Author's Accepted Manuscript

Gene Mutations in Chronic Lymphocytic Leuke-

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Nisar A. Amin, Sami N. Malek

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DOI: http://dx.doi.org/10.1053/j.seminoncol.2016.02.002
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 Gene mutations in Chronic Lymphocytic Leukemia

Nisar A. Amin1 and Sami N. Malek1,2.

Department of Internal Medicine, Division of Hematology and Oncology1, University of

Michigan, Ann Arbor, MI, USA.

Running title: Gene Mutations in CLL

Keywords: Gene mutations, CLL, biology and prognosis

Word Counts: Abstract: 200 , Main Text: 4,500

2
Correspondence should be addressed to: Sami N. Malek, Associate Professor, Department of

Internal Medicine, Division of Hematology and Oncology, University of Michigan, 1500 E.

Medical Center Drive, Ann Arbor, MI 48109-0936. smalek@med.umich.edu. Phone: 734-

763-2194. Fax: 734-647-9654.

1
 Abstract

The recent discovery of genes mutated in CLL has stimulated new research into the role of

these genes in CLL pathogenesis. CLL cases carry approximately 5-20 mutated genes per

exome, a lower number than detected in many human tumors. Of the recurrently mutated

genes in CLL, all are mutated in 10% or less of patients when assayed in unselected CLL

cohorts at diagnosis. Mutations in TP53 are of major clinical relevance, are often associated

with del17p and gain in frequency over time. TP53 mutated and associated del17p states

substantially lower response rates, remission duration and survival in CLL. Mutations in

NOTCH1 and SF3B1 are recurrent, often associated with progressive CLL that is also IgVH

unmutated and ZAP70 positive and are under investigation as targets for novel therapies and

as factors influencing CLL outcome. There are estimated 20-50 additional mutated genes

with frequencies of 1-5% in CLL; more work is needed to identify these and to study their

significance. Finally, of the major biological aberration categories influencing CLL as a

disease, gene mutations will need to be placed into context with regard to their ultimate role

and importance. Such calibrated appreciation necessitates studies incorporating multiple CLL

driver aberrations into biological and clinical analyses.

2
A general introduction: Chronic lymphocytic leukemia (CLL) is the most common leukemia

in the Western world. It has a varied clinical course and multiple biological and clinical

parameters influence the disease. Major biological phenomena driving the disease are CLL

cell-intrinsic (for instance, genomic aberrations, aberrant BCR signaling, epigenetic

aberrations or stable expression changes) or CLL cell-extrinsic (for instance, a dependency on

antigen-stimulated BCR signaling, cell to CLL cell interaction or cytokine to CLL cell

interaction, collectively referred to as the microenvironment). Of the genomic changes

underlying CLL pathogenesis, acquired genomic copy number changes (aCNAs) are well

described1-4. With regard to gene mutations in CLL, our knowledge is undergoing rapid

expansion and definitive answers regarding the role of recurrent gene mutations or mutated

functional pathways in CLL are available for only a few genes5,6.

The recent advent of massively parallel sequencing technologies combined with

advances in targeted DNA enrichment (exon capture reagents) and bioinformatics analysis

tools has allowed for the identification of multiple recurrently mutated genes in CLL. These

mutated genes lay the foundation for studies into the biological and clinical consequences of

such mutations and complement overall studies of factors influencing CLL disease behavior.

Most of our current knowledge of gene mutations in CLL is the result of sequencing of CLL

exomes using whole exome sequencing or WES (exome: the sum of all coding exons of all

genes); very few CLL cases subjected to whole genome sequencing have been reported and

more work in this area is needed to derive meaningful conclusions5-7. The reagents used to

capture and enrich exonic DNA from sheared total genomic DNA isolated from CLL cells are

evolving and in addition to exons may also contain capture probes for genomic DNA

encoding for microRNAs or other non-protein coding RNAs. The depth of sequencing (the

3
number of times any particular nucleotide is part of a sequencing trace or tag) determines the

sensitivity of mutation detection, and in general the higher the depth of coverage the better the

quality characteristics of the study. Due to multiple technical reasons, gaps in sequencing

coverage nonetheless still exist, which are complicated further by the inability of modern

software pipelines to call all true mutations (especially insertion/deletion mutations or indels)

with high confidence. Therefore, WES studies do not detect all true exome mutations in CLL.

CLL exomes carry approximately 5-20 somatically acquired mutated genes per

individual CLL case, much fewer than many other solid cancers5-7. Importantly, the

somatically acquired mutation status of sequence variants in genes, similar to other studies of

genomic changes in cancer, necessitates confirmation of the absence of these candidate

mutations in paired non-tumorous DNA (T-cell-, buccal cell- or skin-derived DNA), a

necessary step not always performed in published studies.

Of the somatically acquired mutated genes in CLL, some genes are recurrently

targeted and constitute the basis for discussions in this review. However, the majority of genes

are not recurrently or are infrequently targeted;it remains unclear on statistical grounds alone

if they could exert a biological function in affected cells (the drivers) or not (the passengers).

Our current ability to separate drivers from passengers in CLL is limited by lack of suitable

experimental systems. Gene mutations with experimentally verified biological effects on the

afflicted CLL cell, for instance, affecting CLL cell proliferation, apoptosis thresholds, CLL

cell to micro-environmental interactions, genomic or epigenetic instability or chemotherapy

resistance could be labeled as gene drivers, while genes that lack any detectable such effects

could be labeled as passengers (passengers are genes subjected to random mutagenesis

without demonstrable effects on the fitness or biological properties of afflicted cells). Others

4
have used practical cut-offs for mutation frequencies (15%) as indicators of drivers status but

such criteria are too stringent for CLL where gene mutations frequencies are 10% or less at

diagnosis.

