Jumping Translocations are Recurrent Abnormalities Associated with Genetic Instability and an Aggressive Disease State in Chronic Lymphocytic Leukemia

Transcription

1 Jumping Translocations are Recurrent Abnormalities Associated with Genetic Instability and an Aggressive Disease State in Chronic Lymphocytic Leukemia Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Cecelia R. Miller Graduate Program in Allied Medical Professions The Ohio State University 2012 Master's Examination Committee: Tammy Bannerman, Advisor Kathy Waller Huey-Jen Lin Nyla A. Heerema

2 Copyright by Cecelia R. Miller 2012

3 Abstract Jumping translocation (JT) is a rare cytogenetic aberration that can occur in hematologic malignancy. It involves the translocation of the same fragment of donor chromosome onto two or more recipient chromosomes typically in different cell lines. Here, for the first time, JTs are established as a type of recurrent abnormality occurring in chronic lymphocytic leukemia (CLL). The cytogenetic characteristics of JTs in CLL, the characteristics of the patient population where they are seen, and correlations with other clinicopathological findings are addressed. Twenty-six patients were identified with JTs at some period during their care. There were a total of 32 donor breakpoints with six patients exhibiting two unique donor breakpoints involved in JTs at the same time. Strikingly, 15 of the donor breakpoints were in 17p11.2. A total of 93 JTs were identified. Forty-one of these JTs resulted in dicentric or pseudodicentric chromosomes, 31 of which had 17p11.2 donor breakpoints. Patients with JTs in CLL appear to develop the disease at an early age and experience an aggressive disease state. These patients develop complex karyotypes and frequently show loss of the tumor suppressor gene TP53, both of which are considered poor prognostic indicators. The biology of JT formation is unclear and warrants further investigation. ii

4 Dedication To David you have my heart. iii

5 Acknowledgments To the entire MLS division, your Coordinated Graduate Program has far exceeded all my expectations and I am proud to have been your student. Dr. Lin, thank you for being a part of this project, for all your advice, and for helping me learn to teach others. Dr. Waller, thank you for the guidance to get this project started, and for all your advice, teachings, and support along the way. Dr. Heerema, thank you for being a mentor to me and for all the guidance you ve given me. Your enthusiasm for cytogenetics has been inspirational. Dr. Bannerman, well what can I say? It looks like we made it through after all! It has been a wonderful experience and I m glad I had you to support me along the way. To my mother, father, and brothers, thank you for all the love and support you have given over the years. To my husband, David, thank you for having dinner ready the nights I had class late and for getting coffee going in the morning. All those little things you do have really helped me along the way. There are no words sufficient to describe how much your support means to me. iv

6 Vita A.A. Health, University of Rio Grande B.S. Microbiology The Ohio State University MLS Coordinated Master s Program, The Ohio State University Fields of Study Major Field: Allied Medical Professions v

7 Table of Contents Page Abstract...ii Dedication...iii Acknowledgements...iv Vita...v List of Tables...viii List of Figures...ix Chapter 1: Introduction...1 Background and Significance of the Problem...1 Objectives...3 Research Questions...3 Definition of Terms...3 Limitations...4 Chapter 2: Review of Literature...6 Chronic Lymphocytic Leukemia...6 Cytogenetic Abnormalities in CLL...7 Jumping Translocations Chapter 3: Methodology...12 Culture and Harvesting Techniques...12 Conventional Cytogenetic Analyses...13 Fluorescence in situ hybridization...13 Chapter 4: Jumping translocations are recurrent abnormalities associated with genetic instability and an aggressive disease state in chronic lymphocytic leukemia...15 Abstract...15 Introduction...16 Methods...17 Results...19 Discussion...22 Acknowledgements...27 vi

8 Authorship...27 References...28 Chapter 5: Summary and Conclusions...42 Results Summary...42 Extended Discussion...42 Implications for Further Research...45 References...47 Appendix Appendix A Karyotypes of patients with jumping translocations s in CLL...56 vii

9 List of Tables Table 1. Jumping translocation breakpoints for the 17p11.2 donor group...32 Table 2. Jumping translocation breakpoints for the miscellaneous donor group...35 Table 3. Clinicopathological data for patients with jumping translocations in CLL...38 Table A.1. Karyotypes of patients with jumping translocations in CLL.56 viii

10 List of Figures Figure 1. Partial karyotypes showing JTs seen in CLL ix

11 Chapter 1 Introduction Background and Significance of the Problem Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western World. Chronic lymphocytic leukemia generally is considered an indolent disease; however, the clinical course of the disease is highly variable. While many patients do not require treatment for years others exhibit aggressive disease and have a poor prognosis (Byrd et al. 2004). Various clinical and biological parameters are used to predict disease aggressiveness; including clinical stage, expression of CD38 and ZAP70, unmutated IgVH, and cytogenetics (Chiorazzi et al. 2005). Chromosomal abnormalities identified by cytogenetic analysis are one important prognostic factor in the risk stratification of CLL. Currently cytogenetic analysis is predominately performed on interphase cells using a fluorescent in situ hybridization (FISH) panel that targets the most common abnormalities seen in CLL. With this technique it has been shown that approximately 80% of patients will have an abnormality (Dohner et al. 2000). This FISH panel is limited to detecting only abnormalities occurring in chromosome regions complementary to the DNA probes used. Importantly, using a FISH panel as the sole cytogenetic diagnostic tool is often insufficient to detect a complex karyotype, a poor prognostic indicator 1

