Detection of notch1 c.7544_7545deICT mutation in chronic lymphocytic leukemia using conventional and real-time polymerase chain reaction
Aim: To evaluate real-time polymerase chain reaction (PCR) assay system for detection of NOTCH1 c.7541_754delCT mutation in chronic lymphocytic leukemia (CLL) patients. Material and Methods: A total of 325 CLL patients were included in the study. Screening for NOTCH1 c.7544_7545delCT was performed u...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Cite this: | Detection of notch1 c.7544_7545deICT mutation in chronic lymphocytic leukemia using conventional and real-time polymerase chain reaction / N.I. Bilous, I.V. Abramenko, A.A. Chumak, I.S. Dyagil, Z.V. Martina // Experimental Oncology. — 2016 — Т. 38, № 2. — С. 112–116. — Бібліогр.: 28 назв. — англ. |
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irk-123456789-1380012018-06-18T03:03:51Z Detection of notch1 c.7544_7545deICT mutation in chronic lymphocytic leukemia using conventional and real-time polymerase chain reaction Bilous, N.I. Abramenko, I.V. Chumak, A.A. Dyagil, I.S. Martina, Z.V. Original contributions Aim: To evaluate real-time polymerase chain reaction (PCR) assay system for detection of NOTCH1 c.7541_754delCT mutation in chronic lymphocytic leukemia (CLL) patients. Material and Methods: A total of 325 CLL patients were included in the study. Screening for NOTCH1 c.7544_7545delCT was performed using conventional PCR-based amplification refractory mutation system (ARMS) method. All 33 samples harboring c.7544_7545delCT allele and 5 negative cases as control were submitted to real-time PCR. Results: Specificity and sensitivity of two PCR techniques were comparable. NOTCH1 c.7544_7545delCT mutation was found by ARMS in 10.1% of CLL patients, which is consistent with the data of other studies. However, the results of ARMS PCR in a minority of cases (2.15%) were doubtful and required reinvestigation. Real-time PCR, being less time-consuming, showed advantage in the assessment of the amplification’s specificity (using the melting curve analysis). It also allows the quantitative assessment of NOTCH1-mutated clone. Conclusion: NOTCH1 c.7544_7545delCT mutation resulting in removal of the C-terminal PEST domain, deregulation of NOTCH1-dependent signaling pathways, has negative influence on prognosis of CLL and efficiency of therapy with anti-CD20 monoclonal antibodies. Real-time PCR allows the fast and reliable detection of c.7544_7545delCT mutation and can be used for the screening of this molecular lesion in CLL patients. 2016 Article Detection of notch1 c.7544_7545deICT mutation in chronic lymphocytic leukemia using conventional and real-time polymerase chain reaction / N.I. Bilous, I.V. Abramenko, A.A. Chumak, I.S. Dyagil, Z.V. Martina // Experimental Oncology. — 2016 — Т. 38, № 2. — С. 112–116. — Бібліогр.: 28 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138001 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions Bilous, N.I. Abramenko, I.V. Chumak, A.A. Dyagil, I.S. Martina, Z.V. Detection of notch1 c.7544_7545deICT mutation in chronic lymphocytic leukemia using conventional and real-time polymerase chain reaction Experimental Oncology |
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Aim: To evaluate real-time polymerase chain reaction (PCR) assay system for detection of NOTCH1 c.7541_754delCT mutation in chronic lymphocytic leukemia (CLL) patients. Material and Methods: A total of 325 CLL patients were included in the study. Screening for NOTCH1 c.7544_7545delCT was performed using conventional PCR-based amplification refractory mutation system (ARMS) method. All 33 samples harboring c.7544_7545delCT allele and 5 negative cases as control were submitted to real-time PCR. Results: Specificity and sensitivity of two PCR techniques were comparable. NOTCH1 c.7544_7545delCT mutation was found by ARMS in 10.1% of CLL patients, which is consistent with the data of other studies. However, the results of ARMS PCR in a minority of cases (2.15%) were doubtful and required reinvestigation. Real-time PCR, being less time-consuming, showed advantage in the assessment of the amplification’s specificity (using the melting curve analysis). It also allows the quantitative assessment of NOTCH1-mutated clone. Conclusion: NOTCH1 c.7544_7545delCT mutation resulting in removal of the C-terminal PEST domain, deregulation of NOTCH1-dependent signaling pathways, has negative influence on prognosis of CLL and efficiency of therapy with anti-CD20 monoclonal antibodies. Real-time PCR allows the fast and reliable detection of c.7544_7545delCT mutation and can be used for the screening of this molecular lesion in CLL patients. |
