Relative Prognostic Significance of ZAP-70 and IGHV1-69 Expression in Chronic Lymphocytic Leukemia,

Abstract Abstract 3891 The leukemia B cells of patients (pts) with chronic lymphocytic leukemia (CLL) express a restricted repertoire of immunoglobulin heavy chain variable region (IGHV) genes. Moreover, certain IGHV genes appear over-represented in this repertoire. Among these, IGHV1-69 was first identified as most frequently expressed, being used by the CLL cells of nearly 20% of all pts (PNAS, 86:5913–7, 1989). CLL cells most frequently express IGHV1-69 without somatic mutations and often with restricted D and JH segment use, providing for stereotypic motifs in the heavy chain third complementarity determining region (HCDR3). We addressed whether there were peculiar biologic or clinical features of pts with CLL that used IGHV1-69. For this, we studied 452 pts identified in a cohort of 2,866 followed by the CLL Research Consortium (CRC) found to have CLL cells that express IGHV1-69. This accounted for 16% of all pts. We found that 420 of 452 CLL samples (93%) express IGHV1-69 without somatic mutation (≥98% sequence homology with germline IGHV1-69), which is in significant contrast to the frequency use of UM IGHV among CLL samples that do not use IGHV1-69. As noted for CLL pts in general, there is a strong association between mutation status and clinical behavior. Among pts that use IGHV1-69, the 32 of 452 pts that use MU IGHV1-69 had a highly indolent clinical course, with a median time from diagnosis to initial treatment (TFS) of 17.3 years. This was significantly longer than the median TFS of 2.8 yrs for the 452 pts that used UM IGHV1-69 (p<0.0001). Among the pts that used UM IGHV1-69 we identified a stereotypic HDCR3 motif shared by more than one patient in 249/452 (55%) of the cases. Multivariate analysis failed to discriminate any significant differences in the median TFS of pts that used IGHV1-69 that had a stereotypic CDR3 motif versus pts who had an idiosyncratic HCDR3 (2.9 yrs vs 3.0 yrs, respectively p=0.14). Interphase FISH for common cytogenetic aberrations in CLL were available for 281 of the 452 cases. Among these, 58% of the cases had deletions at 17p (18%), 11q (23%), or trisomy 12 (17%). The remaining cases had no detectable chromosomal abnormalities (24%) or isolated deletion of 13q (18%). The CLL cells of all 452 pts were examined for ZAP-70. There was a strong association between the expression of ZAP-70 and use of UM IGHV1-69. However, the association between ZAP-70 expression and use of UM IGHV1-69 was not absolute. Only 70% with UM IGHV1-69 were ZAP-70 positive, as were 12.5% that used M IGHV1-69. Of all 452 pts that expressed IGHV1-69, 300 (66%) had ZAP-70 positive CLL cells. These pts had a median TFS of 2.3 yrs, which was significantly shorter than that of the remaining 152 pts with ZAP-70-negative CLL, who had a median TFS of 4.3 yrs (p<0.0001). Moreover, of the 420 pts that used UM IGHV1-69, 296 (70%) had CLL cells that expressed ZAP-70; these pts had a median TFS of 2.3 yrs. This was significantly shorter than the median TFS of pts with CLL cells that express UM IGHV1-69, but were ZAP-70 negative 4.1 yrs (p<0.0001). A Cox regression model revealed that although the presence of detectable chromosomal aberrations was associated to a shorter median TFS, ZAP-70 was a stronger predictor of short TFS (HR for 13q =1.1 p =0.03, HR for trisomy 12 =1.2 p =0.03, HR for 11q =1.6 p =0.03, HR for 17p =1.8, p =0.03) (HR for ZAP-70 positive = 1.8, p=0.0004). The Cox regression model was used to assess the associations of ZAP-70, and the use or not of the UM IGHV1-69 gene with TFS (p-value <0.05 were considered as significant). We investigated these associations using a previously published cohort of characterized 705 CLL pts (Blood.2008;112:1923). The HR associated with the expression of ZAP-70 (HR=3.2) (p<0.0001) was significantly higher than if either the UM IGHV1-69 or the cases with UM IGHV other than IGHV1-69 were incorporated into the model (HR=1.9 and HR=1.6 respectively, p=0.001). We conclude that cases that use IGHV1-69 are peculiar in that they more frequently use UM IGHV and appear to have a higher frequency of adverse cytogenetic features than CLL cases at large. In addition, we found that CLL-cell expression of ZAP-70 can segregate pts that use UM IGHV1-69 into subgroups with disparate clinical behavior, despite the fact that all patients use the same IGHV gene. Moreover, multivariable analyses revealed that ZAP-70 was strongest predictor of short TFS among all other considered prognostic parameters in this distinctive cohort of pts. Disclosures: Kipps: Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

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Chronic Lymphocytic Leukemia Cells With Trisomy 12 Home To The Bone Marrow In a CXCR4-Independent Manner and Are Prone To Proliferate In Vitro

Blood ◽

10.1182/blood.v122.21.870.870 ◽

2013 ◽

Vol 122 (21) ◽

pp. 870-870

Author(s):

Evelyn Hutterer ◽

Elisabeth Hinterseer ◽

Sylvia Ganghammer ◽

Gabriele Brachtl ◽

Daniela Asslaber ◽

...

