensive, NGS-based patient stratification attempts to predict patient outcome in a single sequencing run. DisclosuresBecq:Illumina: Employment. He:Illumina: Employment. Ross:Illumina: Employment. Bentley:Illumina: Employment. Pettitt:Celgene: Research Funding; Gilead: Research Funding; Roche: Research Funding; GSK/Novartis: Research Funding; Napp: Research Funding; AstraZeneca: Research Funding; Chugai: Research Funding. Hillmen:Novartis: Research Funding; Gilead Sciences, Inc.: Honoraria, Research Funding; Alexion Pharmaceuticals, Inc: Consultancy, Honoraria; F. Hoffmann-La Roche Ltd: Research Funding; Celgene: Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Schuh:Giles, Roche, Janssen, AbbVie: Honoraria.

B-chronic lymphocytic leukemia showed triple transformation, to diffuse large B cell, CD20-negative, and T-cell neoplasm during ofatumumab treatment: a case report

BackgroundChronic lymphocytic leukemia (CLL) is a mature lymphoid neoplasm currently categorized as an indolent type of malignant lymphoma. CLL progresses slowly over years, but it eventually transforms to a more aggressive lymphoma such as the diffuse large B-cell (DLBCL) type, also known as Richter’s syndrome.Case presentationWe treated a 69-year-old Japanese male who was histologically diagnosed with Richter’s syndrome after 6 years of CLL. His lymphadenopathy had systemically progressed for years, with lymphocyte counts of less than 10,000 cells/μL and a disease status of Rai classification stage I and Binet classification B. He had high fever and hepatosplenomegaly upon Richter’s transformation. The patient was treated with ofatumumab for refractory CLL, which relieved his febrile lymphadenopathy. He received a total of 11 ofatumumab courses and achieved partial remission. On the day of the 12th course of ofatumumab, his disease relapsed with febrile lymphadenopathy. Computed tomography revealed multiple liver masses and systemic lymphadenopathy, while a liver biopsy confirmed T-cell lymphoma. Concomitantly, CD20-lacking CLL cells were detected in his peripheral blood and bone marrow, and pathological examination of his left cervical lymph node biopsy showed CD20-positive DLBCL. The final diagnosis was three different types of lymphoma pathologies: (1) CD20-positive DLBCL of the lymph nodes, (2) CD20-lacking CLL of the peripheral blood and bone marrow, and (3) peripheral T-cell lymphoma (PTCL) of the liver. He received intravenous and oral dexamethasone therapy as palliative care. He died because of the rapid progression of abdominal masses 2 months after the diagnosis of triple transformation CLL. An autopsy revealed aggressive PTCL with aggressive systemic involvement of the liver, spleen, gall bladder, pericardium, bone marrow, and mediastinal–paraaortic–intraceliac lymph nodes. T-cell receptor study of an autopsy specimen supported the diagnosis of PTCL that spread to the intraceliac organs and lymph nodes. We concluded that his pathogenicity progressed to a mixture of triple lymphoma as a result of double malignant transformations, which included PTCL from CLL, CD20-negative CLL, and CD20-positive DLBCL by Richter’s transformation.ConclusionsOur case provides information on the biology of CLL, to transform from a low-grade chemosensitive status to a malignant chemoresistant status.

Genomic characterization of chronic lymphocytic leukemia (CLL) in radiation-exposed Chornobyl cleanup workers

BackgroundChronic lymphocytic leukemia (CLL) was the predominant leukemia in a recent study of Chornobyl cleanup workers from Ukraine exposed to radiation (UR-CLL). Radiation risks of CLL significantly increased with increasing bone marrow radiation doses. Current analysis aimed to clarify whether the increased risks were due to radiation or to genetic mutations in the Ukrainian population.MethodsA detailed characterization of the genomic landscape was performed in a unique sample of 16 UR-CLL patients and age- and sex-matched unexposed general population Ukrainian-CLL (UN-CLL) and Western-CLL (W-CLL) patients (n = 28 and 100, respectively).ResultsMutations in telomere-maintenance pathway genes POT1 and ATM were more frequent in UR-CLL compared to UN-CLL and W-CLL (both p < 0.05). No significant enrichment in copy-number abnormalities at del13q14, del11q, del17p or trisomy12 was identified in UR-CLL compared to other groups. Type of work performed in the Chornobyl zone, age at exposure and at diagnosis, calendar time, and Rai stage were significant predictors of total genetic lesions (all p < 0.05). Tumor telomere length was significantly longer in UR-CLL than in UN-CLL (p = 0.009) and was associated with the POT1 mutation and survival.ConclusionsNo significant enrichment in copy-number abnormalities at CLL-associated genes was identified in UR-CLL compared to other groups. The novel associations between radiation exposure, telomere maintenance and CLL prognosis identified in this unique case series provide suggestive, though limited data and merit further investigation.

