Next-Generation Sequencing in Chronic Lymphocytic Leukemia Neus Villamor,a Armando López-Guillermo,b Carlos López-Otín,c and Elías Campoa,d The use of next-generation sequencing (NGS) has expanded our knowledge of the genomic alterations in chronic lymphocytic leukemia (CLL) and provides new tools for analyzing leukemic clonal architecture. Recent studies have demonstrated substantial differences in genomic alterations between mutated and unmutated IGHV subgroups, which reflect distinct molecular pathways and mutagenic mechanisms in the pathogenesis of the disease. The mutational profile of CLL can be characterized by a relatively low number of somatic mutations per case, few recurrent mutations at moderate frequency (5%–15%) and a long tail of recurrent lower frequency somatic mutations. Functional and clinical studies of novel mutations have uncovered new mechanisms involved in the pathogenesis of the disease, revealing new insights into CLL molecular evolution that could ultimately translate into improvements in the management of patients. The clonal architecture of CLL shows striking heterogeneity between patients, which could have important clinical implications. In summary, NGS studies of CLL are expanding our fundamental knowledge on the molecular mechanisms involved in the pathogenesis of the disease and offering new perspectives for the clinical management of the patients. Semin Hematol 50:286–295. C 2013 Elsevier Inc. All rights reserved.

C

hronic lymphocytic leukemia (CLL) is the most common lymphoproliferative disorder in adults in Western countries. The disease arises from a subpopulation of mature CD5þ B cells that proliferate and accumulate in the body with progressive infiltration of peripheral blood, bone marrow, and tissues.1,2 The clinical course of CLL is highly heterogeneous, with some patients having a normal life expectancy while others show progressive disease that can be fatal. During the evolution of the disease approximately 1% of patients per year develop a transformation to a diffuse large B-cell lymphoma (DLBCL), an event that confers a poor prognosis. The clinical heterogeneity of CLL has been related to characteristics of patients at diagnosis and to the biology of leukemic cells.2,3 Classically, the mutational status of the

a

Unitat d’Hematopatologia, Servei d’Anatomia Patològica, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain. b Servei d’Hematologia, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain. c Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain. d Universitat de Barcelona, Barcelona, Spain. Conflicts of interest: The authors declare that they have no conflicts of interest or competing financial or personal relationships that could inappropriately influence the content of the article. Address correspondence to Neus Villamor, MD, Unitat d’Hematopatologia, Hospital Clínic, Villarroel 170, 08036-Barcelona, Spain. E-mail: [email protected] 0037-1963/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1053/j.seminhematol.2013.09.005

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variable region of the immunoglobulin heavy chain (IGHV) distinguishes two major subgroups of CLL according to the presence of high or low number of somatic mutations. These mutations are physiologically introduced in the germinal center cells as a mechanism to generate a repertoire of highaffinity antibodies. The putative cell of origin of the different subtypes of CLL is still unresolved, but recent epigenetic and transcriptomic analysis suggest that CLL carrying mutated IGHV may derive from a distinct CD5þ post-germinal center memory B cell, whereas unmutated IGHV CLL would originate from an antigen-experienced B cell that maintains an epigenetic imprint of naïve CD5þ B cells.4,5 Clinical correlations have shown that chromosomal alterations have an important impact on the prognosis of the disease, thus providing a hierarchical classification of patients.1–3 Although these alterations provide information for patient stratification according to the risk for progression, both clinical heterogeneity in the evolution of the disease and the differential response to therapy are not completely explained by these parameters, suggesting that additional factors related to the host (eg, comorbidities and genetic background), microenvironment, and somatic alterations may influence outcome. The recent development of new generation of sequencing technologies (next-generation sequencing [NGS]) and their systematic application to human cancer can provide characterization of somatic events across the complete genome, exome (all annotated exons) or transcriptome (all RNA transcripts) in a single experiment. NGS can be used to detect changes in single nucleotides, small insertions or deletions (indels), or larger structural alterations including

Seminars in Hematology, Vol 50, No 4, October 2013, pp 286–295

Next-generation sequencing in CLL deletions, amplifications, and translocations.6,7 Several subtypes of lymphoid neoplasms have been recently investigated, including CLL,8–10 hairy cell leukemia (HCL),11 DLBCL,12–14 Burkitt lymphoma (BL),15,16 and plasma cell myeloma.17 Although the number of cases examined in most of these tumors is still relatively low, which limits the capacity to draw definitive conclusions, the findings provide new insights into the pathogenesis of the diseases with potential clinical implications.18