Keyword(s):

Bone Marrow ◽

Chronic Lymphocytic Leukemia ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Dependent Manner ◽

Independent Manner ◽

Atypical Cell ◽

Trisomy 12 ◽

Inside Out

Abstract Trisomy 12 (tri12) is a frequent chromosomal aberration in chronic lymphocytic leukemia (CLL) associated with atypical cell morphology, high in vivo tumor proliferation activity and a predisposition to Richter’s transformation. Tri12 harboring CLL cells express increased levels of the negative prognostic marker CD49d, the α4 subunit of the integrin very late antigen 4 (VLA-4), which we previously identified as a key regulator of CLL cell homing to bone marrow (BM). During this process, inside-out activation of VLA-4 upon CXCR4 binding to endothelially displayed CXCL12 is thought to upregulate the adhesive properties of VLA-4 and augment the arrest of CLL cells on the VCAM-1 presenting vessels. Here, we investigated the functional interplay of VLA-4 and CXCR4 in CLL carrying tri12. We first found that the upregulation of CD49d expression in this subset (MFIR CD49d 9.8±5.3 (n=22) vs. 2.7±3.9 (n=126), p<0.0001) was paralleled by their reduced CXCR4 expression (MFIR CXCR4 11.8±7.2 (n=22) vs. 22.7±14.2 (n=126), p=0.0003). Using short term adoptive transfers, we compared the ability of tri12 and no tri12 CLL cells to home to the BM of NOD/SCID mice. 5-10x106 CLL cells were injected into tail vein and homing was evaluated after 3 hours. Based on their more frequent CD49d high phenotype, we observed increased homing rates (homed human CLL cells per 106 injected cells per 106 acquired murine cells) of tri12 compared to no tri12 CLL (225±160 (n=7) vs. 90±117 (n=20), p=0.025). However, when comparing CD49d+ tri12 and CD49d+ no tri12 subsets, we did not observe any significant differences in their homing capacity. To further study CXCL12/CXCR4 function in BM homing, we pretreated mice with either the novel CXCL12 antagonist NOX-A12 or the CXCR4 inhibitor AMD3100 prior to CLL cell injection. While homing of no tri12 CLL cells (n=3, in duplicates) was reduced by both pretreatments (homing rates 137 vs 38 vs 30), the homing capacity of tri12 CLL cells (n=3, in duplicates) was not affected. We next tested whether VLA-4 expressed on these cells was able to undergo CXCL12-induced activation and support cell arrest under shear conditions. To this end, we perfused CLL cells over VCAM-1 or VCAM-1/CXCL12 substrates and analyzed rates and categories of cell tethering at a single cell level by videomicroscopy. CXCL12 induced the arrests of no tri12 CLL cells (n=3) on VCAM-1 under shear flow in a CXCR4 and VLA-4 dependent manner. In contrast, tri12 CLL cells (n=3) robustly tethered to VCAM-1 in the absence of the chemokine, and interactions could not be further enhanced by additional CXCL12 nor could they be abrogated by use of AMD3100. This failure of CXCR4-induced adhesion was not based on a general defect in CXCR4 functionality as in vitro chemotaxis of tri12 CLL cells (n=5) towards CXCL12 was fully maintained. To detect potential differences in VLA-4 affinity regulation, we used a conformationally sensitive antibody that recognizes epitopes induced by VLA-4 ligation, and an LDV-containing VLA-4 specific ligand to probe resting integrin affinity. Also, we used a small fluorescent ligand to study rapid VLA-4 affinity changes during inside-out chemokine induced activation. On resting tri12 CLL, VLA-4 exhibited an affinity state similar to that observed on circulating lymphocytes, and tri12 CLL cells failed to undergo the rapid affinity up-regulation triggered by CXCL12 pretreatment, in keeping with tethering experiments. Next, we investigated whether the tumor microenvironment has a different influence on the behavior of the tri12 subset. Therefore we subjected the cells to in vitro co-cultures mimicking the lymphoid proliferation centers. Basal levels of the early activation marker CD69 were similar in tri12 CLL compared to no tri12 cases. Tri12 CLL, however, underwent stronger activation when cultured in presence of accessory cells (%CD69+ cells 60.0±18.5 (n=4) vs. 17.7±20.1 (n=19), p=0.008). Moreover, in several setups, proliferation rates of these cells were increased, irrespective of the proliferative stimulus and detection method used. In summary, our results provide a mechanistical basis at least in part explaining the peculiar and clinical features of the tri12 CLL subset. In light of the specific migratory and proliferative properties of tri12 cells and novel agents targeting particularly these functions, our findings may also imply therapeutical consequences. Disclosures: Greil: NOXXON Pharma AG: Research Funding. Hartmann:NOXXON Pharma AG: Research Funding.

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A Study of Ribonucleotide Reductase mRNA Expression and Its Prognostic Role in Patients with Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2021-150149 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4678-4678

Author(s):

Sevastianos Chatzidavid ◽

Christina-Nefeli Kontandreopoulou ◽

Panagiotis T Diamantopoulos ◽

Nefeli Giannakopoulou ◽

Panagiota Katsiampoura ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Mrna Expression ◽

Board Of Directors ◽

Ribonucleotide Reductase ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Line Treatment ◽

