In vivo intraclonal and interclonal kinetic heterogeneity in B-cell chronic lymphocytic leukemia

Abstract Clonal evolution and outgrowth of cellular variants with additional chromosomal abnormalities are major causes of disease progression in chronic lymphocytic leukemia (CLL). Because new DNA lesions occur during S phase, proliferating cells are at the core of this problem. In this study, we used in vivo deuterium (2H) labeling of CLL cells to better understand the phenotype of proliferating cells in 13 leukemic clones. In each case, there was heterogeneity in cellular proliferation, with a higher fraction of newly produced CD38+ cells compared with CD38− counterparts. On average, there were 2-fold higher percentages of newly born cells in the CD38+ fraction than in CD38− cells; when analyzed on an individual patient basis, CD38+2H-labeled cells ranged from 6.6% to 73%. Based on distinct kinetic patterns, interclonal heterogeneity was also observed. Specifically, 4 patients exhibited a delayed appearance of newly produced CD38+ cells in the blood, higher leukemic cell CXC chemokine receptor 4 (CXCR4) levels, and increased risk for lymphoid organ infiltration and poor outcome. Our data refine the proliferative compartment in CLL based on CD38 expression and suggest a relationship between in vivo kinetics, expression of a protein involved in CLL cell retention and trafficking to solid tissues, and clinical outcome.

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Prognostic Significance of PET/CT in Patients with Chronic Lymphocytic Leukemia (CLL) Treated with Frontline Chemoimmunotherapy

Cancers ◽

10.3390/cancers12071773 ◽

2020 ◽

Vol 12 (7) ◽

pp. 1773 ◽

Cited By ~ 1

Author(s):

Marika Porrazzo ◽

Emanuele Nicolai ◽

Mara Riminucci ◽

Candida Vitale ◽

Marta Coscia ◽

...

Keyword(s):

Multivariate Analysis ◽

Chronic Lymphocytic Leukemia ◽

Negative Impact ◽

Prognostic Significance ◽

Lymphocytic Leukemia ◽

Independent Effect ◽

Proliferating Cells ◽

Richter Syndrome ◽

Increased Risk ◽

Pet Ct

The role of positron emission tomography/computed tomography (PET/CT) in identifying Richter Syndrome (RS) is well established, while its impact on the survival of patients with chronic lymphocytic leukemia (CLL) has been less explored. The clinical characteristics and PET/CT data of 40 patients with a biopsy-proven CLL who required frontline chemoimmunotherapy, FCR (fludarabine, cyclophosphamide, rituximab) in 20 patients, BR (bendamustine, rituximab) in 20, were retrospectively analyzed. Standardized uptake volume (SUVmax) values ≥ 5 were observed more frequently in patients with deletion 11q (p = 0.006) and biopsies characterized by a rate of Ki67 positive cells ≥ 30% (p = 0.02). In the multivariate analysis, the presence of large and confluent PCs emerged as the only factor with a negative impact on progression-free survival (PFS), and overall survival (OS). Deletion 11q also revealed a significant and independent effect on PFS. SUVmax values ≥ 5 showed no statistical impact on PFS while in multivariate analysis, they revealed a significant adverse impact on OS (median survival probability not reached vs. 56 months; p = 0.002). Moreover, patients with higher SUVmax values more frequently developed Richter Syndrome (p = 0.015). Our results show that higher SUVmax values identify CLL patients with a pronounced rate of proliferating cells in the lymph-node compartment, inferior survival, and an increased risk of developing RS.

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In Vivo Labeling of Dividing Chronic Lymphocytic Leukemia B Cells Suggests That CXCR4 and CD5 Define the Clonal Subfraction That Recently Proliferatead and Emigrated from a Solid Tissue

Blood ◽

10.1182/blood.v112.11.358.358 ◽

2008 ◽

Vol 112 (11) ◽

pp. 358-358 ◽

Cited By ~ 1

Author(s):

Carlo calissano ◽

Rajendra Damle ◽

Gregory Hayes ◽

Elisabeth J Murphy ◽

Marc Hellerstein ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Cell Contact ◽

