Normalized Serum EGF Levels as a Potential Biomarker in Non-Small Cell Lung Cancer: The Role of Platelets

Abstract

Background: Literature reports contradictory findings regarding the capacity of serum EGF concentrations ([EGF]) to differentiate non-small cell lung cancer (NSCLC) patients from healthy individuals. Therefore, the diagnostic capacity of [EGF], suggestive of dependency on this growth factor in NSCLC patients (tumors) and hence indicative of possible response to therapies directed to EGF/EGFR, is still an open question. Inconsistencies likely derive from the lack of harmonization and standardization in methodologies for blood and sera processing.Methods: A cohort of NSCLC patients was evaluated at diagnosis (25) and after first-line-therapy (18/25). Sera were collected 1h and 4h after phlebotomy, controlling the variables influencing [EGF]. EGF was quantified by ELISA. Platelets count was also estimated. The values obtained for several combined and/or normalized by platelets count, variables, were compared to those in selected cohorts of healthy controls.Results: We found differences between healthy individuals and NSCLC patients in the accessibility of EGF to circulation, but not in the total available EGF stock. Indeed, we observed a higher fraction of free EGF in the circulation of patients and consequently a lower amount of EGF stored in platelets. Interestingly, an aberrant relationship between EGF and platelets counts was also observed, especially in patients with thrombocytosis. Moreover, several EGF-related variables, with enough accuracy for discrimination, were identified. Particularly, those variables normalized by platelets count made more evident the differences between patients and healthy controls. Therefore, they might be potential biomarkers in NSCLC.Conclusion: Our results suggest the increase in free/accessible EGF in blood circulation as relevant to the biology of NSCLC, most likely because it reflects a higher accessibility of this growth factor for the tumor. They also suggest some of the study variables to be further evaluated on its predictive value, to select good responders to CIMAvax-EGF® or other therapies targeting the EGF/EGFR system.

Full text

Normalized Serum EGF Levels as a Potential Biomarker in Non-Small Cell
Lung Cancer: The Role of Platelets
González-Pérez Idania1*, Cáceres Lavernia Haslen Hassiul2, Rodríguez Pedro Camilo3, Carr Pérez Adriana1 and León Monzón Kalet1
1Department of Systems Biology, Center of Molecular Immunology, 216 Street and 15th Avenue, Atabey, Siboney, Playa, Havana City, Cuba
2Department of Oncology, Hermanos Ameijeiras Hospital, San Lázaro Street, Havana City, Cuba
3Department of Clinical Trials, Center of Molecular Immunology, 216 Street and 15th Avenue, Atabey, Siboney, Playa, Havana City, Cuba
*Corresponding author: González-Pérez Idania, Department of Systems Biology, Center of Molecular Immunology, 216 Street and 15th Avenue, Atabey, Siboney,
Playa, P.O. Box 16040, Zip Code-11600, Havana City, Cuba, Tel: 2022964810; E-mail: idania.gp@gmail.com
Rec Date: September 05, 2018 Acc Date: October 12, 2018, Pub Date: October 19, 2018
Citation: Idania GP, Hassiul CLH, Camilo RP, Adriana CP, Kalet LM (2018) Normalized Serum EGF Levels as a Potential Biomarker in Non-Small Cell Lung Cancer: The
Role of Platelets. J Mol Biomark Diagn 9: 402.
Abstract
Background: Literature reports contradictory findings regarding the capacity of serum EGF concentrations
([EGF]) to differentiate non-small cell lung cancer (NSCLC) patients from healthy individuals. Therefore, the
diagnostic capacity of [EGF], suggestive of dependency on this growth factor in NSCLC patients (tumors) and hence
indicative of possible response to therapies directed to EGF/EGFR, is still an open question. Inconsistencies likely
derive from the lack of harmonization and standardization in methodologies for blood and sera processing.