Before embarking on a granular gene-by-gene discussion of gene mutations and the

aggregate, albeit limited, knowledge of functions and consequences of such mutations in CLL,

we wish to first discuss and emphasize various broader topics of research into mutated genes

in CLL.

What is the effect of a CLL-associated gene mutation on protein function? Gene

mutations that shorten or elongate the affected proteins or that alter the amino acid sequence

(non-sense mutations that introduce stop codons or insertion/deletion [indel] mutations that

often change the reading frame [also called frameshift mutations]) often inactivate the affected

protein. However, this is not always the case and altered proteins may have augmented

function or even gain-of-function. Even more difficult is the prediction of the effect of

missense mutations (mutations that change the codons for individual amino acids) on protein

function; functional assays are needed to ascertain specific effects. Even within genes that are

recurrently targeted by function- or activity-altering mutations, some of the missense

mutations may be functionally silent.

Caution is advised when interpreting reports of gene groupings by pathways and

subsequent extrapolations of their effects on such a pathway (activating or inactivating)

because mutations in individual genes grouped by association with pathways may still have

opposing effects on such pathways and at times be 1) activating, 2) inactivating or 3)

inconsequential. Finally, activating gene mutations often are mono-allelic while inactivating

5
mutations are either 1) bi-allelic, or 2) associated with gene loss of the wild type allele, or 3)

possibly associated with epigenetic down-regulation of the wild type allele, or 4) mono-allelic

and possibly haploinsufficient (definition: one intact gene copy is insufficient to carry out

complete gene function).

What is the gene mutation frequency in the general CLL population in individual

CLL-associated recurrently mutated genes? The literature offers rather different accounts of

mutations frequencies for various genes in CLL. Other than statistical sampling errors, what

accounts for the differences between studies? Assuming that direct Sanger sequencing of

PCR products generated from genomic DNA templates isolated from almost pure CLL tumor

cell preparations is used, a clonal mutation would need to be present in 10-20% of input DNA

to be detected reliably. Once targeted re-sequencing approaches employing next generation

techniques are used and depending on the depth of coverage, mutation detection is largely

determined by the error rate of the sequencing process and may reach 0.1% variant allele

frequency (VAF: the number of mutated sequences or tags divided by all overlapping

sequenced tags spanning a variant) detection. Technical differences between studies including

lack of verification of somatic mutation status may account for some of the observed

frequency differences.

It appears however, that the strongest factor influencing published mutation

frequencies are biases introduced through use of CLL sample collections that do not reflect

the CLL population in general. Often, instead of studying prospectively and consecutively

enrolled CLL patient cohorts sampled at diagnosis for measurements, various cohorts or other

patient sample collections are employed, at times involving mixtures of untreated and relapsed

6
cases. Similarly, samples from patients on therapeutic trials when obtained shortly before

therapy initiation will not allow for accurate estimations of gene mutation frequencies in CLL

in general, but will ascertain frequencies in CLL patients that have progressive disease and

that survived until the need of therapy arose. One can relatively safely assume that most

published gene mutation frequencies in CLL are higher than true frequencies in unselected

CLL populations at diagnosis.

What do we know about the biological consequences of gene mutations in CLL?

While most of the specifics are covered below, it is fair to state that firm knowledge is limited

but evolving. In large part, this is due to both these mutation being relatively recently

discovered as well as the fact that few experimental model systems that faithfully recapitulate

aspects of CLL pathogenesis exist. Therefore, with the exception of mutations in TP53 or

ATM, caution is necessary in extrapolating mechanisms of mutated genes studied in other

cancers to CLL or to simply arrange CLL-associated gene mutations into functional groups or

pathways without demonstration that such pathways are 1) altered and 2) critically important

to CLL pathogenesis. Model systems involving untransformed CLL precursors or cell lines

resembling CLL or mouse models faithfully mimicking human CLL are needed to further

advance knowledge in this area8,9.

Ultimately, reductionistic ex vivo studies into mechanisms need to be reconciled with

the clinical behavior of CLL cases afflicted with particular mutations under study to identify

mechanisms relevant to CLL as a disease. Such studies need larger patient numbers than

usually studied in single centers.

7
 Are recurrent gene mutations in CLL strongly affecting the aggressiveness and

ultimately the prognosis of CLL? With the exception of TP53 mutations, the answer is

probably No although larger populations of patients will be needed to definitively determine

this. While most reports of gene mutations in CLL are inclusive of some clinical outcome

data, caution is needed before widespread acceptance and adoption of such outcome findings

is recommended; this is especially true if counseling patients based on gene mutation data is

attempted. What is the origin of inter-study variability of proposed effects of individual gene

mutations on patient outcome? Again, biases, introduced through patient selection accounts

for much of the problem; technical shortcomings and non-comparable therapies between

studies complicate interpretations. For instance, use of potent chemoimmunotherapy (CIT)

programs versus other therapies likely affects the prognostic power of mutated genes. Further

complicating matters is the fact that some mutations (NOTCH1, SF3B1) are highly

overrepresented in progressive CLL that tends to be IgVH unmutated and ZAP70 protein

expression positive, two parameters that strongly associate with progressive CLL as defined

by short time from diagnosis to therapy (time-to-first-treatment or TTFT), thus necessitating

large patient samples for comprehensive multivariate analyses.

One common line of reasoning infers from elevated mutation frequencies in relapsed

or refractory CLL that gene mutations are contributory to relapse or chemotherapy

refractoriness. Such argumentation is challenged by the fact that association and causality are

not the same thing and that all biomarkers associated with progressive CLL are enriched in

these CLL subsets.

