Keywords cell adhesion - cardiovascular diseases - risk factors - cytokines - adipokines -
stress - psychological - generalized anxiety disorder
Introduction
Breast cancer (BC) is the most common cancer among women worldwide.[1 ] Onco-vascular risk factors are implicated in both cancer and cardiovascular disease
pathogenesis.[2 ]
[3 ]
[4 ]
[5 ] Specific factors, such as leptin, adiponectin, and cell adhesion molecules (CAMs)
such as sICAM-1 and sVCAM-1, are involved in the pathogenesis of BC.[2 ] Neoadjuvant chemotherapy (NACT) in BC is administered before surgical treatment
of patients with locally advanced malignant tumors and may impact the oncovascular
risk factors.[6 ] NACT affects shared pathways in cancer and cardiovascular disease, increasing long-term
CV risk in BC patients. Various studies suggested that chemotherapeutic regimens such
as docetaxel, doxorubicin, and cyclophosphamide, etc., cause impaired vascular function
through different mechanisms.[7 ]
Adiponectin is an insulin-sensitizing hormone produced by adipocytes that maintains
metabolic homeostasis. Serum adiponectin levels show an inverse relationship with
BC development and with the biologically aggressive phenotype of the tumor.[3 ] In BC, adiponectin manifests pro-apoptotic, anti-proliferative, and anti-migratory
effects.[8 ]
Leptin is an anorexigenic hormone that suppresses appetite through a hypothalamic
central feedback mechanism. It is mainly secreted by adipose tissue. In BC, leptin
signaling regulates the expression of vascular endothelial growth factor, E-cadherin,
survivin, cyclin D1, p53, and other tissue factors. All these molecules are involved
in inflammation, angiogenesis, proliferation, invasion, and migration.[4 ] The adiponectin/leptin ratio is inversely related to the cell survival and tumor
proliferation of BC.[9 ]
Vascular endothelial growth factors (VEGFs), known as CAMs, are expressed on endothelial
surfaces. Both ICAM-1 and VCAM-1 play significant roles in the process of BC pathogenesis;
sICAM-1 and sVCAM-1 are their soluble forms. The activated endothelial cells can secrete
these soluble forms into the bloodstream. The serum concentrations of sVCAM-1 and
sICAM-1 are directly related to the pathogenesis of BC and vascular endothelial dysfunction.
These VEGFs have pro-angiogenic actions.[10 ]
A higher rate of depression is seen in BC patients undergoing chemotherapy.[11 ] The higher levels of pro-inflammatory cytokines released from tissue damage during
chemotherapy can promote pain, depression, and fatigue. The chemotherapy causes a
decline in estrogen. Estrogen has a role in increasing the brain's sensitivity to
serotonin. The decrease in estrogen may also cause depression. Anxiety co-exists with
depression in BC and may be increased by chemotherapy.
Brachial artery flow-mediated dilatation (baFMD) can evaluate endothelial function,
which can be assessed by noninvasive ultrasonography. Several chronic conditions can
affect endothelial function, like hypertension, smoking, hypercholesterolemia, diabetes,
and obesity. Mental stress, sleeplessness, and hormonal changes can acutely affect
endothelial function. Trastuzumab-mediated endothelial dysfunction was observed in
a study conducted by Aamer et al among BC patients.[12 ] There is only scanty information available about the stress levels and endothelial
dysfunction among BC patients who receive NACT.
In the present study, we compared the serum onco-vascular risk factors, stress levels,
and vascular endothelial function before and after NACT among a cohort of locally
advanced primary BC patients to assess the effect of chemotherapy.
Materials and Methods
Study Design
This prospective analytical study was performed in the Department of Biochemistry
in collaboration with the Departments of Medical Oncology and Radio diagnostics of
our Institute. The participants of the study were recruited from the outpatient Department
of Medical Oncology.