Prognostic Role ◽

Advisory Committees ◽

Trisomy 12

Abstract Background Ribonucleotide Reductase (RNR) is responsible for converting ribonucleotides to deoxyribonucleotides required for DNA replication and repair. RNR consists of two subunits, termed subunit 1 (RRM1) and 2 (RRM2). Imbalance in the regulation of RNR activity and control of dNTPs' pool leads to genomic instability and increases mutation rate. RNR expression has been associated with prognosis in pancreatic, non-small-cell lung, breast, and biliary tract cancer. However, RNR expression in chronic lymphocytic leukemia (CLL) and its possible prognostic role have not been investigated yet. Aim In this study we evaluate the possible prognostic role of RNR expression in CLL. Method The study comprised patients with immunophenotypically confirmed disease at the time of sample collection. Peripheral whole blood samples were collected from 84, 27, 15, and 9 patients before treatment, after one, two, and three lines of treatment respectively. RNA extraction and reverse transcription were carried out using standard protocols. A Taqman based real-time PCR was performed on a CFX96 RT-PCR system (Bio-Rad Laboratories, Hercules, CA, USA). For both the housekeeping and target genes, a Taqman primer/probe mix was used according to the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). RRM1 and RRM2 mRNA levels were expressed as an RRM1-2/GAPDH ratio. Western blot analysis was performed to quantify the RRM1 protein levels in a random sample of 41 patients. Antibodies used were: RRM1 #3388, β-actin #4967 and anti-rabbit IgG HRP-conjugated #7074 (Cell Signaling Technology, Danvers, MA, USA). Detection was done using the ECL western blotting reagents. Statistical analysis was conducted to study the possible correlations between the variables. All reported p values are two-tailed. Statistical significance was set at p<0.05 and analyses were conducted using SPSS statistical software (version 22.0). Results From 135 CLL patients included in the study 56.3% were female and the median age at diagnosis was 64 years. Peripheral blood was collected in 84 treatment-naïve patients (62.2%). Median follow up was 6.66 years (3.47 ─ 11.13) and median time from diagnosis until 1st line treatment was 23.1 months (IQR: 5.8 - 56.5 months). Out of 135 patients, 69 (51,1%) received 1 st line treatment and 35 patients (25,9%) 2 nd line treatment with median time between the two treatment lines being 26.5 months (IQR: 7.8 - 40.8 months). Furthermore, 48.5%, 33.8%, 12.3%, 3.1% and 2.3% of the patients had Rai score 0, I, II, III, IV respectively. The median mRNA expression of RRM1 was 0.04 (IQR: 0 - 0.09) and of RRM2 was 0.01 (IQR: 0 - 0.1). RRM1 mRNA expression was significantly higher in patients without anemia (p=.025) and without lymphadenopathy (p=.002). Higher values of ESR (r=-.30; p=.028), LDH (r=-.20; p=.026) and Rai score (r=-.18; p=.037) were associated with lower expression of RRM1 mRNA. In addition, TP53 gene deletion detected by FISH was associated with higher RRM1 mRNA expression (p=.036). Significantly higher RRM2 mRNA expression was reported in patients without lymphadenopathy (p=.021) and Rai score 0 (p=.003). Moreover, higher was the expression of RRM2 mRNA in cases with Trisomy 12 (p=.050). In samples collected before treatment, higher values of RRM1 mRNA expression were statistically significantly associated with lower RAI score (r=-.30; p=.005) and longer time periods between the first two lines of treatment (r=.95; p=.050). Western blot analysis confirmed detection of RRM1 protein but statistical correlation was not carried out due to lack of material from the whole group of patients. Conclusion For the first time, mRNA expression of RRM1 and RRM2 is studied in patients with CLL. These results show RNR involvement in the pathophysiology of CLL. RRM1 and RRM2 mRNA higher expression found in 17p deletion and trisomy 12 cases respectively may be consistent with the existence of a methylation-depended mechanism proposed by other studies. Therefore, these results demonstrate RNR's potential role as a prognostic factor, and make it a probable therapeutic target. A study including a larger number of cases could further confirm our results. Figure 1 Figure 1. Disclosures Kyrtsonis: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/Genesis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria; Sanofi: Membership on an entity's Board of Directors or advisory committees. Panagiotidis: Abbvie: Research Funding; Pfizer: Research Funding; Janssen: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Takeda: Research Funding; Sandoz: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Research Funding; Astellas: Research Funding. Viniou: Sandoz: Research Funding; Takeda: Research Funding; Novartis: Honoraria, Research Funding; Sanofi: Research Funding; Janssen: Honoraria, Research Funding; Pfizer: Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Roche: Research Funding; Astellas: Research Funding; Celgene: Research Funding.

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Chronic Lymphocytic Leukemia Subset Expressing Mutated IGHV3-23 Has Peculiar Clinical and Biological Features.

Blood ◽

10.1182/blood.v114.22.1256.1256 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1256-1256

Author(s):

Riccardo Bomben ◽

Michele Dal-Bo ◽

Dania Benedetti ◽

Daniela Capello ◽

Francesco Forconi ◽

...

Keyword(s):