Genetic landscape of ultra-stable chronic lymphocytic leukemia patients

BackgroundChronic lymphocytic leukemia (CLL) has a heterogeneous clinical course. Beside patients requiring immediate treatment, others show an initial indolent phase followed by progression and others do not progress for decades. The latter two subgroups usually display mutated IGHV genes and a favorable FISH profile. Patients and methodsPatients with absence of disease progression for over 10years (10–34) from diagnosis were defined as ultra-stable CLL (US-CLL). Forty US-CLL underwent extensive characterization including whole exome sequencing (WES), ultra-deep sequencing and copy number aberration (CNA) analysis to define their unexplored genetic landscape. Microarray analysis, comparing US-CLL with non-US-CLL with similar immunogenetic features (mutated IGHV/favorable FISH), was also carried out to recognize US-CLL at diagnosis. ResultsWES was carried out in 20 US-CLL and 84 non-silent somatic mutations in 78 genes were found. When re-tested in a validation cohort of 20 further US-CLL, no recurrent lesion was identified. No clonal mutations of NOTCH1, BIRC3, SF3B1 and TP53 were found, including ATM and other potential progression driving mutations. CNA analysis identified 31 lesions, none with known poor prognostic impact. No novel recurrent lesion was identified: most cases showed no lesions (38%) or an isolated del(13q) (31%). The expression of 6 genes, selected from a gene expression profile analysis by microarray and quantified by droplet digital PCR on a cohort of 79 CLL (58 US-CLL and 21 non-US-CLL), allowed to build a decision-tree capable of recognizing at diagnosis US-CLL patients. ConclusionsThe genetic landscape of US-CLL is characterized by the absence of known unfavorable driver mutations/CNA and of novel recurrent genetic lesions. Among CLL patients with favorable immunogenetics, a decision-tree based on the expression of 6 genes may identify at diagnosis patients who are likely to maintain an indolent disease for decades.

Coexistence of chronic myeloid leukemia and diffuse large B-cell lymphoma with antecedent chronic lymphocytic leukemia: a case report and review of the literature

BackgroundChronic lymphocytic leukemia and chronic myeloid leukemia are the most common types of adult leukemia. However, it is rare for the same patient to suffer from both. Richter’s transformation to diffuse large B-cell lymphoma is frequently observed in chronic lymphocytic leukemia. Purine analog therapy and the presence of trisomy 12, and CCND1 gene rearrangement have been linked to increased risk of Richter’s transformation. The coexistence of chronic myeloid leukemia and diffuse large B-cell lymphoma in the same patient is extremely rare, with only nine reported cases. Here, we describe the first reported case of concurrent chronic myeloid leukemia and diffuse large B-cell lymphoma in a background of chronic lymphocytic leukemia.Case presentationA 60-year-old Saudi man known to have diabetes, hypertension, and chronic active hepatitis B was diagnosed as having Rai stage II chronic lymphocytic leukemia, with trisomy 12 and rearrangement of the CCND1 gene in December 2012. He required no therapy until January 2016 when he developed significant anemia, thrombocytopenia, and constitutional symptoms. He received six cycles of fludarabine, cyclophosphamide, and rituximab, after which he achieved complete remission.One month later, he presented with progressive leukocytosis (mostly neutrophilia) and splenomegaly. Fluorescence in situ hybridization from bone marrow aspirate was positive for translocation (9;22) and reverse transcription polymerase chain reaction detected BCR-ABL fusion gene consistent with chronic myeloid leukemia. He had no morphologic or immunophenotypic evidence of chronic lymphocytic leukemia at the time. Imatinib, a first-line tyrosine kinase inhibitor, was started. Eight months later, a screening imaging revealed new liver lesions, which were confirmed to be diffuse large B-cell lymphoma.ConclusionsIn chronic lymphocytic leukemia, progressive leukocytosis and splenomegaly caused by emerging chronic myeloid leukemia can be easily overlooked. It is unlikely that chronic myeloid leukemia arose as a result of clonal evolution secondary to fludarabine treatment given the very short interval after receiving fludarabine. It is also unlikely that imatinib contributed to the development of diffuse large B-cell lymphoma; rather, diffuse large B-cell lymphoma arose as a result of Richter’s transformation. Fludarabine, trisomy 12, and CCND1 gene rearrangement might have increased the risk of Richter’s transformation in this patient.