Sample Size
The sample size was calculated based on the most important clinical outcome parameter,
baFMD. Lee et al reported a decrease in baFMD after chemotherapy.[13 ] They reported a decrease in baFMD from11.21 ± 4.45 before chemotherapy to 6.11 ± 2.13
after chemotherapy. The sample size was calculated using www.OpenEpi.com . Based on the above observation, the initial estimate was 17 in the group of study
with a 99% CI and a power of 95%. With an expected dropout of 25%, the sample size
was corrected to 25. Julia et al reported an increase in generalized anxiety disorder
and depression in a group of 32 BC patients receiving chemotherapy.[14 ] Hence, to adjust for multiple comparisons before and after chemotherapy, we chose
a larger sample size of 40. This sample size was found to be sufficient for the other
parameters of the study as well.
Inclusion and Exclusion Criteria
Newly diagnosed postmenopausal locally advanced BC (LABC) patients between the age
group of 50 to 65 years and having haemoglobin levels >10 g/dL and who were planned
for NACT, were recruited from the Department of Medical Oncology of our Institute.
Patients having a BMI > 28 kg/m2 and BMI < 18 kg/m2 , and patients having liver and kidney disease and metastasis were excluded. The age
group was restricted to postmenopausal women of 50 to 65 years. Although this reduces
the generalizability of the study, it was expected to avoid the influence of female
reproductive hormones on the study outcome.
Methods
Patients were explained briefly about the study protocol, and written informed consent
was obtained. The standard NACT delivered at our center consisted of three cycles
of FEC (five fluorouracil, epirubicin, cyclophosphamide) and four cycles of docetaxel
75 mg per m2 dose (all cycles were delivered in 3 weekly intervals).[15 ] Patients who were HER2 positive received neoadjuvant trastuzumab (8 mg per kg in
cycle 1 and 6 mg per kg in subsequent cycles) along with the docetaxel cycles (once
in 3 weeks). Our objective was to assess the cumulative effect of NACT on cardiac
function and not its immediate acute effects.
Assessment of Stress Levels
The stress levels were assessed by standardized questionnaires such as PSS (perceived
stress scale),[16 ] PHQ-9 (Patient Health Questionnaire-9),[17 ] and GAD-7 (generalized anxiety disorder-7).[18 ]
Assessment of Blood Levels of Oncovascular Factors
Serum biomarkers: 5 mL of fasting blood samples were collected from study participants
before the first cycle of NACT. The post samples were collected approximately 2 to
3 days after the last chemotherapy to avoid acute drug effect bias. However, the samples
were collected within 1 week. The blood samples were taken before and after the seven
cycles of NACT (approximately 5–6 months). The blood samples were taken on days when
NACT was not administered. The first blood sample was taken on the day before the
start of the cycles of chemotherapy, and the second sample was taken on days after
the last chemotherapy when the patient came for follow-up. Serum samples were stored
at −20°C within 30 minutes of blood collection. Serum adiponectin, sICAM, and sVCAM
were estimated using commercial ELISA kits from Ray Biotech, USA. Serum leptin level
was estimated using the DBC ELISA kit.
Endothelial function: brachial artery FMD (baFMD) was performed in the department
of radiodiagnosis at baseline and after seven cycles of chemotherapy. The baFMD technique
assessed endothelial function using Color Doppler (Esaote MyLab9 eXP, Italy). Before
the baFMD assessment, participants rested for a minimum of 15 minutes; then, basal
measurement was performed in the distal third of the upper arm using a USG probe with
a frequency range of 3 to 7 MHz. After the basal brachial artery diameter (BBAD) measurement,
a BP cuff was positioned on the arm 1 cm distal to the antecubital fossa to stimulate
forearm ischemia. The BP cuff pressure was raised >50 mm Hg compared with the systolic
blood pressure and kept for 5 minutes, and then the BP cuff was deflated. After deflation,
the brachial artery diameter was measured at 30-second intervals for 2 minutes. Among
the four diameter values (30 seconds, 1 minute, 1 minute 30 seconds, and 2 minutes),
peak values were taken as postocclusion brachial artery diameter (POBAD). baFMD was
calculated using this formula, baFMD (%) = (POBAD − BBAD)/BBAD ×100.[12 ]
[13 ]
[19 ]
Statistical Analysis
The Kolmogorov–Smirnov test was used to check the normal distribution of the data.