PATTERNS OF SOMATIC MUTATIONS IN CLL To date, a series of studies have reported the sequence data of 10 whole genomes (WG) and more than 250 whole exomes (WES) of CLL.9,10,19–22 These studies have provided the first insight into genes and pathways targeted by recurrent somatic mutations and have identified potential mechanisms contributing to its mutagenic process. WG analysis has shown that CLL genomes carry approximately 1,000 somatic mutations per tumor in nonrepetitive regions of the genome. This mutational load corresponds to an average of 0.6–0.9 mutations per megabase (Mb) and 10–20 nonsynonymous mutations per case (range, 2–76). This number of mutations is similar to that reported in acute myeloid leukemia but lower than in myeloma (1.3 mutations per Mb)17 or DLBCL (3.2 mutations per Mb).12–14 The most common change in CLL, as in other tumors, is the C-T transition in the context of CpG dinucleotides. IGHV-mutated CLL shows a particular nucleotide substitution characterized by overrepresentation of thymine in the preceding base to adenine in A-C transversions, when compared to the expected prevalence in non-repetitive sequences in wildtype genome. They also contain lower A-C substitutions in GpA dinucleotides than expected by chance.9 Thus, IGHV-mutated CLL had a significantly higher proportion of A-C/T-G mutations than IGHV-unmutated CLL (16 ⫾ 0.2% v 6.2 ⫾ 0.1%).9 This finding has been subsequently confirmed by WE analysis in two independent series.19,20 The pattern and context of these A-C mutations are consistent with the frequent error introduced by DNA polymerase η when it is recruited to repair DNA breaks, a hallmark of the normal process of somatic hypermutation (SH). Therefore, the differences observed between CLL with mutated and unmutated IGHV could be a consequence of an additional imprint of the germinal center microenvironment, reflecting the different cell origin of the two subtypes of CLL. The number of mutations observed in CLL cases increases with age and appears not to significantly differ between untreated and treated patients.19,22

REPERTOIRE OF SOMATIC MUTATIONS IN CODING REGIONS NGS studies of CLL have revealed only a few genes mutated with frequencies in the range of 10%–15% but a large number of genes mutated at lower frequencies

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(2%–5%).10,19,20 A relatively similar heterogeneous profile of mutations has also been observed in DLBCL and plasma cell myeloma.17 Contrary to this pattern, other tumors have a predominant gene that is mutated in virtually all cases, usually with identical or a small set of recurrent mutations, which indicate the presence of a strong driver of oncogenesis. This is the case of the BRAF V600E detected in HCL11 or the MYD88 L265P in Waldenström macroglobulimenia.23 Although many mutated genes occur at low frequency in many tumors, they tend to cluster in common pathogenic pathways. An additional feature of recurrent mutations is the transversal distribution of certain mutations across entities, although generally at diverse frequency. For example, MYD88 mutation has been found recurrently mutated in activated B-cell subtype of DLBCL (29% of cases),24 mucosa-associated lymphoid tissue lymphoma (MALT) (9%),24 and CLL (3%).9 WES analysis in CLL have identified more than 1,000 genes with somatic mutations that predict possible functional consequences.10,20 However, the number of genes recurrently mutated in two or more patients is around 10 times lower and most of them are observed at frequencies below 3%–5%. The distribution of recurrent mutated genes shows a bias in the two IGHV subtypes of the disease with some genes found predominantly or exclusively in CLL with unmutated IGHV CLL (NOTCH1, SF3B1, XPO1, or POT1) while others are mostly found in mutated IGHV CLL (MYD88, CHD2, or KLHL6), suggesting that the different behavior of these two subtypes of CLL may be related to the activation of different molecular mechanisms.25–27 The imprint of the germinal center SH machinery is observed in the pattern of mutations of some genes (ie, KLHL6),20 but the extent and biological impact of this mutational mechanism need additional studies. The molecular heterogeneity of CLL is further demonstrated by the distribution pattern of mutated genes in cohorts analyzed by WES. The most commonly mutated genes in the study of the International Cancer Genome Consortium (ICGC) were NOTCH1 (12%) and SF3B1 (10%) followed by POT1 (5%), CHD2 (5%), and LRP1B (5%),9,20 whereas in the two overlapping series from the Dana-Farber Cancer Institute, the most common were SF3B1 (14%), TP53 (13%), NOTCH1 (10%), MYD88 (8%), and ATM (8%).10,19 The comparison of clinical features of these three series of patients shows marked differences regarding the median age, representation of adverse prognostic parameters, and inclusion of samples after treatment (Table 1). These features can influence the distribution of the mutated genes. In this sense, the frequency of ATM, TP53, and SF3B1 mutations were higher in post-treatment samples.10,19 The heterogeneity of the molecular alterations in CLL is further reinforced by the small number of common recurrent mutations observed in these studies. Intriguingly, when the 100 most frequently mutated genes are considered, a large number of patients still do not show mutations in any (Figure 1). Even though the number of mutated genes is