Methods: A cohort of NSCLC patients was evaluated at diagnosis (25) and after first-line-therapy (18/25). Sera
were collected 1 h and 4 h after phlebotomy, controlling the variables influencing [EGF]. EGF was quantified by
ELISA. Platelets count was also estimated. The values obtained for several combined and/or normalized by platelets
count, variables, were compared to those in selected cohorts of healthy controls.
Results: We found differences between healthy individuals and NSCLC patients in the accessibility of EGF to
circulation, but not in the total available EGF stock. Indeed, we observed a higher fraction of free EGF in the
circulation of patients and consequently a lower amount of EGF stored in platelets. Interestingly, an aberrant
relationship between EGF and platelets counts was also observed, especially in patients with thrombocytosis.
Moreover, several EGF-related variables, with enough accuracy for discrimination, were identified. Particularly, those
variables normalized by platelets count made more evident the differences between patients and healthy controls.
Therefore, they might be potential biomarkers in NSCLC.
Conclusion: Our results suggest the increase in free/accessible EGF in blood circulation as relevant to the
biology of NSCLC, most likely because it reflects a higher accessibility of this growth factor for the tumor. They also
suggest some of the study variables to be further evaluated on its predictive value, to select good responders to
CIMAvax-EGF® or other therapies targeting the EGF/EGFR system.
Keywords: Non-small cell lung cancer; Epidermal growth factor;
Epidermal growth factor receptor; Platelets; Stratication; Diagnostic
biomarker; Predictive biomarker
Abbreviations: NSCLC: Non-Small Cell Lung Cancer; EGF:
Epidermal Growth Factor; EGFR: Epidermal Growth Factor Receptor;
SNP: Single Nucleotide Polymorphism
Introduction
Lung cancer (LC) is the leading cause of cancer deaths worldwide
[1]. Unlike most cancers, which have witnessed steady increases in
survival rates, advances have been slow in LC, for which the 5-year
survival rate is about 18% [2]. In Cuba, malignant tumors of trachea,
bronchia and lung are a health concern as well, with about 4000 newly
cases every year and 5720 deaths in 2017 [3]. However, for non-small
cell lung cancer (NSCLC), the most frequent LC type, new
immunotherapies have shown a great potential for patients in
advanced stages [4]. Moreover, personalized medicine is providing
hope by treating patients with drugs that are eective based on specic
characteristics of their tumors [5]. e Epidermal Growth Factor
(EGF), known to stimulate the growth of several types of epithelial
tissue, possesses strong mitogenic activity on tumor cells that converts
this ligand in an attractive target for designing antitumor strategies [6].
CIMAvax-EGF® [7] is a proven eective Cuban therapeutic vaccine
for advanced NSCLC, which induces anti-EGF antibodies that
recognize the EGF in circulation, preventing its binding to EGFR, and
then disrupting the associated signal transduction cascade in cancer
patients and ultimately cell proliferation. Studies of serum EGF
concentrations ([EGF]) in Cuban patients treated with this vaccine
revealed that high [EGF] are a factor of bad prognosis for NSCLC and
at the same time a predictive biomarker of CIMAvax-EGF® ecacy
[8-10]. However, the capacity of those concentrations to discriminate
between NSCLC patients and healthy individuals (its diagnostic value),
suggestive of dependency on EGF in NSCLC patients (tumors) and
hence indicative of possible response to therapies directed to this
growth factor or its receptor, is still an open question. ere are only a
few reports available on this topic, some of which have published
discrepant ndings [11,12]. is inconsistency of results has been
presumably caused by the lack of harmonization and sometimes of
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ISSN: 2155-9929
Journal of Molecular Biomarkers and
Diagnosis
Idania et al., J Mol Biomark Diagn 2018, 9:5
DOI: 10.4172/2155-9929.1000402
Research Open Access
J Mol Biomark Diagn, an open access journal
ISSN: 2155-9929
Volume 9 • Issue 5 • 1000402

standardization in the methodologies used for blood processing and
sera collection. Essentially, the majority of reports do not consider the
known dependency between [EGF] and the time of sera separation
[13,14]. Neither the type of tubes employed for blood collection, which
aects clotting times and thus the release of EGF by platelets [15-17],
not even the temperature at which blood coagulates, which also
impacts this process. Dierences in the selection of controls and
patients (the lack of control of confounding factors) have also
contributed to discrepant results [12].