Relevance of CD49d and CD38 expressions as predictors of disease progression in chronic lymphocytic leukemia

Background Chronic lymphocytic leukemia is a heterogeneous disease characterized by a highly variable clinical course. The majority of cases do not require therapy and show identical survival rate with their chronic lymphocytic leukemia-free counterparts of the same age. However, other patients may suffer from an aggressive course of the disease with early need for therapy and shorter overall survival. Objective The aim of this study was to investigate the expression pattern and prognostic value of the adhesion molecules, CD49d and CD38, in chronic lymphocytic leukemia (CLL) patients. Moreover, we attempted to investigate their correlation with trisomy 12, which could help in the clarification of the distinctive clinical features of the trisomy 12+ CLL subset. Patients and methods Seventy newly diagnosed chronic lymphocytic leukemia patients and 50 age-matched and sex-matched controls were included in this study. Patients were subjected to full history taking, clinical examination and abdominal ultrasonography. Laboratory investigations included complete blood count, cytogenetic analysis for the presence of trisomy 12, and flow cytometric assessment. Results A significant positive correlation was detected between CD49d expression and CD38 expression. CD49d showed positive correlation with poor prognostic parameters that included organomegaly, high count of white blood cells, lymphocyte count and trisomy 12. CD38 showed a significant positive correlation with high white blood cell count, lymphocyte count, and splenomegaly. In addition, trisomy 12+ chronic lymphocytic leukemia patients showed significant higher lymphocyte count and splenomegaly. Conclusion Our data confirm the suggested role of CD38 and CD49d as predictors for poor clinical outcome of CLL patients. Moreover, trisomy 12+ CLL cells exhibit a high CD49d expression that may account for the unique clinical characteristics of this group.

Prognostic significance of BCL6 and KI67 in patients with chronic lymphocytic leukemia

Background Chronic lymphocytic leukemia (CLL) is a heterogeneous disease; its prognosis depends on the disease stage at diagnosis as well as the presence or absence of high-risk markers to determine the treatment strategy. Aim To evaluate the expression of BCL6 and Ki67 in patients with CLL and to assess their clinical prognostic significance in relation to other established prognostic markers such as Zeta-associated protein of 70-Kd (ZAP70) and β2 microglobulin (β2M). Participants and methods BCL6 and Ki67 were measured using flow cytometry in thirty newly diagnosed CLL patients who presented to the Hematology units in Alexandria Main University Hospital and the Medical Research Institute, Alexandria University, they were also measured in 20 healthy age-matched and sex-matched controls. Results A statistically significant difference was found between cases and controls for both BCL6 and Ki67 expressions (P Conclusion BCL6 expression and Ki67 expressions were positively correlated with the established prognostic markers (clinical staging, LDH, β2M, and ZAP70), so both of them may be considered as prognostic factors in patients with CLL.

Second lung malignancy and Richter syndrome in chronic lymphocytic leukemia: case report and literature review

BackgroundChronic lymphocytic leukemia (CLL) is the most frequent lymphoproliferative disease. Transformation into Richter disease and occurrence of second malignancies involving the lungs are rare complications. The hallmarks of any thoracic involvement are still unknown.Case presentationWe report a case of a 56-year-old male patient, with history of tobacco smoking, who presented with recurrent hemoptysis, fatigue and weight loss. Physical examination was normal except a slightly enlarged supraclavicular lymph node. Chest x-ray revealed a mediastinal widening due to enlarged paratracheal nodes and a left parahilar infiltrate. Blood tests showed a hyperlymphocytosis and a biological inflammatory syndrome. CT scan showed bilateral mediastinal and axillary lymphadenopathy, as well as left supraclavicular lymphadenopathy, with a left upper lobe alveolar attenuation and a solitary contralateral pulmonary nodule. Examination of Virchow’s node and bone marrow biopsies confirmed metastasis of a pulmonary adenocarcinoma, as well as chronic lymphocytic leukemia with Richter’s transformation. The clinical course was unfavorable since the first days of therapy as the patient passed away in a matter of a few days.ConclusionsSteady surveillance of CLL patients and systematic screening for second solid tumors, particularly lung cancer, and Richter’s transformation seem to be relevant more than ever. Early diagnosis might help us understand the pathways leading to these complications and adapt therapy.

