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ONCH-2210; No. of Pages 14 ARTICLE IN PRESS

Critical Reviews in Oncology/Hematology xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Critical Reviews in Oncology/Hematology

journal homepage: www.elsevier.com/locate/critrevonc

Chronic lymphocytic leukaemia

Lydia Scarfò a,b , Andrés J.M. Ferreri a,∗ , Paolo Ghia a,b
a
Department of Onco-Haematology, Division of Experimental Oncology, IRCCS San Raffaele Hospital, Milan, Italy
b
University Vita-Salute San Raffaele, Milan, Italy

Contents

1. General information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1.1. Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1.2. Incidence and prevalence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1.3. Risk factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1.4. Referral . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Pathology and biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.2. Immunophenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.3. Genomic aberrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.4. Immunogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.5. Next generation sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.1. IWCLL 2008 diagnostic criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.2. Clinical presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.3. Differential diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.4. Monoclonal B cell lymphocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Staging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4.1. Clinical staging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5. Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5.1. Natural history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5.2. Traditional prognostic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5.3. Novel prognostic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5.3.1. Biological prognostic factors include . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.1. Treatment guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.2. Response criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.3. Treatment options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.4. First-line treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.5. Treatment for relapsed and refractory CLL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.6. Experimental agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.6.1. Other kinase inhibitors in advanced clinical development are . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.6.2. Promising novel drugs acting with different pathogenesis-related mechanisms are . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.7. Allogeneic stem cell transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.8. Significance of minimal residual disease (MRD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
7. Late sequelae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
7.1. Infection risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
7.2. Second cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
8. Follow-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

∗ Corresponding author.
E-mail address: ferreri.andres@hsr.it (A.J.M. Ferreri).

http://dx.doi.org/10.1016/j.critrevonc.2016.06.003
1040-8428/© 2016 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Scarfò, L., et al., Chronic lymphocytic leukaemia. Crit Rev Oncol/Hematol (2016),
http://dx.doi.org/10.1016/j.critrevonc.2016.06.003
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2 L. Scarfò et al. / Critical Reviews in Oncology/Hematology xxx (2016) xxx–xxx

a r t i c l e i n f o a b s t r a c t

Article history: Chronic lymphocytic leukaemia (CLL) is the most common leukaemia among the adults in the West-
Received 10 February 2015 ern World. CLL (and the corresponding nodal entity small lymphocytic lymphoma, SLL) is classified as
Received in revised form 1 May 2016 a lymphoproliferative disorder characterised by the relentless accumulation of mature B-lymphocytes
Accepted 14 June 2016
showing a peculiar immunophenotype in the peripheral blood, bone marrow, lymph nodes and spleen.
CLL clinical course is very heterogeneous: the majority of patients follow an indolent clinical course with
Keywords:
no or delayed treatment need and with a prolonged survival, while others experience aggressive disease
Chronic lymphocytic leukaemia
requiring early treatment followed by frequent relapses. In the last decade, the improved understanding
Leukaemias
Lymphoproliferative disorders
of CLL pathogenesis shed light on premalignant conditions (i.e., monoclonal B-cell lymphocytosis, MBL),
defined new prognostic and predictive markers, improving patient stratification, but also broadened the
therapeutic armamentarium with novel agents, targeting fundamental signaling pathways.
© 2016 Elsevier Ireland Ltd. All rights reserved.

1. General information
1.1. Definition
According to World Health Organization (WHO) classification,
published in 2008, chronic lymphocytic leukaemia (CLL) is defined
as a lymphoproliferative disorder, composed by monomorphic
round B lymphocytes involving peripheral blood (PB), bone marrow
(BM) and lymphoid organs (Swerdlow et al., 2008). Chronic lym-
phocytic leukaemia lymphocytes are clonal CD19-positive B cells
characterised by a peculiar immunophenotypic profile, coexpress-
ing CD5 and CD23, along with low density of CD20 (CD20 dim).
The less frequent non-leukaemic cases, where lymph node involve-
ment is prevalent, in absence of cytopenias due to BM infiltration
and with less than 5 × 109 /L B lymphocytes in the PB, are classi-
fied as small lymphocytic lymphomas (SLL) (Swerdlow et al., 2008;
Hallek et al., 2008; Shanafelt et al., 2010). This condition represents
a different clinical manifestation of the same disease and follows
the same management guidelines as CLL. Cases with less than
5 × 109 /L B lymphocytes, with the typical CLL-like immunopheno-
typic profile and no other signs or symptoms of lymphoproliferative
disorders, should be classified as CLL-like monoclonal B cell lym-
phocytosis (CLL-like MBL), a recently defined entity (see Section
3.4) (Shanafelt et al., 2010).
and/or non-Hodgkin lymphomas, NHL). Relatives of CLL patients
have a 2-to-8-fold increase in the risk of developing CLL and a 2-
fold increase of NHL, compared to the general population (Slager
et al., 2014; Slager et al., 2013; Goldin et al., 2009). Up to 10% of
CLL cases reported two or more individuals affected by CLL in the
same family, defining the condition of “familial” CLL, that share
the same clinical and biological profile of sporadic cases. The dis-
ease is rarer in the Eastern world (China, Korea, and Japan) and
this lower incidence is maintained in migrants and their progeny
(Boggs et al., 1987; Pan et al., 2002). Finally, linkage studies and,
more recently, genome wide association studies identified more
than 20 susceptibility loci, where many candidate genes, involved
in B-cell biology and apoptotic pathway, are located (Di Bernardo
et al., 2008; Crowther-Swanepoel et al., 2010; Berndt et al., 2013;
Crowther-Swanepoel and Houlston, 2010).
Different studies suggested a link between CLL diagnosis and
occupational and lifestyle factors, with people living or working on
a farm being at higher risk of CLL development, while sun exposure
having a protective role (Slager et al., 2014). An association between
hepatitis C (HCV) infection and CLL development has also been
noted, though not being CLL-specific, as HCV infection is associated
with a wide variety of lymphoproliferative disorders (Marcucci and
Mele, 2011).

1.2. Incidence and prevalence 1.4. Referral

Chronic lymphocytic leukaemia represents the most common
leukaemia among adults in Western Countries. Incidence rate is
similar in Europe and US and ranges between 4 to 6 cases per
100,000 person per year. In US about 15,720 new cases of CLL,
and more than 12,000 in EU, are expected in 2014. The incidence
increases by age, with more than 70% of patients being older than
65 years at diagnosis. Though the median age at diagnosis is 72
years, in the last decades CLL is more often diagnosed also in
younger individuals, with almost 15% of patients of 55 years old
or younger. There is a gender predisposition, as men are more fre-
quently affected by CLL than women (male:female ratio of 1.5-2:1)
(Howlader et al., 2016).
1.3. Risk factors
Several of the epidemiological studies aimed at identifying risk
factors for CLL/SLL occurrence, but no acquired causative factors
leading to CLL development have been identified so far. A genetic
predisposition to disease development has been ascertained and it
is supported by several lines of evidence. First of all, the strongest
and most consistent risk factor for CLL/SLL occurrence is repre-
sented by a family history of haematological malignancies (CLL
It has been recently demonstrated that the management of CLL
patients by disease-specific well trained haematologists results
in longer time to first treatment and a significant improve-
ment in progression-free and overall survival (Shanafelt et al.,
2012). Chronic lymphocytic leukaemia patients referred to qual-
ified haematology/oncology centres are more likely to undergo
prognostic testing, to receive updated treatment and be enrolled
in clinical trials. These issues are particularly relevant for high-
risk patients (see Section 5.3) whose adverse prognosis could be
improved only through the access to novel experimental agents.
2. Pathology and biology
2.1. Morphology
Peripheral blood smears in CLL cases show a high number of
small lymphocytes with scanty cytoplasm and clumped chromatin.
The presence of smudge cells or Gumprecht nuclear shadows (i.e.,
ruptured CLL cells) is a frequent finding, related to the characteristic