A paired t -test was used for normally distributed data, and a Wilcoxon signed-rank test was
used for nonnormally distributed data. The McNemar test was used for nominal data
analysis. p -Value < 0.05 is considered statistically significant. To control for Type I error
due to multiple comparisons, we applied the Bonferroni correction. The adjusted significance
threshold was calculated by dividing the standard α level (0.05) by the number of
comparisons (n ). The effect size was calculated using Cohen's d formula.[20 ]
Ethical Approval
All procedures performed in studies involving human participants were in accordance
with the ethical standards of the institutional and/or national research committee
and with the 1964 Helsinki Declaration and its later amendments or comparable ethical
standards. The study was approved by the Institute Ethics Committee (JIP/IEC/202/129,
dated June 23, 2021). Written consent was obtained from the patients in the study.
Results
Baseline Characteristics
Between August 2021 and May 2022, 40 patients with BC were recruited. The mean age
was 55 years ([Table 1 ]). The stage was T3 in 33% and T4 in 33% of patients, and 68% had node-positive disease.
The proportion of patients with hormone-positive and triple-negative disease was 80
and 28%, respectively.
Table 1
Baseline and treatment characteristics of the patients
Parameters
n (%)
Age in years (mean ± SD)
55 ± 5
Menopausal status
Premenopausal
0 (0)
Postmenopausal
40 (100)
T-stage:
T2
11 (28)
T3
13 (33)
T4
13 (33)
N-stage
N1
16 (40)
N2
11 (28)
ER positive
18 (45)
PR positive
14 (35)
HER2 positive
10 (25)
Triple-negative
11 (28)
Abbreviations: ER, estrogen receptor; HER2, human epidermal growth factor receptor-2;
PR, progesterone receptor.
Effect of NACT on BMI, Lipid Profile, and Fasting Blood Glucose Levels
Among BC patients statistically significant increase in blood pressure, BMI, total
cholesterol, triglycerides (TGs), LDL-cholesterol, and non-HDL-cholesterol was observed
after the NACT. There was a trend toward an increase in the fasting blood glucose
levels, which did not reach statistical significance. ([Table 2 ]). However, the overall change in blood pressure range did not reach the clinically
significant range of hypertension. The overall change in fasting blood glucose also
did not reach the diagnostic range of diabetes mellitus.
Table 2
Comparison of blood pressure, BMI, lipid profile, and fasting blood sugar (FBS) level
among breast cancer patients before and after NACT
Parameters
Before chemotherapy; n = 40
After chemotherapy; n = 40
p -Value
Systolic blood pressure (mm Hg)
122.1 ± 8.4
128 ± 10.2
<0.01[a ]
Diastolic blood pressure (mmHg)
80.8 ± 3.1
82.4 ± 3.9
0.018[a ]
BMI (kg/m2 )
24.8 (21–26.7)
24 (20.4–26.1)
<0.01[a ]
FBS (mg/dL)
106 (94–140)
120 (102–163)
0.08
Total cholesterol (mg/dL)
203 ± 46
216 ± 54
0.022[a ]
LDL-cholesterol (mg/dL)
133 ± 33
143 ± 36
0.035[a ]
TGs (mg/dL)
172 (117–216)
197 (143–261)
0.005[a ]
HDL-cholesterol (mg/dL)
36 (30–47)
34 (30–44)
0.467
Non-HDL cholesterol (mg/dL)
164 ± 45
179 ± 50
0.008[a ]
VLDL-cholesterol (mg/dL)
34 (23–43)
38 (27–52)
0.021[a ]
a
p -Value < 0.05 is considered statistically significant.
Effect of NACT on Serum Leptin, Adiponectin, sICAM, sVCAM, and baFMD Levels
There was a significant (p < 0.01) decrease in the leptin levels and baFMD after chemotherapy among the BC patients.