Gene Ontology ◽

Chronic Lymphocytic Leukemia ◽

Tumor Suppressor ◽

Heavy Chain ◽

Tumor Suppressor Genes ◽

Lymphocytic Leukemia ◽

Clinical Behavior ◽

Qrt Pcr ◽

Biological Features ◽

Mutational Status

Abstract Abstract 1256 Poster Board I-278 Introduction In the last years, the B cell receptor (BCR) has become a key molecule in chronic lymphocytic leukemia (CLL), given the correlation between mutational status of immunoglobulin heavy chain variable (IGHV) genes and disease prognosis. Recently, a fraction of CLL has been shown to preferentially express specific IGHV genes, often in a non-random combination with homologous heavy chain complementarity-determining region-3 (HCDR3) and peculiar light chains. Some of these stereotyped BCR mark CLL subsets with peculiar clinical behavior regardless of IGHV mutations. These data suggest a role for BCR in defining the clinical and biological features of CLL, also beyond the mutational status of IGHV genes. Patients and Methods A HCDR3-driven clustering of 1,426 IG sequences (1,398 patients) was performed using ClustalX(1.83). Time to treatment (TTT) intervals, Rai staging, IGHV mutational status, CD38, ZAP-70, and karyotype abnormalities evaluated by FISH were available for 617 patients. Gene expression profiling (GEP) and quantitative real-time PCR experiments (QRT-PCR) were performed on purified CLL cells. Results IGHV3-23 was totally absent in 71 identified stereotyped clusters despite being the second most frequently used IGHV gene, such distribution was significantly skewed (p<0.0001), compared with the distribution of IGHV genes belonging to stereotyped BCR clusters observed in our series. Although 109/134 IGHV3-23 were mutated (M), alignment of IGHV sequences revealed a high degree of conservation in the context of the 13 AA positions involved in superantigen binding by IGHV3 subgroup genes, suggesting that the majority of M IGHV3-23 cases maintained the capacity to mediate superantigen recognition and binding. Median TTT (73 months) of 43 M IGHV3-23 CLL was significantly shorter than median TTT (253 months, p=0.0153) of 333 M CLL, as well as of 326 M CLL in which 7 cases belonging to the bad prognosis IGHV3-21/IGLV3-21 cluster were excluded (253 months, p=0.0082). Multivariate Cox proportional hazard analyses selected IGHV3-23 usage (p=0.029), Rai stage (p<0.0001) and FISH group (p<0.0001) as independent markers of disease progression for 376 M CLL, and for the cohort in which 7 M CLL from the IGHV3-21/IGLV3-21 cluster were excluded. Comparing 5 M IGHV3-23 and 22 M non-IGHV3-23 CLL for their differential GEP, 212 genes were selected, 108 up-regulated and 104 down-regulated in M IGHV3-23 CLL. Using the “Gene-Ontology Tree Machine” platform, a set of growth/tumor suppressor genes (PDCD4, TIA1, RASSF5), all down-regulated in M IGHV3-23 CLL, was constantly found in several gene-ontology categories related to apoptosis. QRT-PCR confirmed a significant down-regulation of these genes in 15 M IGHV3-23 compared to 35 M non-IGHV3-23 CLL. Given the notion that PDCD4 and TIA1 are among the genes under control of miR-15a and miR-16-1 a “Gene Set Enrichment Analysis” carried out on the 212 differentially expressed genes, confirmed that M IGHV3-23 samples were significantly deprived in genes whose expression is under control of miR-15a and miR-16-1. Accordingly, QRT-PCR experiments performed on 15 M IGHV3-23 and 35 M non-IGHV3-23 CLL revealed significant higher levels of both miR-15a (p=0.0007) and miR-16-1 (p=0.0031) in M IGHV3-23 cases. No difference was found in the distribution of patients with 13q14 deletion between M IGHV3-23 CLL and M non-IGHV3-23 CLL (p=0.19). Considering the cases used for microRNA expression experiments (data available in 47/50 cases), 8/15 M IGHV3-23 CLL bore the 13q14 deletion in more than 20% of nuclei, against 19/32 cases in the group of M non-IGHV3-23 CLL (p=0.94). Conclusion Expression of IGHV3-23 marks a subset of M CLL with a worse prognosis; such a peculiar clinical behavior may be related to superantigen stimulation combined with down-regulation of specific growth/tumor suppressor genes and up-regulation of miR-15a and miR-16-1. Disclosures No relevant conflicts of interest to declare.

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Characterisation of a New Clinical Presentation of Chronic Lymphocytic Leukemia with Symptomatic Nasopharyngeal Mucosa Involvement

Blood ◽

10.1182/blood-2021-144958 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3741-3741

Author(s):

Thimali Ranaweera Arachchige ◽

Antoine Diep ◽

Ambroise David ◽

Melchior Le Mene ◽

Virginie Eclache ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Head And Neck ◽

Board Of Directors ◽

Median Time ◽

Chemokine Receptors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Advisory Committees ◽

Migratory Capacity ◽

Trisomy 12

Abstract Patients with chronic lymphocytic leukemia (CLL) are prone to infectious complications, including Ears, Nose and Throat (ENT) infections due to the humoral immunodepression and/or to the immunosuppression related to the therapy. However, specific CLL infiltration in non-lymphoid regions of the head and neck causing ENT symptoms but unrelated to an infection is not well described. Extra-nodal localizations of CLL cells, including involvement of the mucosa of the rhinopharynx is uncommon and poorly reported. Here, we retrospectively analyzed the clinical, histopathologic and molecular features of 25 CLL patients with specific head and neck involvement. To date, this is the largest cohort reporting this new entity of CLL also named Nasal Associated Lymphoid Tissue (NALT) CLL. All patients had proven CLL prior to symptoms. Median time between ENT manifestation and CLL was 3 years [1-11 years]. Symptoms included chronic coughing (44%), antero-posterior nasal discharge (44%), nasal congestion (33%) and pharyngitidis (33%). ENT examination evidenced cervical lymphadenopathies in 68 % of cases, a granular aspect of the mucosa of the pharynx in 56%, enlarged tonsil (37%) or adenoids (37%). All patients underwent a biopsy of the mucosa of the nose or the throat. Histology and immunochemistry analysis demonstrated an infiltration of small lymphocytes CD20+, CD5+ and CD23+, consistent with a phenotype of CLL. The infiltration was diffuse in 50% of biopsies and perivascular in 38%. Patients with NALT-CLL had a poor prognosis: the majority was IGHV unmutated (n=18/25, 72%). Furthermore, they all required a treatment according to IWCLL criteria few time after the first ENT symptoms (median time 2 years), indicating that NALT-CLL is associated with a more progressive disease. To characterize the genetic background of NALT-CLL, we analyzed the cytogenetic data and NGS sequencing of 13 CLL-associated mutations from peripheral CLL cells. The karyotype was normal in only 2/24 cases (8%) and complex (>3 abnormalities) in 5/24 cases (20%). Half of the patients had a trisomy 12(12/24; 50%), while 13q, 17p and 11q deletions were found in 29%, 8% and 4% respectively. Eleven patients harbored one mutation (44%) while 9 patients had 2 to 5. Mutation of the NOTCH1 pathway was found in half of the cases (11/25 NOTCH1 and 2/25 FBXW7 mutated cases). TP53 and SF3B1 mutations occurred respectively in 20% (5/25 cases) and in 12% (3/25 cases). To gain insight into the molecular mechanism associated with involvement of the rhinopahrynx, we studied the expression of 70 genes related to cell migration and cell adhesion pathways using a qPCR array in the peripheral CLL cells of patients with NALT (n=4) compare to no NALT-CLL (n=4). Genes significantly up regulated with a fold change >2 included the chemokine receptors CCR7 (p=0.05) and CCR5 (p=0.04), the receptor involved in leukocyte trafficking CXCR3 (p=0.01) or the chemoattractant chemokine-like factor CKLF. By targeted qPCR, we confirmed the up regulation of CCR7 (p=0.002), CXCR3 (p=0.04) and CCR5 (p=0.03) in the cohort NALT-CLL patient (n=25) as compared to 20 age-matched CLL patients without head and neck symptoms (77% with unmutated IGHV, 30% with NOTCH1 mutation and 30% with trisomy 12). Up regulation of those targets was independent of the presence of NOTCH1/trisomy12 aberration or of the IGHV mutation status. These results suggest an increased migratory capacity of the leukemic CLL cells into the rhynopharynx mucosa related to a higher expression of these receptors involved in cell trafficking and migration. In line with these results, immunohistochemistry analysis of 5 patients with nasal involvement showed a strong staining of the CCR7 marker on the membrane of CLL cells infiltrating the mucosa. Interestingly, the staining of CCL21, the cognate ligand of CCR7, was positive in the vessels of the mucosa, suggesting that the recruitment and the transendothelial migration of CLL cells into the mucosa occur through a local secretion of CCL21 by the vessels. In summary, we report here a new presentation of CLL associated with symptomatic and specific ENT localization. CLL cells are predominantly IGHV unmutated, harbor NOTCH1 mutation and/or trisomy12 and show a higher expression of the chemokine receptors CCR7, CCR5 and CXCR3. We are currently studying the expression of those receptors by flow cytometry and the enhanced migratory capacity toward CCL21 through an in vitro chemotaxis assay. Disclosures Letestu: AbbVie: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Janssen: Research Funding, Speakers Bureau. Cymbalista: Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; ASTRA ZENECA: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Lilly-LOXO: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Biological and Clinical Features of Patients with Chronic Lymphocytic Leukemia Bearing Trisomy 12