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Table 1. Recurrent Somatic Mutated Genes Detected by Whole-Exome Sequencing and Clinical

Characteristics of the Patients Quesada et al20

Wang et al10

Landau et al19,a

(n¼105)

(n¼91)

(n¼160)

Gene NOTCH1 SF3B1 POT1 CHD2 LRP1B* ATM CSMD3 MUC2 SI* ASXL1* LPHN3* NXF1 SFRS1 SLC4A1* XPO1 MYD88 RIMS2

% 12 10 5 5 5 4 4 4 4 3 3 3 3 3 3 3 3

Median age Untreated (%) Del17p (%) UM-IGHV (%)

62 years 100% 1% 45%

Gene TP53* SF3B1 MYD88 ATM FBXW7 NOTCH1 ZMYM3* DDX3X* MAPK1 FAT4 MUC2 DST* CSMD1*

% 15 15 10 9 4 4 4 3 3 5 4 4 4

54 years 66% 19% 49%

Gene SF3B1 TP53* NOTCH1 MYD88 ATM XPO1 CHD2 POT1 HIST1H1E* NRAS BCOR* ZMYM3* RIPK1 SAMHD1 KRAS MED12* ITPKB DDX3X* EGR2*

% 14 13 10 8 8 4 4 3 3 3 3 3 3 3 2 2 2 2 2 54 years 79% 13% 38%

a

Includes 81 patients from Wang et al. *Genes mutated in both ICGC and Dana-Farber Institute cohorts. NOTE. Genes in bold are recurrently mutated in all three WES analysis; underlined genes are recurrently mutated in two different WES studies

very high, they tend to cluster in a limited number of pathways that usually include one of the genes mutated at higher frequency and several low recurrent mutated genes.10,19,20 These pathways include NOTCH signaling; mRNA splicing, processing, and transport; innate inflammatory mechanisms; DNA damage response and cell cycle control; Wnt signaling; B-cell receptor signaling; and chromatin modification.19,20 The clinical and biological relevance of most of these mutated genes is still unknown since they have recently been identified by NGS and await further functional characterization. Nevertheless, the functional implications and clinical impact of some of them, particularly NOTCH1, SF3B1, and BIRC3, has been already evaluated in relatively large series of patients.

NOTCH1 MUTATIONS NOTCH1 has been found to be recurrently mutated in 10%–15% of CLL patients in independent analyses, and

thus represents one of the more frequently mutated genes in CLL.8,9,28 NOTCH1 encodes a class I transmembrane protein that serves as a ligand-activated transcription factor regulating cell differentiation, proliferation, and apoptosis. Upon activation, NOTCH1 undergoes several proteolytic cleavages that result in the translocation of the intracellular portion to the nucleus where it activates transcription of multiple target genes. The mutations of NOTCH1 observed in CLL generate a premature stop codon, resulting in a NOTCH1 protein lacking the C-terminal domain that contains a PEST degradation sequence. Removal of this region leads to a more stable and active isoform of the protein in CLL cells, thus inducing a higher expression of genes of the NOTCH1 signaling pathway.9 The majority of NOTCH1 mutations (96%) are located in exon 34 affecting the PEST region of the protein (Table 2 and Figure 2A). Thirty-two different mutations of NOTCH1 have been described with a hotspot corresponding to a 2-bp deletion resulting in a frameshift

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Figure 1. Percentage of patients with mutation in at least one of the first 91 more frequently mutated genes in Quesada et al.20

