1.1. Aim: Febrile neutropenia, a fatal side effect, can be appropriately treated if we can predict its risk of development before starting chemotherapy. The Nicotinamide Phosphoribosyltransferase (NAMPT) activity and Sirtuin 1 (SIRT1) gene expression have been reported to be essential for identifying the neutrophils mediated by granulocyte colony-stimulating factor.
1.2. Methods: We measured the activity of the NAD+-SIRT 1 pathway and Vitamin B3 (VB3) levels in cancer patients and examined correlations with grade 4 neutropenia due to chemotherapy.
1.3. Results: A total of 21 chemo-naïve small and non-small cell lung cancer patients were enrolled. We obtained blood samples before the first treatment, 2 and 4 days after starting treatment, at the nadir, and before starting the second course of treatment. NAMPT activity before starting treatment was significantly lower in the group with grade 4 neutropenia (N=9) than in those without neutropenia (N=12) (0.69 vs 1.63 ng/mL, p=0.003). SIRT1 gene expression and plasma concentrations of VB3 did not significantly differ between these two groups. On comparison with healthy volunteers, NAMPT activity was significantly higher in cancer patients, however no significant differences were noted between the grade 4 neutropenia group and grade 0-3 neutropenia group.
1.4. Conclusion: Our finding suggests that NAMPT activity before treatment may be a predictive factor for neutropenia with cancer chemotherapy. Further studies will be needed to confirm the role of the NAD+-SIRT1 pathway in cancer patients receiving chemotherapy, and evaluate the association between NAMPT activity and patient background.
2. Keywords: NAMPT activity; Neutronenia; SIRT1 gene expression
cancer chemotherapy frequently causes treatment delays, dose reduction, and
Febrile Neutropenia (FN) [1,2]. Several studies
have examined the proportion and prevalence of neutropenia and FN with various
types of cancers [3-5], and confirmed the
patient risk factors for FN are elderly age, advanced disease state, poor
performance status or nutritional status, and presence of medical comorbidities
. However, little is known about the
predictive markers for severe neutropenia, thereby hampering the ability to
estimate the disease severity before starting chemotherapy. Scokowa et al.
reported the Nicotinamide Phosphoribosyltransferase (NAMPT) activity and the
Sirtuin 1 (SIRT1) gene expression-noticing NAD+-SIRT1
pathway to be essential for differentiating the neutrophils mediated by
Granulocyte Colony-Stimulating Factor (G-CSF) in healthy individuals and in
individuals with congenital neutropenia .
The treatment of
healthy individuals with high doses of vitamin B3 (nicotinamide; VB3), a
substrate of NAMPT, induced neutrophilic granulocyte differentiation . We therefore hypothesized that the NAD+-SIRT1 pathway might be controlled in cancer
patients who developed severe neutropenia after starting chemotherapy. An
appropriate response to febrile neutropenia, which is a fatal side effect, can
be ensured at the risk of developing neutropenia can be predict before starting
In order to
identify the predictive factors of neutropenia and suggest preventative and
therapeutic regimens for severe neutropenia. We herein analyzed the predictive
value of NAMPT activity, SIRT1 gene expression, and VB3 levels for neutropenia
in cancer patients, with concurrent measurements in healthy volunteers. This
study was supported by efforts
from the promotion plan for the platform of human resource development for
A total of 21
chemo-naïve small and non-small cell lung cancer patients newly diagnosed at
Shimane University Hospital were enrolled between October 2013 and August 2014.
The eligibility criteria for the chemotherapy regimen were cisplatin and
pemetrexed (±bevacizumab), etoposide, docetaxel,
vinorelbine or irinotecan, carboplatin and paclitaxel (±bevacizumab),
etoposide or nab- paclitaxel and docetaxel monotherapy. The major exclusion
criteria were obvious bone marrow metastasis and the administration of
corticosteroids, except as an antiemetic treatment.
toxicities were assessed using the National Cancer Institute Common Terminology
Criteria for Adverse Events (NCI-CTCAE) version 4.0, and determined during the
first course of chemotherapy. This study was reviewed and approved by the
relevant institutional review boards of the Shimane University (IRB number: 1347),
and written informed consent was obtained from all patients and healthy
volunteers prior to participation.