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Bilous, N.I. Abramenko, I.V. Chumak, A.A. Dyagil, I.S. Martina, Z.V. |
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Bilous, N.I. Abramenko, I.V. Chumak, A.A. Dyagil, I.S. Martina, Z.V. |
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Bilous, N.I. |
| title |
Detection of notch1 c.7544_7545deICT mutation in chronic lymphocytic leukemia using conventional and real-time polymerase chain reaction |
| title_short |
Detection of notch1 c.7544_7545deICT mutation in chronic lymphocytic leukemia using conventional and real-time polymerase chain reaction |
| title_full |
Detection of notch1 c.7544_7545deICT mutation in chronic lymphocytic leukemia using conventional and real-time polymerase chain reaction |
| title_fullStr |
Detection of notch1 c.7544_7545deICT mutation in chronic lymphocytic leukemia using conventional and real-time polymerase chain reaction |
| title_full_unstemmed |
Detection of notch1 c.7544_7545deICT mutation in chronic lymphocytic leukemia using conventional and real-time polymerase chain reaction |
| title_sort |
detection of notch1 c.7544_7545deict mutation in chronic lymphocytic leukemia using conventional and real-time polymerase chain reaction |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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2016 |
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| citation_txt |
Detection of notch1 c.7544_7545deICT mutation in chronic lymphocytic leukemia using conventional and real-time polymerase chain reaction / N.I. Bilous, I.V. Abramenko, A.A. Chumak, I.S. Dyagil, Z.V. Martina // Experimental Oncology. — 2016 — Т. 38, № 2. — С. 112–116. — Бібліогр.: 28 назв. — англ. |
| series |
Experimental Oncology |
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| fulltext |
112 Experimental Oncology 38, 112–116, 2016 (June)
DETECTION OF NOTCH1 c.7544_7545delCT MUTATION
IN CHRONIC LYMPHOCYTIC LEUKEMIA USING CONVENTIONAL
AND REAL-TIME POLYMERASE CHAIN REACTION
N.I. Bilous*, I.V. Abramenko, A.A. Chumak, I.S. Dyagil, Z.V. Martina
SI “National Research Center for Radiation Medicine of the National Academy of Medical Sciences
of Ukraine”, Kyiv 04050, Ukraine
Aim: To evaluate real-time polymerase chain reaction (PCR) assay system for detection of NOTCH1 c.7541_754delCT mutation
in chronic lymphocytic leukemia (CLL) patients. Material and Methods: A total of 325 CLL patients were included in the study.
Screening for NOTCH1 c.7544_7545delCT was performed using conventional PCR-based amplification refractory mutation
system (ARMS) method. All 33 samples harboring c.7544_7545delCT allele and 5 negative cases as control were submitted
to real-time PCR. Results: Specificity and sensitivity of two PCR techniques were comparable. NOTCH1 c.7544_7545delCT
mutation was found by ARMS in 10.1% of CLL patients, which is consistent with the data of other studies. However, the results
of ARMS PCR in a minority of cases (2.15%) were doubtful and required reinvestigation. Real-time PCR, being less time-con-
suming, showed advantage in the assessment of the amplification’s specificity (using the melting curve analysis). It also allows the
quantitative assessment of NOTCH1-mutated clone. Conclusion: NOTCH1 c.7544_7545delCT mutation resulting in removal of the
C-terminal PEST domain, deregulation of NOTCH1-dependent signaling pathways, has negative influence on prognosis of CLL
and efficiency of therapy with anti-CD20 monoclonal antibodies. Real-time PCR allows the fast and reliable detection
of c.7544_7545delCT mutation and can be used for the screening of this molecular lesion in CLL patients.
Key Words: chronic lymphocytic leukemia, NOTCH1 deletion, amplification refractory mutation system, sequencing, real-time PCR.