(p.P2515Rfs*4) that truncates the protein. This change represents 80%–85% of all NOTCH1 mutations described thus far. One mutation in the homodimerization domain (HD), p.V1722M, was acquired in the transformation to DLBCL of a CLL that already carried a p.P2515Rfs*4.8 The association of these two NOTCH1 mutations in transformation to DLBCL could be relevant because they act synergistically and are associated with a more aggressive disease in patients with T-cell acute lymphoblastic leukemia.29 Similarly, a recent study has described the translocation dic(9;14)(q34;q32) fusing IGH and NOTCH1 associated with an increased NOTCH1 expression in a DLBCL transformed from a CLL that already carried the common p.P2515Rfs*4, suggesting that it could be involved in the progression of the disease.30 NOTCH1 mutations have been more frequently found in CLL with adverse prognosis parameters such as

unmutated IGHV and elevated expression of ZAP-70 and CD38.9,25,28,31–34 These patients are more likely to have high levels of circulating lactate dehydrogenase and β2-microglobulin.34 NOTCH1 mutations are also associated with trisomy 12,28,31–36 particularly when this chromosomal alteration is the only genetic aberration.36 Patients with NOTCH1 mutations have a more aggressive disease as they have more advanced clinical stage and higher risk for progression than unmutated patients.28,32,34 NOTCH1 confers an increased risk of transformation to DLBCL (hazard ratio  5)34,37 with a cumulative incidence at 10 years of 31%, which is independent from unmutated IGHV and TP53.34,37 Most studies have found a shorter time to treatment (TTT) and overall survival (OS) in patients carrying NOTCH1 mutations,25,28,31–34,38 with similar effect for the different types of NOTCH1 mutations.28,34 However, the independent prognostic impact of these mutations from

Table 2. Frequency, Distribution, and Type of NOTCH1 Mutations

Authors

Patients

Technique

Exon(s)

N

Mutateda

p.P2515Rfs*4b

Wang et al10 Oscier et al25 Villamor et al34,c Shedden et al33 Rossi et al40 Del Guidice et al31,d Di Ianni et al48 Balatti et al35,e Mansouri et al38

U&T U U U&T D NS NS NS D

WES HRM Sanger Sanger Sanger Sanger Sanger Sanger Sanger

All 34 34 34 34 34 26,27,34 34 34

91 466 565 257 637 104 43 263 360

4 (4.4%) 49 (10.5%) 63 (11.1%) 21 (8.2%) 71 (11.1%) 25 (24%) 2 (4.6%) 29 (11%) 17 (4.7%)

100% 84% 86% 62% 82% 88% 100% 93% NS

2,786

281 (10.1%)

83.7%

Total a

Eleven mutations were in the TADD region (4.2%). p.P2515Rfs*4: 221/261 cases with available data. c Includes and expands the cases identified in Puente et al.9 d CLL cases selected by the presence of trisomy 12. e Seventy-seven cases of cases included were selected by the presence of trisomy 12. Abbreviations: U, untreated; T, treated; D, at diagnosis; NS, not specified; HRM, high-resolution melting. b

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Figure 2. Distribution and frequency of mutations in (A) NOTCH1 gene and (B) SF3B1 gene. Numbers indicate amino acid position, yellow stars indicate truncating mutation, and blue stars indicate missense mutation. (Refer to Seminars in Hematology online for color figure.)

other parameters associated with aggressive disease (ie, IGHV mutational status) is not completely clear. Several studies have found that both NOTCH1 and IGHV contribute independently to worse OS,25,28 but this finding has not been corroborated in other studies.33,34 NOTCH1 mutations are detected at lower frequencies in monoclonal B-cell lymphocytosis (MBL) (3.2%)39 and at diagnosis (5%–8%)8,38 than in patients who progress (10%–16%),25,34 are refractory to treatment (21%),8 or undergo transformation to DLBCL (31%).8 These findings are not unexpected due to the association of NOTCH1 mutation with adverse clinicobiological parameters. This raises the question whether a NOTCH1 mutation is a late and acquired event in CLL or if its higher incidence reflects the disease evolution of patients with NOTCH1 mutations. Acquisition of NOTCH1 mutations has been reported in transformation to DLBCL

in five of 16 patients,8 and in one of four patients who become refractory to chemotherapy.28 In contrast, a sequential study of 200 patients identified only one patient who increased the size of a NOTCH1 mutated clone to levels detectable by Sanger sequencing after 9 years of stable disease.34 Moreover, the allelic frequency analysis of NOTCH1 in sequential samples from 202 patients showed no significant variation over time.40 These data suggest that acquisition of NOTCH1 mutations in the evolution of the disease, although possible, most likely is a rare event in CLL patients. The potential impact of NOTCH1 mutations in response to treatment and outcome after treatment is still not well understood. In patients recruited to the randomized UK LFR CLL4 trial (chlorambucil v fludarabine v fludarabine plus cyclophosphamide [FC]), response to treatment was not related to the presence of NOTCH1