In this work, [EGF] and platelets counts were quantied in 25
NSCLC patients, using previously standardized methodologies for
blood processing and sera separation [14]. Platelet´s contribution to
[EGF] was studied in patients, showing a clear dierence to what is
reported for healthy individuals. Several combined and platelets-
normalized EGF-related variables were proposed and evaluated on its
capacity to discriminate between patients and healthy individuals. We
identied variables with a clear diagnostic value and a simple
biological interpretation. Moreover, we used them in an attempt to
infer the EGF-dependency in tumors of individual patients. We suggest
to further evaluate some of these variables, on its predictive value to
select good responders to treatment with CIMAvax-EGF® vaccine or
other therapies targeting the EGF/EGFR system.
Materials and Methods
Study design
e [EGF] were assessed in NSCLC patients at the time of diagnosis
(To) and 4-6 weeks aer the rst-line-therapy (T1).
Phlebotomy and sera separation
ese procedures were performed according to González-Pérez et
al. [14]. Each phlebotomy provided two sera, separated at 1 h and 4 h
aer venipuncture, and therefore two [EGF]: [EGF]1h and [EGF]4h,
respectively.
Patient and control cohorts: Ethical aspects
Naive patients, with a cytohistological conrmation of NSCLC, a
performance status ECOG<3, and a measurable disease according to
RECIST version 1.1 [18], were recruited from October 2014-May 2016,
at Hermanos Ameijeiras Hospital (HAH) in Havana. ose with brain
metastasis and chronic diseases other than hypertension and heart
disease were not enrolled in the study. Staging was determined
according to the 7th edition of the TNM system [19]. A group of 25
patients was evaluated at diagnosis (NSCLC1) and a subgroup of 18
was additionally evaluated aer treatment (NSCLC2) (Table 1 and
Supplementary Table S1). erapy consisted on 3-6 cycles of Cisplatin
or Carboplatin, administered with Etoposide, Vinblastine or Paclitaxel,
every 21 days, in combination or not with radiotherapy (23-60 Gy in
20 fractions over 4 weeks, Table 1). Two healthy control cohorts,
balanced by age and gender to match the NSCLC groups (HC1 and
HC2 in Supplementary Table S1 and S2), were randomly picked out
from a panel of healthy Cuban donors [14]. e members of the third
control group HC3 were selected from the same panel, based on the
availability of their platelets count.
e study was approved by the Ethical Committee of HAH. e sera
and patient´s data were collected in compliance with Helsinki
Declaration.
Primary variables
Platelets count and serum EGF concentrations were considered
primary variables. Based on a previous study [14], the EGF measured
at the rst hour of coagulation [EGF]1h, is known to be the EGF more
accessible to circulation. erefore, in this study the [EGF]1h is
interpreted as a good estimate of the actual concentration of free EGF
in blood circulation (Supplementary Figure S1). Conversely, the EGF
measured at 4 h [EGF]4h is very close to the concentration plateau
achieved by the progressive EGF release by platelets during aggregation
(Supplementary Figure S1). Consequently, the [EGF]4h is interpreted as
the average total stock of EGF in the blood sample of an individual.
Measurement of primary variables
[EGF]: e [EGF] were assessed with the UMELISA EGF® kit [20],
whose standard is calibrated against the WHO IS EGF 91/530
(NIBSC). Additionally, in a previous study we veried a very good
correlation between the UMELISA kit and the widely used Human
EGF Immunoassay Quantikine® ELISA (R&D Systems, Minneapolis,
MN, USA) (Supplementary Figure S2).