4.2. Analysis of the NAD+-SIRT1
We obtained blood
samples from 21 patients before the first course chemotherapy, 2 and 4 days
after starting chemotherapy, at the nadir, and before starting the second
course of chemotherapy. We analyzed Complete Blood Cell Counts (CBCs),
including number of neutrophils. The plasma NAMPT activity was measured by
ELISA (Visfatin EIA Kit; Cayman Chemical, Ann Arbor, MI, USA), and VB3 levels
were measured by bioassay (SRL, Tokyo, Japan). The mononuclear cell fraction
was obtained from a density gradient using a cell preparation tube (BD
Vacutainer® CPT; Becton, Dickinson
and Company, NJ, USA). Reverse transcription polymerase chain reaction was
carried out to measure SIRT1 gene expression at each point. We also sampled
plasma and mononuclear cell fractions to assess NAMPT activity and SIRT1 gene expression
in 19 healthy volunteers.
4.3. Statistical Analysis
All data were
assessed using Student’s t-test. P-values <0.05 were considered to indicate
a statistically significant difference. All analyses were conducted using the
SPSS software program version 19 (IBM, Okinawa, Japan).
5.1. Patient Characteristics
characteristics are summarized in (Table 1). Twenty-one
patients with a median age of 64 years were enrolled in this study. Almost 70%
were male. The histological classifications were 3 SCLCs and 18 NSCLCs, and
clinical or pathological stage was adjuvant setting (stage IIA-IIIA) in 6 and
recurrent or metastatic in 15. Nine patients had grade 4 neutropenia. (Figure 1) shows the change in the number of
neutrophils after chemotherapy. The neutrophil count temporarily rose under the
influence of a corticosteroid 2 to 4 days after starting therapy, before
subsequently reaching its nadir.
5.2. Assessment of NAD+-SIRT1
NAMPT activity at
before starting chemotherapy was significantly lower in the group with grade 4
neutropenia (N=9) than in those without neutropenia (N=12) (0.69 vs 1.63 ng/mL,
p=0.003) (Figure 2). With regard to SIRT1 gene
expression, while a notable increase in expression was observed at the
neutrophil nadir in the G0-3 neutropenia group, no significant differences were
noted between this group and the grade 4 neutropenia group (Figure 3). The plasma
concentrations of VB3 did not significantly differ between these two groups
(data not shown). On comparison with healthy volunteers, the NAMPT activity was
significantly higher in cancer patients, but no statistical differences were
noted between the grade 4 neutropenia group and healthy volunteers (Table 2).
This is the first
report to examine the association between the NAD+-SIRT1
pathway and neutropenia in cancer patients.
The purpose of this study was to clarify the role of the NAD+-SIRT1 pathway in risk of developing neutropenia after starting cancer
chemotherapy. We demonstrated the following three
points. First, the NAMPT activity was significantly lower in severe neutropenia
patients before starting chemotherapy than in those without neutropenia.
Second, the SIRT1 gene expression among patients with grade 0 to 3 neutropenia
markedly increased in the nadir phase. Third, the NAMPT activity in advanced
lung cancer patients was higher than in healthy volunteers.
NAMPT is a key
biosynthetic enzyme converting nicotinamide to nicotinamide adenine
mononucleotide, the main source of NAD+8).
An elevation of the NAMPT and NAD+
levels in extracellular and intracellular (myeloid cells) environments promotes
the overexpression and activation of SIRT1 . SIRT1
activation may therefore stimulate increased binding of these factors to
promoters of genes encoding G-CSF and G-CSF receptor. NAD+-dependent SIRTs regulate a variety of
biological responses, such as the stress response, metabolism, aging and cell
Given our present
findings, we hypothesize that NAMPT induces a G-CSF autoregulatory loop via the
following sequential mechanism: The decrease in the neutrophil number after
starting chemotherapy induces activation of the NAD+-SIRT1 pathway controlled by NAMPT. This positive
autoregulatory system proceeds relatively smoothly in cancer patients with
NAMPT activity before chemotherapy and it also recognizes an over expression of
SIRT1 in the nadir phase. On the other hand, the production of NAD+ is late or absent in patients with a low NAMPT
activity who may develop severe neutropenia without recognizing any over
expression of SIRT1.