Please cite this article in press as: Scarfò, L., et al., Chronic lymphocytic leukaemia. Crit Rev Oncol/Hematol (2016),
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ONCH-2210; No. of Pages 14 ARTICLE IN PRESS
L. Scarfò et al. / Critical Reviews in Oncology/Hematology xxx (2016) xxx–xxx
Table 1
Immunophenotype in CLL and other lymphoproliferative disorders.
Markers CLL MCL SMZL
CD19 + + + +
CD5 + + +/−
CD20 dim + +
CD23 + – +/−
FMC7 – + +/−
Light chain dim Bright Bright
Heavy chain IgM/IgD IgM/IgD IgM/IgD
CD200 + – +/−
CLL: chronic lymphocytic leukaemia; MCL: mantle cell lymphoma; SMZL: splenic
marginal zone lymphoma; B-PLL: B-cell prolymphocytic leukaemia.
fragility of the cell membrane, and was considered a hallmark of this
disease (Matutes and Polliack, 2000). Prolymphocytes (larger cells
with prominent central nucleolus) can be admixed to small lym-
phocytes in variable proportions, but usually represent less than
55% of the cells (Swerdlow et al., 2008). An increased (between 10%
and 55%) percentage of prolymphocytes is frequently associated
with a more aggressive clinical course (Swerdlow et al., 2008).
Chronic lymphocytic leukaemia diagnosis does not require
lymph node biopsy nor bone marrow aspirate/biopsy, since a
typical immunophenotype performed on the peripheral blood is
sufficient for a conclusive diagnosis. If lymph node biopsy is per-
formed for clinical reasons (SLL cases, differential diagnosis), nodal
involvement by CLL/SLL shows a pseudofollicular pattern where
regularly-spaced pale areas (called “proliferation centres”), com-
posed by larger cells, are divided by a background of smaller and
darker cells (Ponzoni et al., 2011). Bone marrow infiltration may be
interstitial, nodular and/or diffuse, and in most cases the lymphoid
infiltrate represents >30% of the cellularity (Hallek et al., 2008).
2.2. Immunophenotyping
Peripheral blood immunophenotype is required to confirm CLL
diagnosis in any case of unknown lymphocytosis. First of all, flow
cytometry should be used to assess the clonality of B-cell expansion,
showing the evidence of light chain restriction (i.e. either kappa
or lambda typically at low levels of expression). Chronic lympho-
cytic leukaemia cells express B-cell markers, like CD19, along with
low levels of CD20, and are positive for CD5 and CD23. The use
of these markers is needed for the differential diagnosis with oth-
ers B-cell lymphoproliferative disorders, in particular to exclude
the diagnosis of mantle cell lymphoma, that is positive for CD5 but
negative for CD23. Recently CD200 has also been demonstrated to
be instrumental in the differential diagnosis between CLL and other
lymphoproliferative disorders (in particular MCL), being constantly
expressed in CLL cases and absent in MCL (Alapat et al., 2012).
Chronic lymphocytic leukaemia cells are usually negative for FMC7
and express low levels of surface immunoglobulins (in most cases
IgM and/or IgD, rarely IgG or IgA) (Table 1).
2.3. Genomic aberrations
At variance with other lymphoproliferative disorders (e.g., fol-
licular lymphoma or mantle cell lymphoma), in most of which
a distinctive genomic hallmark is found, there is no CLL-specific
genomic aberration. Traditional cytogenetic evaluation through
chromosome banding was not useful in detecting genomic aberra-
tions in CLL cases due to the low mitotic rate. Recently, conventional
G-banding cytogenetics (CGC) has been combined with different B-
cell mitogens in cultures. These combinations are generally able
to obtain a high mitotic index in CLL cells, allowing the identi-
fication of chromosomal translocations and complex karyotypes.
Further studies are currently ongoing to define the best combina-
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http://dx.doi.org/10.1016/j.critrevonc.2016.06.003
3
tion of mitogens and the additional prognostic and predictive value
of chromosomal aberrations detected by CGC (Rigolin et al., 2012).
B-PLL The application of fluorescence in situ hybridization (FISH) tech-
nique demonstrated that genomic aberrations can be identified in
+/− more than 80% of CLL cases; in most cases they involve loss or gain
+ of genetic materials while translocations are rare (Dohner et al.,
– 2000). The four most frequent genomic alterations are independent
+
prognostic factors at multivariate analysis and include:
Bright
IgM/IgD *deletion of the long arm of chromosome 13 (del13q14): it
+/− is found in 50% of CLL cases and it is usually associated with
favourable prognosis when isolated. Two non-coding microRNA
genes, miR-15a and miR16-1, are located in this region and have
been demonstrated to play a tumour suppressor function through
BCL2 regulation (Cimmino et al., 2005);
*trisomy 12 (+12): detected in 10–20% of cases, often as unique
alteration, in the original hierarchical prognostic model by Dohner
et al.,(2000) was classified as an intermediate prognosis marker.
This alteration remains ill-defined, though potential candidate
genes upregulated through a gene dosage effect have been identi-
fied and include P27, CDK4, HIP1R, MYF6 and MDM2 (Winkler et al.,
2005). Recent next generation sequencing studies have demon-
strated an association with NOTCH1 mutation (see Section 2.5) (Del
Giudice et al., 2012; Balatti et al., 2012; Lopez et al., 2012);
*deletion of the long arm of chromosome 11 (del11q22-q23):
found in 5–20% of CLL patients, it is highly variable in size and it is
associated with dismal prognosis. The ataxia-teleangectasia (ATM)
gene, a known tumour suppressor gene, is located in this region and
is affected by the deletion (Stankovic et al., 1999). Larger deletions
include also other potentially relevant genes like BIRC3, recently
identified as genetic abnormality in refractory CLL cases (Rossi et al.,
2012a);
*deletion of the short arm of chromosome 17 (del17p13): this
alteration is usually detected in less than 10% of CLL cases at diag-
nosis but its prevalence increases up to 30% in refractory cases and
over the disease course (Gaidano et al., 2012). This deletion involves
the tumour suppressor gene TP53. Patients bearing del17p belong
to the highest risk category and usually follow a dismal clinical
course.
2.4. Immunogenetics
Chronic lymphocytic leukaemia cells express on their surface
the B-cell receptor (BCR), where a key component is represented
by surface immunoglobulins (IG). Surface IG expression is crucial
for survival and functioning of normal B cells and of many B-cell
lymphoproliferative disorders.
The analysis of IG gene rearrangements in CLL showed that
about 50% of cases bear unmutated IG genes almost identical to
the germ-line sequence (i.e., the percentage of identity of the
heavy chain variable gene, IGHV, to the corresponding germ-
line gene is >98%), while 50% of cases showed the presence of
somatic mutations (identity <98%) (Fais et al., 1998). This finding
has relevant pathogenetic implications, as the process of somatic
hypermutation takes place after antigen encounter in the lymph
node microenviroment and suggested that the antigenic trigger
could represent a tumour driver in CLL cases. In addition, the
IGHV mutational status (mutated vs. unmutated) carries an inde-
pendent prognostic relevance, with unmutated cases following
a more aggressive clinical course (Hamblin et al., 1999; Damle
et al., 1999). Further efforts in the immunogenetic area have also
supported a plausible role for antigen recognition in CLL patho-
genesis, demonstrating that unrelated CLL cases worldwide may
express highly homologous BCRs (called “stereotyped”), where