Platelets count
Blood collected into EDTA-containing test-tubes vacutainer (Becton
Dickinson, UK) was assayed in hematological analyzer Mindray
BC-3200 (impedance method). We used the reference interval
150-400 × 109 platelets/L, employed at HAH and commonly accepted
in clinical laboratories [21], with thrombocytosis dened as a counting
above 400 × 109 platelets/L. Note that, although data reported seem to
indicate an associated to aging decrease in platelets count and also
statistically signicant dierences between counting in women and
men [22-24], these dierences have no practical importance [25] and
do not account for separate norms for women and men [26].
Moreover, these dierences usually oscillate in the range of variability
of platelet’s count estimation (≤ 6% for Mindray BC-3200, according to
Peng et al. [27]).
Combined variables
e calculated ratio r=[EGF]1h/[EGF]4h is interpreted as the EGF
fraction from the total stock which is available in circulation. en, the
dierence d=[EGF]4h-[EGF]1h is interpreted as a measure proportional
to the EGF stored in platelets (not available to circulation).
Normalized variables
We dened several EGF-related variables normalized by platelets
count, expressed in grams of EGF per platelet. e [EGF]1h/platelets/L
is interpreted as the average EGF contributed to circulation per
platelet. e [EGF]4h/platelets/L is interpreted as the average total
stock of EGF per platelet. Finally, d/platelets/L is interpreted as the
average EGF stored per platelet (not in circulation).
Discriminatory capacity of dierent variables
e capacity of variables to discriminate between patients
(NSCLC1, n=25) and healthy controls (HC3, n=15) was assessed by the
nonparametric method for univariate analysis ROC (Receiver
Operating Characteristic) [28,29]. e discrimination accuracy
achieved by each studied variable was evaluated by the area under
curve (AUC) and the associated p value. For variables with higher/
lower values in patients than in controls, the AUC represents the
Citation: Idania GP, Hassiul CLH, Camilo RP, Adriana CP, Kalet LM (2018) Normalized Serum EGF Levels as a Potential Biomarker in Non-
Small Cell Lung Cancer: The Role of Platelets. J Mol Biomark Diagn 9: 402. doi:10.4172/2155-9929.1000402
Page 2 of 11
J Mol Biomark Diagn, an open access journal
ISSN: 2155-9929
Volume 9 • Issue 5 • 1000402

probability that a randomly evaluated patient will have a lower/higher
value than a randomly evaluated control. e p value tests the null
hypothesis that the AUC is truly equal to 0.50 (indicating the test is not
better at diagnosing than chance). AUC values >0.70 with p<0.05
identify the variables with signicant discriminatory capacity. A
signicant p-value in pairwise comparisons of AUCs means the
variable with the highest AUC diagnoses signicantly better than the
others.
Inference of EGF-dependency in NSCLC
e EGF-dependency of the tumors in dierent patients was
inferred from their stratication by those variables that were found
discriminatory in ROC analysis. An optimal cut-o value C(>/<)
(decision threshold) was determined for each variable, by maximizing
simultaneously sensitivity- Se (%) and specicity-Sp (%) [30]. It was
assumed that patients correctly identied by the ROC test (above C>/
below C<) are those with values more anomalous for the evaluated
variable, being therefore correctly discriminated with respect to
healthy individuals. Consequently, it was inferred that the variables
able to better achieve the discrimination might be those chosen to
evaluate the stratication of patients, regarding their potential
response to treatment with therapies targeting the EGF/EGFR system.
Statistics
Unpaired Student’s t-test was used to compare means (α=0.05).
Confounding factors were controlled by design (matching) and by
analysis (stratication). Statistical correlations and some associations
were assessed with the Pearson´s correlation coecient-R and by
ANOVA or contingency analysis. e statistical packages Statgraphics
Centurion XVI, version 16.1.11 and GraphPad Prism 5 were used.
Results
Serum EGF levels and patient´s clinicopathological
characteristics
Table 1 shows the clinicopathological characteristics of the 25
NSCLC patients included in our study, the majority of which classied
into metastatic state (60% stage IV) or stage III (36%). ese high
percentages were expected, because NSCLC is mostly diagnosed at
advanced stages. e majority of patients (2/3) was 60 years or older
(mean age 62.52 years), which was expected because LC mainly occurs
in older people. e predominant skin color was white (76%), in
correspondence with its higher presentation inside our population
(>64% according to the latest Cuban census about population and
housing [31]). e proportion of white skin with other (3:1), the ratio
men:women, and the predominant ECOG, were very similar to those
found in a previous Cuban study [10].