Chronic lymphocytic leukemia (CLL) is the most
common leukemia of the adult population in Europe
and the US. Several prognostic markers have been
reported, which correlate with the clinical course
and guide treatment decisions. Mutations in the
NOTCH1 gene have recently been identified as new
genetic alterations associated with shorter time-
to-first-treatment and progression-free survival
(PFS) [1–4]. Furthermore, clinical resistance to the
anti-CD20 monoclonal antibodies in CLL patients with
mutated NOTCH1 was found in some clinical trials,
which manifested as a lack of benefit from the addi-
tion of rituximab to fludarabine-cyclophosphamide,
or ofatumumab to chlorambucil [5–8].
The NOTCH1 gene encodes for a transmem-
brane receptor, which functions as a ligand-activated
transcription factor. Upon binding with ligands
of the Jagged or Delta families, a cascade of pro-
teolytic cleavages take place that result in the release
of NOTCH1’s intracellular domain from the membrane,
translocation to the nucleus and subsequent activa-
tion or repression of target genes through interaction
with other transcription factors such as CBF-1 [9].
The NOTCH1 signalling is involved in critical cellular
processes such as proliferation, apoptosis and diffe-
rentiation [10]. Alterations in NOTCH1 signalling have
been reported in different diseases including several
cancers [11].
In CLL NOTCH1 mutations are detected in 8–15%
cases regardless of clinical phase of disease and are
represented mostly by a recurrent 2-bp frameshift
deletion (c.7541_7542delCT) localized in the exon 34.
This deletion results in removal of the C-terminal PEST
domain, which regulates protein turnover to the ubiqui-
tin-proteasome complex for subsequent degradation,
thereby leading to accumulation of constitutively active
protein and deregulation of NOTCH1-dependent path-
ways [12]. Constitutive activation of NOTCH1 signalling
is considered to contribute to apoptosis resistance and
increased survival of CLL cells [13, 14]. For detection
of NOTCH1 mutations conventional DNA sequencing
techniques are generally used. Besides, Rossi
et al. [1] have designed polymerase chain reaction
(PCR)-based amplification refractory mutation system
(ARMS) method for screening of the most common
NOTCH1 c.7541_7542delCT mutation.
T h e a i m o f t h i s p a p e r w a s t o e v a l u -
ated real-time PCR assay system for detection
of NOTCH1 c.7541_7542delCT mutation. We have not
found any reports of the using real-time PCR for this
purpose in the available literature.
MATERIALS AND METHODS
A total of 325 CLL patients referred to the State
Institution “National Research Center for Radiation
Medicine of the National Academy of Medical Sciences
of Ukraine” (NRCRM) were included in study. CLL
diagnosis was established according to standardized
criteria of International Working Group on CLL [15].
Immunophenotyping of peripheral blood cells was
performed in Laboratory of Immunocytochemistry,
Department of Clinical Immunology, NRCRM. The sta-
ging was obtained according to both the Rai and Binet
systems [16, 17]. The study was approved by the
Submitted: March 17, 2016.
*Correspondence: E-mail: nbilous@yahoo.com
Abbreviation used: ARMS – amplification refractory mutation
system; CLL – chronic lymphocytic leukemia; PCR – polymerase
chain reaction; PFS – progression-free survival.
Exp Oncol 2016
38, 2, 112–116
Experimental Oncology 38, 112–116, 2016 (June) 113
Ethical Committee of NRCRM and all patients gave
informed consent.
Molecular studies were performed on DNA sam-
ples extracted from peripheral blood mononuclear
cells with the QIAamp Blood Mini Kit (Qiagen, United
Kingdom). In all observed CLL patients the presence
of NOTCH1 c.7544_7545delCT mutation was investi-
gated by ARMS using primers and PCR parameters
developed by Fabbri et al. [14] with very little modifica-
tions. In brief, two forward primers (specific for the mu-
tant allele, ForMUT: 5՛-TCCTCACCCCGTCCCGA-3՛; for
both mutant and wild-type alleles, ForC: 5՛-GTGACCG-
CAGCCCAGTT-3՛) and common reverse primer (Rev,
5՛-AAGGCTTGGGAAAGGAAGC-3՛) were used. PCR re-
action was performed with 50 ng of DNA in a 30 μL of fi-
nal volume reaction mixture conta ining 15 ml 2X PCR
Master Mix (Fermentas, Lithuania) and 0.1; 0.4 and
0.5 μM of ForC, ForMUT and Rev primers, respec-
tively. PCR conditions were: 95 °C for 3 min followed
by 30 cycles at 95 °C for 30 s, 59 °C for 40 s, 72 °C for
40 s with a final extension at 72 °C for 40 s in Bio-Rad
C1000 thermal cycler. Amplified PCR products were
separated on 2% agarose gel and visualized after
staining with ethidium bromide. The 283 bp band indi-
cated the wild-type allele was observed in all samples,
whereas the additional band of 183 bp was found only
in NOTCH1-mutated cases.