Proliferating Cells ◽

Solid Tissue ◽

Group A ◽

Group B ◽

Time Point

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is due, in most of the cases, to the slow progressive invasion of phenotypically homogeneous monoclonal B cells. Several studies are clarifying that B-CLL cells preferentially proliferate in the context of lymphoid solid tissues where they receive accessory signals (chemokines, cytokines, cell to cell contact). Deuterium (2H) incorporation into newly synthesized DNA can be used as a direct in vivo measure of cell division. Using this technique in B-CLL, it has been found that although the fraction of cells dividing each day is small (0.1 – ~2% of the clones), the absolute number of proliferating cells is sizeable (~109 – 1012/day). The study also elucidated a relationship between proliferation and disease activity. By using this same approach, we have tried to refine our understanding of this small percentage of proliferating cells. Thirteen patients drank deuterated water (2H2O) for 6–12 weeks (“labeling period”) and were followed for 24 weeks (“washout period”). Blood was drawn during and at the end of labeling (1st, 2nd time points) and at the end of washout (3rd time point). Based upon proliferation kinetics patients could be divided into 2 groups (Group A and Group B). Four/13 patients (Group A) showed unusual kinetic curves with a strikingly delayed appearance of labeled cells in the blood as compared to Group B patients. This suggested that B-CLL cells of these patients were retained longer at the site of proliferation. Therefore, the two groups were compared for expression of a panel of chemokine receptors (CCR1, CCR2, CCR4, CCR5, CCR7, CXCR1, CXCR3, CXCR4, and CXCR5) by flow cytometry. Only CXCR4 was found at significant higher densities in Group A compared to Group B (P = 0.002). CXCR4 expression was then used to identify B-CLL sub-populations enriched in cells displaying Ki67 and MCM6, two cell cycle related markers. We observed a relationship between CXCR4 and CD5 where CXCR4dim cells tended to be CD5bright and vice-versa with the majority of the cells falling in an intermediate category (CXCR4intCD5int). The CXCR4dimCD5bright subpopulation contained significantly more Ki67+ and MCM6+ cells. Based on this observation, B-CLL cells of 9 patients were sorted into CXCR4brightCD5dim, CXCR4intCD5int, CXCR4dimCD5bright and 2H was measured for each fraction. A hierarchy of 2H enrichment in the fractions was found: CXCR4dimCD5bright > CXCR4intCD5int >CXCR4brightCD5dim. These differences were statistically significant during labelling period (1st and 2nd time point) but completely disappeared at the end of washout (3rd time point). The 2H enrichment measured in the unfractionated clones at the 1st, 2nd and 3rd time point was similar (0.45±0.15 vs. 0.53±0.16 vs. 0.45±0.07, P = ns), suggesting a redistribution of label among the fractions, presumably due to a change of phenotype over time. In vitro studies have suggested that CXCR4 has a crucial role in trafficking and survival of B-CLL cells, and it is emerging as a powerful predictor for bone marrow infiltration and clinical evolution. CXCR4 surface expression is downregulated after encountering its ligand SDF-1. Our data indicate a relationship between CXCR4 levels and B-CLL cells kinetics. Moreover, since CLL cell subpopulations defined by relative CXCR4 and CD5 levels differ in the number of proliferating cells, we suggest that these markers ca be used to define cells that have emigrated recently (CXCR4dimCD5bright) or much earlier (CXCR4brightCD5dim) from the solid tissue in which they divided. The results strongly suggest, in vivo, the important role of the stroma and SDF-1-producing cells for B-CLL cell proliferation providing indications for disease control strategies.

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Fluorescent in situ hybridization and cytogenetic studies of trisomy 12 in chronic lymphocytic leukemia

Blood ◽

10.1182/blood.v81.10.2702.2702 ◽

1993 ◽

Vol 81 (10) ◽

pp. 2702-2707 ◽

Cited By ~ 68

Author(s):

SM Escudier ◽

JM Pereira-Leahy ◽

JW Drach ◽

HU Weier ◽

AM Goodacre ◽

...