12 (Haferlach et al. 2007). In addition, it has been demonstrated that chromosomal abnormalities are frequently detected by conventional cytogenetic analysis in patients with normal FISH results (Haferlach et al. 2007; Rigolin et al. 2012). Historically, identification of chromosomal aberrations by traditional metaphase cytogenetics has been limited by the low mitotic activity of CLL cells in culture. The recent introduction of CpG oligodeoxynucleotides (CpG-ODN) to CLL cultures has significantly improved the ability to obtain informative consistent karyotypic results from patients with CLL (Dicker et al. 2006; Heerema et al. 2010). Chromosomal abnormalities are reported to be reliable prognostic indicators in CLL (Juliusson et al. 1990). When recurrent abnormalities are found it suggests that the aberrant chromosome region may contain genes of interest for studying the genetic basis of the leukemia. Due to the difficulties in culturing CLL tumor cells and standard use of a FISH panel for patient cytogenetic analysis there is a lack of data characterizing additional abnormalities found in these patients. Jumping translocations are rare cytogenetic abnormalities seen constitutionally and in hematologic malignancy. Jumping translocations have been reported most frequently in multiple myeloma, followed by acute myeloid leukemia and acute lymphoblastic leukemia (Berger and Bernard 2007). There has been a single report of a JT in CLL (Callet-Bauchu et al. 1999). Jumping translocations are often secondary aberrations considered indicative of genomic instability (Batanian et al. 1998). JTs have been associated with aggressive disease (Manola et al. 2008; Najfeld et al. 2010) and poor prognosis (Fan et al. 2000; Shippey et al. 1990; Shikano et al. 1993). In addition, the 2

13 biology of this type of abnormality is not well understood and requires further investigation. Objectives There has been only one report of a jumping translocation in CLL described in the literature. This study will establish that JTs are a recurrent abnormality in CLL and describe new information regarding the potential significance of this type of abnormality in CLL. This study will also contribute significant data on the subject of JTs, which currently has limited data available. Research Questions What are the cytogenetic characteristics of jumping translocations in CLL? What are the characteristics of patient population where these jumping translocations are seen? Does the presence of jumping translocations show any correlation with other clinicopathological findings? Definition of Terms Cytogenetics - The study of chromosomes. Clinical cytogenetics is concerned with the relationship between chromosomal abnormalities and pathologic conditions. Fluorescent in situ hybridization (FISH) - a cytogenetic technique used to detect and localize the presence or absence of specific DNA sequences on chromosomes using 3

14 fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence complementarity. Chronic lymphocytic leukemia (CLL) A neoplastic disease characterized by the proliferation and accumulation of morphologically mature but immunologically dysfunctional B lymphocytes. Jumping translocation (JT) A rare chromosomal abnormality characterized by the same fragment of donor chromosome translocating with two or more recipient chromosomes typically in different cell lines. Time to treatment (TTT) Time elapsed between diagnosis and when treatment begins for the first time. CpG-oligodeoxynucleotide (CpG-ODN) a short single-stranded synthetic or bacterial DNA molecule that contains a cytosine followed by a guanine. When the CpG motifs are unmethlyated, they act as immunostimulants. Limitations Twenty metaphases are analyzed per case. While this is often enough to detect any major abnormality some rare recipient chromosome breakpoints may go undetected. Cases with JTs which do not fit database search criteria may go undetected. Detection of chromosomal abnormalities by conventional metaphase analysis on CLL samples prior to the use of CpG-OGD was limited. Therefore the patient samples in this study were taken after the induction of CpG use in the laboratory, beginning in late 4

15 2007. Since many patients are recent diagnoses there is insufficient data to obtain complete information on time to treatment and overall survival. The results of this study are indicative of a patient population seen at a large regional quaternary care center. These results may not applicable to other CLL populations or across the CLL population spectrum. 5

16 Chapter 2 Review of Literature Chronic Lymphocytic Leukemia Chronic lymphocytic leukemia (CLL) is a clonal proliferation of neoplastic B lymphocytes. Chronic lymphocytic leukemia is the most common adult leukemia in the Western World, affecting mainly elderly individuals. Chronic lymphocytic leukemia has an extremely variable clinical course. While many patients do not require treatment for years others exhibit aggressive disease and have a poor prognosis (Byrd et al. 2004). The standard system of staging the disease to estimate prognosis in the US is the Rai system. Based on the clinical symptoms of the patient they are categorized in either early (Rai 0), intermediate (Rai I/II) or advanced (Rai III/IV) stage disease with median estimated survival times of greater than 10 years, 5-7 years, and 1-3 years (Rai et al. 1975). However, clinical staging of a patient does not determine if and at what rate the disease will progress. A second challenge encountered with this disease is choosing when to initiate treatment and which therapeutic option is best. To try to address these issues a number of clinical and biological parameters can be used, including clinical patient characteristics, laboratory parameters, expression of CD38 and ZAP70, unmutated IgVH, and cytogenetics (Chiorazzi et al. 2005; Byrd et al. 2004). Recent research has focused on the genetics of the disease since this information may be able to provide insight to the 6