A number of human malignant diseases have been
shown to overexpress NAMPT, including lung, esophageal, and cervical cancers as
well as lymphoma [9-13]. Further, several
previous studies have reported that SIRT1 overexpression was associated with a
poor prognosis in cancer patients. For example, Li et al. showed that SIRT1
expression in lung adenocarcinoma tissue as assessed using immunohistochemistry
was significantly associated with a high pathological stage [9,10]. Similar findings were noted in the present
study, as SIRT1 expression measured using mononuclear cells, was found to be
high in lung cancer patients, particularly those in stage IV, compared with
healthy volunteers. Given that the SIRT1 expression decreased in patients under adjuvant
conditions with completely resected lung cancer, severe neutropenia may thus develop more easily
under adjuvant conditions than in patients with metastasis.
associated with the present study include the small size of the study
population and the varied patient background. Further studies with larger, more
homogeneous patient populations are needed to confirm whether or not the NAMPT
activity and SIRT1 expression represent useful predictive markers of severe
neutropenia. In addition, the utility of VB3 prophylaxis in preventing
neutropenia and the role of SIRT1 gene expression stimulation in promoting
cancer progression are unclear and warrant further examination.
We herein showed that the NAMPT activity before treatment may be a predictive factor for neutropenia with cancer chemotherapy. The role of the NAD+-SIRT1
pathway in cancer patients receiving chemotherapy should therefore be
examined further in future studies, and the association
between the NAMPT activity and patient
background should also be evaluated.
We thank all of
the patients, their families and the volunteers. We also thank Ms. Michiko
Horiguchi for assistance and technical advice.
All authors have no conflict of interest to
This study was
supported by MEXT’s Promotion Plan for the Platform of Human Resource
Development for Cancer Project.
This study was
presented at ASCO 2016, abstract published only. “A clinical study on activity
of NAMPT and SIRT1 gene expression in neutropenia with cancer chemotherapy.” J
Clin Oncol 34, 2016 (suppl; abstr e21639).
Figure 1: Neutrophil count after chemotherapy. The neutrophil count of grade 0-3 neutropenia patients (A) and grade 4 neutropenia patients (B). Data represented each patient in the line of the different color.
Figure 2: NAMPT activity before and after cancer chemotherapy. Data shown mean±SE. All data were assessed using Student’s t-test.
Figure 3: SIRT1 gene expression before and after cancer chemotherapy. Data shown mean±SE. All data were assessed using Student’s t-test.
median age ,years (range)
Gender Male / Female
Type of cancer (SCLC/NSCLC)
Clinical orpathological stage (IIA/IIB,IIIA/IV)
ECOG performance status (0/1/2/3)
Chemotherapy regimen (N) CDDP/VNR (6), CDDP/PEM/BEV (5),DTX monotherapy(3), CDDP/GEM (3), CDDP/VP-16(2), CBDCA/PTX/BEV (1) , CBDCA/VP-16 (1)
Abbreviations: SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer; CDDP, cisplatin; VNR, vinorelbine; PEM, pemetrexed; BEV, bevacizumab; DTX, docetaxel; GEM, gemcitabine; VP-16, etoposide; PTX, paclitaxel; CBDCA, carboplatin
Table 1: Patient characteristics
Healthy volunteers (N=19)
cancer patients (N=21)
G0-3 neutropenia (N=12)
G4 neutropenia ( N=9)
stage IIA-IIIA(adjuvant setting,N=6)
Table 2: Comparison of NAMPT activity between lung cancer patients and healthy volunteers.