Blood ◽

10.1182/blood.v120.21.3907.3907 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3907-3907

Author(s):

Paolo Strati ◽

Lynne V. Abruzzo ◽

William Wierda ◽

Susan Lerner ◽

Susan M. O'Brien ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Board Of Directors ◽

Median Time ◽

Research Funding ◽

Progressive Disease ◽

Clinical Course ◽

Lymphocytic Leukemia ◽

Interphase Nuclei ◽

Advisory Committees ◽

Trisomy 12

Abstract Abstract 3907 Cytogenetic abnormalities are among the most important predictors of clinical course and response to therapy in patients (pts) with chronic lymphocytic leukemia (CLL). Conventional chromosome banding (CBA) and fluorescence in situ hybridization (FISH) analyses detect abnormalities in 40–50% and 80% of pts, respectively. Trisomy 12 (+12), observed in ∼20% of CLL pts by FISH, is associated with atypical morphology and immunophenotype, and a more aggressive clinical course. We, therefore, review the clinical characteristics and outcome of 312 CLL pts with +12 evaluated at our center between 1988 and 2011. FISH analysis for common abnormalities associated with CLL was performed on interphase nuclei obtained from cultured bone marrow cells using a multi-color probe panel designed to detect deletions of 11q22.3 (ATM), 13q14.3 (D13S319), 13q34 (LAMP1), 17p13.1 (TP53) and trisomy 12 (12p11.1-q11) (Abbott Molecular, Abbott Park, IL). Survival curves were calculated using Kaplan-Meier estimates and compared using the log-rank test. Differences were considered significant for p < 0.05. Patient characteristics at diagnosis are presented in Table 1. Of 215 pts assessed by both CBA and FISH, 105 were positive for +12 by both analyses and 110 were positive only by FISH. By CBA (112 pts, including 7 assessed only by CBA), +12 was the sole abnormality in 52 pts (47%); +12 was associated with +19 in 17 pts (16%), with del(14q) in 9 pts (8%), with +18 in 8 pts (7%), with +8 in 3 pts (3%), with del(13q) in 3 pts (3%), with t(14;19)(q32;q13) in 3 pts (3%) and with other abnormalities in 17 pts (13%). By FISH (287 pts), +12 was the sole abnormality in 225 pts (78%) and was associated with del(13q) in 62 pts (22%). The median number of interphase nuclei positive for +12 by FISH was 47% (range, 5–93%). One-hundred-eighty-seven pts (60%) needed treatment, with a median Time-To-Treatment (TTT) of 46 months (range, 35–56). The TTT was significantly shorter in pts with Rai stage III-IV disease, splenomegaly, lymphadenopathy, B2m > 4 mg/L, CD38+, ZAP70+, +12 detected by both CBA and FISH, and +12 associated with del(14q) or t(14;19). All 187 pts with progressive disease received treatment: 105 with an FCR-based regimen, 28 with rituximab(R)-based therapy (R+ GM-CSF or R+ methylprednisolone), and 28 with investigational drugs (Lenalidomide, R+ lenalidomide, GS101, or Ibrutinib). Overall response rate was 98%, 89% and 96%, respectively, whereas complete remission rate was 87%, 11% and 36%, respectively. Fifty-five pts failed first-line treatment; their median Failure-Free Survival (FFS) was 27 months (range, 0–87). The FFS was significantly longer in pts who received FCR-based regimens (p<0.001)(Fig 1). The median overall survival (OS) has not been reached, and only 33 pts have died. The OS was significantly shorter in pts older than 65 years, with ALC > 30,000, and with a median +12 positivity in >30% of interphase nuclei by FISH. A trend toward longer OS was observed for pts with +12 associated with +19 (p=0.07). Richter's Syndrome (RS) and second malignancies (SM) were the leading causes of death (5 and 13 of 33 deaths, respectively). RS was reported in 12 pts (4%), after a median time of 36 months from diagnosis. SM was reported in 31 pts (10%), after a median time of 30 months from diagnosis. At the time of diagnosis of SM, 13 patients had received a therapy for CLL and 18 were untreated. In conclusion, pts with CLL and +12 have unique laboratory and clinical features. A high proportion develops progressive disease and requires treatment. Among available therapies, FCR-based regimens are associated with a longer FFS. A high rate of SM is observed in pts with +12, including in pts who have not received prior treatment Disclosures: Wierda: Abbott Laboratories: Research Funding. O'Brien:Avila: Research Funding; Bayer: Consultancy; Bristol-Myers Squibb: Research Funding; Gilead Sciences: Consultancy, Research Funding; Celgene: Consultancy; Cephalon: Consultancy; CII Global Research Foundation: Membership on an entity's Board of Directors or advisory committees; Genentech BioOncology: Research Funding; Genzyme: Consultancy; GlaxoSmithKline: Consultancy; MorphoSys: Consultancy; Novartis: Consultancy; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy; Seattle Genetics, Inc.: Consultancy; Sigma Tau Pharmaceuticals: Consultancy; Talon: Research Funding; The Medal Group: Speakers Bureau.