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291

mutations in any of the three therapeutic arms.25 However, NOTCH1 was a prognostic factor for OS from randomization independent from IGHV mutational status and other adverse parameters.25 Moreover, preliminary data suggest that the depth of response in patients with NOTCH1 mutated CLL is lower and the relapse rate after achieving complete response is higher than in patients with unmutated NOTCH1.34 Therefore, prospective studies addressing the impact of NOTCH1 in response to treatment will be of great interest.

SFB31 MUTATIONS One of the most interesting findings of the genomic studies in CLL has been the discovery of highly recurrent somatic mutations in SF3B1 (10%–15%) and the potential relevance of other alterations in the splicing machinery and RNA processing and transport in the pathogenesis of the disease.10,20,21 SF3B1 encodes a core component of the spliceosome involved in the binding of the U2 snRNP to the branchpoint close to the 3′ splicing sites and ensures the fidelity of the splicing process. The predominant SF3B1 mutation is K700E that accounts for approximately 45% of cases, followed by G742D (  15%), and K666 (  10%). The remaining 30% are represented by mutations at 22 different positions (Table 3 and Figure 2B). Most of these mutations cluster in the C-terminal HEAT-repeat domain of SF3B1 supposed to disrupt the binding of the protein to some cofactor that, in turn, might decrease the splicing fidelity in specific genes. This hypothesis was confirmed by the identification of several genes with anomalous spliced transcripts in cases with SF3B1 mutation.10,20 The frequency of SF3B1 mutations at diagnosis is approximately 5%–7%26,38,40 and its frequency increases in samples obtained at progression (17%)25 or after treatment (12%–24%)10,41 (Table 4). The clinicobiological characteristics of patients with SF3B1 mutations are not fully established. Patients with SF3B1 mutations present in advanced clinical stage20,26 and with adverse biological factors such as unmutated IGHV,10,20,41 high ZAP-70,26,41 or del11q.10 However, not all associations

have been confirmed in other studies.20,25,26,41 Additionally, no relationship between SF3B1 mutations and transformation to DLBCL has been found.26,37 Patients with SF3B1 mutation have a shorter TTT10,20,26,41 and a lower OS20,25,26,41 than unmutated patients. In most studies, SF3B1 mutations have an impact on OS that is independent of the status of either TP53 or IGHV.25,26,38 An interesting observation is that SF3B1 mutations are more frequent in cases refractory to fludarabine (17%) lacking TP53 mutation.26 In the randomized UK LRF CLL4 trial, no differences in the overall response rate and progressionfree survival (PFS) were observed according to the presence of SF3B1 mutations, but PFS of patients in the FC arm carrying SF3B1 mutations and wild-type TP53 was significantly shorter.25 In line with these findings, a high frequency of SF3B1 mutations (26%) were observed in patients included in the German CLL study group (GCLLSG) CLL3X trial, in which high-risk patients (ie, fludarabine-refractory or del17p) were treated with reduced intensity allogeneic transplant.42 Therefore, SF3B1 mutations could be useful in the identification of patients refractory to fludarabine, particularly among those with wild-type TP53. In addition to SF3B1, exome studies have revealed mutations in different genes of the spliceosome subunits and RNA transport machinery. Ramsay et al21 have identified 52 mutations in 29 genes involved in these pathways. These alterations were found in 44 of 140 (31%) CLL patients and virtually all were predicted to have functional effect including several frameshifts, premature stop codons, or missed splicing sites.21 Four genes involved in RNA splicing (SF3B1, SFRS1/SRSF1, U2AF65/U2AF2, and BRUNOL/CELF4), and three from the RNA export machinery (NXF1, XPO1, and DDX3X) were among the 60 genes recurrently mutated in CLL at a higher rate than expected.20 Interestingly, all mutations in genes of the RNA transport machinery were found in CLL with unmutated IGHV.21 Although the high frequency of mutations in these pathways suggests a role in the pathogenesis of the disease, the functional implications and clinical significance are still not fully understood.