Next-generation sequencing and FISH studies reveal the appearance of gene mutations and chromosomal abnormalities in hematopoietic progenitors in chronic lymphocytic leukemia

BackgroundChronic lymphocytic leukemia (CLL) is a highly genetically heterogeneous disease. Although CLL has been traditionally considered as a mature B cell leukemia, few independent studies have shown that the genetic alterations may appear in CD34+ hematopoietic progenitors. However, the presence of both chromosomal aberrations and gene mutations in CD34+ cells from the same patients has not been explored.MethodsAmplicon-based deep next-generation sequencing (NGS) studies were carried out in magnetically activated-cell-sorting separated CD19+ mature B lymphocytes and CD34+ hematopoietic progenitors (n = 56) to study the mutational status of TP53, NOTCH1, SF3B1, FBXW7, MYD88, and XPO1 genes. In addition, ultra-deep NGS was performed in a subset of seven patients to determine the presence of mutations in flow-sorted CD34+CD19− early hematopoietic progenitors. Fluorescence in situ hybridization (FISH) studies were performed in the CD34+ cells from nine patients of the cohort to examine the presence of cytogenetic abnormalities.ResultsNGS studies revealed a total of 28 mutations in 24 CLL patients. Interestingly, 15 of them also showed the same mutations in their corresponding whole population of CD34+ progenitors. The majority of NOTCH1 (7/9) and XPO1 (4/4) mutations presented a similar mutational burden in both cell fractions; by contrast, mutations of TP53 (2/2), FBXW7 (2/2), and SF3B1 (3/4) showed lower mutational allele frequencies, or even none, in the CD34+ cells compared with the CD19+ population. Ultra-deep NGS confirmed the presence of FBXW7, MYD88, NOTCH1, and XPO1 mutations in the subpopulation of CD34+CD19− early hematopoietic progenitors (6/7). Furthermore, FISH studies showed the presence of 11q and 13q deletions (2/2 and 3/5, respectively) in CD34+ progenitors but the absence of IGH cytogenetic alterations (0/2) in the CD34+ cells. Combining all the results from NGS and FISH, a model of the appearance and expansion of genetic alterations in CLL was derived, suggesting that most of the genetic events appear on the hematopoietic progenitors, although these mutations could induce the beginning of tumoral cell expansion at different stage of B cell differentiation.ConclusionsOur study showed the presence of both gene mutations and chromosomal abnormalities in early hematopoietic progenitor cells from CLL patients.

Chronic lymphocytic leukemia with clinical debut as neurological involvement: a rare phenomenon and the need for better predictive markers

BackgroundChronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries. The frequency of symptomatic central nervous system (CNS) involvement is unknown but thought to be a rare phenomenon. Currently there are no known risk factors for CNS involvement.Case presentationWe describe a clinically staged low-risk CLL case that presented with symptomatic CNS involvement and progressed rapidly to death. Evaluation of the surface adhesion molecules identified a markedly altered expression pattern of the integrin, CD49d, and the tetraspanin, CD82, in the index case when compared to similar low-risk CLL cases. We found that the early Rai clinical stage CLL patients showed linear correlation for the co-expression of CD82 and CD49d. In contrast, this unique index case with CNS involvement, which has the same Rai clinical stage, had a significantly lower expression of CD82 and higher expression of CD49d.ConclusionsThese data suggest that the expression profile of CD49d and CD82 may represent potential biomarkers for patients with increased propensity of CNS involvement. Moreover, this study illustrates the critical need for a better mechanistic understanding of how specific adhesion proteins regulate the interactions between CLL cells and various tissue sites.

CD73, a potential diagnostic marker in Egyptian patients with chronic lymphocytic leukemia

Objective This work aimed to study CD73 expression in chronic lymphocytic leukemia (CLL) patients and its correlation with other disease characteristics. Background CLL is a common B-lymphoproliferative disorder, which can be diagnosed in most cases by morphology and flowcytometry. It displays heterogeneity in clinical presentation, course, and response to treatment, which is likely due to the diversity of the biological nature of the disease. Diagnostic difficulty might arise in some patients due to atypical morphology and/or immunophenotype. CD73 is an extracellular enzyme that hydrolyses adenosine monophosphate to adenosine and is expressed on a subset of T and B lymphocytes. A number of studies have shown CD73 as a contributing factor in the multistep process of carcinogenesis by inducing favorable environment for tumor growth. However, it was shown in some studies to induce apoptosis. Patients and methods CD73 expression on both B and T lymphocytes was measured in 25 cases of newly diagnosed CLL and 15 healthy controls. The levels of expression were correlated to other disease characteristics. Results CD73 expression on CD19 + B lymphocytes was significantly lower in CLL patients compared with controls (mean 3.74 vs. 29.35%, respectively, P Conclusion CD73 expression is a potential diagnostic marker in CLL. This can be particularly relevant in cases with atypical immunophenotype using the current CLL scoring system. CD73 may have a role in CLL pathogenesis as low expression can lead to failure of termination of immune response with subsequent proliferation and clonal expansion of the involved clone.