Keyword(s):

In Situ Hybridization ◽

Chronic Lymphocytic Leukemia ◽

Median Survival ◽

Chromosomal Abnormalities ◽

Residual Disease ◽

Clonal Evolution ◽

Lymphocytic Leukemia ◽

Cytogenetic Studies ◽

Trisomy 12

Abstract Cytogenetic studies (CG) of 475 chronic lymphocytic leukemia (CLL) cases showed trisomy 12 in 6.1% or 26% of patients with abnormal karyotypes. Fluorescence in situ hybridization (FISH) detected trisomy 12 in 35% of 117 CLL patients. Only 34.6% of cases detected by FISH were detected by CG. Twelve patients had low levels of trisomic cells (4% to 11%) relative to clonal B cells (47.5% to 86%), suggestive of clonal evolution. Untreated patients with trisomy 12 were predominantly male (P < .05) and had an increased incidence of splenomegaly (P < .03). Patients with trisomy 12 were more likely to be previously treated and had advanced Binet stage compared with those without trisomy 12. The median survival was shorter in patients with trisomy 12 (7.8 years) and patients with other chromosomal abnormalities without trisomy 12 by FISH (5.5 years) than in patients with diploid karyotypes (14.4 years). The response to fludarabine was similar to that of patients with diploid karyotypes, but there was a trend for earlier disease progression. FISH detected residual disease in all patients with trisomy 12 in complete (n = 6) or partial remission (n = 4). As few as 1 trisomic cell in 5,000 was detected by performing FISH on fluorescence-activated cell sorter-sorted cells. Trisomy 12 was absent in T cells in patients with trisomy 12. We conclude that FISH identifies trisomy 12 approximately 2.6 times more often than CG, readily identifies minimal residual disease, and predicts for a shorter median survival.

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In Vivo Labeling of Newly Synthesized DNA Suggests That the CD38+ Fraction Is Enriched in Proliferating Cells within a Clone of Chronic Lymphocytic Leukemia B Cells.

Blood ◽

10.1182/blood.v108.11.27.27 ◽

2006 ◽

Vol 108 (11) ◽

pp. 27-27 ◽

Cited By ~ 3

Author(s):

Carlo Calissano ◽

Rajendra Damle ◽

Taraneh Banapour ◽

Denise Cesar ◽

Marc Hellerstein ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Gas Chromatography Mass Spectrometry ◽

Deuterated Water ◽

Proliferating Cells ◽

Cd38 Expression ◽

Accessory Cells ◽

The Difference

Abstract B-cell chronic lymphocytic leukemia (B-CLL) has been considered for years as a slowly progressive invasion of phenotypically homogeneous, monoclonal B cells that divide rarely and accumulate because of a primary apoptotic defect. Recent studies are clarifying that the leukemic clone can be comprised of subpopulations of cells expressing different surface markers suggestive of intraclonal diversity. Indeed, it is now widely accepted that activation signals (i.e., antigenic stimulation, interaction with accessory cells, cytokines) can push B-CLL cells into the cell cycle or rescue them from apoptosis. Unanswered questions are whether all or only a subset of cells in the clone participates in these proliferative events and to what extent those cells that have divided can be identified. We have tried to answer this by labeling those cells that divide in vivo with the non-radioactive stable isotope deuterium (2H) in the form of deuterated water. Nine B-CLL patients with leukemic clones comprising 1.7 – 97.8% CD38+ cells drank daily, for 12 weeks, an aliquot of 2H2O. CD19+CD5+CD38+ and CD19+CD5+CD38− cells were isolated from PBMC drawn after 8 weeks (labeling period) and 24 weeks (washout period) and the incorporation of 2H into equal amounts of genomic DNA was measured by gas chromatography/mass spectrometry. At week 8, the mean 2H enrichment in the CD38+ population was higher than in the CD38− fraction (0.862 ± 0.24 vs. 0.445 ± 0.13; P = 0.034). Enhanced 2H enrichment in the CD38+ population was detected in every patient with CD38+/CD38− ratios ranging from 1.15 to 5.58. In 4 patients, the CD38+/CD38− ratio was above 2.0. In contrast, at week 24, 12 weeks after the cessation of 2H2O intake, 2H enrichment in DNA in the CD38+ vs. the CD38− population had changed. In 7 of 9 patients, the CD38+/CD38− enhancement ratios diminished, remaining essentially constant for the other 2 patients. The difference of CD38+/CD38− mean ratios at week 8 and week 24 was significant (2.254 ± 0.12 vs. 1.124 ± 0.13; P = 0.035). Moreover, the incorporation of 2H into CD38+ cells fell in 6 of 9 patients, with reciprocal increases in label incorporation in the CD38− cells for 8 of 9 patients. Thus, the differences in 2H incorporation into DNA at weeks 8 and 24 suggest either that the life spans of the two subpopulations differ (i.e., CD38+ cells may be shorter lived) or that expression of CD38 may be a dynamic process in vivo changing from positive to negative in individual cells over time after replication. These in vivo findings indicate that, within the same B-CLL patient, the leukemic subpopulation that expresses CD38 contains more cells that have recently undergone cell division than the CD38− subpopulation. This may relate to the clinical correlation of CD38 expression with poor clinical outcome. The 2H2O labeling technique provides a safe in vivo method to further fractionate the proliferative leukemic compartment as a means to better understand the pathophysiology of B-CLL and possibly provide an important target for therapy.