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Clonal Evolution In Patients With Previously Untreated Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v122.21.1643.1643 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1643-1643

Author(s):

Sameer A. Parikh ◽

Kari G. Rabe ◽

Stephanie A. Smoley ◽

Anne E. Wiktor ◽

Curtis A. Hanson ◽

...

Keyword(s):

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

Mayo Clinic ◽

Research Funding ◽

Clonal Evolution ◽

Clinical Importance ◽

Lymphocytic Leukemia ◽

Risk Category ◽

Time Interval ◽

Trisomy 12

Abstract Background Although the clinical importance of del 17p13 in patients with chronic lymphocytic leukemia (CLL) is well recognized, the implications of when this defect is identified are less clearly defined. In particular, the distinction between the identification of del 17p13 at the time of diagnosis and secondary acquisition of del 17p13 during the course of the disease are poorly characterized. Methods We identified all patients with CLL cared for at the Mayo Clinic between 1/1/2000 and 12/31/2012 who underwent baseline fluorescence in-situ hybridization (FISH) testing prior to receiving treatment. The Results of repeat FISH testing were reviewed to identify cases with clinically ascertained clonal evolution. Results A total of 1757 patients with newly diagnosed CLL were seen at Mayo Clinic prior to receiving treatment. Among these, 1243 had FISH testing performed prior to treatment and within 36 months of diagnosis. The median time from diagnosis to initial FISH was 0.8 months (11.5 to 35.4 months). The Results of baseline FISH testing among these patients is as follows: 486 (39%) had del 13q14, 234 (19%) had trisomy 12, 115 (9%) had del 11q23, 59 (5%) had del 17p13, 15 (1%) had other (e.g., del6q) and 334 (37%) had no abnormalities detected. Among these patients, 344 (28%) underwent repeat FISH testing during the course of their disease. Repeat FISH testing was typically performed for clinical suspicion of karyotype evolution or prior to therapy selection in patients with a long time interval between first prognostic FISH and initiation of treatment. Among these 344 patients, 41 (12%) acquired new cytogenetic abnormalities on repeat FISH. Classification of these 344 patients by the Dohner classification at diagnosis and time of follow-up FISH is shown in Table 1. Among the 41 pts who acquired a new FISH detectable genetic abnormality, the newly acquired defect resulted in a change in Dohner FISH risk category for 39 patients including 15/41 (37%) who acquired a del 17p13. Baseline clinical and prognostic characteristics of patients who developed clonal evolution to those who did not are shown in Table 2. Patients with unmutated immunoglobulin heavy chain (IGHV) gene mutation status (p<0.0001) and CD49d (p=0.003) expression were more likely to experience clonal evolution. Among the 319 patients who did not have del 17p13 on baseline FISH, 15 (5%) acquired del 17p13 during the course of their disease. The overall survival from the date del 17p13 was identified is shown for those with del 17p13 at diagnosis (n=59) compared to those who acquired del 17p13 later in the course of the disease (n=15) in Figure 1A. Similarly, among 339 patients who did not have del 6q23 on baseline FISH, 9 (3%) acquired del 6q23 during the course of their disease. The overall survival from the date del 6q23 was identified is shown for those with del 6q23 at diagnosis (n=11) and those who acquired del 6q23 later in the course of the disease (n=9) in Figure 1B. Conclusion Acquired cytogenetic evolution is clinically ascertained by FISH during the course of the disease in approximately 12% of patients. The newly acquired defects in these patients result in a change in Dohner classification for in >90% of these patients including ∼37% who acquire del 17p13. The clinical implications of del 17p13 is influenced by the timing of ascertainment with markedly shorter survival for those who acquire del 17p13 during the course of the disease relative to those with this defect at diagnosis. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

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Surface IgM Levels Independently Influence Clinical Behavior and Associate with Altered Phenotype and Genetics in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v124.21.830.830 ◽

2014 ◽

Vol 124 (21) ◽

pp. 830-830

Author(s):

Samantha J Drennan ◽

Annalisa D'Avola ◽

Ian Tracy ◽

Isla Henderson ◽

Marta Larrayoz ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Kinase Inhibitors ◽