17 biology of the disease as well as define mechanisms that are responsible for its clinical behavior. Genomic aberrations identified by cytogenetic analysis are pathologically and clinically important in CLL. Cytogenetic Abnormalities in CLL Genomic aberrations provide insight into the pathogenesis of CLL as well as allow for identification of patients with distinct clinical features. Approximately 80% of CLL patients have a genomic aberration which can be detected by FISH of interphase cell nuclei with the disease specific probe set for target regions on 13q, 11q, 12 centromere, 17p, and 6q. Using interphase cytogenetics, Dohner et al. found an occurrence of 55% for deletion 13q, 18% for deletion 11q, 16% for trisomy 12, 7% for deletion 17p, and 6% for deletion 6q. The 17p deletion, 11q deletion, trisomy 12, normal karyotype and 13q deletion had respective median survival times of 32, 79, 114, 111, and 133 months (Dohner et al. 2000). These abnormalities are considered hierarchical, with patients being classified based on the most deleterious abnormality present. Cytogenetic aberrations are also used to predict response to various types of therapy. Grever et al. demonstrated that patients with deletion of 17p or deletion of 11q have a significantly inferior progression free survival after initial response to therapy with either fludarabine or fludarabine plus cyclophosphamide treatment (Grever et al. 2007). Inferior progression free survival has also been noted with rituximab and fludarabine treatment (Byrd et al. 2006). Karyotype complexity ( 3 chromosomal abnormalities) has also been described as an adverse prognostic indicator in CLL (Dierlamm et al. 1997; Juliusson et al. 1990). Haferlach et al. 7

18 described a significant association between complex aberrant karyotypes and 17p deletions (Haferlach et al. 2007). When 17p is deleted there is a loss of the tumor suppressor gene TP53 that resides at 17p13.1. The TP53 protein is involved in arresting cell cycle or inducing apoptosis in the presence of DNA damage (Vogelstein et al. 2000). A loss of this gene is associated with increased drug resistance and decreased progression free survival and overall survival time (Dohner et al. 1995). This loss may also lead to genomic instability causing patients to show a greater number of chromosome abnormalities compared to undeleted cases (Haferlach et al. 2007). When there is a loss of one allele of TP53, it is common for the second allele to have a mutation, rendering it inactive as well (Dohner et al. 1995). Deletion of 17p13 is detected by FISH in approximately 7% of B-CLLs (Dohner et al. 2000; Haferlach et al. 2007). This TP53 deletion is frequently associated with structural abnormalities such as unbalanced translocations or isochromosomes occurring at or near 17p11.2 resulting in the loss of the 17p arm (Fabris et al. 2008; Fink et al. 2006). The loss of 17p appears to be a major outcome in whole-arm chromosomal translocations in hematologic malignancies and often occurs as part of a complex karyotype (Adeyinka et al. 2007). Fluorescent in situ hybridization is predominately used to detect cytogenetic abnormalities in CLL because it does not require dividing cells. It is difficult to grow CLL tumor cells in culture, since the cells are naturally being arrested at the GoG1 phase of the cell cycle. In cell culture the cells only somewhat respond to traditionally used B- cell mitogens and the detection of abnormal clones occur in approximately 30-50% of 8

19 cases (Dierlamm et al. 1997; Geisler et al. 1997). The recent use of CpGoligodeoxynucleotides (ODN) has significantly improved the abnormality detection rate of metaphase cytogenetics (Decker et al. 2000; Muthusamy et al. 2011). The CpG-ODNs are short (19-25 bases) synthetic or bacterial single stranded DNA in which the CpG motifs are unmethylated. The CpG motifs stimulate cellular immune responses through Toll-like receptor-9 mediation. This stimulation induces cellular activation that leads to S-phase entry. The use of FISH alone in CLL cytogenetic analysis does not detect karyotype complexity, an important prognostic indicator. It also does not permit the identification of the specific type of abnormality present. Using FISH methodology without conventional metaphase cytogenetics hinders the ability to detect new recurrent abnormalities that could be significant in understanding the biology of this disease. For example, Rigolin et al. found in a population of 492 CLL patients 8.9% had an abnormal karyotype with normal FISH results. The abnormal karyotype correlated with shorter time to treatment and shorter survival. In addition, 14 patients (~3%) with normal FISH were found to have a complex karyotype by metaphase analysis (Rigolin et al, 2012). Jumping Translocations Jumping translocation (JT) is a rare cytogenetic aberration that can occur in hematologic malignancy. It involves the translocation of the same fragment of donor chromosome onto two or more recipient chromosomes typically in different cell lines. Jumping translocations have been reported in various hematologic malignancies, predominately multiple myeloma, acute lymphoblastic leukemia, and acute myeloid 9