Evaluation of Integrated CLL Scoring System (ICSS) in 420 Patients with Chronic Lymphocytic Leukemia

BACKGROUNDChronic Lymphocytic Leukemia (CLL) is one of the most common hematological malignancies in Western countries. The disease is characterized by heterogeneous clinical course and outcome. During the last 15 years several clinical, biological and molecular prognostic factors have been identified, validated and some of them are currently used in patients’ and treatment management. To improve the predictive accuracy of these markers, they have been combined into prognostic indexes (W. Wierda, JCO 2011, D. Rossi, Blood 2012, J. Bahlo, Haematologica 2015). Werecently proposed the Integrated CLL Scoring System (ICSS) based on cytogenetic abnormalities by FISH, IGHV mutational status and CD38 expression from 212 patients (A. Visentin et al, Clin Lymph Myeloma & Leuk 2015).The aim of this study was to validate the prognostic power of our index into a larger series of 420 CLL patients.METHODS420 CLL patients referred to the Hematology Unit of Padua University Hospital from 1989 to 2015 were recruited in this study. According to ICSS, patients were classified as: low-risk, those patients with 13q deletion or normal FISH, IGVH mutated and CD38<30%; high-risk, subjects with 17p or 11q deletion and/or IGVH unmutated and CD38>30%; intermediate-risk, all remaining patients. Treatment free survival (TFS) was calculated as time from diagnosis to treatment (event), death or last known follow-up (censored). Overall survival (OS) was calculated from the date of diagnosis to death for any cause (event) or last known follow-up (censored). TFS and OS were compared with log-rank test and plotted using Kaplan-Meier method. The predictive accuracy of ICSS was evaluated by the Harrel’s concordance index (c-index); a value >0.5 implies a good predictive ability.RESULTSThe median age of our cohort was 62 years; 64% were male and 85% were Binet stage A at diagnosis. Cytogenetic analysis by FISH showed that 41 patients harbored 17p deletion, 50 11q deletion, 236 13q deletion, 44 trisomy 12 and 49 had normal FISH. 236 (56%) patients had IGHV gene homology >98% (i.e. mutated IGHV) and 103 (25%) expressed more then 30% of CD38.According to ICSS 202 (48%) subjects were classified as low-risk, 83 (20%) intermediate-risk and 135 (32%) high-risk.After a median follow-up of 81 months, the median TFS for ICSS classes of risk were 211, 70 and 27 months (log-rank test, p<0.0001, Figure 1A). The estimated 10-year TFS were 61%, 37% and 10% for low, intermediate and high-risk patients. The median OS were 213 and 136 months for intermediate and high-risk, while it was not reached for low-risk patients (Log-rank test, p<0.0001, Figure 1B). After 10 years from diagnosis the estimated OS were 88%, 79% and 57%, respectively. These data were confirmed by a multivariate analyses. In fact, high-risk patients had 5.3 and 4.0 times risk of start treatment and death than low-risk subjects, respectively (p<0.0001). This model was statistically internally validated, showing c-indexed of 0.712 and 0.693 for TFS and OS, respectively.In multivariate analyses, variables confirmed to predict adverse prognosis were male gender (p=0.0183), age>65years (p<0.0001), Rai III-IV (p=0.0025), Binet C (p=0.0002), 17p deletion (p=0.0002), TP53 abnormalities (p=0.0051), unmutated IGVH (p<0.0001), CD38>30% (p=0.0044) and high-risk ICSS (p<0.0001).CONCLUSIONSWe herein provide evidence of the prognostic power and feasibility of ICSS into a large population of CLL patients. The use of this prognostic index could help physician into follow-up schedule, since high-risk patients should be monitored more often given the estimated increased risk of progression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Role of microRNAs in regulation of the TNF/TNFR gene superfamily in chronic lymphocytic leukemia

BackgroundChronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. The biology of this disease remains elusive. In the present times, targeted molecular therapy defines the treatment. This has made understanding the elusive biology and molecular mechanisms of the disease more relevant. MicroRNAs (miRNAs) are non-encoding RNAs that play important gene-regulatory roles in neoplastic processes. As in silico studies list nearly hundreds and more target genes for each miRNA, it is of interest to see if there are genes that have already been implicated in this disease. ObjectiveThe purpose of this study was to identify genes regulated by miRNAs that are independently implicated in the pathogenesis of CLL in previously published literature. Material and methodsEight miRNAs were selected. Genes implicated in the biology of CLL in association studies and massively parallel sequence studies were selected. TargetScanHuman 6.2, a computational program that predicts target genes of miRNAs on the basis of sequence analysis, was used to link the miRNAs to genes of interest. ResultsThe genes targeted by miR-15a, miR-223, miR-29a, and miR181a included IL10RA, BCL2, BCL6, DDX3X, and FBXW7. The most significant link observed was that several gene members of the TNF/TNFR superfamily were targets of miR-15a, miR-29a, and miR-181a. ConclusionMiRNAs implicated in the pathogenesis of CLL regulate genes that have independently been shown to play a role in CLL. The link between miRNAs and the TNF/TNFR superfamily is especially exciting. Understanding the molecular basis of these links in future studies may pave the way for using these miRNAs as therapeutic targets in CLL.

Ibrutinib as Initial Therapy for Patients with Chronic Lymphocytic Leukemia

BackgroundChronic lymphocytic leukemia (CLL) primarily affects older persons who often have coexisting conditions in addition to disease-related immunosuppression and myelosuppression. We conducted an international, open-label, randomized phase 3 trial to compare two oral agents, ibrutinib and chlorambucil, in previously untreated older patients with CLL or small lymphocytic lymphoma.MethodsWe randomly assigned 269 previously untreated patients who were 65 years of age or older and had CLL or small lymphocytic lymphoma to receive ibrutinib or chlorambucil. The primary end point was progression-free survival as assessed by an independent review committee.ResultsThe median age of the patients was 73 years. During a median follow-up period of 18.4 months, ibrutinib resulted in significantly longer progression-free survival than did chlorambucil (median, not reached vs. 18.9 months), with a risk of progression or death that was 84% lower with ibrutinib than that with chlorambucil (hazard ratio, 0.16; P<0.001). Ibrutinib significantly prolonged overall survival; the estimated survival rate at 24 months was 98% with ibrutinib versus 85% with chlorambucil, with a relative risk of death that was 84% lower in the ibrutinib group than in the chlorambucil group (hazard ratio, 0.16; P=0.001). The overall response rate was higher with ibrutinib than with chlorambucil (86% vs. 35%, P<0.001). The rates of sustained increases from baseline values in the hemoglobin and platelet levels were higher with ibrutinib. Adverse events of any grade that occurred in at least 20% of the patients receiving ibrutinib included diarrhea, fatigue, cough, and nausea; adverse events occurring in at least 20% of those receiving chlorambucil included nausea, fatigue, neutropenia, anemia, and vomiting. In the ibrutinib group, four patients had a grade 3 hemorrhage and one had a grade 4 hemorrhage. A total of 87% of the patients in the ibrutinib group are continuing to take ibrutinib.ConclusionsIbrutinib was superior to chlorambucil in previously untreated patients with CLL or small lymphocytic lymphoma, as assessed by progression-free survival, overall survival, response rate, and improvement in hematologic variables. (Funded by Pharmacyclics and others; RESONATE-2 ClinicalTrials.gov number, NCT01722487.)

Idelalisib Treatment Is Associated with Improved Cytopenias in Patients with Relapsed/Refractory iNHL and CLL

