16 www.futuremedicine.com Stephan Stilgenbauer Stephan Slgenbauer is Associate Professor and Deputy Chairman at the Department of Internal Medicine III at the University of Ulm, Germany. His research focus is on the molecular pathogen- esis and translaon into novel treatment strategies in hemato- logical malignancies. He has authored numerous arcles in jour- nals such as the New England Journal of Medicine, Lancet, Nature Medicine, Proceedings of the Naonal Academy of Sciences USA, Journal of Clinical Oncology, Blood, Leukemia, Oncogene and Haematologica. Thorsten Zenz Thorsten Zenz received his MD from the University of Freiburg, Germany, in 1996. Aſter beginning his training in internal medicine at the University of Freiburg, he worked as a research fellow at the Kimmel Cancer Center in Philadelphia, PA, USA. In 2001, he joined the Hematology/Oncology Department at the University of Ulm, Germany, and has completed his clinical training in internal medicine and hematology/oncology. In recent years, his main interest has been the invesgaon of genec defects leading to refractory chronic lymphocytic leukemia and strategies to overcome drug resistance. The work has contributed to clinically relevant risk models in chronic lymphocyc leukemia. He is currently an Aending Physician at the Department of Hematology, Oncology and Rheumac Disease of the University of Heidelberg as well as the Department of Translaonal Oncology at the National Center for Tumor Diseases (NCT)/German Cancer Research Center (DKFZ), where he heads the Secon ‘Lymphoma Research’. About the Authors For reprint orders, please contact: [email protected]
Stephan StilgenbauerStephan Stilgenbauer is Associate Professor and Deputy Chairman at the Department of Internal Medicine III at the University of Ulm, Germany. His research focus is on the molecular pathogen-esis and translation into novel treatment strategies in hemato-logical malignancies. He has authored numerous articles in jour-nals such as the New England Journal of Medicine, Lancet, Nature Medicine, Proceedings of the National Academy of Sciences USA, Journal of Clinical Oncology, Blood, Leukemia, Oncogene and Haematologica.
Thorsten ZenzThorsten Zenz received his MD from the University of Freiburg, Germany, in 1996. After beginning his training in internal medicine at the University of Freiburg, he worked as a research fellow at the Kimmel Cancer Center in Philadelphia, PA, USA. In 2001, he joined the Hematology/Oncology Department at the University of Ulm, Germany, and has completed his clinical training in internal medicine and hematology/oncology. In recent years, his main interest has been the investigation of genetic defects leading to refractory chronic lymphocytic leukemia and strategies to overcome drug resistance. The work has contributed to clinically relevant risk models in chronic lymphocytic leukemia. He is currently an Attending Physician at the Department of Hematology, Oncology and Rheumatic Disease of the University of Heidelberg as well as the Department of Translational Oncology at the National Center for Tumor Diseases (NCT)/German Cancer Research Center (DKFZ), where he heads the Section ‘Lymphoma Research’.
Stephan Stilgenbauer & Thorsten ZenzA number of characteristics of chronic lymphocytic leukemia set it apart from other cancers [1–3]. The most prominent pathogenetic mechanisms include genomic aberrations targeting critical genes (e.g., miRNAs, TP53,
ATM and NOTCH1); antigen drive and stereotyped B-cell receptors; and microenvironmental stimulation [1–4]. While the precise sequence of events is currently unclear, our growing understanding of chronic lymphocytic leukemia biology is evolving to the translation of understanding to therapeutic exploitation. In this chapter, we give an overview of genetic lesions in chronic lymphocytic leukemia.
IGHV mutation status 18
17p deletion & TP53 mutation 18
11q deletion 19
Trisomy 12 19
13q deletion 19
Other recurrent aberrations in CLL 20
Exploiting the genetic profile of CLL 23
Chapter 2
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IGHV mutation statusDifferent levels of somatic hypermutation of the IGHV (and IGKV) separates chronic lymphocytic leukemia (CLL) patients into two prognostic (and biological) subgroups (IGHV mutated and unmutated CLL) [5,6]. In addition to the variable degree of mutations in the immunoglobulin variable regions, CLL samples d isp lay a remarkable
overrepresentation of particular IGHV gene repertoires (e.g., IGHV1–69, IGHV4–34, IGHV3–23 and IGHV3–21) [7]. This limited repertoire has been termed ‘stereotyped’ and suggests that restricted antigen selection contributes to CLL pathogenesis. Alternatively, the targeting of a B-cell subset with restricted IGHV repertoire may lead to stereotyped IGHV usage.