Cox Regression ◽

Conflicts Of Interest ◽

Dominant Role ◽

Critical Role ◽

Lymphocytic Leukemia ◽

Clinical Behavior ◽

Notch1 Mutations

Abstract The tumor B-cell receptor (BCR) is the key to survival and proliferation in chronic lymphocytic leukemia (CLL) and is now a therapeutic target of very effective BCR-associated kinase inhibitors. CLL cells are typically characterized by a variable state of anergy, defined by low surface IgM (sIgM) levels and signaling ability with consequent relatively low proliferation rate. The subset with unmutated IGHV (U-CLL) is generally less anergic with a poorer prognosis than that with mutated IGHV (M-CLL), and appears more sensitive to BCR-inhibitors (Byrd et al., NEJM, 2013). However it remains unclear if the variable anergy reflects the nature of the cell of origin and/or the functional state of the BCR, thereby determining clinical behavior. In this study we investigated if variations of BCR sIgM levels and signaling correlate with clinical behavior of CLL and assessed the phenotypic and genetic associations with the variations. The study included samples at diagnosis or prior to treatment from 222 patients with sIgM/D CLL diagnosed according to the iWCLL2008 criteria (99 months median follow up). sIgM levels on the CD19+/CD5+ CLL cells and signaling [% intracellular Ca2+ mobilization (iCa2+)] were determined using soluble F(ab’)2 polyclonal antibodies (Mockridge at al, Blood, 2007). sIgM levels were analyzed for their association with i) survival, ii) phenotype (CD38, ZAP70 and BCR regulators CD5, CD19, CD20, CD22) and iii) genetic lesions (Del13q, Trisomy12, Del11q/ATM, Del17p/TP53, or NOTCH1 and SF3B1 mutants). Time from diagnosis to progression requiring first treatment (TTFT) was used as primary endpoint, while overall survival (OS) was used as a secondary endpoint to avoid the influences of chemotherapy. Best cut-offs for progression requiring treatment were determined with ROC and Youden’s T-tests (treatment as a state variable). Levels of sIgM (5th-95th percentile 12-378, median 52) varied markedly between patients. Levels were higher at more advanced stage of disease and sIgM expression (MFI>56) and signaling (iCa2+>6%) associated with significantly more rapid TTFT. Although sIgD levels and signaling also associated with shorter TTFT, multivariate Cox regression adjusted for IGHV status, sIgM levels, sIgM signaling, sIgD levels and sIgD signaling revealed that only U-IGHV (p<0.001, 95% CI 1.64-3.97) and high sIgM levels (p=0.004, 95% CI 1.24-3.16) were independent predictors of shorter TTFT. Also, high sIgM levels and not high sIgD associated with shorter OS, despite chemotherapy. These data confirmed the specific influence of anergy which drives down sIgM and not sIgD, and is associated with less aggressive tumor behavior in CLL. By focusing on sIgM, a multivariate analysis adjusted for IGHV status, sIgM levels, CD38 and ZAP70 confirmed U-IGHV (p=0.002, 95% CI 1.35-3.56) and high sIgM (p=0.003, 95% CI 1.25-3.04) as the only independent predictors of shorter TTFT. Consistently, high sIgM levels associated with a more aggressive CLL even when U-CLL or M-CLL were investigated separately. Phenotypic analyses showed that high sIgM CLL had higher CD20, CD38, ZAP70, and CD22, than low sIgM CLL. Genetic analysis revealed that CLL subsets harboring Trisomy12 or Del17p/TP53 had significantly higher sIgM levels and signaling capacity than Del13q, even when U-CLL or M-CLL were analyzed separately. Trisomy12 U-CLL cases were enriched for NOTCH1 mutations. Consistently, U-CLL with NOTCH1 mutations (with or without Trisomy12) expressed higher sIgM than Del13q U-CLL. This study has several biological and clinical implications. First, it supports the critical role of sIgM in CLL-associated anergy. Our previous studies had shown dynamic variations of sIgM on circulating CLL cells following (auto)antigen engagement in tissue (Mockridge et al., Blood, 2007; Coelho et al. Blood, 2013). The subpopulation with higher sIgM is potentially the most dangerous and the most sensitive to kinase-inhibitors. Second, it documents the dominant role of sIgM levels on cell signaling and on tumor progression in CLL, with relevance even within U-CLL and M-CLL. Third, this study suggests influences of altered genetics on sIgM levels. However BCR-inhibitors overcome the influence of genetic lesions like Del17p/TP53. This points to a dominant role of reversed sIgM anergy in the behavior of CLL in vivo and claims the need of studies to verify sensitivity to kinase-inhibitors in CLL patients with different sIgM levels. Disclosures No relevant conflicts of interest to declare.

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Characterization of Long Non-Coding RNAs in Chronic Lymphocytic Leukemia: Evidence for Association with Disease Progression in Trisomy 12 Patients

Blood ◽

10.1182/blood.v124.21.3281.3281 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3281-3281

Author(s):