20 leukemia (Berger and Bernard 2007). Jumping translocations have also been reported as constitutional abnormalities, though their behavior differs from the common trends described in hematologic malignancy. Jumping translocation donor breakpoints occur most often in centromeric regions, frequently at 1q. A review by Berger and Bernard found that of 108 published JTs (hematologic and constitutional) 52% of the recipient breakpoints were located in telomeric regions, 30% in centromeric regions, and 18% outside of these two regions. The repetitive nature of the telomeric and centromeric regions may favor their increased involvement in these recombinations (Berger and Bernard 2007). One proposed mechanism on the formation of JTs is that they occur due to instability of highly decondensed pericentromeric heterochromatin. Sawyer et al. proposed this based on cases showing decondensation occurring in 1q of multiple myeloma patients who developed JTs. The authors suggest that the decondensation is indicative of hypomethylation of the region. The causative agent of the hypomethylation is unknown (Sawyer et al. 1998). One proposed causative agent is viral-induced immunosuppression. Sawyer et al. suggests immunosuppression can cause hypomethylation and lead to centromeric instability and JTs. Chromosome 1 is considered the most likely target for decondensation due to its large constitutive heterochromatin region (Sawyer et al. 1995). Jumping translocations may further be facilitated by the presence of shortened telomeres (Wan et al. 2004). Telomeres, repetitive DNA at the ends of chromosomes, maintain chromosome integrity. Shortened telomeres have been associated with 10

21 chromosome instability (M kacher et al. 2007; Counter et al. 1992). Gray et al. proposed telomere loss or length reduction to be the reason this region is heavily involved as recipient chromosomes; and that unstable shortened telomeres capture a telomere-bearing JT donor segment to stabilize the chromosome (Gray et al. 1997). Zou et al. found in an AML patient that cells with an abnormal clone had significantly shorter telomeres than normal cells. Cells that had a JT had telomeres slightly shorter than those seen in abnormal clones without a JT (Zou et al. 2012). It has also been suggested that JTs may be triggered by viral infection (Andreasson et al. 1998). Jumping translocations have been documented in human SV-40 infected cell lines (Meisner et al. 1988; Hoffschir et al. 1992). However, there is no definitive data to support this claim in JTs described in vivo (Berger and Bernard 2007). A single case of a jumping translocation has been reported in CLL. Callet-Bauchu et al. described a case with a donor fragment of 17p11 jumping to recipient breakpoints 15p11, 20p11, and 22p11, forming dicentric chromosomes (Callet-Bauchu et al. 1999). 11

22 Chapter 3 Methodology Records, from October 2007 to July 2011, of CLL patients were reviewed for karyotypes containing JTs. Diagnosis of CLL was made by physicians based on WHO criteria. Clinicopathological data were reviewed for each confirmed case of a JT. Institutional review-board approved; written informed consent was obtained from all patients participating in these studies. Culture and Harvesting Techniques Cells from bone marrow or peripheral blood (2.0 X10 6 cells/ml) were incubated in RPMI 1640 medium (Fischer Scientific, Houston, TX) with 2% L-Glutamine (Gibco Invitrogen, Carlsbad, CA), supplemented with 20% fetal bovine serum (Hyclone Laboratories, Logan, TX), and 2% penicillin and streptomycin (Gibco Invitrogen). The mitogens used included: pokeweed mitogen (PWM, 10μg/mL, Sigma Aldrich, St. Louis, MO), phorbol 12-myristate 13-acetate (PMA, 40ng/ml, Sigma Aldrich) and CpG ODN 685 (20 μg/ml, synthesized by Sigma Aldrich). CpG ODN 685 alone (20 μg/ml) as a mitogen was also used in a second culture. The mitogens were added to the cultures, and the cells were incubated for 72 hours at 37 C in a 5% CO 2 incubator. Samples were then harvested. Colcemid (Gibco Invitrogen) was added to arrest cells in metaphase by interfering with spindle formation. Hypotonic solution (0.075M KCl) was added to swell cells and allow the chromosomes to be dispersed. Carnoy s fixative (3:1 ratio of reagent 12

23 grade methanol to acetic acid) was added to denature and precipitate protein as well as cause fixation of the chromosomes. The fixed cell suspension was then dropped on glass slides for G-banding and FISH preparation. Conventional Cytogenetic Analyses Slides for conventional chromosome analysis of metaphases were G-banded, a staining procedure which creates sequential light and dark bands along the chromosomes that can be used to identify and differentiate the chromosomes with microscopy. Slides were bathed in a dilute trypsin solution to help remove histone and non-histone proteins then were stained using Wright stain. Metaphase spreads images were captured at 1000X total magnification using Applied Spectral Imaging software. Twenty metaphases were completely analyzed whenever possible. Karyotypes were described following the ISCN 2009 standard (Shaffer et al. 2009) except single cell abnormalities were reported when they demonstrated a JT. Fluorescence in situ hybridization Fluorescence in situ hybridization (FISH) was also done on the mitogen-stimulated cultures. In the laboratory, we have found no difference in detection of any abnormalities when comparing FISH results from mitogen-stimulated versus unstimulated samples, although the stimulated samples sometimes have a higher frequency of an aberration (data not shown). FISH probes for the CLL FISH panel D13S319 (13q14.3), D12Z3 (chromosome 12 centromere), ATM (11q22.3) and p53 (17p13.1) (Abbott Molecular, Des Plaines, IL) as well as probes for BCL6 break-apart (3q27), MYB (6q23), MYC 13