BIRC3 MUTATIONS Table 3. SF3B1 Mutations in CLL (from arti-

cles reported in Table 4) Mutation

%

K700E G742D K666E/M/N/Q/R/T H662D/Q/N K622D/V I704F/N Other

43% 17% 10% 4% 3% 3% 20%

BIRC3 encodes a protein member of the inhibitor of apoptosis (IAP) family that inhibits apoptosis and regulates nuclear factor-κB (NF-κB) activity. BIRC3 mutations were initially identified by WGS8 and confirmed in an extended series of CLL patients.27 These mutations predict for an inactive protein generated by premature stop codons that eliminate or truncate the C-terminal ring domain. Additionally, deletions of BIRC3 at 11q23 have been observed by single-nucleotide polymorphisms (SNPs) array, resulting in lower expression of the protein.27 According to the predicted loss of function, these genetic alterations seem to be associated with the activation of the non-canonical NF-κB pathway. The frequency of BIRC3 mutations is

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Table 4. Frequency, Distribution, and Type of Mutations of SF3B1

Authors 10

Wang et al Oscier et al25,a Quesada et al20,b Rossi et al40 Schwaerderle et al41,c Mansouri et al38

Patients

Technique

Exon(s)

N

Mutated

K700E

U&T U U D U&T D

WES Sanger Sanger Sanger Sanger Sanger

All 14,15,16 14,15,16,18 14,15,16,18 14,15,16,18 14,15,16

91 437 279 637 545 360

14 73 27 43 36 13

50% 51% 33% 42% 55% NS

2,349

206 (8.8%)

Total

(15.4%) (16.7%) (9.7%) (6.7%) (6.6%) (3.6%)

43%

a

Patients analyzed at progression. One hundred five patients analyzed also by WES. c In 298 cases only exons 14 and 15 were analyzed. Abbreviations: U, untreated; T, treated; D, at diagnosis. b

almost undetectable in MBL and uncommon at diagnosis (0%–5%).10,20,27 Although these mutations are not associated with the progression of the disease or transformation to DLBCL, they have been frequently found (25%) in patients refractory to fludarabine with wild-type TP53. Interestingly, TP53 and BIRC3 alterations were mutually exclusive, suggesting that they may be alternative mechanism conferring resistance to fludarabine. BIRC3 anomalies detected at diagnosis had an adverse impact on the survival of the patients that appear to be independent from other adverse factors such as TP53 abnormalities.27

INTEGRATING NEW MUTATIONS IN PROGNOSTIC MODELS The clinical independent impact of new mutations found by NGS in CLL suggests that these alterations could be incorporated into new prognostic models to improve risk stratification of CLL patients.38,40 In a recent study, Rossi et al40 revised the prognostic value of traditional and new parameters in 637 newly diagnosed CLL patients. High-risk patients were defined by abnormalities in TP53 and BIRC3 with a 10-year OS of 29%. An intermediate-risk group, with a 10-year OS of 37%, consisted of patients with del11q22-q23, NOTCH1, and SF3B1 mutations. Low-risk patients, with a 10-year OS of 57%, harbored trisomy 12 or no apparent genetic prognostic factor, while patients with a very low risk (10-year OS: 69.3%) were those with isolated del13q14. This proposed hierarchical system to classify patients should be validated in additional prospective series.

RECURRENT MUTATIONS AT LOW FREQUENCY IN CLL Genome sequencing studies have discovered a long list of additionally mutated genes at low frequencies that could play a role in the pathogenesis of the disease in subsets of patients. The oncogenic role and clinical impact of most of these genes is unknown, but recent studies are revealing