CDR3 (complementarity-determining region-3) sequences, usually
extremely variable, are characterised by shared amino acid motifs.
Cases expressing stereotyped BCRs collectively account for 30% of
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all CLL and some major subsets carry also an independent prog-
nostic significance (Agathangelidis et al., 2012; Vardi et al., 2014;
Stamatopoulos et al., 2007).
2.5. Next generation sequencing
The recent application of massively sequencing technologies
started unravelling the high genetic and epigenetic heterogeneity
in CLL. Whole-exome sequencing (WES) demonstrated the genetic
variability of the disease, but also revealed novel common gene
mutations, potentially involved in natural history of the disease
(Puente et al., 2011; Wang et al., 2011; Quesada et al., 2011; Rossi
et al., 2012d). The most frequent somatic mutations were found
to be involved in critical cellular pathways including: DNA damage
and cell cycle control (TP53, ATM, BIRC3) (Rossi et al., 2012a), mRNA
processing (SF3B1) (Rossi et al., 2011), NOTCH signalling (NOTCH1)
(Rossi et al., 2012b; Villamor et al., 2013a), and inflammatory path-
ways (MYD88) (Gruber and Wu, 2014; Villamor et al., 2013b). Some
lesions are mutually exclusive while others co-segregate, suggest-
ing distinct evolutionary pathways on one side, and synergistic
effects on the other side (Baliakas et al., 2015). Four alterations
showed a prognostic role in larger cohorts and have been proposed
to be incorporated in prognostic nomograms:
*NOTCH1: recurrently mutated in 10–15% of CLL patients,
the gene encodes for a class I transmembrane protein acting
as ligand-activated transcription factor involved in cell differen-
tiation, proliferation and apoptosis. More than 90% of NOTCH1
mutations are located in exon 34 causing disruption of the PEST
domain and are frequently associated with trisomy 12, unmutated
IGHV gene, aggressive clinical course and increased risk of trans-
formation to diffuse large B-cell lymphoma (so called Richter’s
transformation) (Rossi et al., 2012b; Del Poeta et al., 2013; Rossi
et al., 2012c; Mansouri et al., 2013).
*SF3B1: the mutation is found in 5–7% of cases at diagnosis, but
its frequency increases in progressive cases and at relapse (up to
20%) (Wang et al., 2011; Quesada et al., 2011; Rossi et al., 2012d;
Rossi et al., 2011). SF3B1 is a component of the spliceosome and
the mutations usually involve the C-terminal HEAT-repeat domain
and impair the protein binding. SF3B1 mutations are associated
with dismal clinical course and fludarabine-refractoriness.
*BIRC3: it is involved in apoptosis inhibition and NF␬B (nuclear
factor ␬B) regulation. The mutations generate stop codons elim-
inating the C terminal ring domain. BIRC3 mutations are more
frequent in fludarabine-refractory CLL patients and are mutually
exclusive with TP53 mutations (Rossi et al., 2012a).
*TP53: TP53 mutations are related to short survival and
chemorefractoriness in CLL patients; 80–90% of cases bearing
del17p have a TP53 mutation in the remaining allele, causing a
complete functional inactivation of p53 pathway. These mutations
are more frequent in unmutated IGHV gene cases and indepen-
dently predict a worse clinical outcome (Zenz et al., 2008; Zenz
et al., 2009; Zenz et al., 2010a). The prevalence of TP53 mutations
are low at diagnosis, but increase over disease course. Recent stud-
ies demonstrated that also tiny TP53 mutated subclones (below the
threshold of Sanger sequencing and detected only by ultra deep
next-generation sequencing) are important drivers of the disease
course, associated with an aggressive clinical course and poor sur-
vival. These small TP53 mutated subclones are usually predominant
at the time of CLL relapse and predict the development of chemore-
fractoriness (Rossi et al., 2014).
Genome studies also revealed a wide intraclonal heterogene-
ity, with different CLL subpopulations being present in variable
proportion in the same patient over time (Landau et al., 2013;
Schuh et al., 2012). In general the number of genomic alterations
tends to increase over disease course and it is strongly influenced
by treatments, frequently conditioning clonal evolution. Mutated
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http://dx.doi.org/10.1016/j.critrevonc.2016.06.003
subclones, already detectable in minor proportion at diagnosis or
before starting therapy, become more prevalent in the leukaemic
populations at relapse, likely conditioning a more aggressive course
of the disease (Gruber and Wu, 2014; Rossi et al., 2014).
3. Diagnosis
3.1. IWCLL 2008 diagnostic criteria
According to the International Workshop on Chronic Lympho-
cytic Leukaemia (IWCLL) 2008 (Hallek et al., 2008), updating the
National Cancer Institute-Working Group (NCI-WG) 1996 guide-
lines (Cheson et al., 1996), CLL diagnosis requires the following
criteria:
1. the presence of at least 5 × 109 B lymphocytes/L in the peripheral
blood;
2. a peculiar immunophenotypic profile as detected by flow cytom-
etry, showing:
a clonal light chain restriction (either kappa or lambda);
b CD5 expression;
c CD23 expression;
d low levels of CD20, CD79b and surface immunoglobulin expres-
sion.
Small lymphocytic lymphoma diagnosis requires less than
5 × 109 B lymphocytes/L in the peripheral blood and the presence of
lymphadenopathies and/or splenomegaly and, whenever possible,
should be confirmed by histopathology evaluation.
The presence of less than 5 × 109 B lymphocytes/L in the
peripheral blood, in the absence of lymphadenopathies or hep-
atosplenomegaly or disease-related cytopenias or B symptoms,
defines the condition known as monoclonal B-cell lymphocytosis.
In the absence of cytopenias, bone marrow evaluation is no
longer required for CLL/SLL diagnosis, while it is part of the pre-
treatment staging evaluation and it is required to confirm complete
remission at the end of treatment.
3.2. Clinical presentation
Most patients are asymptomatic at diagnosis and the disease is
detected due to increased lymphocyte count at blood evaluations
performed for unrelated reasons. In some cases the clinical pre-
sentation is characterised by palpable lymphadenopathies and/or
splenomegaly that warrant further investigations.
B symptoms (fever of unknown origin higher than 100.5 ◦ F or
38.0 ◦ C for ≥2 weeks without other evidence of infection; uninten-
tional weight loss of 10% or more within the previous 6 months; or
night sweats for more than 1 month without evidence of infection)
are rarely present. Advanced stage patients can show fatigue and
intolerance to physical exercise due to anemia, secondary to bone
marrow infiltration, while the presence of bleeding manifestations
due to low platelet count is very rare.
Chronic lymphocytic leukaemia per se confers a higher vul-
nerability to infections, explained by immune defects in humoral
and cell-mediated immunity. Abnormalities in T-cell subsets and
neutrophil-monocyte functions, along with hypogammaglobuline-
mia, are frequently detected. The latter might occur over the disease
course and is more pronounced in advanced stages (Morrison,
2010). Disease-associated reduced immune function does not
improve after effective treatment and it is actually worsened by
the majority of available immunochemotherapeutic regimens. Bac-
terial infections involving upper and lower respiratory tract and
urinary tract are the most frequent manifestations, though an
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Table 2 Table 3
Differential diagnosis for CLL. Rai staging system.