24 break-apart (8q24), IgH/CCND1 fusion (14q32/11q13), IgH break-apart (14q32) (Abbott Molecular) and (6q21) (Poseidon, Amsterdam, The Netherlands) were used. Hybridization was according to the manufacturers directions. Two hundred cells/probe were analyzed, 100 by each of two independent observers. FISH control values were calculated using the Beta-inverse function. Each case was compared for consistency of FISH results with conventional karyotyping results. FISH using centromere probes (Abbott Molecular) was done on metaphases to confirm some of the dicentric chromosomes. Slides for metaphase FISH had coverslips removed and were pretreated to remove contaminating chemicals and Wright stain. Probe hybridization was then carried out according to the manufacturer s directions. 14

25 Chapter 4 Jumping translocations are recurrent abnormalities associated with genetic instability and an aggressive disease state in chronic lymphocytic leukemia Cecelia R. Miller, 1,2 Frederick Racke, 2 Andrew McFaddin, 2 Heather Breindenbach, 2 Huey-Jen Lin, 1 Kathy Waller, 1 Tammy Bannerman, 1,2 John C. Byrd, 3,4 and Nyla A. Heerema 2 1 Division of Medical Laboratory Science, School of Health and Rehabilitation, The Ohio State University, Columbus, Ohio; 2 Department of Pathology, The Ohio State University, Columbus, Ohio; 3 Division of Hematology-Oncology, College of Medicine, The Ohio State University, Columbus, Ohio; 4 Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio. Abstract Jumping translocation (JT) is a rare cytogenetic aberration that can occur in hematologic malignancy. It involves the translocation of the same fragment of donor chromosome onto two or more recipient chromosomes typically in different cell lines. Here, for the first time, we establish JTs are a type of recurrent abnormality occurring in chronic lymphocytic leukemia (CLL). We address the cytogenetic characteristics of JTs in CLL, the characteristics of the patient population where they are seen, and correlations with other clinicopathological findings. We identified 26 patients with JTs. There were a total of 32 donor breakpoints with six patients exhibiting two unique donor breakpoints involved in JTs at the same time. Strikingly, 15 of the donor breakpoints were in 17p11.2. A total of 93 JTs were identified. Forty-one of these JTs resulted in dicentric or pseudodicentric chromosomes, 31 of which had 17p11.2 donor breakpoints. Patients with 15

26 JTs in CLL appear to develop the disease at an early age and experience an aggressive disease state. These patients develop complex karyotypes and frequently show loss of the tumor suppressor gene TP53, both of which are considered poor prognostic indicators. The biology of JT formation is unclear and warrants further investigation. Introduction Jumping translocation (JT) is a rare cytogenetic aberration involving the translocation of the same fragment of donor chromosome onto two or more recipient chromosomes typically in different cell lines. They have been reported as acquired abnormalities in malignancy and as constitutional abnormalities, though with differing behavior. Their presence in malignancies has been associated with genetic instability, an aggressive disease state, and poor prognosis. 1-3 Jumping translocations have been reported most frequently in lymphoid disorders such as multiple myeloma, acute lymphoblastic leukemia and non-hodgkin lymphoma, but has also been described in other hematologic malignancies such as acute myeloid leukemia. In previous reports, donor breakpoints show a preference to centromeric and heterochromatic regions, while recipient breakpoints most often occur in telomeric and subtelomeric regions. The most commonly occurring donor chromosome segment is 1q. 4 Other donor chromosomes have been described, including chromosomes 3, 7, 9, 11, and The recipient chromosomes appear to be random. The etiology of JT formation remains to be fully deciphered. Several possible scenarios leading to JT formation have been considered including chromosome 16

27 instability, viral infection, pericentromeric heterochromatin decondensation, and shortened telomeres Chronic lymphocytic leukemia (CLL) is a clonal proliferation of neoplastic B lymphocytes. Chronic lymphocytic leukemia is the most common adult leukemia in the Western World. Chronic lymphocytic leukemia generally is considered an indolent disease. However, the clinical course of the disease is highly variable. While many patients do not require treatment for years, others exhibit aggressive disease and have a poor prognosis. 15 A number of clinical and biological parameters predict disease aggressiveness, including clinical stage, expression of CD38 and ZAP70, unmutated IgVH, and cytogenetics. 16 Chromosomal abnormalities identified by metaphase cytogenetic analysis and FISH are one important prognostic factor in the risk stratification of CLL. Historically, identification of chromosomal aberrations by traditional metaphase cytogenetics has been limited by the low mitotic activity of CLL cells in culture. The recent introduction of CpG oligodeoxynucleotides (CpG-ODN) to CLL cultures has significantly improved the ability to obtain informative karyotypic results from patients with CLL. 17 Here we describe for the first time a series of JTs in CLL. We present 26 CLL patient cases with karyotypes containing this abnormality. To our knowledge only a single mention of this phenomenon occurring in a CLL patient has been reported previously