the functional mechanisms of some of them and the distinctive clinical features of patients carrying such mutations. MYD88 (myeloid differentiation primary response 88), a critical adaptor element in the interleukin-1 (IL1R) and Toll-like receptors (TLR) signaling pathway, has been found to be recurrently mutated in 3%–10% of CLL patients.9,10 MYD88 is recruited to the cytoplasmic portion of the TLRs and activates IRAK4, which, in turn, phosphorylates and activates IRAK1 and subsequently tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) resulting in the activation of NF-κB. The most common MYD88 mutation is L265P, but other mutations have occasionally been found (L258P, V217F, M232T).10,40 These mutations occur in heterozygosis and affect highly phylogenetically conserved residues.9,24 MYD88 L265P acts as an activating mutation on CLL cells, binding larger amounts of IRAK1 than the wild type and inducing higher activation of the downstream effectors STAT3 and NF-κB p65 subunit.9 After stimulation of IL1R or TLR, CLL cells carrying the L265P mutation secrete larger amounts of interleukin-1 receptor antagonist (ILRA), interleukin-6 (IL6), and chemokine ligands 2, 3, and 4 (CCL2, CCL3, and CCL4) that play a role in the supportive microenvironment of CLL cells and protect the cells from spontaneous apoptosis. Preliminary analyses of patients carrying MYD88 mutations suggest that they may correspond to a particular clinical subgroup. CLL is a disease of older individuals with a median age at diagnosis of 70 years. Strikingly, patients with mutated MYD88 are mostly diagnosed under the age of 50 years and virtually all cases have mutated IGHV.9,10 The high frequency of MYD88 mutations observed by both Wang et al10 and Landau et al19 probably reflects the younger age of the patients in these cohorts (median age, 54 years). Further clinical studies are needed to clarify the clinical relevance of these mutations. POT1 (protection of telomeres 1) encodes for a protein of the shelterin complex, a multiprotein structure that protects the telomeres. Exome sequencing and expanded validation studies have found POT1 mutations in 1%–5% of the

Next-generation sequencing in CLL patients,10,20 but they reach 9% in the subset of CLL with unmutated IGHV.43 The majority of POT1 mutations are located in the oligonucleotide/oligosaccharide binding (OB1 and OB2) folds in highly phylogenetically conserved residues. Mutated POT1 normally interacts with other members of the shelterin complex (TRF1, TRF2, RAP1), but the mutations observed in CLL affect key residues that destabilize or abrogate binding of POT1 to telomeric single-stranded DNA. POT1 mutations are heterozygous and most likely function as a dominant-negative inducing a dysfunctional telomeric phenotype characterized by elongated telomeres and unprotected telomere ends that lead to chromosomal alterations (fusion of sister chromatids, multi-telomeric signals suggestive of telomeric fragility and unresolved mitotic chromosomal structures).43 POT1-mutated CLLs are associated with adverse diagnostic biological features such as advanced Binet stages, unmutated IGHV, and high expression of ZAP-70 by the leukemic cells. SI (sucrose-isomaltase) is a type II transmembrane glycoprotein essential for processing dietary carbohydrates that is mutated in 2%–4% of CLL cases.10,20,44 SI is composed of two highly similar sucrase and isomaltase subunits originating from a single polypeptide precursor through proteolytic cleavage. It is glycosylated in the Golgi apparatus before sorting its mature protein form in the luminal pole of the intestinal cells. The role of SI in cancer has been investigated in colorectal malignancies and is upregulated in adenomas and correlates with progression to frank carcinoma.45 The SI heterozygous mutations observed in CLL induce loss of its enzymatic function due to a defective maturation pattern along the secretory pathway; this results in its accumulation in the endoplasmic reticulum and reduced normal expression of the mature form in the cell surface.44 CLL cells with mutated SI had a differential gene expression profile affecting mainly metabolic pathways with upregulation of several processes known to participate in cancer metabolic reprogramming, suggesting that this mechanism may participate in the pathogenesis of the disease. Finally, a reciprocal and recurrent RNA chimera between YPEL5 and PPP1CB has been recently identified by paired-end transcriptome sequencing.46 Chimeric transcripts appear to be specific to CLL leukemic cells and were detected by quantitative polymerase chain reaction in 95% of patients. Additional studies confirming this finding and analyzing the cellular mechanism of this chimeric transcript are desirable.

HIERARCHICAL ARCHITECTURE OF CLL SUBCLONES AND CLINICAL EVOLUTION Two recent studies have analyzed the subclonal composition of CLL and its modulation during the evolution of disease.19,22 Schuh et al22 studied three patients with intermediate-risk CLL at five different time points (pretreatment and after different sequential treatments).