To confirm specif icity of ARMS PCR for
NOTCH1 c.7544_7545delCT detection, 5 DNA samples
which were detected as positive and 5 negative samples
were analysed by direct Sanger sequencing. Additi-
onally, 7 cases which were doubtful with ARMS PCR also
were sequenced. For this, PCR amplification was per-
formed on 50 ng of DNA using abovementioned ForC
and Rev primers (0.5 μM each) and 2X PCR Master Mix
(Fermentas, Lithuania) in a total volume of 25 μL, and
the same as for ARMS cycling conditions. Amplicons
were spin column purified with PCR purification kit (Pro-
mega, USA) and sequenced directly using Rev primers
and BigDye Terminator Cycle Sequencing Reaction Kit
(Perkin Elmer, USA). Analysis was performed in an auto-
mated DNA sequencer ABI-310 (Applied Biosystem,
USA) as reported earlier [18]. Data were compared with
NOTCH1 germline sequence (RefSeq NM_017617.3).
Real-time PCR for NOTCH1 c.7544_7545delCT
mutation was developed using the same ForMUT and
Rev primers as for ARMS method and 2X Absolute Blue
qPCR SYBR Green Mix (Thermo Scientific, USA) on the
Bio-Rad IQ Real-time System. PCR reaction was per-
formed in a final volume of 25 μL with 0.175 μL of each
primer and 50 ng DNA. PCR conditions were: 95 °C for
15 min followed by 40 cycles at 95 °C for 15 s, 60 °C for
30 s, 72 °C for 30 s. The program was ended with
a dissociation curve analysis to verify the product and
identify the presence of spurious PCR bands or primer
dimmers. Each sample was assayed in duplicate. PCR
standard curve was generated by a serial dilution of the
NOTCH1-mutated DNA sample (50; 10; 2; 0.4; 0.08 ng).
Amplification of the reference β-microglobulin (B2M)
gene (forward primer 5՛-CGGGCATTCCTGAAGCTGA-3՛
and reverse primer 5՛-GGATGGATGAAACCCAGACA-
CATAG-3՛) was used as the internal control. The reaction
mix without DNA template was used as the negative
control. Levels of NOTCH1 c.7544_7545delCT in sam-
ples relative to the reference gene were evaluated based
on the value of delta threshold cycles (∆CT).
All 33 samples harboring c.7544_7545delCT and
10 cases lacking c.7544_7545delCT by ARMS were
submitted to real-time PCR.
Statistics were performed using the SPSS 17.0 soft-
ware package (SPSS, USA). Data shown are the means
plus or minus standard deviations, and medians.
The comparisons were analyzed with t-test. Correla-
tions of ∆CT and hematologic parameters of patients
were assessed using Pearson’s correlation. Kaplan —
Meier curves and log rank test were used to determine
PFS for NOTCH1-mutated cases. p-value less than
0.05 was considered statistically significant.
RESULTS AND DISCUSSION
NOTCH1 c.7544_7545delCT mutation was found
by ARMS in 33 (10.1%) of 325 CLL patients (Fig. 1).
This frequency was comparable with data from other
CLL cohorts [19–21]. Results of ARMS reaction were
apparent in 31 cases, but in 2 cases a weak mutational
signals of a typical mobility were observed. Besides
this, in 5 patients weak bands with mobility which was
a little different from the 183 bp band of NOTCH1-
mutated positive control were revealed. In repeated
studies, these bands have been not identified, and
cases were regarded as “presumably negative”.