28 Methods Diagnosis of CLL was made based on WHO criteria. Records of CLL patients were reviewed for karyotypes containing JTs. Clinicopathological data were reviewed for each confirmed case. Institutional review-board approved, written informed consent was obtained from all patients participating in these studies. Conventional cytogenetic analyses Cells from bone marrow or peripheral blood (2.0x10 6 cells/ml) were incubated in RPMI 1640 medium (Fischer Scientific, Houston, TX) with 2% L-Glutamine (Gibco Invitrogen, Carlsbad, CA), supplemented with 20% fetal bovine serum (Hyclone Laboratories, Logan, TX), and 2% penicillin and streptomycin (Gibco Invitrogen). The mitogens used included: pokeweed mitogen (PWM, 10μg/mL, Sigma Aldrich, St. Louis, MO), phorbol 12-myristate 13-acetate (PMA, 40ng/ml, Sigma Aldrich) and CpG ODN 685 (20 μg/ml, synthesized by Sigma Aldrich). CpG ODN 685 alone (20 μg/ml) as a mitogen was also used in a second culture. The use of CpG ODN enhances detection of chromosomal abnormalities in CLL and gives consistent karyotypic results. 19,20 The mitogens were added to the cultures, and the cells were incubated for 72 hours under standard laboratory conditions. All samples were harvested, fixed, G-banded using trypsin, and stained with Wright stain according to standard laboratory procedures. Twenty metaphases were completely analyzed whenever possible. Karyotypes were described following the ISCN 2009 standard 21 except single cell abnormalities were reported when they demonstrated a JT. Fluorescence in situ hybridization (FISH) 18

29 FISH was also done on the mitogen-stimulated cultures. FISH probes for the CLL FISH panel D13S319 (13q14.3), D12Z3 (chromosome 12 centromere), ATM (11q22.3) and p53 (17p13.1) (Abbott Molecular, Des Plaines, IL) as well as probes for BCL6 break-apart (3q27), MYB (6q23), MYC break-apart (8q24), IgH/CCND1 fusion (14q32/11q13), IgH break-apart (14q32) (Abbott Molecular) and (6q21) (Poseidon, Amsterdam, The Netherlands) were used. Hybridization was according to the manufacturer s directions. Two hundred cells/probe were analyzed, 100 by each of two independent observers. FISH control values were calculated using the Beta-inverse function. Each case was compared for consistency of FISH results with conventional karyotyping results. Additionally, FISH using centromere probes (Abbott Molecular) was done on metaphases to confirm some of the dicentric chromosomes. Results A total of 26 cases of JTs were identified from a patient population of 878 CLL patients seen from October 2007 to July Previous specimens were examined prior to this if the patient had evidence of JT. Karyotype designations for the 26 patients are provided in Supplementary Table 1. Examples of JTs in CLL patients are shown in Figure 1. There were a total of 32 donor breakpoints with six patients exhibiting two unique donor breakpoints involved in JTs at the same time. Twenty-four donor breakpoints occurred in centromeric regions, six between the peri-centromeric and subtelomeric regions, and two donor segments were of unknown material. Strikingly, 15 of the donor breakpoints occurred in 17p11.2. For analysis purposes we separated 19

30 patients based on their donor breakpoint, one group representing breakpoints in 17p11.2 and a second miscellaneous group including all other breakpoints (Tables 1 & 2). A total of 44 recipient breakpoints were identified in the 17p11.2 donor breakpoint group (Table 1). Of the recipient breakpoint locations 28/44 (63.6%) occurred in centromeric regions, 8/44 (18.2%) occurred between the pericentromeric and subtelomeric regions, 2/44 (4.6%) occurred in subtelomeric regions. Six of the 44 (13.6%) recipient breakpoints were unknown. These JTs resulted in the formation of 29 dicentric chromosomes and two pseudodicentric chromosomes. Twenty-six of the dicentric chromosomes had centromeric recipient breakpoints, one had a breakpoint along the chromosome arm, and two had telomeric recipient breakpoints. The two pseudodicentric chromosomes had recipient breakpoints between the pericentromeric and subtelomeric regions. Multiple repeat recipient breakpoints locations were identified in this group including; 18p11.2 (6), 8p11.2 (3), 8q11.2 (2), 14p11.2 (2), 12p11.2 (2), 17p11.2 (2), and 20p11.2 (2). All donor breakpoints not occurring at 17p11.2 were compared in a second miscellaneous group. This group had a total of 17 donor breakpoints in at least 11 different chromosomes (Table 2). Two repeat donor breakpoints were identified; 18p11.2 (2) and 4q12 (2). Additional donor breakpoints were 1p32, 8p21, 9q12, 11q21, 12p11.2, 13p11.2, 13q14, 13q21, 14p11.2, 15p11.2, 19p13.3, and two breakpoints of unidentified chromatin. In total in this group 9/17 (52.9%) donor breakpoints occurred in centromeric regions, 6/17 (35.3%) between the pericentromeric and subtelomeric regions, and 2/17 (11.8%) were not identified. 20