MCL SMZL B-PLL Stage Characteristics Median survival

Pleomorphic PB Presence of villous >55% 0 only lymphocytosis in the peripheral blood >150 months
infiltration, lymphocytes in prolymphocytes and bone marrow infiltration
predominantly some cases (peripheral blood I presence of lymphadenopathies 101 months
represented by (peripheral blood smear) II presence of hepatosplenomegaly 71 months
medium size smear) III presence of anemia (defined as Hb <11 g/dl) 19 months
lymphocytes with IV presence of thrombocytopenia 19 months
more abundant (Plt <100,000/mm3 )
cytoplasm
Usually CD5+, but Rarely CD5 and Usually CD5
CD23 negative CD23 positive positive, CD23
(never in the bone negative, FMC7 Table 4
marrow) bright Revision of the Rai staging system.
Cyclin D1 positive Usually Usually
Stage Characteristics
characterised by characterised by
splenomegaly prominent Low risk only lymphocytosis in the peripheral blood and
splenomegaly bone marrow infiltration (same as stage 0)
Intermediate risk presence of lymphadenopathies (same as stage I)
and/or hepatosplenomegaly (same as stage II)
High risk presence of anemia (same as stage III) and or
increased risk of viral reactivation (e.g., herpes zoster infection) has
thrombocytopenia (same as stage IV)
been also reported. Advanced disease is frequently characterised
by worsening infectious complications, also due to previous treat-
ments and representing the main cause of death.
Chronic lymphocytic leukaemia is associated with autoim- laboratory abnormality – not an overt disease – should be con-
mune manifestations, in most cases directed against hematopoietic sidered; on the contrary, cases with higher B lymphocyte count
cells. Autoimmune haemolytic anemia (AIHA) is the most fre- (>0.5 × 109 L) carry a risk of progression into CLL requiring treat-
quent (10–25%) autoimmune manifestation, followed by immune ment of 1–2% per year (Shanafelt et al., 2009; Rossi et al., 2009;
thrombocytopenia (ITP, 1–5%). Pure red cell aplasia (PRCA) and Molica et al., 2011; Scarfò et al., 2012; Dagklis et al., 2009; Fazi et al.,
autoimmune neutropenia are very rare disease-related manifes- 2011). For this reason, international guidelines and expert panels
tations (<1% and 0.2% of cases, respectively) (Hodgson et al., 2011). recommend that subjects with MBL should be followed up lifelong
It should be noted that autoantibodies against red blood cells or with complete blood cell count and clinical evaluation every 6–12
platelet membrane antigens are synthesised by non-malignant B months (Shanafelt et al., 2010)
cell clones and have been related to a profound immune system
dysregulation due to CLL (Zent and Kay, 2010; Strati and Caligaris- 4. Staging
Cappio, 2011). Paraneoplastic pemphigus, acquired angioedema,
and cold agglutinin disease are rare non-haematologic autoim- 4.1. Clinical staging
mune complications of CLL (Dearden, 2008).
At variance with other leukaemias (in particular, acute myeloid Two staging systems are currently applied in CLL patients to
leukaemia), the high absolute lymphocyte count (i.e., hyperleuko- define disease burden and treatment indication: Rai (Rai et al.,
cytosis, frequently detected in CLL patients) does not cause signs 1975) and Binet (Binet et al., 1981) staging system. These two time-
or symptoms of leukostasis and does not require therapeutic inter- honoured systems have many advantages as they are easy to define,
vention. being based only on complete blood cell count (CBC) and physical
examination, and they have been shown to be powerful prognostic
3.3. Differential diagnosis indicators.
1. Rai staging system: the original classification proposed in
The presence of a high lymphocyte count prompts for two main 1975 differentiates 5 groups based on progressively shorter overall
differential diagnosis: survival (Table 3).
*reactive conditions: viral infections, usually characterised by The system has been later revised and risk categories has been
the transiency of this phenomenon, in some cases (e.g., EBV, CMV adapted as shown in Table 4.
infections) diagnosed by serological evaluation; 2. Binet staging system: it defines 3 different risk category
*other lymphoproliferative disorders. based on the number of lymphoid sites involved at physi-
The latter category include lymphomas in leukaemic phase, e.g., cal exam (cervical, axillary and inguinal nodes, hepatomegaly,
mantle cell lymphoma (MCL), splenic marginal zone lymphoma splenomegaly) and the CBC values (Table 5).
(SMZL), follicular lymphoma (FL) and other leukaemias (such as
hairy cell leukaemia – HCL, and B-cell prolymphocytic leukaemia – 5. Prognosis
B-PLL). This distinction is based on several laboratory evaluations,
including – but not limited to – morphology, flow cytometry and 5.1. Natural history
FISH analysis (Table 2).
The clinical course of CLL patients is extremely heterogeneous
3.4. Monoclonal B cell lymphocytosis and the rough data of a median overall survival of 10 years is not
able to capture the clinical variety of the disease, as survival ranges
This recently defined condition is diagnosed based on the from few months to decades. On the one hand, about one third of
absence of disease related signs or symptoms, no palpable lym- patients never require treatment, are regularly followed up over
phadenopathies nor hepatosplenomegaly and the presence of a time, and die “with” CLL rather than “because of” the disease. On
CLL-like clonal B-cell population with less than 5 × 109 B lympho- the other hand, about the same proportion has to be treated at, or
cytes/L. In the case of lower clonal B cell counts (<0.5 × 109 L), a shortly after, diagnosis due to anemia, thrombocytopenia and/or