One may postulate that gene mutations that cause progressive or aggressive CLL may

be acquired over time in individual CLL patients, as has already been demonstrated for TP53

8
mutations. One experimental approach to study this is in paired CLL samples procured

longitudinally and before and after therapy (paired diagnosis and relapsed samples)10. Such

research is under way and will further inform us about gene candidates that influence CLL

clinical behavior.

Which gene mutations cause resistance to chemotherapy in CLL? The answer is

linked to the definition of therapy resistance (in vivo, ex vivo) and is influenced by the actual

therapies or experimental assays used. Clearly, most relevant are in vivo data using modern

chemoimmunotherapy programs; data based on single agent alkylator drugs may allow for

detection of weak effects but are limited in their general applicability. In this context it may

be relevant that practice differences appear to exist between US and European centers and

physicians with single agent alkylator drugs like chlorambucil seemingly being more often

used in Europe.

In vivo, cellular resistance is indirectly measured by a short progression free survival

(PFS) following frontline therapy, which is correlated with the degree of minimal residual

disease (MRD) present after completing therapy. Using short PFS or a complete lack of

therapy response as surrogates for partial or complete therapy resistance, only TP53 mutations

and to a lesser degree, mutations in SF3B1 are recognized as adverse. ATM mutations in the

setting of an 11q deletion may also qualify although we still lack unbiased somatic ATM

mutation data for large CLL cohorts to reach definitive conclusions and to separate ATM

effects from 11q effects. With regard to survival, which is a more complicated but also more

important clinical endpoint, only TP53 mutations are established to be adverse.

9
 Ex vivo chemotherapy or external radiation cell kill assays are used to measure ex vivo

cell intrinsic sensitivity or resistance11. Results of such assays are influenced by assay

conditions as well as cell co-culture and cytokine conditions. The resulting data from such

assays are hypothesis-generating and valuable but often are performed in small sample

numbers and alone should probably not be used to definitively label genes as resistance-

inducing unless matched with clinical data.

While it is clear that TP53 mutations cause strong in vivo and ex vivo alkylator and

nucleoside analog therapy resistance, including absolute resistance to radiation-induced

apoptosis, data for all other genes are largely not supportive of a strong chemotherapy

resistance phenotype11,12. Rather, published data for SF3B1 mutations or NOTCH1 mutations

support a mild phenotype13-15. The concept of thresholds and insult strength to overcome such

a threshold is rarely mentioned in the literature. Given that a CLL cell can either commit

apoptosis or repair itself, what resistance threshold conferred on CLL cells by mutations is

required to be clinically meaningful?

Finally, cell systems with engineered alterations in CLL-associated gene mutations are

needed to convincingly demonstrate that such mutations alone can alter chemotherapy

efficacy, as measured for instance through dose response curves following drug treatment in

isogenic cell lines (lines harboring an engineered mutation in a gene of interest versus the

parenteral line that is wild type for such a gene) and carrying TP53 wild type genes.

Which subclonal gene mutations are biologically or clinically important? CLL, like

all cancers, is not comprised of one uniform cell population. Rather, dominant clones cause

the initial disease-associated symptoms and an unknown number of subclones exists that may

10
or may not rise to clonal dominance over time. Clonal mutations in pure tumor cell

populations should be present in 50% (mono-allelic) or 100% (bi-allelic) of all sequencing

reads, while subclonal mutations are reflected by VAFs <50%. In real life, VAFs of <50%

may still be fully clonal if tumor cell contaminations and technical reasons are accounted for.

A uniformly accepted definition of a subclone is lacking, and therefore, could comprise all

mutations with VAFs <50%, for instance VAFs of 0.1%, 1%, 5% or 25%. If subclones rise to

clonal dominance, such dominance may arise before or after therapy10; however, if subclones

do not rise to clonal dominance it is harder to imagine how they may affect the disease and the

affected patients?

One of the contemporary questions regarding subclonal dynamics centers on the

importance of subclones as variables influencing CLL as a disease16. While acquired clonal

dominance of TP53-mutated CLL cells especially after therapy (and often associated with

substantial increases in genomic complexity) is established, it remains unclear if other genes

frequently serve a similar subclonal driver role or if their rise to clonal dominance follows

other established drivers (in a sense relegating subclones as defined by gene mutation VAFs

to passenger status in specific settings; for instance a NOTCH1 mutation gaining in VAF

occurring in the setting of an a TP53 mutations gaining in VAF in the same patient)10,17,18.

Similar concepts of course apply to all other molecular alterations that exist at subclonal

levels in CLL.

Therefore, unless established drivers (TP53 mutations, del17p, del11q, IgVH status, a

complex karyotype or SNP array-based genomic complexity and others) are included in

modeling trying to isolate the effects of novel candidate mutated subclonal candidate driver

11
genes, we cannot be certain of what roles novel genes play in this context. This is an area of

ongoing active research.

Which gene mutations increase the likelihood for Richter’s transformation in CLL?

The transformation of CLL to large cell lymphoma or rarely Hodgkin lymphoma is commonly

referred to as Richter’s transformation. Such transformation occurs in a few percent of CLL

patients per year; it is likely that multiple distinct molecular avenues to transformation exist.

Factors that have been implicated as playing a role are TP53 mutations/del17p (~50%

frequency in Richter’s DLBCL), certain BCR stereotypes, NOTCH1 mutations (~20-30%

frequency in some series but not others), trisomy 12 (~30%), c-Myc gene translocations,

CDKN2A gene(s) deletion (~30%) and possible genomic instability due to unknown factors19-
21
. However, none of these factors, where measured in prospective CLL cohorts followed for

sufficient time to ascertain RS frequencies, have a high positive predictive value for

transformation to occur--thus limiting clinical counseling based on such aberrations.