31 A total of 49 recipient breakpoints were found in the miscellaneous group. The recipient breakpoint locations were 18/49 (36.7%) in telomeric regions, 6/49 (12.2%) in centromeric regions, 19/49 (38.8%) between the pericentromeric and subtelomeric regions, and 6/49 (12.2%) unknown. Eight dicentric chromosomes were formed by these JTs and two pseudodicentric chromosomes. All dicentric chromosomes had a centromeric donor breakpoint. Six of the dicentric chromosomes had a centromeric recipient breakpoint, two had a telomeric recipient breakpoint, and two occurred with unidentified recipient breakpoints. Both pseudodicentric chromosomes had recipient breakpoints between the pericentromeric and subtelomeric regions. One had a centromeric donor breakpoint and the other a donor breakpoint between the pericentromeric and subtelomeric region. Repeat recipient breakpoint locations in the miscellaneous group were found at chromosome bands 5q35 (2), 12q24.1 (2), 17p11.2 (2), and 18q23 (2). Six cases, cases 3, 7, 8, 18, 23, and 25, had two unique donor breakpoints involved in JTs at the same time. Each JT was grouped based on its donor breakpoint location. Three of the six patients with two unique donor breakpoints had a breakpoint involved as both donor and recipient in the JTs. Interphase FISH analysis showed loss of TP53 in 22 of the patients. One patient had loss of TP53 confirmed by FISH on metaphases, bringing the total number of patients with TP53 loss to 23 (Table 3). FISH analysis of cases 2, 22, and 26 showed no loss of TP53. The second most common abnormality seen by FISH was loss of 13q14.3 in 17 patients. 21

32 Pertinent clinical information is shown in Table 3. The median patient age at time of diagnosis for all patients was 56 years with a range of 37 to 78 years. The median time to treatment (TTT) was 12 months with a range of 0 to 88 months. The male to female ratio was 1.36:1. At diagnosis 88% of patients for whom data were available (n=24) were Rai stage 0 or 1. The kappa to lambda light chain restriction ratio was 1:1.78, with one case indeterminate. At the time of this writing, ten patients were deceased. The median survival time for these patients after loss of TP53 was detected was 12 months. When comparing the two donor breakpoint groups, patients with 17p11.2 donor breakpoints and a second JT were categorized with 17p11.2 donor group. The miscellaneous donor breakpoint group (n=11) had a median age at diagnosis of 50 years, a median TTT of 18 months, and contained two deceased patients. The 17p11.2 donor breakpoint group (n=15) had a median age at diagnosis of 59.5 years, a median TTT of 12 months, and contained eight of the deceased patients. Discussion Here for the first time we describe a series of JTs occurring in CLL. We present 26 cases from a population of 878 patients seen at our institution between October 2007 and July This patient population is indicative of a large regional quaternary care center. The prevalence of this abnormality may be slightly underestimated in this patient population due to the difficulties in detecting jumping translocations that appear as rare nonclonal abnormalities. The majority of patients with JTs in CLL experienced an aggressive disease course with a short median time to treatment (TTT) of 12 months. Rai stage at diagnosis was 2 or less for 96% of the patients for whom data were available 22

33 (n=24). The patients median age at time of diagnosis was 56 years, markedly younger than the overall CLL population median age at diagnosis of 72 years. 22 All but one patient was under the age of 72 at diagnosis. Complex karyotypes were seen in all patients, with additional abnormalities occurring with the JT in all but one case. Twentythree patients had unbalanced JTs. Two cases (Cases 6 and 16) had both balanced and unbalanced JTs. One case (Case 2) exhibited a balanced JT as the sole abnormality. Jumping translocation as a sole abnormality is extremely rare; with few previously reported occurrences Jumping translocations were found in five patients before they began any treatment. Using only FISH analysis, in place of metaphase cytogenetics in CLL, would not detect these abnormalities and their associated karyotype complexity. Jumping translocations are rare cytogenetic aberrations found in hematologic malignancy, most often lymphoid disorders such as multiple myeloma, acute lymphoblastic leukemia and non-hodgkin lymphoma. 4 In the literature a single case of a JT occurring in a patient with CLL was described by Callet-Bauchu, et al. 18 with 17p11 as the donor segment forming dicentric chromosomes with recipient breakpoints at 20p11, 22p11, and 15p11. While data regarding JT breakpoints across all hematologic malignancies is highly heterogeneous, certain trends have emerged. Breakpoints of donor chromosomes most often occur in centromeric regions, with a preference towards 1q. 4 No recipient chromosome has been reported to have preferential involvement in these translocations. However, JT recipient breakpoints have been found most often in telomeric regions. While the underlying mechanism behind JT formation is not yet 23

34 known, the highly repetitive nature of the pericentromeric and telomeric regions may make these regions prone to involvement in the rearrangements. 1,11 Jumping translocations in CLL have a heterogeneous presentation that differs from trends seen in other hematologic malignancies. In our miscellaneous group, donor breakpoints occurred most frequently in centromeric regions, though none at 1q. Repeat donor breakpoints were seen in two cases at 18p11.2 and in two cases at 4q12. Recipient breakpoints occurred in telomeric regions and between pericentromeric to subtelomeric regions at about the same rate. Centromeric breaks occurred much less frequently. Dicentric chromosomes occurred with 18p11.2 donor breakpoints, a 12p11.2 donor breakpoint, as well as the acrocentric chromosome donor breakpoints of 13p11.2, 14p11.2, and 15p11.2. A total of 15 cases had 17p11.2 as a donor segment, indicating a preferential involvement of this band as a donor breakpoint for JTs in CLL. Donor breakpoints at 17p11.2 most frequently associated with centromeric recipient breakpoints. The jumping translocations involving 17p11.2 often resulted (31/44 donor recipient pairs) in the formation of dicentric or pseudodicentric chromosomes; 26 of the 28 had centromeric recipient breakpoints, one recipient breakpoint was along the chromosome arm, and both telomeric recipient breakpoints resulted in dicentric chromosome formations. Multiple repeat recipient breakpoints seen within the 17p11.2 group suggest that recipient breakpoint locations in CLL JTs may not be entirely random. The most frequent recipient breakpoint, 18p11.2, occurred six times. Dicentric chromosomes, most frequently dicentric (17;18), have been previously reported as a recurrent abnormalities in CLL. 18,26 24