Several groups have demonstrated that CLL subsets share structural features. The ‘stereotyped’ B-cell receptors occur in up to 30% of patients and are more common in CLL with unmutated IGHV. This has also been suggested to have distinct clinical features. Patients with stereotyped IGHV3–21 usage have been suggested to have inferior overall survival, which may be independent of IGHV mutational status [8,9].
The study of the VH mutational status has greatly helped our understanding of CLL and our ability to separate prognostic subgroups in CLL.
17p deletion & TP53 mutationDeletions of the short arm of chromosome 17 (17p13) are found in 4–8% of patients with CLL (first-line treatment situation). The 17p deletion virtually always includes band 17p13 where the key tumor suppressor TP53 is located. CLL with 17p deletion is associated with refractoriness to chemotherapy and very poor outcome [10–12]. Among cases with 17p deletion, the majority show mutations in the remaining TP53 allele (>80%), which suggests that
p53 is the pathogenetically implicated in the phenotype associated with 17p deletion. Interestingly, even 17p cases without TP53 mutation show downregulation of p53 targets, similar to TP53 mutant cases, suggesting that the p53 pathway is inactivated.
Mutations of TP53 are found in approximately 4–50% of patients with CLL – depending on the clinical situation (i.e.,
TP53: gene encoding for p53 tumor protein, a key mediator of treatment resistance in
chronic lymphocytic leukemia.
ATM: ataxia teleangiectasia mutated gene, affected by 11q deletion.
IGHV: immunoglobulin heavy-chain variable gene segments, the mutation status of these is of prognostic value in chronic lymphocytic leukemia.
The clinical course of chronic lymphocytic leukemia is largely determined by biological
disease characteristics.
IGHV mutation status and its surrogate markers such as ZAP-70 predict the rate of disease progression among early-stage patients.
17p deletion and TP53 mutation predict for nonresponse to treatment with conventional chemotherapy (chlorambucil, fludarabine, FC, bendamustine, fludarabine, cyclophosphamide, rituximab, pentostatin, cyclophosphamide and rituximab.
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early stage vs refractory) – and have been associated with very poor prognosis (‘ultrahigh risk’) in a number of studies [1]. In cases without 17p13 deletion, TP53 mutations are much rarer, but have a similarly profound impact on outcome (for a detailed discussion please refer to Chapter 7 of this book). Recent evidence suggests that TP53 mutations carry a disease specific profile in CLL and heterogeneity may exist regarding prognostic impact of different mutations [13,14].
11q deletionApproximately a quarter of patients with treatment indications carry the 11q deletion. Cohorts of patients with early-stage CLL will show considerably smaller incidences (~10%) [1]. The deleted region harbors the ataxia telangiectasia mutated gene in almost all cases. A subset of cases showed biallelic inactivation of ataxia telangiectasia mutated by mutation of the remaining allele and these cases appear to have a poorer prognosis [15]. Nonetheless, the majority of cases with 11q deletion do not show the inactivation of the other ataxia telangiectasia mutated allele by mutation, suggesting that additional genes could be targeted by the deletion.
Patients with 11q deletion have a more rapid progression of disease, as measured by shorter treatment-free intervals and shorter survival times.
Trisomy 12Trisomy 12 is a frequent aberration in CLL (10–20%). Genes involved in the pathogenesis of CLL with trisomy 12 are unknown, but a gene dosage effect has been described. The initially described association of trisomy 12 with poorer outcome is not confirmed in current trials [16]. CLL with trisomy 12 rarely show TP53 mutations – a finding that may contribute to the benign course [13].