What is the interrelation of gene mutations and acquired genomic complexity in

CLL? A complex karyotype or SNP array-based genomic complexity is a very adverse trait

for survival in CLL4,22. Which gene mutations associate with genomic complexity? An

established fact is the strong association of TP53 mutations with genomic complexity with

~50% of CLL cases with ≥3 aCNAs harboring such mutations. No other gene is significantly

associated with genomic complexity in CLL (although some genes may be involved in

specific cases, an area of active research). It is unclear what permissive factors exist in CLL

allowing for genomic complexity to emerge and persist.

12
 Do gene mutations initiate CLL or prime for CLL development at the hematopoietic

stem cell level? Hematopoietic stem cell (HSCs) isolated from CLL patients have a

heightened propensity to differentiate into B-cells in mouse xenografts23. More recently,

CLL-associated driver mutations have been identified in HSC-containing cell fractions

isolated by FACS from CLL patient’s bone marrow samples24. It is therefore possible that,

akin to AML, CLL driver mutations pre-dispose HSCs to future CLL development at some

small stochastic frequency with final transforming events occurring at later stages of

differentiation25. This constitutes an active research area.

A discussion of specific gene mutations in CLL:

TP53 mutations: TP53 mutations are the most clinically adverse mutations in CLL and no

other gene mutation in CLL is of equal negative prognostic significance26. The incidence of

TP53 mutations in unselected CLL cohorts is relatively low at diagnosis (estimated at ~ 5%-

7%) 27 but rises with time and especially after therapy. The biology and clinical significance

of TP53 mutations has been extensively reviewed and therefore the reader is referred to recent

reviews and literature1,28-31.

Of major importance for TP53-mutated CLL, which responds comparatively poorly to

all current therapies, has been development of targeted therapies, like ibrutinib or idelalisib32-
35
. Use of these agents is changing the clinical approach to these patients and has motivated

discussion regarding the appropriate use of peripheral blood or marrow transplantations in this

high-risk CLL subset36. Mechanisms of resistance to targeted agents in this CLL subset will

be of future research interest.

13
NOTCH1 mutations: NOTCH1 mutations were discovered through WES and targeted

sequencing approaches and are present in ~5-10% of CLL at diagnosis and at a higher

frequency in relapsed patients7,37. The most common NOTCH1 mutation is a recurrent

particular dinucleotide deletion (c.7541_7542het_delCT; p.2514fs) targeting and altering the

PEST domain, which physiologically regulates stability of this protein. NOTCH1-mutated

CLL frequently carry trisomy 12 suggesting a molecular cooperation38-40 and are IgVH

unmutated (UM) and ZAP70 positive in >80% of cases. Mutations in FBXW7 detected in a

few percent of CLL may also activate or pre-activate the NOTCH1 pathway. In addition,

various missense mutations in NOTCH1 have been identified at low frequencies; of note

however, missense mutations in NOTCH1 may be subject to false positive and negative

mutation calls unless SNPs are excluded through analysis of paired non-tumorous DNA.

The critical biological consequences of mutated NOTCH1 in CLL remain unknown.

Stabilization of the NOTCH1 protein has been suggested by some but not other studies 7,15, as

well as phosphorylation of the NOTCH1 intracellular domain in NOTCH1 mutated CLL

cells41. It is likely that mutated NOTCH1 proteins exerts some enhanced pro-survival or

activation signal on afflicted CLL cells especially in the LN-microenvironment and after

engagement by NOTCH1 ligands13,42 and it is possible that this could be exploited

therapeutically.

Ex vivo some degree of resistance of CLL cells carrying NOTCH1 mutations to

chemotherapy has been measured but this does not translate into substantial clinical resistance

in patients treated with modern chemoimmunotherapy (CIT) programs39,43. In less intensively

treated patients, some modest shortening of PFS is documented44. As previously outlined, the

14
vast majority of NOTCH1 mutated CLL cases are also IgVH UM and ZAP70 +, which may

explain associations with prognosis, especially if TTFT as opposed to OS is considered as an

endpoint. In multivariate analysis and in the setting of CIT, NOTCH1 mutations are not

selected as an independent factor39,43. In aggregate, published data do not support a major role

for NOTCH1 mutations in negatively affecting prognosis in CLL but mild effects may need

further study and such effects have been incorporated into novel prognostic scoring systems
45
.

Of interest is the reported association of NOTCH1 mutations with Richter’s

syndrome7,19,20,46. Clearly, Richter’s transformation severely shortens the life of most such

afflicted patients and in this scenario a subset of NOTCH1-mutated CLL patients do poorly47.

This is in need of further study.

SF3B1 mutations: SF3B1 mutations were discovered through WES and are present in ~5-

10% of CLL at diagnosis5,6,48,49 and at higher frequencies in relapsed disease. Mutations in

SF3B1 usually are mono-allelic missense mutations targeting hotspots (in particular amino

acid residues #700 and 741). The genetic data suggest a gain of function and possibly

oncogenic role. SF3B1 is part of a spliceosome complex that is involved in 3’-splice site

recognition. Consequently, aberrant transcripts have been detected through RNA-seq-based

approaches in SF3B1-mutated CLL, including genes involved in phosphorylation-mediated

control of cell cycle progression but a critical target has not yet been functionally validated50.

In some studies but not others, there is an enrichment of SF3B1 mutations in 11q CLL

suggesting a collaborative effect5,50. Given that the vast majority of CLL harboring 11q

deletions and SF3B1 mutations are also IgVH UM, the later may explain co-enrichment in

15
selected cohorts. Further, a role of aberrantly spliced and truncated ATM in mutant SF3B1

biology has been proposed but is controversially discussed14,50.