35 Unlike the majority of JTs previously reported in hematologic malignancy, our 17p11.2 donor breakpoints rarely associated with telomeres. Multiple breakpoint cluster regions have been identified in the centromeric region of 17p. 27,28 The 17p11.2 region is characterized by multiple large palindromic low copy repeats (LCR) which may favor its increased involvement in rearrangements. 29 Region specific LCRs generally contain DNA blocks of ~10-400kb with at least 95-97% homology. Low copy repeats are heavily represented in pericentromeric and subtelomeric regions and may result in unstable genomic regions that are prone to nonallelic homologous recombination (NAHR). 30 LCRs have been proposed as target regions for JTs in a constitutional case. 31 These characteristics of the 17p11.2 region as well as its prevalence as a recurrent rearrangement site in CLL may explain its increased involvement in JTs in CLL. Further characterization of the breakpoint locations in 17p11.2 as well as recurrent recipient breakpoints would be useful in determining the nature of these recombinations. Twenty-three of twenty-six of our cases showed significant loss of TP53, located at 17p13.1, by interphase or metaphase FISH. This loss occurred before or concurrently with the JT. TP53 is involved in arresting cell cycle or inducing apoptosis in damaged cells. 32 The loss of TP53 in CLL patients is associated with increased drug resistance and decreased progression free survival and overall survival time. 33 This loss may also lead to genomic instability causing patients to show a greater number of chromosome abnormalities compared to undeleted cases. 34 When there is a loss of one allele of TP53, it is common for the second allele to have a mutation, rendering it inactive 25

36 as well. 33 Jumping translocations in CLL were found to frequently involve 17p11.2 as a breakpoint, usually resulting in a dicentric chromosome. In six cases in the miscellaneous donor breakpoint group dicentric chromosomes with 17p11.2 breakpoints occurred without being directly involved in the JT. These translocations resulted in significant loss of TP53 for these patients. Loss of TP53 in CLL is commonly due to rearrangements involving 17p Jumping translocations appear to be a recurring type of rearrangement involving this region. Across all donor and recipient breakpoints in these JTs, fourteen repeat breakpoints were seen. The repeat breakpoints in our CLL JT cases suggest that certain regions are more prone to involvement in these rearrangements, particularly 17p11.2. Repeat breakpoint locations were most often in centromeric regions, with only two in telomeric regions and one in a chromosome arm. The high prevalence of breakpoints occurring in centromeric or telomeric regions suggests that genomic architecture may make these regions susceptible to involvement in JTs. In addition, the high prevalence of dicentric chromosomes with centromeric breakpoints could in part be due to increased stability in mitosis when the centromeres are in close proximity to each other. 35 Chronic lymphocytic leukemia patients with JTs exhibit highly complex karyotypes indicative of chromosome instability. Our findings suggest that JTs are a type of recurrent abnormality occurring in CLL, often in conjunction with dicentric chromosomes involving 17p. These JTs appear to behave non-randomly; sequence homology between the repetitive elements in centromeric and telomeric regions as well as regions containing LCR may mediate these translocations. Jumping translocations in 26

37 CLL commonly occur in addition to other chromosomal abnormalities and thus contribute to a complex karyotype. Thus, JTs may be a manifestation of genetic instability. Our patients with JTs frequently had loss of TP53 and in some cases these occurred concurrently. Both complex karyotype and loss of TP53 are poor prognostic indicators in CLL. Patients with JTs develop disease at an earlier age than the average CLL population and progress to requiring treatment more quickly. Overall, patients with JTs in CLL appear to have a poor outcome; though whether this is due to the complex karyotype, loss of TP53, the JT, or a combination of these factors is not clear. Further investigation into the mechanisms behind JT formation is warranted. Acknowledgements The authors thank the cytogenetic technologists at The Ohio State University for technical assistance. C.M. is a MS candidate at The Ohio State University and this work is submitted in partial fulfillment of the requirement for the MS. Authorship Contribution: N.A.H., F.R., and C.M. conceived the study; C.M., N.A.H. and T.B. had primary responsibility for the manuscript; T.B., N.A.H.,H.J.L.,K.W., and J.C.B. approved this project; C.M., F.R., A.M., H.B., and N.A.H. collected and analyzed data; J.C.B provided patient and clinical data; and all authors checked and approved the final version of the manuscript. Conflict-of-interest disclosure: The authors declare no competing financial interests. 27

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