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Table 5
Binet staging system.
Stage Characteristics
A no anemia (Hb > 10 g/dl) or thrombocytopenia
(Plt >100 000/mm3 ) and up to 2 lymphoid sites
involved
B no anemia (Hb > 10 g/dl) or thrombocytopenia
(Plt >100 000/mm3 ) and more than 2 lymphoid
sites involved
C presence of anemia (Hb < 10 g/dl) and/or
thrombocytopenia (Plt <100,000/mm3 )
regardless the number of lymphoid sites
involved
bulky lymphadenopathies and/or splenomegaly and has a reduced
life expectancy (around 2–3 years). Finally, roughly one third of
patients develop disease-related signs and symptoms and require
treatment at variable time-length from the diagnosis.
Available treatments can often induce disease remission, but
they are not able to reach the cure and CLL still remains an incurable
disease in virtually all cases. According to published data, derived
from alkylating-based chemotherapeutic approaches, there is no
benefit in early treatment, and recent biological studies support this
notion giving us some hints that early chemotherapy could actually
be detrimental and be involved in the selection of resistant clones
occurring at disease relapse.
Nowadays there are no prognostic factors able to discriminate
CLL patients who will eventually progress at diagnosis. For this
reason, long-term follow up is advised once a CLL diagnosis is estab-
lished. Prognostication in CLL is an active research field and many
studies tried to define not only prognostic markers able to pre-
dict the clinical course at diagnosis, but also predictive markers
able to predict response to treatment thus potentially influencing
treatment choices.
5.2. Traditional prognostic factors
Traditional prognostic factors include clinical parameters, lab-
oratory data and biological factors that have been demonstrated
to be differentially present in low and high-risk disease (Furman,
2010; Chiorazzi, 2012).
Among traditional clinical prognostic factors, Rai and Binet stag-
ing systems and lymphocyte doubling time (LDT, i.e., the time
required for doubling the absolute lymphocyte count) are the most
widely applied and are also reported as indication for treatment.
Early stages, according to Rai and or Binet classifications, have
longer overall survival, while a LDT less than 6 or, in some series, 12
months is frequently associated with disease progression (Molica
et al., 1990). Several clinical and laboratory factors have been
claimed in the past as relevant prognostic factors, but many of them
(e.g., pattern of bone marrow infiltration, ␤2 microglobulin, serum
timidin-kinase, soluble CD23) are related to the disease burden and
can be confounding at diagnosis.
5.3. Novel prognostic factors
In the last 15 years biological markers have been identified
allowing a risk stratification of CLL patients at diagnosis and they
currently are in widespread use in clinical practice. It should be
emphasised that, among them, only the presence of del17p by FISH
and TP53 mutations, assessed at disease progression, influence clin-
ical management of CLL patients (Stilgenbauer and Zenz, 2010).
5.3.1. Biological prognostic factors include
1. FISH analysis: the four most frequently detected alterations
bear also a prognostic value (Dohner et al., 2000):
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a Del13q14: this aberration carries a favourable prognostic value,
when isolated, as CLL patients bearing this sole abnormality show
a better prognosis than those with normal (i.e., without FISH
detected abnormalities) karyotype;
b Trisomy 12: it is associated with a less favourable clinical course
and with a somewhat shorter survival than patients with normal
FISH panel;
c Del11q22-23: patients carrying this abnormality often show
bulky lymphadenopathies and aggressive clinical course, with
reduced overall survival;
d Del17p13: as mentioned, this alteration involves the tumour
suppressor gene TP53, and in more than 80% of cases is associ-
ated with TP53 mutation on the remaining allele. This aberration
is associated with a dire clinical outcome, being linked to
fludarabine-refractoriness, treatment resistance and early dis-
ease relapse (Zenz et al., 2010b).
2. IGHV gene mutations: several studies clearly demonstrated
an association between unmutated IGHV genes and poor clinical
outcome. This prognostic value was reported for the first time
15 years ago; though nowadays the test for IGHV gene mutation
analysis is quite fast, reliable and widely available, being rou-
tinely applied, at that time the technique applied for IGHV gene
sequencing was considered expensive and cumbersome, and differ-
ent surrogate markers, more easily determined by flow-cytometry
analysis (e.g., CD38 (Damle et al., 1999), CD49d (Gattei et al., 2008),
and ZAP70 (Crespo et al., 2003)), have been proposed, but none of
them have replaced the relevance of the IGHV mutational status.
3. TP53 mutation: though the mutation of TP53 is frequently
combined with del17p, about 5% of untreated CLL patients with
progressive disease requiring treatment bear a TP53 mutation
without a del17p. Recent guidelines strongly suggest to integrate
TP53 mutation analysis in the diagnostic work-up of CLL patients
before starting treatment together with del17p assessed by FISH
(Pospisilova et al., 2012).
CD38 (a cell surface protein involved in the interaction with the
microenvironment), CD49d (the integrin alpha 4 subunit of VLA-4
receptor), and ZAP70 (an intracellular kinase) have been proposed
as independent prognostic markers, based on several prospective
clinical trials and retrospective data collection studies demonstrat-
ing that a higher level of CD38 (more than 7, 20 or 30% in different
series) (Ghia et al., 2003; Malavasi et al., 2011), of CD49d (more than
30%) (Dal Bo et al., 2014) and of ZAP70 (more than 20%) (Rassenti
et al., 2004) were associated with worse prognosis. Technical issues
impaired the standardisation of ZAP70 (an intranuclear marker)
detected by flow cytometry; this marker is not any longer included
in the work up evaluation.
Novel gene mutations that appear to be relevant in the
relapsed/refractory disease setting have been detected by next gen-
eration sequencing along with the above-cited TP53 mutations, and
include:
1. NOTCH1: its mutations showed a negative impact in overall
survival in several cohorts and were associated with an increased
risk of Richter’s transformation (Fabbri et al., 2011). CLL patients
with NOTCH1 mutations did not benefit from the addition of rit-
uximab to fludarabine and cyclophosphamide combination (see
Section 6) (Stilgenbauer et al., 2014), while a longer progression-
free survival was demonstrated when treated with alemtuzumab
(Schnaiter et al., 2013);
2. SF3B1: its mutation showed a negative effect on overall sur-
vival (Rossi et al., 2011);
3. BIRC3: its mutations are associated with a very dismal prog-
nosis, similar to that of TP53 mutated patients (Rossi et al., 2012a).
It should be noted that, for patients requiring treatment,
response to therapy is per se the most powerful prognostic indi-
cator for survival. Patients obtaining a complete response to
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Table 6 *progressive disease (PD): increase of 50% or more in the disease

Treatment indications.
Stage
Early stage disease (Rai 0, Binet A)
Intermediate stage (Rai I and II, Binet B)
Advanced stage (Rai III and IV, Binet C)
Management
Monitoring without receiving
therapy until disease
progression
Monitoring until signs or
symptoms of active disease
Treatment required
burden.
Clinically beneficial responses include CR and PR, while SD and
PD should be considered treatment failures.
Table 7 reports the extended IWCLL 2008 criteria for defining
the quality of responses.
6.3. Treatment options

The treatment choice requires a careful evaluation of different

treatment usually fare better than those with a partial response. As
more effective treatment options are now available, recent clinical
trials have demonstrated that reaching so-called minimal resid-
ual disease (MRD) negativity predicts longer progression-free and
overall survival (Ghia, 2012) (see Section 6.8).
6. Treatment
6.1. Treatment guidelines
The main issues in the management of CLL patients are related
to the timing (when?) and to the choice (how?) of treatment (Ghia
and Hallek, 2014).
International guidelines clearly specify that treatment indica-
tion is based on the presence of active disease (Table 6).
The following signs and/or symptoms define active disease
(Hallek et al., 2008):
*Progressive bone marrow failure, demonstrated by occur-
rence or worsening of anemia and/or thrombocytopenia due to
bone marrow infiltration;
*Bulky disease, demonstrated by massive (>10 cm) or rapidly
progressive lymphadenopathies and/or massive (>6 cm bellow left
costal margin) or rapidly progressive splenomegaly;
*Uncontrolled autoimmune cytopenias, defined as autoim-
mune anemia and/or thrombocytopenia not responsive to steroid
treatment;
*Rapid LDT, defined by being <6 months or by an >50% increase
in ≤2 months;
*Presence of B symptoms, including unintentional weight loss
≥10% in the last 6 months, fever >38.0 ◦ C for ≥2 weeks without
infections, drenching night sweats for >1 month, extreme fatigue
(ECOG performance status 2 or worse).
Guideline interpretation should be carefully done and some
caveats should be highlighted:
*in patients with <30 × 109 lymphocytes/L, the LDT should not
be used as single parameter to define disease progression requiring
treatment;
*the absolute lymphocyte count (ALC) has no value in defining
treatment indication as CLL patients can reach markedly elevated
ALC without experiencing signs or symptoms of leukostasis;
*the presence of hypogammaglobulinemia and/or of a mono-
clonal component and/or frequent infection complications is not
relevant in defining treatment indication.
6.2. Response criteria
Standardised response criteria include the following categories
(Hallek et al., 2008):
*complete response (CR): complete disappearance of disease at
clinical level (including bone marrow evaluation);
*partial response (PR): reduction of 50% or more in the disease
burden, with CLL being still detectable at clinical evaluation;
*stable disease (SD): patients who did not achieve at least a PR
and did not fulfil PD definition (i.e., disease burden showed less
than 50% increase or reduction);
factors including:
1. patient fitness status based on comprehensive geriatric assess-
ment (i.e., the comorbidity burden and the performance status,
along with the functional and mental status and the need for
caregiver);
2. the genetic profile (i.e., the presence vs. absence of TP53 alter-
ations, detected by FISH and/or by mutation analysis);
3. the disease status (first-line treatment vs. ≥2 line of treatment;
relapse vs. refractoriness to last treatment) (Ghia and Hallek,
2014).
6.4. First-line treatment
a Fit or go-go patients: CLL patients are defined as “fit” or “go-
go” by a normal renal function (creatinine clearance >70 mL/min)
and no or few comorbidities (usually rated on a comorbid-
ity index of rating scale – CIRS – score of 6 or less). These
subjects, if experiencing progressive disease, are candidates to
receive a chemoimmunotherapy combination with fludarabine,
cyclophosphamide and rituximab (FCR), currently considered the
standard of care. After preliminary results derived from phase II
studies in US (Keating et al., 2005), thanks to a European phase III
study (CLL8) (Ghia and Hallek, 2014) chaired by the German CLL
Study Group (GCLLSG), this treatment has been demonstrated to
reach high response rate (overall response rate 90%, with com-
plete response 40%) and to confer a significant advantage in
terms of progression-free and overall survival in comparison to
the standard regimen FC (fludarabine and cyclophosphamide),
becoming the new standard of care. Updated results based on
longer follow up have shown that a relevant proportion of
patients remain without detectable disease 10 years after the
end of treatment. This impressive result comes at a price, as
the randomised study described a higher incidence of grade 3–4
neutropenia (though apparently not associated with an increase
in the infection risk) (Hallek et al., 2010), and follow up stud-
ies described a more frequent occurrence of bacterial and viral
infections for 2 years after treatment completion (Tam et al.,
2008). In addition, there are some concerns on the potential toxi-
city of this combination for bone marrow precursors and, though
most clinical trials did not show an increase in the risk of ther-
apy related myelodysplasia and/or acute myeloid leukaemia, the
occurrence of late onset cytopenias (mainly neutropenia) is not
rarely reported (Strati et al., 2013a). It is now actively debated
if the standard treatment scheme, including 6 courses, can be
safely reduced to 3 or 4 cycles based on the minimal residual
disease (MRD) evaluation aiming at reducing toxicity without
compromising efficacy (Strati et al., 2014). Preliminary results
from a retrospective analysis recently suggested that three FCR
courses can be effective if achieving MRD status and prompted
randomised MRD-guided prospective clinical trials. It is worth
noting that most patients enrolled in the CLL8 phase III trial were
younger than 70 years, and the tolerance in fit patients older than
70 years was quite poor, requiring early treatment stop and/or
dose reduction due to adverse events in many cases (Hallek
et al., 2010). Alternative treatment options for first-line treatment