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Investigating the allelic frequency of multiple mutations, they identified the founder clone and distinct subclones in each patient that indicate five different patterns of clonal evolution: (1) subclones with initial high frequency remaining high or (2) decreasing after treatment; (3) initial low frequency remaining low or (4) increasing after treatment; and (5) appearance of new clones undetectable at diagnosis. Each patient showed different subclonal dynamics and not all patients displayed the five evolving patterns. One of the patients maintained different subclones with stable evolution over time, despite receiving multiple therapies. A second patient had a similar equilibrium of different subclones but eventually, after several relapses, a subclone outcompeted the others at the time of disease progression. Finally, a third patient showed dramatic changes in clone composition after subsequent chemotherapies in which different minor subclones present at diagnosis became the predominant clone at each relapse. The list of mutated genes in the founder clone was unique to each patient and involved genes recurrently mutated in CLL (SF3B1, SAMHD1, and MED12) that were associated with 5–10 non-recurrent mutations that could be passenger mutations. The integrative analysis of high-depth coverage sequencing data and DNA copy number alterations allowed Landau et al19 to estimate the fraction of cancer cells carrying different mutations. Driver mutations were classified as clonal and subclonal according to their presence in all tumor cells or small subsets. Clonal mutations were considered to represent early events in the evolution of the disease, whereas subclonal mutations were likely to be acquired late in the evolution of the disease. In this study, MYD88 mutations, 13q allelic deletions, and trisomy 12 were identified as clonal in 80%–100% of the samples, while TP53, ATM, and SF3B1 mutations were more often subclonal. Interestingly, clonal mutations appear to be restricted to B-cell malignancies, whereas subclonal mutations occur in genes frequently found mutated across tumors.19 The analysis of sequential samples has led to the proposal of a progression model of the disease in three periods. In the first preneoplastic phase, lymphoid cells accumulate passenger mutations in proportion to the age of the patient. The acquisition of a mutation in a B-cell driver gene will promote the expansion of a founder clone that will continue accumulating other mutations and will generate different subclones. These heterogeneous subclonal populations may persist in a relative balanced equilibrium over time. In some cases, a third period may be opened with the emergence of a dominant clone under intrinsic or external selective influences. One of the most powerful extrinsic influences is chemotherapy, which can select for the expansion of a clone already present in a minor subpopulation in early phases of the disease; the emerging clones usually carry mutations in genes that confer resistance to chemotherapy. Moreover, the study of Landau et al19 reveals the clinical implications of a more refined understanding of clonal evolution in CLL. The authors

N. Villamor et al

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suggested that the presence of subclones adversely impact the outcome of patients, who had shorter failure-free survival times. Rossi et al40 evaluated the clonal evolution of TP53, NOTCH1, SF3B1, MYD88, and BIRC3 mutations in sequential samples from 202 patients, six of them with transformation to DLBCL. NOTCH1 and MYD88 mutations were only acquired in two and one of the 196 CLLs, respectively, and only one of these NOTCH1 mutations emerged after treatment. On the contrary, seven of nine TP53 mutations, six of SF3B1 mutations, and four of four BIRC3 mutations were acquired in relapsed samples. These observations support the idea that NOTCH1 and MYD88 mutations seem to be early events in the evolution of CLL, whereas TP53, SF3B1, and BIRC3 mutations appear to be mainly selected after treatment.

CONCLUSIONS AND FUTURE DIRECTIONS Genome sequence analysis of CLL with NGS has substantially expanded our knowledge of the genetic alterations in CLL and now offers new tools for understanding the evolution of CLL.47 These studies have identified a high number of genes and pathways altered in different subgroups of patients. The initial functional and clinical studies of the most common and novel mutations have already revealed new mechanisms involved in the pathogenesis of the disease and have identified subgroups of patients with different prognosis and response to treatment. This information should provide new biomarkers and potential therapeutic targets to improve the management of patients. However, the heterogeneous landscape of mutations in CLL, the absence of a clear driver mutation in a substantial number of cases and the still unanswered questions on the clinical impact of common mutations, in spite of the already large number of patients investigated, reveal a disease of unprecedented genetic complexity. The relevance of the hierarchical clonal distribution in the evolution of the disease provides a conceptual framework to integrate genetic and microenvirontmental influences with clinical and biological implications.47 The understanding and translation to the clinic of these insights is challenging and will require further studies integrating genomic data in well-characterized clinical studies.

Acknowledgments The ICGC CLL-Genome Project is funded by Spanish Ministerio de Economía y Competitividad (MINECO) through the Instituto de Salud Carlos III (ISCIII) and Red Temática de Investigación del Cáncer (RTICC) (RD12/ 0036/0023 and RD12/0036/0023) del ISCIII. Carlos López-Otín is an investigator of the Botin Foundation and Elías Campo an ICREA- Acadèmia researcher. We are grateful to the members of our groups for their contribution to the studies of the consortium, N. Villahoz and M. C. Muro for their excellent work in the coordination of the CLL Spanish Consortium, and Silvia Martin, Cristina

Capdevila and Miguel Osuna for their excellent technical assistance.

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