BACKGROUNDChronic lymphocytic leukemia (CLL) and indolent non-Hodgkin lymphoma (iNHL) are B-cell malignancies associated with neutropenia, anemia and thrombocytopenia.The etiology of impaired hematopoiesis is not well understood, however the bone marrow leukemia/lymphoma cell infiltrates that often occur with these diseases is thought to contribute. In studies evaluating the selective PI3Kd inhibitor idelalisib (IDL) in B-cell malignancies, hematologic responses across all 3 lineages were observed in a majority of patients (pts) with baseline (BL) cytopenias.This post hoc analysis reports hemogram changes in pts with relapsed or refractory (R/R) CLL/iNHL treated with IDL in two pivotal studies and describes trends in hematologic parameters over time during IDL treatment in pts with or without BL cytopenias.METHODSIn phase 3 study 312-0116 (NCT01539512), frail pts with R/R CLL were randomized to receive a combination of 8 doses of rituximab (R) with IDL 150 mg BID or placebo (P). In phase 2 study 101-09 (NCT01282424), pts with refractory iNHL received IDL 150 mg BID. In both studies, IDL was continued until disease progression (PD) or unacceptable toxicity. Trial inclusion criteria allowed enrollment of pts with BL cytopenias of any grade (CLL) or grade ˂3 (iNHL).Hematologic profiles for pts in each study were categorized as normal or abnormal (any grade of cytopenia) at BL. In the iNHL study, pts with PD at the first assessment were excluded from this analysis to avoid confounding by underlying uncontrolled disease.RESULTSA total of 345 pts participated in the 2 trials. The overall response rates of IDL-treated patients in the CLL and iNHL studies were 81% and 57%, respectively.For pts with CLL on IDL+R (n=110), BL cytopenias (grade ≥1) included anemia (76%), thrombocytopenia (62%), and neutropenia (34%). For pts with iNHL on IDL-mono (n=115), BL cytopenias included anemia (50.4%), thrombocytopenia (64.3%), and neutropenia (75.7%). We present changes in hemoglobin (Hgb), platelet (PLT), or neutrophil (ANC) counts over time (BL, peak, and time to peak) in pts with an abnormal BL hemogram in Table 1.Median hematologic lab values over time were unchanged in pts with normal hemograms at BL. Among patients with BL cytopenia, anemia and thrombocytopenia improved over time in pts with CLL and iNHL while on treatment with IDL. For pts with CLL, the magnitude of improvement was larger for pts in the IDL+R arm compared to those in the P+R arm. For pts in the IDL+R arm, median peak values of Hgb and PLT were observed within 6 months of IDL initiation. For pts with iNHL, median peak values for Hgb and PLT were observed within 3 months. ANC remained stable over time in CLL, and increased in iNHL.CONCLUSIONSIdelalisib treatment was associated with improvement in Hgb and PLT counts in this population of pts with R/R CLL or iNHL. In those pts with BL anemia or thrombocytopenia, peak improvement in Hgb or PLT values occurred early in treatment (≤3 months for iNHL and ≤6 months for CLL). [Display omitted] DisclosuresO'Brien:Gilead: Research Funding. Davies:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Flinn:Cephalon, Inc; Teva Pharmaceutical Industries Ltd; Genentech, inc; Gilead: Research Funding. Gopal:Spectrum, Pfizer, BioMarin, Cephalon/Teva, Emergent/Abbott. Gilead, Janssen., Merck, Milennium, Piramal, Seattle Genetics, Giogen Idec, BMS: Research Funding; Gilead, Spectrum, Pfizer, Janssen, Seattle Genetics: Consultancy; Millennium, Seattle Genetics, Sanofi-Aventis: Honoraria. Kipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor. Salles:Celgene Corporation; Roche and Gilead Sciences: Research Funding; Calistoga Pharmaceuticals, Inc.; Celgene Corporation; Genentech, Inc.; Janssen Pharmaceutica Products, L.P.; Roche: Consultancy; Celgene Corporation; Roche: Speakers Bureau. Newcomb:Gilead: Employment. Waldapfel:Gilead: Employment. Zhang:Gilead: Employment. Stilgenbauer:Gilead: Honoraria, Research Funding.

CD47 agonist peptides induce programmed cell death in refractory chronic lymphocytic leukemia B cells via PLCγ1 activation: evidence from mice and humans.

BackgroundChronic lymphocytic leukemia (CLL), the most common adulthood leukemia, is characterized by the accumulation of abnormal CD5+ B lymphocytes, which results in a progressive failure of the immune system. Despite intense research efforts, drug resistance remains a major cause of treatment failure in CLL, particularly in patients with dysfunctional TP53. The objective of our work was to identify potential approaches that might overcome CLL drug refractoriness by examining the pro-apoptotic potential of targeting the cell surface receptor CD47 with serum-stable agonist peptides.Methods and FindingsIn peripheral blood samples collected from 80 patients with CLL with positive and adverse prognostic features, we performed in vitro genetic and molecular analyses that demonstrate that the targeting of CD47 with peptides derived from the C-terminal domain of thrombospondin-1 efficiently kills the malignant CLL B cells, including those from high-risk individuals with a dysfunctional TP53 gene, while sparing the normal T and B lymphocytes from the CLL patients. Further studies reveal that the differential response of normal B lymphocytes, collected from 20 healthy donors, and leukemic B cells to CD47 peptide targeting results from the sustained activation in CLL B cells of phospholipase C gamma-1 (PLCγ1), a protein that is significantly over-expressed in CLL. Once phosphorylated at tyrosine 783, PLCγ1 enables a Ca2+-mediated, caspase-independent programmed cell death (PCD) pathway that is not down-modulated by the lymphocyte microenvironment. Accordingly, down-regulation of PLCγ1 or pharmacological inhibition of PLCγ1 phosphorylation abolishes CD47-mediated killing. Additionally, in a CLL-xenograft model developed in NOD/scid gamma mice, we demonstrate that the injection of CD47 agonist peptides reduces tumor burden without inducing anemia or toxicity in blood, liver, or kidney. The limitations of our study are mainly linked to the affinity of the peptides targeting CD47, which might be improved to reach the standard requirements in drug development, and the lack of a CLL animal model that fully mimics the human disease.ConclusionsOur work provides substantial progress in (i) the development of serum-stable CD47 agonist peptides that are highly effective at inducing PCD in CLL, (ii) the understanding of the molecular events regulating a novel PCD pathway that overcomes CLL apoptotic avoidance, (iii) the identification of PLCγ1 as an over-expressed protein in CLL B cells, and (iv) the description of a novel peptide-based strategy against CLL.