A peculiar enrichment of SF3B1 mutations in CLL subsets defined by the sequence of

the rearranged Ig genes (stereotype subset #2) has been uncovered and unraveling a reason for

this association is a future research area51-53. Overall, much work is needed to identify the

mechanism(s) how mutated SF3B1 exerts its effect on CLL cells. The related question

whether SF3B1 mutations exert their effect early or late in CLL pathogenesis is also largely

unanswered.

SF3B1 mutations are associated with a modest shortening of PFS in modern CIT-

treated CLL patients43. Independent effects on OS are either not detected or detected in the

setting of weaker therapies or unreported therapies 44,54,55. Ex vivo, partial chemotherapy

resistance has been documented14. Efforts at targeting aberrant SF3B1 function

therapeutically are under way.

POT1 mutations: Mutations in POT1, a shelterin complex protein associated with telomeres,

occur in 3-5% of unselected CLL, and like mutations in NOTCH1 or SF3B1 are enriched in

IgVH UM CLL. These mutations cluster in a specific domain of POT1. POT1 has also been

identified as a genomic susceptibility locus for CLL56.

One study measured increased chromosomal fusions and breakage events in POT1

mutated CLL cells, in line with reasoning that telomere dysfunction causes chromosomal

instability in CLL57,58. Unpublished results in 20 CLL with high SNP 6.0 array-based

genomic complexity and wild type TP53 did not detect POT1 mutations (Malek, Sami

16
unpublished) and it would be interesting to determine if CLL with complex karyotypes and

TP53 wild type status carry POT1 mutations at elevated frequencies.

Experimentally, genome editing of POT1 allowing for the introduction of CLL-

associated POT1 mutations into suitable cell line models may allow for more direct

measurements of effects of POT1 mutation on genomic stability. The prognostic effects of

POT1 mutations are in need of further study.

EGR2 mutations: EGR2 mutations are recurrent in CLL and occur at an estimated frequency

of ~5%; the true frequency in CLL at diagnosis is unknown24. EGR2 is a downstream target

of BCR activation but the precise role in B-cells and CLL cells is unknown.

MYD88 mutations: Mutation in the innate immune system Toll-like receptor signal transducer

MYD88 are detected in 2-3% of CLL7. These mutations target a hotspot amino acid (L265P)

and are overrepresented in IgVH mutated CLL. Intuitively, it is somewhat surprising that

these mutations, despite reported activation of NFKB and cytokine secretion following TLR

engagement, are not associated with CLL disease progression but instead associate with non-

aggressive CLL 45,59. It remains unclear why CLL selects for these mutations but it has been

suggested that they constitute early genetic events in CLL pathogenesis16.

WNT pathway mutations: The WNT pathway is activated in some CLL cases due to over

expression of pathway components or gene mutations or epigenetic changes (LEF1) or

unknown mechanisms. Sporadic mutations in members of this extended pathway have been

described and some have been functionally studied60-62. Whether an activated WNT pathway

17
is a good target for therapies in CLL awaits further study63. Missense mutation in the FAT1

gene have been reported in fludarabine-refractory CLL; functional studies have not yet been

reported64.

Deletion 11q-associated mutations (ATM, BIRC3): 11q deletions in CLL are of varying

length but always remove one copy of the ATM gene65. The residual ATM gene is mutated in

a subset of cases66 but in the majority of cases is wild type and not haplo-insufficient67.

Curiously, despite 15 years of study, we still do not know the mutation frequency of ATM in

unselected CLL cohorts, which is largely due to the large size of the gene and the need for

analysis of paired normal DNA as ATM has many SNPs. Estimating the ATM mutation

frequency in CLL without confirmation of somatic mutation status inflates true mutation

frequencies.

BIRC3, a gene mono allelically removed in long 11q deletions, is infrequently mutated

in CLL at diagnosis and more study is needed to fully appreciate the effect of this gene on

CLL biology or clinical progression68,69. Mutations in BIRC3 and mono allelic deletions of the

gene as part of 11q deletions are frequently grouped and reported together (and coined BIRC3

disruptions). Given that large 11q deletions span greater than one hundred genes, such

inclusive terminology needs separation of BIRC3 effects from all other putative

haploinsufficient 11q gene candidates.

BTK and PLC-gamma mutations and ibrutinib resistance: The novel BTK inhibitor ibrutinib

is changing the management of CLL34. It is a covalent inhibitor for BTK and targets BTK

p.C481. In patients with clinical resistance to ibrutinib, mutations in p.C481 (p.C481S) have

18
been identified that alter the drug – enzyme interaction and prevent covalent binding70,71.

These mutations are one cause of acquired in vivo resistance to ibrutinib. In addition,

mutations in phospholipase C-gamma (PLC-gamma) have been identified in two patients.

These mutations are functional as measured through enhanced calcium flux in Ig- treated

PLC-gamma mutant transfected DT40 B-cells. What other gene mutations result in acquired

ibrutinib resistance and what non-mutational mechanisms may exist is being actively

investigated.

Other recurrently mutated genes in CLL: The study of gene mutations in CLL is actively

ongoing and more recurrently mutated genes are identified each year. Ultimately, somewhere

between 20-50 such genes will have been identified in CLL, most with mutations frequencies

of 1-5% at diagnosis. The known genes with mutations in CLL have been summarized in a

recent review by JC Strefford and are not further annotated here2. Some of these, for instance

CHD2, FAT1, ZMYM3, NXF1, BCOR, XPO1, MED12, SAMHD1, IRF4, NFKBIE and others

will be studied more intensively in upcoming years24,72-74. As with all mutated genes in CLL,

understanding the reason why each gene is mutated and what it means for CLL cell biology

and effects on clinical characteristics is the challenge. Equally challenging is the integration

of mutation effects with the effects of other well-established drivers on CLL biology and their

interplay and relative importance in shaping CLL clinical behavior.