There was a trend toward an increase in the adiponectin/leptin ratio and sICAM levels
after chemotherapy among the patients ([Table 3 ]; [Fig. 1A, B ])
Fig. 1 Effect of NACT on serum leptin levels and baFMD (%). (A ) On serum leptin levels and (B ) on baFMD (%). *
p -Value < 0.05 is considered statistically significant.
Table 3
Comparison of serum leptin, adiponectin, adiponectin/leptin ratio, sVCAM-1, sICAM-1
levels, and baFMD among breast cancer patients before and after chemotherapy
Parameters
Before chemotherapy; n = 40
After chemotherapy; n = 40
p -Value
Leptin (ng/mL)
30.1 (24.3–40.9)
20.4 (13.1–30.6)
<0.01[a ]
Adiponectin (μg/mL)
58.4 (43.2–80.0)
54.6 (40.0–85.5)
0.528
Adiponectin/Leptin
1.69 (0.95–3.6)
2.73 (1.72–5.76)
0.067
sICAM-1 (ng/mL)
283.1 (231.9–357.0)
323.5 (261.4–395.8)
0.063
sVCAM-1 (ng/mL)
391.6 (314.8–516.4)
395.5 (316.1–541.7)
0.361
baFMD (%)
10.8 (8.3–14.2)
6.5 (2.9–11.4)
<0.01[a ]
Abbreviations: baFMD, brachial artery flow-mediated dilation; sICAM-1, soluble intercellular
adhesion molecule-1; sVCAM-1, soluble vascular cell adhesion molecule 1.
a
p -Value < 0.05 is considered statistically significant.
Effect of NACT on Perceived Stress, Depression, and Anxiety among BC Patients
There was a significant increase in perceived stress, depression, and anxiety among
BC patients after chemotherapy ([Table 4 ]; [Fig. 2A–C ]).
Fig. 2 Effect of NACT on stress parameters. (A ) Effect of NACT on stress level (PSS score), (B ) effect of NACT on depression (PHQ-9 score), and (C ) effect of NACT on anxiety (GAD-7 score). *
p -Value < 0.05 is considered statistically significant.
Table 4
Comparison of PSS, PHQ-9, and GAD-7 scores among breast cancer patients before and
after chemotherapy
Questionnaire
Reference range
Before chemotherapy; n = 40
After chemotherapy; n = 40
p -Value
PSS (score)
0–13: low stress 14–26: moderate stress
27–40: high stress
7 (5–10)
9 (7–14)
0.007[a ]
PHQ-9
0–4: minimal depression
5–9: mild depression
10–14: moderate depression 15–19: moderately severe depression
20–27: severe depression
4 (2–5)
8 (4–11)
<0.01[a ]
GAD-7
0–4: minimal anxiety
5–9: mild anxiety 10–14: moderate anxiety
15–21: severe anxiety
2 (2–4)
6 (4–9)
<0.01[a ]
Abbreviations: GAD-7, generalized anxiety disorder-7; PHQ, Patient Health Questionnaire-9;
PSS, perceived stress scale.
a
p -Value < 0.05 is considered statistically significant.
There was a significant decrease in serum leptin level after NACT with a medium effect
size; additionally, there was a significant increase in anxiety and depression after
NACT with a larger effect size ([Table 5 ]).
Table 5
Effect size of significant parameters with 95% confidence interval
Parameters
Before chemotherapy; n = 40; 95% confidence interval
After chemotherapy; n = 40; 95% confidence interval
Effective size (Cohen's d )
Leptin (ng/mL)
28.3–46.4
18.2–29.8
0.5
baFMD (%)
9.8–14
6.2–9.3
0.3
PSS (score)
6.8–9.8
9–12.1
0.4
PHQ-9 (score)
3.3–5.2
6.9–10.3
1.0
GAD-7 (score)
2.2–3.6
5.7–8.5
1.16
Abbreviations: GAD-7, generalized anxiety disorder-7; PHQ, Patient Health Questionnaire-9;
PSS, perceived stress scale.
Further, there was a significant increment in the frequency of depression and anxiety
among the BC patients after chemotherapy ([Table 6 ]).