283 bp —
183 bp —
1 2 3 4 5 6 50 bp 6 7 8 9 10 11
Fig. 1. Results of NOTCH1 c.7544_7545delCT mutation de-
tection in CLL patients by ARMS method. The additional band
of 183 bp identify of NOTCH1-mutated cases
In 5 apparently NOTCH1-mutated cases and
in 2 cases with weak specific signal according
to ARMS method, which were analysed also by di-
rect Sanger sequencing, the presence of a typical
2 bp deletion c.7544_7545delCT was confirmed
(Fig. 2). In all cases mutation process involved only
one allele. By contrast, 5 NOTCH1-unmutated cases
by ARMS, and also 5 “presumably negative” cases,
were negative by Sanger sequencing. So, our data
are consistent with the suggestion of Rossi et al.
that ARMS method had 100% specificity in detection
of NOTCH1 c.7544_7545delCT mutation [1].
The next step was the development a SYBR
green-based real- t ime PCR for detect ion
of NOTCH1 c.7544_7545delCT mutation. Sensitivity
and specificity of the real-time PCR was determined
by a serial dilution of the mutant DNA sample from CLL
patient with a predominance of leukemic B-cells (WBC
114 Experimental Oncology 38, 112–116, 2016 (June)
112 • 109/l, 85% of the blood leukocytes were CD19+/
CD5+/CD23+ lymphocytes). Specific amplification
of NOTCH1 deletion was found in all dilutions of DNA, in-
cluding maximal (0.08 ng), which corresponds to appro-
ximately 10% of mutant alleles in the sample. The melting
curve analysis revealed that the amplicon containing
c.7544_7545delCT mutation melted at 91 °C in the form
of a clear single peak. Correlation coefficient of the
standard curve for NOTCH1 was 0.984 with a slope value
of −3.38 (PCR efficiency — 97.6%) (Fig. 3). Correlation
coefficient of the standard curve for B2M was 0.978 with
a slope value of −3.44 (PCR efficiency — 95.0%). Thus,
amplification efficiency of both genes was comparable,
and B2M can be used as a referent for NOTCH1 detec-
tion. These obtained data regarding sensitivity of real-
time PCR for detecting NOTCH1 mutations showed
no significant difference from other methods (ARMS and
direct DNA sequencing, which allow detecting mutations
present in at least 10% of the alleles) [1, 22].
b
a
Fig. 2. Detection of NOTCH1 c.7544_7545delCT mutation
by direct Sanger sequencing: a) wild type NOTCH1 sequencing;
b) NOTCH1 c.7544_7545delCT mutation, the position of 2 bp de-
letion is indicated by arrow
Amplification of the reference B2M gene us-
ing real-time PCR was similar in NOTCH1-mu-
tated and NOTCH1-unmutated cases according
ARMS. The mean ± SD of CT values for B2M was
23.77 ± 0.84 and 24.51 ± 1.24, correspondingly
(p = 0.234).
All 33 CLL cases with c.7544_7545delCT by ARMS
were positive by real-time PCR also. The mean ±
SD of CT values for NOTCH1 was 28.17 ± 1.87, and
median of ∆CT was 3.9 (range 1.4–7.5 cycles), and the
mean ± SD of ∆CT was 3.9 ± 1.4. Specificity of reactions
was confirmed by the melting curve analysis. In the
most cases (26 of 33; 78.8%), ∆CT values ranged from
1.4 to 4.9 cycles, and ∆CT values more than 7 cycles
(7.1 and 7.5 cycles) were observed only in 2 cases
(weak mutational signal by ARMS reaction).
b
c
a
Fig. 3. Melting curve analysis: a) Delta reaction curve vs cycle
number for real-time PCR assay. The NOTCH1 mutant DNA
was diluted by serial dilutions; specific amplification was found
in all dilutions of DNA, including maximal (0.08 ng); b) Disso-
ciation curve for real-time PCR assay. Melting curve analysis
revealed that the c.7544_7545delCT specific amplicon melted
at 91 °C in the form of a clear single peak; c) Standard curve for
real-time PCR assay for NOTCH1 c.7544_7545delCT mutation
We did not observe a specific amplification
in NOTCH1-unmutated cases, including “presumably
negative” cases: in seven cases any amplification was
found during the 40 cycles of the reaction, in other ca-
ses the mean ± SD of CT values for NOTCH1 was 37.04 ±
2.6, median of ∆CT was 10.9 (range 10.4–21.8 cycles),
a clear single peaks in the melting curve were absent
(Fig. 4). Thus, the application of real-time PCR allows
to clearly identify CLL patients with c.7544_7545delCT
NOTCH1 mutation even in cases of doubt by ARMS
reaction. In such cases the use of real-time PCR is ex-
pedient as it has some advantages over DNA Sanger
sequencing (time- and cost-effective).