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http://dx.doi.org/10.1016/j.critrevonc.2016.06.003
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Table 7
IWCLL 2008 Response criteria.
Criteria CR
Group A
Lymphadenopathies None >1.5 cm
Hepatosplenomegaly None
ALC <4000/␮l
Bone marrow Normocellular, 30% lymphocytes, no
B-lymphoid nodules. Hypocellular marrow
defines CRi
Group B
Platelet count >100,000/␮l
Haemoglobin >11 g/dl
Neutrophils >1500/␮l
CR: complete response; PR: partial response; PD: progressive disease; CR: complete response with incomplete bone marrow recovery.
in this category include the use of bendamustine + rituximab
(BR) (Fischer et al., 2012). This combination has been recently
compared with FCR in a randomised GCLLSG trial (CLL10) and
showed a better tolerance for BR, but a lower rate of com-
plete response and an overall shorter progression-free survival
in comparison to FCR though apparently non confirmed among
individuals >65 years of age (Eichhorst et al., 2013).
b Unfit or slow go: “Unfit” or “slow go” patients are characterised
by a relevant comorbidity burden (CIRS score >6) and/or impaired
renal function (creatinine clearance <70 mL/min). This category
includes the vast majority of CLL patients, considering the median
age at diagnosis and the fact that patients might need treat-
ment even years after the diagnosis. In Europe, these patients
were usually treated with the alkylating agent chlorambucil in
monotherapy. This strategy has several advantages, including
oral administration, low cost and good tolerability. Neverthe-
less, this drug is associated with a low rate of response (with
anecdotal or no CRs) and a short PFS and seems to increase the
long-term risk of therapy-related myelodysplasia and/or acute
myeloid leukaemia. Further attempts were pursued to improve
disease control replacing chlorambucil with purine analogues,
in particular fludarabine. Though an increase in response rate
and complete response rate have been demonstrated, elderly
patients treated with fludarabine fare worse than those treated
with chlorambucil in terms of overall survival (Eichhorst et al.,
2009). These findings led to phase III studies testing the combina-
tion of chlorambucil with anti-CD20 antibodies; this strategy has
been able to significantly improve response rate and progression-
free survival, maintaining a favourable side-effect profile (Goede
et al., 2014). The main toxicity associated with anti-CD20 admin-
istration was due to infusion-related reactions, mainly grade 1
and 2, and more frequent with the first or second infusion, and
an increase in grade 3 and 4 neutropenia without increasing
the infection risk. The depth of response was greatly improved
and the combinations of both chlorambucil + obinutuzumab and
chlorambucil + ofatumumab were able to reach minimal residual
disease negative remissions in almost 20% and more than 10% of
cases, respectively (Goede et al., 2014; Hillmen et al., 2013). Based
on these results, both anti-CD20 antibodies have been recently
approved for first-line treatment of CLL patients in combina-
tion with chlorambucil. In particular, a randomized phase III trial
demonstrated that the combination of obinutuzumab plus chlo-
rambucil was able not only to increase the depth of response but
also to prolong the progression-free survival even compared with
the combination of rituximab plus chlorambucil. Considering that
the process of ageing is extremely heterogeneous and that nowa-
days many elderly patients maintain a very good performance
status, more intensive treatment combinations have been tested
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PR PD
Decrease ≥50% Increase ≥50%
Decrease ≥50% Increase ≥50%
Decrease ≥50% Increase ≥50%
50% reduction in marrow infiltrate, or
B-lymphoid nodules
>100,000/␮l or increase ≥50% over baseline Decrease of ≥50% from
baseline secondary to CLL
>11 g/dL or increase ≥50% over baseline Decrease of ≥50% from
baseline secondary to CLL
>1500/␮l or >50% improvement over baseline
in this population. Among them, the most widely applied are FCR-
Lite (Foon et al., 2009), a regimen where the doses of fludarabine
and cyclophosphamide have been reduced to improve tolera-
bility, the combination of pentostatine, cyclophosphamide and
rituximab (PCR) (Kay et al., 2007; Shanafelt et al., 2007), and the
use of bendamustine monotherapy or in combination with ritux-
imab (Fischer et al., 2012; Knauf et al., 2009). Though preliminary
results are promising, these combinations still need to be vali-
dated in randomised, prospective clinical trials in this category
of patients.
c High-risk: this category is mainly represented by CLL patients
bearing del17p and/or TP53 mutations (Stilgenbauer and Zenz,
2010). It should be noted that the presence of these aberra-
tions per se does not represent an indication for treatment, as
a proportion of patients with these aberrations (mainly asso-
ciated with mutated IGHV genes) follow an indolent clinical
course (Best et al., 2009). If these alterations are detected in
patients with progressive disease requiring treatment, they are
associated with a dismal prognosis and there are no standard
treatments able to overcome this adverse clinical course. For this
reason, it is strongly recommended that patients carrying these
abnormalities are enrolled in clinical trials with novel agents, act-
ing independently from the TP53 pathway. For these patients,
allogeneic stem cell transplantation should be considered early
during disease course, as most of the effective treatments are
able to obtain short-lasting responses (mainly PR) at best. Differ-
ent debulking strategies are currently applied in order to reduce
disease burden. In some cases FCR is administered, as a signifi-
cant proportion of these patients respond, though only partially,
but relapse early. Patients can also benefit from alemtuzumab-
based combinations, though the response duration is still short.
The combination of alemtuzumab and high-dose steroids was
found to be very effective in this population with an ORR up
to 90% and a CR rate up to 65% (Pettitt et al., 2006; Pettitt
et al., 2012). More intensive combinations based on the addi-
tion of cyclophosphamide and/or fludarabine to alemtuzumab
have been demonstrated to be associated with unacceptably high
infection rate and treatment-related mortality (Lepretre et al.,
2012). The addition of alemtuzumab or mitoxanthrone to FCR
did not lead to a significant improvement in ORR, while causing
more frequent bone marrow toxicities and infectious complica-
tions (Parikh et al., 2011; Bosch et al., 2009).
More recently, novel kinase inhibitors have been approved
by the Food and Drug Administration (FDA) and the European
Medicines Agency (EMA) in first-line for patients with 17p dele-
tion (if chemotherapy is contraindicated) and they are going to turn
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around the CLL treatment algorithm in this category of patients in
the near future. The approved agents include:
*Ibrutinib: this oral compound (420 mg once daily) irreversibly
inhibits BTK (Bruton-tirosine kinase), a key component down-
stream to the B-cell Receptor (BCR) pathway. BTK activity is
required for calcium release, activation of the NF␬B and NFAT path-
ways. Ibrutinib irreversibly binds to BTK at the C481 residue and
causes kinase function inactivation. It is able to induce modest CLL
cell apoptosis and block of proliferation and B-cell receptor sig-
nalling in vitro. In CLL patients, the compound is administered
at 420 mg once-daily dose. Main side effects include diarrhea,
bleeding manifestations and CYP3A4 interactions contraindicating
the concurrent administration of strong inhibitors or inducers of
cytochrome p450 (O’Brien et al., 2014; Byrd et al., 2013, 2014). It has
also been approved for the use in relapsed/refractory CLL patients
(see Section 6.5).
*Idelalisib: it is an orally administered PI3K␦ inhibitor (150 mg
twice daily). PI3K␦ is the isoform preferentially expressed in normal
and leukaemic B lymphocytes where it is involved in prolifer-
ation and survival. Activation of PI3K requires LYN-dependent
phosphorylation of CD19, a B cell-specific cell surface molecule.
After BCR triggering, the cytoplasmic domain of CD19 is phos-