Table 6
Effect of NACT on frequency of depression, anxiety, and stress among breast cancer
patients
Effect of NACT on the frequency of depression among breast cancer patients
Group
Minimal depression; PHQ-9 Score (0–4)
Mild and above depression; PHQ-9 score ≥ 5
p -Value
Before chemotherapy; n = 40
24
16
0.002[a ]
After chemotherapy; n = 40
10
30
Effect of NACT on the frequency of anxiety among breast cancer patients
Minimal anxiety; GAD-7 score (0–4)
Mild and above anxiety;GAD-7 score ≥ 5
p -Value
Before chemotherapy; n = 40
33
7
<0.01[a ]
After chemotherapy; n = 40
12
28
Effect of NACT on the frequency of stress among breast cancer patients
Minimal stress PSS; score 0–13
Mild and above stress; PSS score >13
p -Value
Before chemotherapy; n = 40
34
6
0.109
After chemotherapy; n = 40
28
12
a
p -Value < 0.05 is considered statistically significant.
Thirty-five percent of the patients shifted to mild and above depression level, and
53% patients shifted to mild and above anxiety level after NACT.
Correlation Analysis Between Various Parameters Before NACT
There was a significant positive correlation between baFMD, GAD-7, PHQ-9, and CAMs
like sICAM-1, sVCAM-1 ([Table 7 ]).
Table 7
Correlation analysis between various parameters before NACT
Parameter
Parameter
r -Value
p -Value
baFMD (%)
GAD-7
0.33
0.038[a ]
PHQ-9
GAD-7
0.56
<0.01[a ]
sICAM-1
sVCAM-1
0.38
0.015[a ]
a
p -Value < 0.05 is considered statistically significant.
Correlation Analysis Between Various Parameters After NACT
There was a significant positive correlation between PSS, PHQ-9, and GAD-7 scores
after NACT ([Table 8 ]).
Table 8
Correlation analysis between various parameters after NACT
Parameter
Parameter
r -Value
p -Value
PSS
PHQ-9
0.63
<0.01[a ]
PSS
GAD-7
0.67
<0.01[a ]
PHQ-9
GAD-7
0.85
<0.01[a ]
a
p -Value < 0.05 is considered statistically significant.
Discussion
The potential risk for cardiovascular disorders, endothelial dysfunction, and psychological
stress is expected to be higher among the BC patients who received NACT. This could
increase the morbidity and mortality of BC patients. This study demonstrates the significant
impact of NACT on serum biomarkers and clinical stress assessment in patients with
LABC. Along with changes in stress levels and elevation of markers associated with
cardiovascular disorders, altered endothelial function was also observed. This is
one of the first studies examining several factors contributing to cardiovascular
diseases.
This study observed a significant decrease in serum leptin levels after NACT among
the BC patients. There are no previous reports of serum leptin levels in LABC patients
undergoing NACT. Tas et al reported decreased serum leptin levels in nonsmall cell
lung cancer after chemotherapy.[21 ] From the available information in the literature, it is impossible to explain the
decrease in leptin after chemotherapy. The leptin production is under the strict control
of pro-inflammatory cytokine action, especially TNF-α and IL-6, through upregulation
of the “ob” gene expression. This may be taken into consideration when providing an
explanation.[14 ] Leptin is considered a cardiovascular risk factor.[22 ] In cross-sectional studies, increased leptin levels have been reported among BC
patients compared with healthy controls.[23 ] Since leptin is elevated in both cardiovascular diseases and cancer, it may be considered
an oncovascular risk factor. The reduced leptin levels observed in the study after
NACT may be reducing CV risk.
Although changes in the adiponectin levels were not found, trends of an increase in
the adiponectin/leptin ratio after chemotherapy among the BC patients were observed
(p = 0.067). According to Gema et al, an Adiponectin/Leptin ratio is considered normal
when equal to or higher than 1.0, where adiponectin and leptin levels are expressed
in µg/mL and ng/mL, respectively. Moderate-medium increased risk is predicted for
a ratio between 0.5 and 1.0, and a severe increase in cardio-metabolic risk is suggested
for a ratio below 0.5.[24 ] Among our patients, the ratio was already more than 1.0 (1.69) before chemotherapy,
which increased to 2.73 after the chemotherapy. This observation may be explained
as part of the ongoing defense mechanism against the toxicity of NACT. It may also
be explained by the effect of chemotherapy in reducing the cancer burden. NACT could
have reduced the baseline inflammatory state.