19
 ACKNOWLEDGEMENT

SM is a Scholar in Clinical Research of the Leukemia and Lymphoma Society (SM) and is

supported by the National Institutes of Health through CA136537 and through the University

of Michigan's Cancer Center Support Grant (5 P30 CA46592).

The authors are grateful to Drs. Jon C. Strefford and Brian Parkin for critical review of the

manuscript.

INDIVIDUAL CONTRIBUTIONS

Nisar Amin and Sami Malek wrote the paper.

CONFLICT OF INTEREST

There is no conflict of interest to disclose.

20
 REFERENCES

1. Malek SN. The biology and clinical significance of acquired genomic copy number

aberrations and recurrent gene mutations in chronic lymphocytic leukemia. Oncogene.

2013;32(23):2805-2817.

2. Strefford JC. The genomic landscape of chronic lymphocytic leukaemia: biological

and clinical implications. Br J Haematol. 2014.

3. Dohner H, Stilgenbauer S, Benner A, et al. Genomic aberrations and survival in

chronic lymphocytic leukemia. N Engl J Med. 2000;343(26):1910-1916.

4. Ouillette P, Collins R, Shakhan S, et al. Acquired genomic copy number aberrations

and survival in chronic lymphocytic leukemia. Blood. 2011;118(11):3051-3061.

5. Wang L, Lawrence MS, Wan Y, et al. SF3B1 and other novel cancer genes in chronic

lymphocytic leukemia. N Engl J Med. 2011;365(26):2497-2506.

6. Quesada V, Conde L, Villamor N, et al. Exome sequencing identifies recurrent

mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia. Nat Genet.

2011;44(1):47-52.

7. Puente XS, Pinyol M, Quesada V, et al. Whole-genome sequencing identifies recurrent

mutations in chronic lymphocytic leukaemia. Nature. 2011;475(7354):101-105.

8. Bertilaccio MT, Scielzo C, Simonetti G, et al. Xenograft models of chronic

lymphocytic leukemia: problems, pitfalls and future directions. Leukemia. 2013;27(3):534-

540.

9. Simonetti G, Bertilaccio MT, Ghia P, Klein U. Mouse models in the study of chronic

lymphocytic leukemia pathogenesis and therapy. Blood. 2014;124(7):1010-1019.

21
10. Ouillette P, Saiya-Cork K, Seymour E, Li C, Shedden K, Malek SN. Clonal evolution,

genomic drivers, and effects of therapy in chronic lymphocytic leukemia. Clin Cancer Res.

2013;19(11):2893-2904.

11. Ouillette P, Fossum S, Parkin B, et al. Aggressive chronic lymphocytic leukemia with

elevated genomic complexity is associated with multiple gene defects in the response to DNA

double-strand breaks. Clin Cancer Res. 2010;16(3):835-847.

12. Mohr J, Helfrich H, Fuge M, et al. DNA damage-induced transcriptional program in

CLL: biological and diagnostic implications for functional p53 testing. Blood.

2011;117(5):1622-1632.

13. Rosati E, Sabatini R, Rampino G, et al. Constitutively activated Notch signaling is

involved in survival and apoptosis resistance of B-CLL cells. Blood. 2009;113(4):856-865.

14. Te Raa GD, Derks IA, Navrkalova V, et al. The impact of SF3B1 mutations in CLL on

the DNA-damage response. Leukemia. 2014.

15. Arruga F, Gizdic B, Serra S, et al. Functional impact of NOTCH1 mutations in chronic

lymphocytic leukemia. Leukemia. 2014;28(5):1060-1070.

16. Landau DA, Carter SL, Stojanov P, et al. Evolution and impact of subclonal mutations

in chronic lymphocytic leukemia. Cell. 2013;152(4):714-726.

17. Rossi D, Khiabanian H, Spina V, et al. Clinical impact of small TP53 mutated

subclones in chronic lymphocytic leukemia. Blood. 2014;123(14):2139-2147.

18. Malcikova J, Stano-Kozubik K, Tichy B, et al. Detailed analysis of therapy-driven

clonal evolution of TP53 mutations in chronic lymphocytic leukemia. Leukemia. 2014.

19. Rossi D, Gaidano G. Richter syndrome. Adv Exp Med Biol. 2013;792:173-191.

22
20. Fabbri G, Khiabanian H, Holmes AB, et al. Genetic lesions associated with chronic

lymphocytic leukemia transformation to Richter syndrome. J Exp Med. 2013;210(11):2273-

2288.

21. Chigrinova E, Rinaldi A, Kwee I, et al. Two main genetic pathways lead to the

transformation of chronic lymphocytic leukemia to Richter syndrome. Blood.

2013;122(15):2673-2682.

22. Kujawski L, Ouillette P, Erba H, et al. Genomic complexity identifies patients with

aggressive chronic lymphocytic leukemia. Blood. 2008;112(5):1993-2003.

23. Kikushige Y, Ishikawa F, Miyamoto T, et al. Self-renewing hematopoietic stem cell is

the primary target in pathogenesis of human chronic lymphocytic leukemia. Cancer Cell.

2011;20(2):246-259.

24. Damm F, Mylonas E, Cosson A, et al. Acquired initiating mutations in early

hematopoietic cells of CLL patients. Cancer Discov. 2014;4(9):1088-1101.

25. Welch JS, Ley TJ, Link DC, et al. The origin and evolution of mutations in acute

myeloid leukemia. Cell. 2012;150(2):264-278.

26. Gaidano G, Ballerini P, Gong JZ, et al. p53 mutations in human lymphoid

malignancies: association with Burkitt lymphoma and chronic lymphocytic leukemia. Proc

Natl Acad Sci U S A. 1991;88(12):5413-5417.