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Evolving View of the In-Vivo Kinetics of Chronic Lymphocytic Leukemia B Cells

Hematology ◽

10.1182/asheducation-2006.1.273 ◽

2006 ◽

Vol 2006 (1) ◽

pp. 273-278 ◽

Cited By ~ 10

Author(s):

Nicholas Chiorazzi ◽

Manlio Ferrarini

Keyword(s):

Cell Death ◽

Chronic Lymphocytic Leukemia ◽

Programmed Cell Death ◽

Clinical Course ◽

Lymphocytic Leukemia ◽

Proliferating Cells ◽

In Vivo Kinetics ◽

Kinetics Of ◽

Cell Chronic Lymphocytic Leukemia

Abstract B-cell chronic lymphocytic leukemia (B-CLL) has long been considered a disease of “accumulation,” due to a presumed defect in programmed cell death. Recent data, however, suggest that B-CLL cells are born at a normal to an accelerated rate, with the rate of proliferation varying among patients. In addition, differences in birth rates, activation state, and inducibility appear to exist among subpopulations of cells within individual leukemic clones. The extent to which such dissimilarities influence clinical course and outcome is still unclear. This review examines the evidence supporting the existence of a proliferative compartment in B-CLL and the role that proliferating cells might play in the progression and evolution of this disease.

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Prospective Evaluation of Clonal Evolution During Long-Term Follow-Up of Patients With Untreated Early-Stage Chronic Lymphocytic Leukemia

Journal of Clinical Oncology ◽

10.1200/jco.2006.06.9492 ◽

2006 ◽

Vol 24 (28) ◽

pp. 4634-4641 ◽

Cited By ~ 175

Author(s):

Tait D. Shanafelt ◽

Thomas E. Witzig ◽

Stephanie R. Fink ◽

Robert B. Jenkins ◽

Sarah F. Paternoster ◽

...

Keyword(s):

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

Chromosomal Abnormalities ◽

Early Stage ◽

Clonal Evolution ◽

Prospective Trial ◽

Lymphocytic Leukemia ◽

Cytogenetic Abnormalities ◽

Long Term Follow Up

Purpose Retrospective studies suggest cytogenetic abnormalities detected by interphase fluorescent in situ hybridization (FISH) can identify patients with chronic lymphocytic leukemia (CLL) who will experience a more aggressive disease course. Other studies suggest that patients may acquire chromosome abnormalities during the course of their disease. There are minimal prospective data on the clinical utility of the widely used hierarchical FISH prognostic categories in patients with newly diagnosed early-stage CLL or the frequency of clonal evolution as determined by interphase FISH. Patients and Methods Between 1994 and 2002, we enrolled 159 patients with previously untreated CLL (83% Rai stage 0/I) on a prospective trial evaluating clonal evolution by FISH. Patients provided baseline and follow-up specimens for FISH testing during 2 to 12 years. Results Chromosomal abnormalities detected by FISH at study entry predicted overall survival. Eighteen patients experienced clonal evolution during follow-up. The rate of clonal evolution increased with duration of follow-up with only one occurrence in the first 2 years (n = 71; 1.4%) but 17 occurrences (n = 63; 27%) among patients tested after 5+ years. Clonal evolution occurred among 10% of ZAP-70–negative and 42% of ZAP-70–positive patients at 5+ years (P = .008). Conclusion This clinical trial confirms prospectively that cytogenetic abnormalities detected by FISH can predict overall survival for CLL patients at the time of diagnosis, but also suggests that many patients acquire new abnormalities during the course of their disease. Patients with higher ZAP-70 expression may be more likely to experience such clonal evolution. These findings have important implications for both clinical management and trials of early treatment for patients with high-risk, early-stage CLL.