Several studies reported higher levels of CAMs among the BC patients compared with
healthy controls.[2 ]
[25 ] CAMs promote tumor vascularisation, serving as pro-angiogenic factors. The migration
of cancer cells and tumor growth are also promoted by sICAM-1 and sVCAM-1 CAMs.[2 ] No reports are available on the effect of NACT on the levels of CAMs among BC patients.
We found trends of an increase in serum sICAM level (p = 0.063) among our BC patients after NACT, with no significant changes in the sVCAM
levels. However, Maria et al reported an increase in the levels of serum CAMs among
BC patients after cancer treatments in the form of surgery and radiotherapy.[26 ] This may be explained by the enhanced release of the CAMs from the endothelial surface
due to cancer therapy.
Elevated serum concentrations of CAMs, sVCAM-1, and sICAM-1 were found in patients
with coronary artery disease.[10 ] sVCAM-1 is a biomarker that predicts the occurrence, development, and preservation
of cardiovascular disease.[26 ] Since these molecules are found to be elevated in both cardiovascular diseases and
cancer, they may be addressed as oncovascular risk factors, a term yet to be coined
in the literature. The pathological basis for the increase in these risk factors in
cancer and coronary artery diseases is far from clear and needs to be further investigated.
Compared with a baFMD value of 40 in healthy Indians,[3 ] there was a significant reduction in the levels of baFMD among our BC patients (10.8).
This further decreased to 6.5 after the chemotherapy, indicating further deterioration
of endothelial function. The only previous study conducted by Lee et al in this regard
reports increased endothelial dysfunction in BC patients who received anthracycline-based
chemotherapy.[19 ] Longer-term follow-up studies have indicated that patients who receive chemotherapy
may have adverse cardiovascular outcomes later in life. Impaired baFMD may be an early
indicator of this problem.
Perceived stress levels, depression, and anxiety may increase among BC patients during
chemotherapy due to the release of pro-inflammatory cytokines from tissue damage.
Depression is also caused by the decrease in estrogen during chemotherapy. In this
study, a significant increase in psychological stress, anxiety, and depression was
observed among the BC patients after NACT. A significant portion of patients progressed
from minimal to mild and above depression and from minimal anxiety to mild and above
anxiety. The only other study in this regard by Reece et al reported high stress and
anxiety levels in BC patients after chemotherapy.[21 ] The inflammation can also promote cardiovascular risk and endothelial dysfunction.
Therefore, all three areas of our study, cardiovascular risk, endothelial dysfunction,
as well as perceived stress, depression, and anxiety, may be interconnected. However,
no correlations were observed between oncovascular risk factors and stress parameters,
probably due to the effect of chemotherapy. However, baFMD (%) correlated with GAD-7.
None of the study's patients had any disease progression during chemotherapy. Therefore,
the biochemical, radiological, and psychological changes observed could be attributed
to the effects of chemotherapy.
Strengths and Limitations
Strengths and Limitations
This was a comprehensive study that assessed the effect of NACT on psychological distress,
endothelial function, and onco-vascular risk factors among patients with BC. One of
the limitations of the study is the smaller sample size.
Future and Generalizability of Research
Future and Generalizability of Research
The same study may be performed in multiple centers to confirm the result and the
generalizability of the findings.
Conclusion
NACT of patients with locally advanced primary BC was associated with increased endothelial
dysfunction, perceived stress, depression, and anxiety, as well as resulting in changes
in onco-vascular risk factors and dyslipidaemia. Future studies may include follow-up
of these patients to understand the recovery or persistence of these comorbidities
after chemotherapy. This study was of an exploratory nature, and there is a need for
larger, multicenter, long-term follow-up.