Real-time PCR also can be used to determine the
size of cell’s clone harboring mutations. Prognos-
tic significance of quantitative assessment of some
disease-associated genetic markers, which reflect
tumor burden, was shown in CLL and other lympho-
proliferative disorders [23–26]. However, as it was found
by high-throughput sequencing and CD19+-selected
cells, NOTCH1 mutations generally do not occur during
Experimental Oncology 38, 112–116, 2016 (June) 115
CLL course and the mutational load in positive cases
tends to be stable over time. Furthermore, all NOTCH1-
mutated cases (in which mutations were present in the
main clone or at low clonal level) had the same adverse
prognostic significance [3]. In our study the analysis
of NOTCH1 mutations was performed on unselected pe-
ripheral blood mononuclear cells. Thus we could evaluate
the size of NOTCH1-mutated clone only approximately.
The content of CD5+/CD23+ lymphocytes in the samples
ranged from 66% to 97%. No correlations between
∆CT values and WBC content (r = −0.105; p = 0.588), the
percentage of CD5+/CD19+ cells (r = −0.075; p = 0.710),
and absolute number of CD5+/CD19+ cells (r = −0.076;
p = 0.700) were found (Fig. 5). Respectively, high CT va-
lues, which indicate the presence of mutations in the
main clone, were observed in patients with relatively low
leukocytosis, and vice versa. We compared PFS using
Kaplan — Meier method and Log-rank test of patients
without NOTCH1 deletion, with presence of NOTCH1 de-
letion on the whole and depending on ∆CT values (< mean
– SD; > mean + SD; intermediate value). PFS was shorter
in all NOTCH1-mutated cases (43 months) compared
with NOTCH1-unmutated cases (49 months; p = 0.036).
Median of PFS was 16 months for patients with ∆CT values
< 2.52 (mean − SD), 49 months for patients with ∆CT valu-
es > 5.34 (mean + SD), and 36 months for patients with
intermediate ∆CT values (p = 0,022). Real-time PCR
using CD5+/CD19+-selected cells from peripheral blood
of CLL patients allows more accurately evaluate influence
of NOTCH1-mutated cell’s burden on PFS.
Fig. 4. Dissociation curve for real-time PCR assay. Melting curve
analysis revealed the NOTCH1 c.7544_7545delCT mutation
specific (indicated by arrow) and unspecific amplicons
CD5+CD19+ cells, %
De
lta
C
T
60 70 80
8
6
4
2
90 100
R2 Linear = 0.006
Fig. 5. Evaluation of correlations of ∆CT and the percentage
of CD5+/CD19+ cells using Pearson’s analysis showed no cor-
relation (r = −0.075; p = 0.710)
In conclusion, we evaluated real-time PCR assay
for detection of NOTCH1 mutations in comparison with
ARMS method. Specificity and sensitivity of both tech-
niques were comparable. NOTCH1 c.7544_7545delCT
mutation was found in 10.1% of CLL patients, which
is consistent with other reports [19–21, 27]. However,
the results of ARMS in a minor number of cases
(7 of 325 cases; 2.15%) were doubtful and required
reinvestigation. Sanger sequencing allows to precisely
identify deletion, but it is quite laborous and more ex-
pensive than conventional PCR-based methods. In this
context real-time PCR being time- and cost-effective,
extends the opportunities for an objective assessment
of the amplification’s specificity and thus might be used
for fast screening for NOTCH1 c.7544_7545delCT mu-
tation. Furthermore, under certain conditions it might
allow a quantitative assessment of NOTCH1-mutated
clone. CLL patients harboring NOTCH1 deletion showed
a significantly shorter PFS in comparison with NOTCH1-
unmutated cases sustaining reported adverse impact
of this alteration on outcome [1, 20, 21]. Besides the
prognostic implications, NOTCH1 mutations might
guide treatment options in the context of advisability
of anti-CD20 monoclonal antibodies addition [5–7],
and also might provide a therapeutic target for
NOTCH1 pathway inhibitors. The use of NOTCH1 inhibi-
tors in combination with chemotherapy is considered
as a promising approach for the treatment of CLL cases
with NOTCH1-activating mutations [28].
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