Higher gene expression variability in the more aggressive subtype of chronic lymphocytic leukemia.

BackgroundChronic lymphocytic leukemia (CLL) presents two subtypes which have drastically different clinical outcomes, IgVH mutated (M-CLL) and IgVH unmutated (U-CLL). So far, these two subtypes are not associated to clear differences in gene expression profiles. Interestingly, recent results have highlighted important roles for heterogeneity, both at the genetic and at the epigenetic level in CLL progression.MethodsWe analyzed gene expression data of two large cohorts of CLL patients and quantified expression variability across individuals to investigate differences between the two subtypes using different measures and statistical tests. Functional significance was explored by pathway enrichment and network analyses. Furthermore, we implemented a random forest approach based on expression variability to classify patients into disease subtypes.ResultsWe found that U-CLL, the more aggressive type of the disease, shows significantly increased variability of gene expression across patients and that, overall, genes that show higher variability in the aggressive subtype are related to cell cycle, development and inter-cellular communication. These functions indicate a potential relation between gene expression variability and the faster progression of this CLL subtype. Finally, a classifier based on gene expression variability was able to correctly predict the disease subtype of CLL patients.ConclusionsThere are strong relations between gene expression variability and disease subtype linking significantly increased expression variability to phenotypes such as aggressiveness and resistance to therapy in CLL.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-014-0125-z) contains supplementary material, which is available to authorized users.

Antibody and plasmablast response to 13-valent pneumococcal conjugate vaccine in chronic lymphocytic leukemia patients--preliminary report.

BackgroundChronic lymphocytic leukemia (CLL) leads to significant immune system dysfunction. The predominant clinical presentation in 50% of patients involves recurrent, often severe, infections. Infections are also the most common (60–80%) cause of deaths in CLL patients. The scope of infections varies with the clinical stage of the disease. Treatment-naive patients typically present with respiratory tract infections caused by encapsulated bacteria Streptococcus pneumoniae and Haemophilus influenzae. Since 2012, the 13-valent pneumococcal conjugate vaccine (PCV13) has been recommended in the United States and some EU countries for pneumococcal infection prevention in patients with CLL (besides the long-standing standard, 23-valent pneumococcal polysaccharide vaccine, PPV23). The aim of this study was to compare the immune response to PCV13 in 24 previously untreated CLL patients and healthy subjects.MethodsBoth groups were evaluated for: the levels of specific pneumococcal antibodies, the levels of IgG and IgG subclasses and selected peripheral blood lymphocyte subpopulations including the frequency of plasmablasts before and after immunization.ResultsAdequate response to vaccination, defined as an at least two-fold increase in specific pneumococcal antibody titers versus pre-vaccination baseline titers, was found in 58.3% of CLL patients and 100% of healthy subjects. Both the CLL group and the control group demonstrated a statistically significant increase in the IgG2 subclass levels following vaccination (P = 0.0301). After vaccination, the frequency of plasmablasts was significantly lower (P<0.0001) in CLL patients in comparison to that in controls. Patients who responded to vaccination had lower clinical stage of CLL as well as higher total IgG, and IgG2 subclass levels. No significant vaccine-related side effects were observed.ConclusionsPCV13 vaccination in CLL patients is safe and induces an effective immune response in a considerable proportion of patients. To achieve an optimal vaccination response, the administration of PCV13 is recommended as soon as possible following CLL diagnosis.