phorylated by LYN, leading to PI3K binding and then production
of the lipid phosphatidylinositol-3,4,5-triphosphate (PIP3). PIP3
activates several BCR signalling components, in particular the ser-
ine/threonine protein kinase AKT. Idelalisib has been approved in
combination with rituximab also for relapsed/refractory CLL beside
in first line treatment of patients carrying deletion 17p (Furman
et al., 2014). Transaminase elevation, drug-related pneumonitis
and colitis (including early and late-onset diarrhea) were reported
as principal side effects. In most cases these adverse events were
reversible after drug interruption and/or corticosteroids treatment
and they usually did not occur again restarting the drug at the same
or at lower dose after complete recovery (Brown et al., 2014; Flinn
et al., 2014).
6.5. Treatment for relapsed and refractory CLL
There is no standard treatment for relapsing patients and the
regimen choice is mainly based on response duration to first-line
treatment, patient fitness and side effects experienced with previ-
ous therapy. Patients with refractory disease and/or unfavourable
genetic abnormalities should be always considered for enrolment
in clinical trials including or not stem cell transplantation.
In everyday practice, patients treated with first-line
immunochemotherapy combinations, experiencing a response
duration of more than 24–36 months, should receive the same
combinations (Ghia and Hallek, 2014). Attention should be paid
to cumulative bone marrow toxicity in patients receiving FCR as
first-line treatment as few data are available.
Patients relapsing within 24–36 months after the end of treat-
ment and refractory patients (defined as those with stable or
progressive disease after treatment or relapsing within 6 months
after completing a fludarabine-based combination) represent a
clinical challenge and should always be considered for enrolment
in clinical trials. Of course, they should not be retreated with the
first-line regimen and evaluation of response should be carefully
performed in order to early interrupt ineffective treatment and
avoid meaningless toxicities. The most frequently applied options
till recently included:
*alemtuzumab-based combinations: the combination of flu-
darabine and alemtuzumab has been tested in phase II clinical trials
where it was able to reach more than 80% of responses and 30% CRs
(Elter et al., 2011; Elter et al., 2005; Kennedy et al., 2002);
*high-dose steroids associated with anti-CD20 monoclonal
antibody: the combination of methylprednisolone (1 g/mq for
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http://dx.doi.org/10.1016/j.critrevonc.2016.06.003
9
3–5 days) in association with dose-dense rituximab (rituximab
375 mg/mq days 1,8,15) every 4 weeks showed valuable but
short-lasting responses in the relapsed/refractory setting including
patients bearing TP53 abnormalities (Castro et al., 2008). Treatment
is complicated by fatigue, hyperglycemia, sinusitis and dyspepsia.
The novel kinase inhibitors ibrutinib and idelalisib (plus ritux-
imab) have also been approved in the relapsed/refractory setting
(Byrd et al., 2014; Furman et al., 2014). They are rapidly effective in
lymph node shrinkage and this reduction is frequently associated
with the occurrence of so-called redistribution lymphocytosis, that
it is thought to be related to a compartment shift of CLL cells.
*Ibrutinib: it showed impressive overall response rate in the
relapsed/refractory setting also in patients with high-risk disease
(up to 88% PFS and 90% OS at 6 months).
*Idelalisib: it showed great efficacy when combined with rit-
uximab, in particular in patients bearing del17p and/or TP53
mutations (Furman et al., 2014).
*Ofatumumab, a second-generation anti-CD20 monoclonal
antibody, has been approved for patients with CLL refractory to
both purine analog and alemtuzumab (so-called double-refractory
CLL) (Wierda et al., 2010). This drug, targeting a different epi-
tope of CD20, demonstrated a slower dissociation rate (making it
more effective against cells characterized by low levels of CD20
expression, like CLL cells) and it is more potent at inducing
complement-dependent cytotoxicity in comparison to rituximab. It
was able to obtain more than 50% of responses in double-refractory
patients, being generally well tolerated, but the median duration of
response was less than 6 months.
6.6. Experimental agents
Beside the recently approved agents, in the last other novel
exciting treatment approaches started being tested in the CLL field,
raising further hope that we are getting closer to a chemo-free
strategy for CLL patients aiming at cure.
6.6.1. Other kinase inhibitors in advanced clinical development
are
*IPI-145: this is an orally available inhibitor of PI3K␥ and ␦ iso-
forms (Flinn et al., 2013), demonstrating up to 50% ORR in phase
I studies and now tested in phase II and III clinical trials (IPI-145,
2014);
*second- and third-generation BTK inhibitors: including CC-292,
ONO-4059, ACP-196;
*CDK inhibitors: the first-generation compound, flavopiridol,
and its next-in line drug, dinaciclib, showed promising response
rates in patients with relapsed disease associated with an increase
in the risk of tumour lysis syndrome occurrence, but their clinical
development for CLL was recently stopped.
6.6.2. Promising novel drugs acting with different
pathogenesis-related mechanisms are
*ABT-199: this is a third-generation BCL2 inhibitor, designed
to overcome the limitations of previous BCL2 family inhibitors. It
is orally available, shows a selective BCL2 inhibition profile with-
out interfering with BCL-XL and therefore not causing relevant
thrombocytopenia. Phase I and II clinical trials were amended
after detecting a high risk of tumour lysis syndrome (rarely seen
in CLL), introducing a lead-in dose followed by a dose-escalation
phase that showed impressive ORR and CR response rate includ-
ing achievement of MRD negativity status in monotherapy in
relapsed/refractory patients (Seymour et al., 2013);
*Lenalidomide: this oral compound, commercially available for
the treatment of multiple myeloma and lymphomas, was suc-
cessfully administered also in CLL patients (Badoux et al., 2011;
Kater et al., 2014; Strati et al., 2013b). The mechanism of action
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is not clearly understood, but it is thought to be based on its
immunomodulatory effects. Main side effects include: neutropenia,
a CLL-specific tumour flare reaction (characterised by the painful
enlargement of nodal sites involved by the disease) and increased
thrombotic risk requiring prophylactic therapy.
6.7. Allogeneic stem cell transplantation
Autologous stem cell transplantation is no longer consid-
ered suitable for the treatment of CLL patients, being associated
with relevant toxicities without significant advantages in terms
of progression-free and overall survival when compared to
immunochemotherapy regimens (i.e., FCR) (Montserrat and
Gribben, 2011; Sutton et al., 2011; Magni et al., 2014). The advent
of novel kinase inhibitors, next-generation monoclonal antibod-
ies and immunomodulatory agents is changing the treatment
paradigm for CLL patients. As information on long-term safety
and efficacy of these novel agents is still lacking, recently updated
EBMT recommendations reported that allogeneic stem cell trans-
plantation should be strongly considered in fit patients showing
refractory disease and/or bearing TP53 aberrations (Dreger et al.,
2014). Patients with high-risk CLL should be offered the chance to
receive novel agents and, when the maximum response is reached,
two main options should be considered:
1. to consolidate the result performing an allogeneic stem cell
transplantation;
2. to continue treatment with novel agents until progression and
delay the transplant procedure at the next-treatment line.
The transplant procedure should be carefully discussed with
the patients, considering that early transplantation in high-risk
CLL is supported by the coexistence of relapsed/refractory CLL
and unfavourable genetic abnormalities (del17, TP53 mutation,
del11q), a younger age and the absence of relevant comorbidities,
and the availability of a well-matched donor. Several studies using