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Clonal Evolution in Chronic Lymphocytic Leukemia: Acquisition of High-Risk Genomic Aberrations Associated with Unmutated VH, Resistance to Therapy, and Short Survival.

Blood ◽

10.1182/blood.v108.11.296.296 ◽

2006 ◽

Vol 108 (11) ◽

pp. 296-296 ◽

Cited By ~ 1

Author(s):

Stephan Stilgenbauer ◽

Sandrine Sander ◽

Lars Bullinger ◽

Axel Benner ◽

Elke Leupolt ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Clonal Evolution ◽

Lymphocytic Leukemia ◽

Single Institution ◽

Need For Treatment ◽

Rate Of Progression ◽

Genomic Aberrations ◽

Adverse Factor

Abstract Genomic aberrations were analyzed by fluorescence in situ hybridization (FISH) at various time points during the disease course of 64 patients with chronic lymphocytic leukemia (CLL) from a single institution. After a median time of 42 (23–73) months, 11 of the 64 (17%) patients acquired the following additional aberrations: del(17p13) (n=4), del(6q21) (n=3), del(11q23) (n=2), +(8q24) (n=1), and evolution from monoallelic to biallelic del(13q14) (n=3). The baseline clinical characteristics of the patients with and without clonal evolution were not significantly different. Remarkably, clonal evolution occurred exclusively among cases with unmutated VH status (Figure 1). The group with clonal evolution showed a higher rate of progression in stage (82% vs. 28%), a greater need for treatment (100% vs. 62%), and a higher death rate (67% vs. 28%). The median survival time after the occurrence of clonal evolution was 22 months. Expansion of the clone with del(17p13) was observed in all patients during chemotherapy (chlorambucil n=4, fludarabine n=3, FC n=2, rituximab n=1) indicating in-vivo resistance to treatment (Figure 2). In multivariate analysis, clonal evolution was identified as independent adverse factor with regard to overall survival. Figure 1 Figure 1. Figure 2 Figure 2.

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Genetic Abnormalities in Chronic Lymphocytic Leukemia: Where We Are and Where We Go

BioMed Research International ◽

10.1155/2014/435983 ◽

2014 ◽

Vol 2014 ◽

pp. 1-13 ◽

Cited By ~ 62

Author(s):

Anna Puiggros ◽

Gonzalo Blanco ◽

Blanca Espinet

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Chromosomal Abnormalities ◽

Clonal Evolution ◽

Gene Mutations ◽

Therapeutic Strategies ◽

Lymphocytic Leukemia ◽

Wide Range ◽

Trisomy 12 ◽

Genomic Aberrations ◽

Next Generation Sequencing Ngs

Chromosomal abnormalities in chronic lymphocytic leukemia (CLL) are detected in up to 80% of patients. Among them, deletions of 11q, 13q, 17p, and trisomy 12 have a known prognostic value and play an important role in CLL pathogenesis and evolution, determining patients outcome and therapeutic strategies. Standard methods used to identify these genomic aberrations include both conventional G-banding cytogenetics (CGC) and fluorescencein situhybridization (FISH). Although FISH analyses have been implemented as the gold standard, CGC allows the identification of chromosomal translocations and complex karyotypes, the latest associated with poor outcome. Genomic arrays have a higher resolution that allows the detection of cryptic abnormalities, although these have not been fully implemented in routine laboratories. In the last years, next generation sequencing (NGS) methods have identified a wide range of gene mutations (e.g.,TP53, NOTCH1, SF3B1,andBIRC3) which have improved our knowledge about CLL development, allowing us to refine both the prognostic subgroups and better therapeutic strategies. Clonal evolution has also recently arisen as a key point in CLL, integrating cytogenetic alterations and mutations in a dynamic model that improve our understanding about its clinical course and relapse.