Renee C. Tschumper ◽

Tait D. Shanafelt ◽

Neil E. Kay ◽

Diane F. Jelinek

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Disease Progression ◽

Research Funding ◽

Clinical Course ◽

Lymphocytic Leukemia ◽

Lncrna Expression ◽

Trisomy 12 ◽

Non Coding Rnas

Abstract BACKGROUND: Chronic lymphocytic leukemia (CLL) is a heterogeneous B cell malignancy with patients being categorized into disease subsets based on several key biologic parameters, e.g., mutation status (mutated, M; or unmutated, UM) of the immunoglobulin heavy chain variable region (IGHV), acquired chromosomal abnormalities, and expression of CD38 and CD49d. Furthermore, about one third of CLL patients express stereotyped B cell receptors and/or may acquire high risk common mutations in genes such as NOTCH1 and SF3B1 suggesting ongoing genetic evolution as drivers of disease development. Critical to this concept, those CLL patients with trisomy 12 (T12) defects have a higher incidence of mutations in NOTCH1 and often have a stereotyped receptor. However, T12 patients may have a variable clinical course that appears to be unrelated to these 2 drivers suggesting an additional, possibly non-coding genetic component that may further impact disease progression in these patients. One potentially relevant genetic factor that could influence T12 clinical course is long non-coding RNAs (lncRNAs). LncRNAs are transcripts longer than 200 nucleotides that can affect a number of cellular processes. Importantly, lncRNAs have been implicated in various cancers including malignant hematopoiesis indicating they could be therapeutic targets and/or clinically useful biomarkers. METHODS: To pursue a role for lncRNAs in T12 we used v3.0 Arraystar Human LncRNA Microarrays to assess the global profile of lncRNA expression in CLL with an emphasis on patients with T12. Two cohorts of 6 patients with T12 were selected for comparison: one defined as progressive with a short time to treatment (TTT) (treatment ≤1 year after diagnosis) and one as indolent (no treatment > 5 years after diagnosis). Each cohort included 3 patients with M and 3 with UM IGHV status. RNA from normal CD5+ and CD5- B cells was included as a control. To compensate for the small sample size in each cohort, a significant difference in lncRNA expression between the groups was defined as a fold change (FC) ≥5.0, p-value ≤0.05 and false discovery rate (FDR) ≤ 0.05. RESULTS: An initial global comparison of CD5+/CD5- normal B cells vs all CLL samples found that 609 lncRNAs were differentially expressed using the criteria listed above with 158 lncRNAs having a FC>10. Notable lncRNAs in this group included: LOC541472 (down in CLL and associated with the IL-6 gene), D63785 (up in CLL and associated with TBC1D3C, an oncoprotein), CTC-459I6.1 (up in CLL and associated with RASGRF2) and AC002480.5 (down in CLL and associated with STEAP1B, shown to be overexpressed in prostate cancer). We next evaluated T12 samples and identified 90 candidate lncRNAs that may discriminate between progressive and indolent T12 cases. Within this group were 11 lncRNAs with a FC > 10, 5 of which have no known associated gene. Of those associated with known genes, 3 were ultra-conserved region encoding lncRNAs down-regulated in progressive T12 patients (TTT ≤1 yr) and linked to hephaestin-like protein 1 precursor, pannexin-1, and tubulin beta-3 chain isoform 1. Of potential high relevance we found that the lncRNA LPP-AS1 was down-regulated in progressive T12 patients (TTT ≤1 yr) and known to be associated with the LIM-containing lipoma preferred partner (LPP) gene (p=0.028; FDR=0.03 and FC=18.3). Looking specifically at IGHV M progressive T12 patients (T12M≤1 yr) vs IGHV M indolent T12 patients (T12M>5 yrs), we again found the LPP-AS1 lncRNA was highly down-regulated in T12M≤1 (p=0.00046; FDR=0.006 and FC=34.5) but it was not found to be differentially expressed in the UM T12≤1 yr vs UM T12>5 yr comparison. The LPP gene has been shown to play a role in cell-cell adhesion, motility and signaling, and is often the fusion partner for the mixed lineage leukemia (MLL) gene in secondary acute leukemia. Furthermore, LLP may play a role in breast cancer cell invasion. LPP-AS1 may be participating in IGHV M T12 progression by affecting LPP and thus influencing migration through the lymph node microenvironment. CONCLUSION: While candidate lncRNAs in T12 CLL need to be validated, the LPP-AS1 lncRNA shows promise as a possible marker and potential treatment target for those patients with T12 and M IGHV that may progress rapidly. Further studies are needed to evaluate the impact of lncRNAs on clinical outcome of T12 CLL patients. Disclosures Shanafelt: Hospiria: Research Funding; Pharmacyclics/Jannsen: Research Funding; Cephalon: Research Funding; Celgene: Research Funding; glaxoSmithKline: Research Funding; Genetech: Research Funding; Polyphenon E Int'l: Research Funding. Kay:Celgene: Research Funding.

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ZAP-70 directly enhances IgM signaling in chronic lymphocytic leukemia

Blood ◽

10.1182/blood-2004-05-1715 ◽

2005 ◽

Vol 105 (5) ◽

pp. 2036-2041 ◽

Cited By ~ 170

Author(s):

Liguang Chen ◽

John Apgar ◽

Lang Huynh ◽

Frank Dicker ◽

Teresa Giago-McGahan ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Heavy Chain ◽

Variable Region ◽

Adenovirus Vector ◽

Lymphocytic Leukemia ◽

Clinical Behavior ◽

Heavy Chain Variable Region ◽

Variable Region Genes ◽

Aggressive Clinical Behavior

Abstract Chronic lymphocytic leukemia (CLL) B cells that express unmutated immunoglobulin heavy-chain variable region genes (IgVH) generally express ZAP-70, in contrast to normal B cells or most CLL cases with mutated IgVH. Following IgM ligation, ZAP-70+ CLL cells had significantly higher levels of phosphorylated p72Syk, BLNK, and phospholipase-Cγ (PLCγ) and had greater[Ca2+]i flux than did ZAP-70–negative CLL cases, including unusual ZAP-70–negative cases with unmutated IgVH. IgM ligation of ZAP-70–negative CLL B cells infected with an adenovirus vector encoding ZAP-70 induced significantly greater levels of phosphorylated p72Syk, BLNK, and PLCγ and had greater[Ca2+]i flux than did similarly stimulated, noninfected CLL cells or CLL cells infected with a control adenovirus vector. We conclude that expression of ZAP-70 in CLL allows for more effective IgM signaling in CLL B cells, a feature that could contribute to the relatively aggressive clinical behavior generally associated with CLL cells that express unmutated IgVH.

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THE ROLE OF THE GENETIC ABNORMALITIES, EPIGENETIC AND microRNA IN THE PROGNOSIS OF CHRONIC LYMPHOCYTIC LEUKEMIA

Experimental Oncology ◽

10.31768/2312-8852.2018.40(4):261-267 ◽

2018 ◽

Vol 40 (4) ◽

pp. 261-267 ◽

Cited By ~ 5

Author(s):

K Tari ◽

Z Shamsi ◽

H Reza Ghafari ◽

A Atashi ◽

M Shahjahani ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Kinase Inhibitors ◽

Bone Marrow Involvement ◽

P53 Mutation ◽

Lymphocytic Leukemia ◽

Gene Promoters ◽

Epigenetic Changes ◽

Genetic Changes ◽

Trisomy 12

Chronic lymphocytic leukemia (CLL) is increased proliferation of B-cells with peripheral blood and bone marrow involvement, which is usually observed in older people. Genetic mutations, epigenetic changes and miRs play a role in CLL pathogenesis. Del 11q, del l17q, del 6q, trisomy 12, p53 and IgVH mutations are the most important genetic changes in CLL. Deletion of miR-15a and miR-16a can increase bcl2 gene expression, miR-29 and miR-181 deletions decrease the expression of TCL1, and miR-146a deletion prevents tumor metastasis. Epigenetic changes such as hypo- and hypermethylation, ubiquitination, hypo- and hyperacetylation of gene promoters involved in CLL pathogenesis can also play a role in CLL. Expression of CD38 and ZAP70, presence or absence of mutation in IgVH and P53 mutation are among the factors involved in CLL prognosis. Use of monoclonal antibodies against surface markers of B-cells like anti-CD20 as well as tyrosine kinase inhibitors are the most important therapeutic approaches for CLL.

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