27. Zainuddin N, Murray F, Kanduri M, et al. TP53 Mutations are infrequent in newly

diagnosed chronic lymphocytic leukemia. Leuk Res. 2011;35(2):272-274.

28. Trbusek M, Malcikova J. TP53 aberrations in chronic lymphocytic leukemia. Adv Exp

Med Biol. 2013;792:109-131.

23
29. Pospisilova S, Gonzalez D, Malcikova J, et al. ERIC recommendations on TP53

mutation analysis in chronic lymphocytic leukemia. Leukemia. 2012;26(7):1458-1461.

30. Zenz T, Habe S, Denzel T, et al. Detailed analysis of p53 pathway defects in

fludarabine-refractory chronic lymphocytic leukemia (CLL): dissecting the contribution of

17p deletion, TP53 mutation, p53-p21 dysfunction, and miR34a in a prospective clinical trial.

Blood. 2009;114(13):2589-2597.

31. Zenz T, Krober A, Scherer K, et al. Monoallelic TP53 inactivation is associated with

poor prognosis in chronic lymphocytic leukemia: results from a detailed genetic

characterization with long-term follow-up. Blood. 2008;112(8):3322-3329.

32. Furman RR, Sharman JP, Coutre SE, et al. Idelalisib and rituximab in relapsed chronic

lymphocytic leukemia. N Engl J Med. 2014;370(11):997-1007.

33. Brown JR, Byrd JC, Coutre SE, et al. Idelalisib, an inhibitor of phosphatidylinositol 3-

kinase p110delta, for relapsed/refractory chronic lymphocytic leukemia. Blood.

2014;123(22):3390-3397.

34. Byrd JC, Furman RR, Coutre SE, et al. Targeting BTK with ibrutinib in relapsed

chronic lymphocytic leukemia. N Engl J Med. 2013;369(1):32-42.

35. Farooqui MZ, Valdez J, Martyr S, et al. Ibrutinib for previously untreated and relapsed

or refractory chronic lymphocytic leukaemia with TP53 aberrations: a phase 2, single-arm

trial. Lancet Oncol. 2014.

36. Dreger P, Schetelig J, Andersen N, et al. Managing high-risk CLL during transition to

a new treatment era: stem cell transplantation or novel agents? Blood. 2014;124(26):3841-

3849.

24
37. Fabbri G, Rasi S, Rossi D, et al. Analysis of the chronic lymphocytic leukemia coding

genome: role of NOTCH1 mutational activation. J Exp Med. 2011;208(7):1389-1401.

38. Balatti V, Bottoni A, Palamarchuk A, et al. NOTCH1 mutations in CLL associated

with trisomy 12. Blood. 2012;119(2):329-331.

39. Shedden K, Li Y, Ouillette P, Malek SN. Characteristics of chronic lymphocytic

leukemia with somatically acquired mutations in NOTCH1 exon 34. Leukemia.

2012;26(5):1108-1110.

40. Lopez C, Delgado J, Costa D, et al. NOTCH1 mutations in chronic lymphocytic

leukemia with trisomy 12. Genes Chromosomes Cancer. 2012.

41. De Falco F, Sabatini R, Falzetti F, et al. Constitutive phosphorylation of the active

Notch1 intracellular domain in chronic lymphocytic leukemia cells with NOTCH1 mutation.

Leukemia. 2014.

42. Onaindia A, Gomez S, Piris-Villaespesa M, et al. Chronic lymphocytic leukemia cells

in lymph nodes show frequent NOTCH1 activation. Haematologica. 2014.

43. Stilgenbauer S, Schnaiter A, Paschka P, et al. Gene mutations and treatment outcome

in chronic lymphocytic leukemia: results from the CLL8 trial. Blood. 2014;123(21):3247-

3254.

44. Oscier DG, Rose-Zerilli MJ, Winkelmann N, et al. The clinical significance of

NOTCH1 and SF3B1 mutations in the UK LRF CLL4 trial. Blood. 2013;121(3):468-475.

45. Rossi D, Rasi S, Spina V, et al. Integrated mutational and cytogenetic analysis

identifies new prognostic subgroups in chronic lymphocytic leukemia. Blood.

2013;121(8):1403-1412.

25
46. Rossi D, Rasi S, Fabbri G, et al. Mutations of NOTCH1 are an independent predictor

of survival in chronic lymphocytic leukemia. Blood. 2011.

47. Villamor N, Conde L, Martinez-Trillos A, et al. NOTCH1 mutations identify a genetic

subgroup of chronic lymphocytic leukemia patients with high risk of transformation and poor

outcome. Leukemia. 2012.

48. Wan Y, Wu CJ. SF3B1 mutations in chronic lymphocytic leukemia. Blood.

2013;121(23):4627-4634.

49. Rossi D, Bruscaggin A, Spina V, et al. Mutations of the SF3B1 splicing factor in

chronic lymphocytic leukemia: association with progression and fludarabine-refractoriness.

Blood. 2011;118(26):6904-6908.

50. Ferreira PG, Jares P, Rico D, et al. Transcriptome characterization by RNA

sequencing identifies a major molecular and clinical subdivision in chronic lymphocytic

leukemia. Genome Res. 2014;24(2):212-226.

51. Agathangelidis A, Ntoufa S, Stamatopoulos K. B cell receptor and antigens in CLL.

Adv Exp Med Biol. 2013;792:1-24.

52. Strefford JC, Sutton LA, Baliakas P, et al. Distinct patterns of novel gene mutations in

poor-prognostic stereotyped subsets of chronic lymphocytic leukemia: the case of SF3B1 and

subset #2. Leukemia. 2013;27(11):2196-2199.

53. Rossi D, Spina V, Bomben R, et al. Association between molecular lesions and

specific B-cell receptor subsets in chronic lymphocytic leukemia. Blood. 2013;121(24):4902-

4905.