Reports about variation of [EGF] in dierent subsets of NSCLC
patients are scarce and controversial. erefore, despite our small
sample size, we report in Table 1 the average [EGF] measured at 1 h
and 4 h, in naive patients with dierent clinicopathological
characteristics. Note that, as it is explained in section Materials and
Methods, [EGF]1h most likely estimates the concentration of free EGF
in circulation, and [EGF]4h corresponds to the total stock of EGF.
Despite the overall small sample size, with the ratios of sampling
obtained and α=0.05, statistical powers > 0.99 were reached in the
signicant dierences found between [EGF] in the groups compared.
Interestingly, there were no dierences between most patient´s strata
regarding [EGF]. As Lemos-González et al. [11], we did not nd
association between [EGF] and the stages III and IV of the disease,
patient´s gender or age, although a tendency to decrease was
appreciated for [EGF]4h in older patients (61-78 years), as it was
previously described for healthy individuals [14,32,33]. However,
signicant dierences were found between T3 and T4 status analyzing
[EGF]4h (p=0.0103), but not when [EGF]1h were compared (p=0.2740).
e evaluation of nodal involvement revealed as well, a tendency of
[EGF] to decrease at N3 level, as compared to N0 and N2, but without
statistical signicance.
Serum EGF levels before/aer rst-line therapy
Figure 1A illustrates that in NSCLC patients [EGF] at 1 h and 4 h
were highly variable, at diagnosis (To), as well as aer rst-line therapy
(T1). EGF concentrations at 4h were higher than those at 1h, on
average (Figure 1A) and also in the majority of patients, with a few
exceptions that showed [EGF]4h<[EGF]1h (Figures 1B and 1C).
Overall, in qualitative terms, EGF concentrations in NSCLC patients
were highly variable, just as reported earlier in healthy individuals
[14,34,35]. Moreover, they usually increased with the time of sera
separation, just as described for normal subjects [13,14].
erapy reduced the average [EGF] in patients, but signicantly
only circulating levels ([EGF]1h, Figure 1A). at decrease was clearly
patient-dependent and not described by a simple relation of
proportionality. erefore, while we found a signicant, although non-
strong, correlation between [EGF] at 4 h and 1 h at diagnosis To
(NSCLC1, n=24: R=0.5120, p=0.0105), in treated patients T1 this
correlation was clearly lost (NSCLC2, n=18: R=0.2506, p=0.3159).
Serum EGF levels in NSCLC patients and controls
Figure 2 shows the direct comparison of [EGF] between NSCLC
patients and groups of healthy individuals well-balanced by age and
gender (HC1 and HC2). ere were no dierences between these
cohorts (Figure 2A) regarding the average total stock of EGF
([EGF]4h), neither before (To) nor aer chemoradiotherapy (T1).
However, the average concentration of free EGF in serum ([EGF]1h)
was signicantly higher in naive- but not in treated-patients (Figure
2B). To further compare healthy controls and NSCLC patients, we used
the combined variables r=[EGF]1h/[EGF]4h and d=[EGF]4h-[EGF]1h,
which oer dierent, but complementary information (see Materials
and Methods). We found very signicant dierences between controls
and patients at diagnosis by the variable r (Figure 2C), which were
stronger than those found by comparing [EGF]1h (Figure 2B).
Additionally, r also caught some dierence aer treatment, although it
was not statistically signicant. e variable d however, captured the
dierence at To similarly to [EGF]1h and also at T1 (Figure 2D).