reduced-intensity conditioning (RIC) allo-SCT have demonstrated
event-free-survival (EFS) and overall survival (OS) rates of 35–45%
and 50–60% at 5 years (Böttcher et al., 2011; Schetelig et al., 2008).
Five-year survival for patients with sensitive and non-bulky disease
is between 54% and 79%, and up to 50% of patients can reach MRD
negativity after transplant. The disease control after allo-SCT is not
influenced by the IGHV gene status or TP53 dysfunction or purine
analogue-refractoriness. Current RIC approaches are less toxic and
recent studies have demonstrated a reduced early mortality (<5%),
but the overall non-relapse mortality is still high (up to 15–30%)
in the CLL setting particularly related to acute and chronic graft
versus host disease (GVHD). Chronic GVHD represents a relevant
concern as it is affecting the quality of life in at least 25% of surviving
patients.
6.8. Significance of minimal residual disease (MRD)
As more effective treatment strategies are becoming available,
the disease eradication becomes an achievable goal. International
guidelines define minimal residual disease negativity as the pres-
ence of less than 1 CLL cell among 10,000 leukocytes (10−4 )
detected through quantitative PCR or multi-colour flow cytome-
try analysis. Several studies demonstrated that MRD negativity is
a powerful predictor of progression-free and overall survival, the
latter in particular when using FCR (Böttcher et al., 2012) or alem-
tuzumab (Lin et al., 2010), and it may become a desirable goal for
the treatment strategy in young fit CLL patients. Nowadays, MRD
evaluation is not recommended in routine clinical practice but it is
included in most recent ongoing clinical trials with a twofold aim:
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1. to validate a surrogate marker for progression-free and overall
survival;
2. to define a reliable parameter able to guide treatment deci-
sions (further treatment vs. early stop vs. maintenance) to avoid
overtreatment on the one side and to delay clinically evident
relapse on the other side.
7. Late sequelae
7.1. Infection risk
Patients affected by CLL are at increased risk of developing
infectious complications and the risk is increased if they receive
treatment for the disease. Increased infectious risk is related to
immune defects inherent to CLL as well as to therapy-related
immunosuppression (Morrison, 2010). The spectrum of infections
changes according to the chosen regimen, e.g., purine analogues,
monoclonal antibodies (alemtuzumab and rituximab), alkylating
agents or combinations of these drugs. Bacterial infections are
the most common, but fungal and herpesvirus infections are also
frequent with the use of these agents. An increased risk for bac-
terial infections and herpetic reactivation has been demonstrated
for up to 2 years after intensive immunochemotherapy combi-
nations like FCR. Though patients with hypogammaglobulinemia
and frequent, relevant infectious episodes benefit from prophy-
lactic intravenous immunoglobulin support, this treatment does
not seem to be cost-effective in patients without clinically rel-
evant infections. Monoclonal antibodies (in particular rituximab
and alemtuzumab) administration is very rarely complicated by
a usually fatal neurologic syndrome called progressive multifocal
leucoencefalopathy, that is a viral disease caused by JC virus reac-
tivation and characterised by a progressive inflammation of the
white matter (Isidoro et al., 2014). The use of vaccination strate-
gies, including vaccines for pneumococcus, influenza, haemophilus,
tetanus, typhoid and diphtheria, has been evaluated in CLL patients.
As immunization responses may be suboptimal due to impaired
antibody production as well as defects in antigen presentation it has
been proposed that responses may be stronger and more persistent
with protein and conjugated vaccines than with polysaccharide
vaccines. As available information is scanty, consensus recommen-
dations on this matter are still lacking.
7.2. Second cancers
Patients affected by CLL have a higher risk of developing
other cancers in comparison to age- and gender-matched con-
trols. An increased incidence of second cancers has been reported,
though the relative contribution of chemoimmunotherapy regi-
mens remains still unknown. As the distribution of second cancers
in CLL is similar to what is found in solid organ or autologous stem
cell transplant recipients (with particularly increased incidence
of malignant melanoma, soft-tissue sarcomas and lung cancer), a
role for the immunologic defects associated with the disease has
been hypothesized. Epidemiological studies found a relative risk of
second cancers of 2.2, with skin, prostate, breast, melanoma, lym-
phoma, gastrointestinal and lung cancers being the most frequent.
Second cancers were more common in older people and in male. In
addition to an increased risk of occurrence, CLL diagnosis seems also
to confer an unfavourable prognosis in patients with second can-
cers. Inferior overall survival and cancer-specific survival has been
demonstrated for breast, colon rectum, kidney, prostate or lung
cancer in patients with pre-existing CLL. In addition to CLL-related
risk, chemotherapy and/or immunochemotherapy combinations
for CLL treatment have been implicated in additional risk. While
purine-analogue single-agent did not seem to be associated with
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L. Scarfò et al. / Critical Reviews in Oncology/Hematology xxx (2016) xxx–xxx
higher risk of developing second cancers (beside the risk related to
CLL), recently it has been demonstrated that the risk of second can-
cers is 2.38 times higher in comparison to the general population in
CLL patients treated with fludarabine, cyclophosphamide and rit-
uximab, the current standard first-line treatment for fit patients
(Benjamini et al., 2014). Among CLL patients who developed a
second cancer after FCR treatment a higher than expected rate
of therapy-related acute myeloid leukaemia/myelodysplastic syn-
drome (t-AML/MDS) and of Richter transformation was detected
(5.1% and 9%, respectively). These patients experienced a short
median overall survival. The features of t-AML/MDS in CLL patients
treated with FCR are consistent with t-AML/MDS related to other
alkylating agents (i.e. more frequent presentation as MDS than as
AML, recurrent unbalanced loss of chromosome 5 and/or 7 or com-
plex karyotype), but showed a shorter latency (median 35 months,
in comparison to historical controls treated with alkylating agents
where the latency is 5–7 years), becoming even shorter (23 months)
in patients with prolonged myelosuppression after FCR, where t-
AML/MDS can directly emerge without complete recovery of blood
cell count (Zhou et al., 2012).
8. Follow-up
There are no clear indications about the frequency and the eval-
uation required for the follow-up of asymptomatic CLL patients.
Patients who are candidates to a “wait and watch” approach should
be evaluated with regular clinical and laboratory follow-up. Based
on to the recently updated IWCLL2008 guidelines (Hallek et al.,
2008), neither bone marrow biopsies nor CT scans are recom-
mended at diagnosis or during follow up, unless the disease is
becoming symptomatic or progressive (Ghia and Hallek, 2014).
After diagnosis, CLL patients are followed up at regular intervals,
being shorter at the beginning, to exclude a rapid increase in the
disease burden. Follow up is based on clinical evaluation and lab
tests (complete blood cell count, kidney and liver function), while
annual abdominal ultrasound is optional.
According to international guidelines additional tests (includ-
ing CT scans and bone marrow evaluation) should be performed at
disease progression.
PET/CT scan is not useful in defining the disease status in CLL
patients but could be applied in subjects with suspected Richter
transformation, aiming at defining the optimal nodal site for biopsy.
Conflict of interest
The authors declare no conflict of interest.
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Please cite this article in press as: Scarfò, L., et al., Chronic lymphocytic leukaemia. Crit Rev Oncol/Hematol (2016),
http://dx.doi.org/10.1016/j.critrevonc.2016.06.003