13q deletionThe 13q14 deletion is the most common structural chromosome aberration in CLL. The deletion is not limited to CLL, but is also observed in other lymphomas, myeloma and prostate cancer. Micro RNAs (miR-15a/miR-16) were identified to be located in the critical region of the 13q14 dele-tion [17,18]. A transgenic mouse model with a targeted deletion of the MIR-15a/16–1 locus (and DLeu2) recapitulates many of
Testing for 17p deletion should be performed before the initiation of therapy.
Patients with chronic lymphocytic leukemia with 17p deletion are candidates for novel treatment approaches and stem cell transplantation in clinical trials.
Some aberrations are associated with poor outcome (particularly deletion 17p and TP53 mutation), others are linked to a favorable outcome (deletion 13q as sole aberration and mutated IGHV).
Abnormalities of key tumor suppressors (ATM, miR-15a/16–1 and TP53) have been identified in chronic lymphocytic leukemia and these are important ‘drivers’ of the disease.
Micro RNA: a class of small (~21 nucleotides) noncoding RNAs of pathogenic importance.
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the features of CLL and lymphoma. The mice develop a monoclonal B-cell lympho-cytosis-like disorder, CLL and lymphoma, which suggests that miR-15a/16–1 have
indeed a pathogenetic role in CLL development. The understanding how the deregulation of the miR leads to cancer is far from complete. Key ele-ments of growth control (apoptosis, cell cycle progression) appear to be regulated.
Other recurrent aberrations in CLLRecurrent translocationsIn contrast to other types of leukemia or B-cell lymphomas, recurrent balanced translocations are rare in CLL [16,19,20]. Translocations with breakpoints in band 14q32, where the immunoglobulin heavy-chain genes are located, are the most common recurrent translocations in CLL. Recently, stimulation with CD40 ligand or CpG oligodeoxynucleotides and IL-2, to increase the frequency of metaphase spreads, suggested that unbalanced translocations occurred in 34% of patients with CLL [19]. In a larger series of over 500 patients, aberrations were detected in 83.0% by chromosome banding (78.4% cases by FISH, which only probes a limited set of regions). CLL was shown to be characterized mainly by genomic imbalances and not balanced translocations. Recurrent reciprocal translocations were rare and mostly target known regions as the Ig locus (in band 14q32, discussed above) or the 13q14 region with concomitant loss of genomic material [20].
ChromothripsisWhile balanced translocations are rare in CLL, recently a novel mechanism of chromosomal rearrangements called chromothripsis has been identified in CLL and other cancers [21]. In the process, up to hundreds of rearrangements occur in a ‘one-off cellular crisis’ [21]. Rearrangements are typically confined to single chromosomes and are characterized by frequent oscillations between one and two copy number states [21]. The rearrangements are likely to occur during a ‘single cellular catastrophe’ [21]. Cancer-causing lesion can emerge from this process as demonstrated for the MYC oncogene.
Gene mutationsRecurrent somatic gene mutations contribute to the pathogenesis and char-acteristics of CLL. Although they occur in significantly higher numbers in solid tumors, recent whole-genome sequencing of CLL samples demonstrated
Chromothripsis: novel mechanism of chromosomal rearrangements during a ‘single
cellular catastrophe’.
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approximately 1000 somatic mutations per tumor [22]. An overview of the most important recurrent somatic gene mutations in CLL is given in
Table 2.1.
Mutations occurring in CLL affect Notch signaling, which is important for various developmental and physiological processes. In CLL, Notch signaling is constitutively activated by mutations. Activation of Notch signaling in CLL cells results in resistance to apoptosis. In T-cell acute lymphoblastic leukemia (T-ALL), more than half of the patients show activating mutations of NOTCH1.
The first studies of NOTCH1 mutations in CLL revealed a heterozygous frameshift deletion of two base pairs (∆CT7544–7545, P2515fs) affecting its PEST domain in approximatley 5% of CLL patients. This deletion leads to a premature stop codon and a lack of the C-terminal domain containing the PEST sequence with a stabilized Notch 1 isoform [22]. The ana lysis of a cohort of CLL patients identified this deletion in approximately 8% of the cases (clustering with IGHV unmutated and cases with TP53 disruption) and the mutation was associated with shorter overall
Table 2.1. Overview of recurrent somatic gene mutations relevant in chronic lymphocytic leukemia.