54. Baliakas P, Hadzidimitriou A, Sutton LA, et al. Recurrent mutations refine prognosis

in chronic lymphocytic leukemia. Leukemia. 2014.

26
55. Jeromin S, Weissmann S, Haferlach C, et al. SF3B1 mutations correlated to

cytogenetics and mutations in NOTCH1, FBXW7, MYD88, XPO1 and TP53 in 1160

untreated CLL patients. Leukemia. 2014;28(1):108-117.

56. Speedy HE, Di Bernardo MC, Sava GP, et al. A genome-wide association study

identifies multiple susceptibility loci for chronic lymphocytic leukemia. Nat Genet.

2014;46(1):56-60.

57. Ramsay AJ, Quesada V, Foronda M, et al. POT1 mutations cause telomere

dysfunction in chronic lymphocytic leukemia. Nat Genet. 2013;45(5):526-530.

58. Britt-Compton B, Lin TT, Ahmed G, et al. Extreme telomere erosion in ATM-mutated

and 11q-deleted CLL patients is independent of disease stage. Leukemia. 2012;26(4):826-830.

59. Martinez-Trillos A, Pinyol M, Navarro A, et al. Mutations in TLR/MYD88 pathway

identify a subset of young chronic lymphocytic leukemia patients with favorable outcome.

Blood. 2014;123(24):3790-3796.

60. Wang L, Shalek AK, Lawrence M, et al. Somatic mutation as a mechanism of

Wnt/beta-catenin pathway activation in CLL. Blood. 2014;124(7):1089-1098.

61. Gutierrez A, Jr., Tschumper RC, Wu X, et al. LEF-1 is a prosurvival factor in chronic

lymphocytic leukemia and is expressed in the preleukemic state of monoclonal B-cell

lymphocytosis. Blood. 2010;116(16):2975-2983.

62. Lu D, Zhao Y, Tawatao R, et al. Activation of the Wnt signaling pathway in chronic

lymphocytic leukemia. Proc Natl Acad Sci U S A. 2004;101(9):3118-3123.

63. Gandhirajan RK, Poll-Wolbeck SJ, Gehrke I, Kreuzer KA. Wnt/beta-catenin/LEF-1

signaling in chronic lymphocytic leukemia (CLL): a target for current and potential

therapeutic options. Curr Cancer Drug Targets. 2010;10(7):716-727.

27
64. Messina M, Del Giudice I, Khiabanian H, et al. Genetic lesions associated with

chronic lymphocytic leukemia chemo-refractoriness. Blood. 2014;123(15):2378-2388.

65. Saiya-Cork K, Collins R, Parkin B, et al. A pathobiological role of the insulin receptor

in chronic lymphocytic leukemia. Clin Cancer Res. 2011;17(9):2679-2692.

66. Austen B, Powell JE, Alvi A, et al. Mutations in the ATM gene lead to impaired

overall and treatment-free survival that is independent of IGVH mutation status in patients

with B-CLL. Blood. 2005;106(9):3175-3182.

67. Ouillette P, Li J, Shaknovich R, et al. Incidence and clinical implications of ATM

aberrations in chronic lymphocytic leukemia. Genes Chromosomes Cancer.

2012;51(12):1125-1132.

68. Rose-Zerilli MJ, Forster J, Parker H, et al. ATM mutation rather than BIRC3 deletion

and/or mutation predicts reduced survival in 11q-deleted chronic lymphocytic leukemia: data

from the UK LRF CLL4 trial. Haematologica. 2014;99(4):736-742.

69. Rossi D, Fangazio M, Rasi S, et al. Disruption of BIRC3 associates with fludarabine

chemorefractoriness in TP53 wild-type chronic lymphocytic leukemia. Blood.

2012;119(12):2854-2862.

70. Furman RR, Cheng S, Lu P, et al. Ibrutinib resistance in chronic lymphocytic

leukemia. N Engl J Med. 2014;370(24):2352-2354.

71. Woyach JA, Furman RR, Liu TM, et al. Resistance mechanisms for the Bruton's

tyrosine kinase inhibitor ibrutinib. N Engl J Med. 2014;370(24):2286-2294.

72. Clifford R, Louis T, Robbe P, et al. SAMHD1 is mutated recurrently in chronic

lymphocytic leukemia and is involved in response to DNA damage. Blood. 2014;123(7):1021-

1031.

28
73. Ramsay AJ, Rodriguez D, Villamor N, et al. Frequent somatic mutations in

components of the RNA processing machinery in chronic lymphocytic leukemia. Leukemia.

2013;27(7):1600-1603.

74. Kampjarvi K, Jarvinen TM, Heikkinen T, et al. Somatic MED12 mutations are

associated with poor prognosis markers in chronic lymphocytic leukemia. Oncotarget. 2014.

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Table 1: Gene mutations in CLL: A few selected open questions

How do gene mutations cooperate with other major biological phenomena influencing CLL as
a disease? What is their relative importance?
Why are gene mutations relatively rare in CLL when compared with other hematological
neoplasms?
What are the critical targets of SF3B1 mutations in CLL? What are the physiological
consequences of SF3B1 mutations in CLL precursor cells or CLL cells?
What are suitable experimental systems to study CLL-associated gene mutations?
Why do CLL cells recurrently select for NOTCH1 mutations?
Which gene mutations and acquired genomic copy number aberrations functionally
cooperate, and how is this accomplished?
Are CLL cells addicted to some of the recurrently mutated genes? Which CLL-associated
mutated genes may be targets for novel therapies?
How many of the recurrently mutated genes affect functional pathways that are critical to CLL
pathogenesis?
Which genes other than TP53 substantially shorten survival in CLL?

30