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Fluorescent in situ hybridization and cytogenetic studies of trisomy 12 in chronic lymphocytic leukemia

Blood ◽

10.1182/blood.v81.10.2702.bloodjournal81102702 ◽

1993 ◽

Vol 81 (10) ◽

pp. 2702-2707 ◽

Cited By ~ 1

Author(s):

SM Escudier ◽

JM Pereira-Leahy ◽

JW Drach ◽

HU Weier ◽

AM Goodacre ◽

...

Keyword(s):

In Situ Hybridization ◽

Chronic Lymphocytic Leukemia ◽

Median Survival ◽

Chromosomal Abnormalities ◽

Residual Disease ◽

Clonal Evolution ◽

Lymphocytic Leukemia ◽

Cytogenetic Studies ◽

Trisomy 12

Cytogenetic studies (CG) of 475 chronic lymphocytic leukemia (CLL) cases showed trisomy 12 in 6.1% or 26% of patients with abnormal karyotypes. Fluorescence in situ hybridization (FISH) detected trisomy 12 in 35% of 117 CLL patients. Only 34.6% of cases detected by FISH were detected by CG. Twelve patients had low levels of trisomic cells (4% to 11%) relative to clonal B cells (47.5% to 86%), suggestive of clonal evolution. Untreated patients with trisomy 12 were predominantly male (P < .05) and had an increased incidence of splenomegaly (P < .03). Patients with trisomy 12 were more likely to be previously treated and had advanced Binet stage compared with those without trisomy 12. The median survival was shorter in patients with trisomy 12 (7.8 years) and patients with other chromosomal abnormalities without trisomy 12 by FISH (5.5 years) than in patients with diploid karyotypes (14.4 years). The response to fludarabine was similar to that of patients with diploid karyotypes, but there was a trend for earlier disease progression. FISH detected residual disease in all patients with trisomy 12 in complete (n = 6) or partial remission (n = 4). As few as 1 trisomic cell in 5,000 was detected by performing FISH on fluorescence-activated cell sorter-sorted cells. Trisomy 12 was absent in T cells in patients with trisomy 12. We conclude that FISH identifies trisomy 12 approximately 2.6 times more often than CG, readily identifies minimal residual disease, and predicts for a shorter median survival.

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Clinico-Biological Implications of Modified Levels of Cytokines in Chronic Lymphocytic Leukemia: A Possible Therapeutic Role

Cancers ◽

10.3390/cancers12020524 ◽

2020 ◽

Vol 12 (2) ◽

pp. 524 ◽

Cited By ~ 3

Author(s):

Alessandro Allegra ◽

Caterina Musolino ◽

Alessandro Tonacci ◽

Giovanni Pioggia ◽

Marco Casciaro ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cellular Proliferation ◽

Innate Immune ◽

Lymphocytic Leukemia ◽

Adaptive Immune ◽

Cytokine Balance ◽

Proliferation And Apoptosis ◽

History Of ◽

Leukemic Clone

B-cell chronic lymphocytic leukemia (B-CLL) is the main cause of mortality among hematologic diseases in Western nations. B-CLL is correlated with an intense alteration of the immune system. The altered functions of innate immune elements and adaptive immune factors are interconnected in B-CLL and are decisive for its onset, evolution, and therapeutic response. Modifications in the cytokine balance could support the growth of the leukemic clone via a modulation of cellular proliferation and apoptosis, as some cytokines have been reported to be able to affect the life of B-CLL cells in vivo. In this review, we will examine the role played by cytokines in the cellular dynamics of B-CLL patients, interpret the contradictions sometimes present in the literature regarding their action, and evaluate the possibility of manipulating their production in order to intervene in the natural history of the disease.

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