Gene Mutation Incidence Comment Ref.
NOTCH1 P2515fs (CLL), various in heterodimerization and PEST domain (other cancers, especially T-ALL)
CLL: 5–20% CLL: associated with unmutated IGHV, poor prognosis
[22,23]
MYD88 L265P CLL: 3% CLL: potential association with mutated IGHV, advanced clinical stage
[22]
BRAF V600E (>90%) CLL: 3%HCL: 70–100%
Among the most common somatic mutations in all cancers
[24; Stilgenbauer
& Zenz,
Unpublished Data]
TP53 Scattered across all exons (mainly DNA-binding domain)
CLL: 4–37% CLL: associated with very poor prognosis (ultrahigh risk), poor outcome, higher genetic complexity
[1]
ATM Scattered across all exons
CLL: 12% (with 11q deletion 36%)
CLL: associated with lower and treatment-free survival
[15,25]
CLL: Chronic lymphocytic leukemia; T-ALL: T-cell acute T lymphoblastic leukemia.
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survival. Strikingly, the ∆CT7544–7545 deletion was shown to be even more prevalent (~20%) in patients with progressive or chemorefractory CLL [23]. A more recent study based on whole-genome sequencing of CLL samples detected NOTCH1 mutations among the four recurrently mutated genes [22]. Taken together, these findings suggest that mutational status of NOTCH1 mutations are prevalent and might contribute to high-risk CLL.
Whole-genome sequencing of CLL samples also revealed an activating recurrent mutation (L265P) in the myeloid differentiation primary response gene 88 (MYD88) in approximately 3% of patients [22]. MYD88 is a protein crucial for interleukin and Toll-like receptor (TLR) signaling. Mutant MYD88 was able to bind highly efficient to IL-1 receptor-activated kinase 1, a protein essential for early responses to TLR stimulation. Importantly, MYD88 mutations were associated with IGHV-mutated and advanced clinical stage, while no correlation with disease progression or overall survival was seen [22]. Interestingly, the L265P mutation was also shown to occur in various lymphomas. For example, 29% of diffuse large B-cell lymphoma (activated B-cell-like subtype) harbor this mutation,
which is essential for the viability of these tumor cells in vitro and contributes to the constitutive activation of NF-kB signaling via assembly with IL-1 receptor-activated kinase 1.
The improved understanding of the clinical course of specific genetic subgroups is
beginning to be translated into specific treatment approaches.
Table 2.2. Summary of current genetic prognostic factors and potential treatment options in chronic lymphocytic leukemia (first-line treatment indication).
Risk Risk factor Treatment approach
Ultrahigh risk(~10–15%)
17p deletion Clinical trial with investigational agent acting independent of p53, allogeneic stem cell transplantationTP53 mutation
Exploiting the genetic profile of CLL Our understanding of genetic and biological mechanisms in CLL has improved risk stratification. The goal is a genotype- and risk-adapted therapy. The growing wealth of genetic aberrations identified in CLL will lead to improved risk models and ultimately to the development of novel agents that act specifically in genetic groups and will prove their clinical importance in the coming years.
Acknowledgements
Supported by the Harald Huppert Stiftung, the MDACC-DKFZ SINF program, the ‘Deutscht. Krebshilfe’, CLL Global research Foundation, and the Helmoltz Society
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organi-zation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, con-sultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
Summary.
� Genomic aberrations can be found in more than 80% of all chronic lymphocytic leukemia patients by the standard FISH test.
� Mutations of key tumor-suppressor genes such as TP53 and ATM have been identified. � Structure and mutation status of the IGHV rearrangement give insight in cellular origin and
pathogenic mechanism. � Genetic parameters (17p deletion, TP53 mutation, IGHV status) are among the strongest
prognostic markers in prospective treatment trials (Table 2.2).
� 17p deletion and TP53 mutation are today used to stratify ‘ultrahigh-risk’ chronic lymphocytic leukemia patients.
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