Summarizing, our results show dierences between healthy
individuals and NSCLC patients regarding the accessibility of EGF to
circulation, but not regarding the total stock of EGF. Certainly, we
observed a higher fraction of free EGF in the circulation of patients (r)
and consequently a lower amount of EGF stored in platelets (d). ese
results suggest that the increase in free/accessible EGF in blood
circulation is relevant to the biology of NSCLC, most likely because it
reects a higher accessibility to this tumoral growth factor.
Citation: Idania GP, Hassiul CLH, Camilo RP, Adriana CP, Kalet LM (2018) Normalized Serum EGF Levels as a Potential Biomarker in Non-
Small Cell Lung Cancer: The Role of Platelets. J Mol Biomark Diagn 9: 402. doi:10.4172/2155-9929.1000402
Page 3 of 11
J Mol Biomark Diagn, an open access journal
ISSN: 2155-9929
Volume 9 • Issue 5 • 1000402

Parameters NSCLC1, To
n (%)b
NSCLC2, To
n (%)b
[EGF]1ha,b
(pg/mL)
[EGF]4ha,b
(pg/mL)
Gender W 5(20) 3(17) 803 ± 228 1017 ± 172
M 20(80) 15(83) 627 ± 80 1010 ± 75
Age (years) 46-60 11(44) 9(50) 702 ± 111 1165 ± 69
61-78 14(56) 9(50) 631 ± 110 902 ± 96
Skin colour White 19(76) 14(78) 648 ± 98 1029 ± 83
Other 6(24) 4(22) 706 ± 102 957 ± 109
Primary tumor status T1 3(12) 3(17) 757 ± 83 987 ± 99
T2 5(20) 5(28) 555 ± 231 980 ± 200
T3 4(16) 2(11) 868 ± 285 1364 ± 110
T4 13(52) 8(44) 618 ± 89 918 ± 77
Nodal status N0 7(28) 5(28) 853 ± 125 1151 ± 81
N1 1(4) 0(0) -- --
N2 6(24) 6(33) 832 ± 188 1198 ± 130
N3 11(44) 7(39) 493 ± 114 853 ± 96
Distant metastasis M0 10(40) 7(39) 660 ± 164 1062 ± 138
M1 10(40) 7(39) 644 ± 112 936 ± 82
M1a 3(12) 2(11) 721 ± 86 1132 ± 73
M1b 2(8) 2(11) 676 ± 27 --
Stage I 0(0) 0(0) -- --
II 1(4) 1(6) -- --
III 9(36) 6(33) 589 ± 165 1014 ± 144
IV 15(60) 11(61) 664 ± 75 974 ± 64
ECOG 1 24(96) 18(100) -- --
2 1(4) 0(0) -- --
Platelets (× 109/L) ≤ 400 16(64) 12(67) 709 ± 93 967 ± 70
>400 9(36) 6(33) 579 ± 140 1100 ± 149
Primary tumor
Size (mm)
≤ 35 7/22(32) 6(33) 685 ± 115 1114 ± 57
>35 15/22(68) 12(67) 653 ± 100 977 ± 87
1st line-therapy CT 25(100) 10(56) -- --
CRT 8(32) 8(44) -- --
# of cycles ≤ 3 8/24(33) 3(17) -- --
4-6 16/24(67) 15(83) -- --
W: Women; M: Men; CT: Chemotherapy; CRT: Chemoradiotherapy; Other: Negroes and mulattos; SME: Standard Mean Error; aMean [EGF] ± SME; bRounded values
Table 1: Demographic and clinicopathological characteristics of NSCLC patients at diagnosis: Its relation with serum [EGF].
Citation: Idania GP, Hassiul CLH, Camilo RP, Adriana CP, Kalet LM (2018) Normalized Serum EGF Levels as a Potential Biomarker in Non-
Small Cell Lung Cancer: The Role of Platelets. J Mol Biomark Diagn 9: 402. doi:10.4172/2155-9929.1000402
Page 4 of 11
J Mol Biomark Diagn, an open access journal
ISSN: 2155-9929
Volume 9 • Issue 5 • 1000402

Figure 1: Variability of EGF concentrations in sera of NSCLC
patients before/aer chemoradiotherapy: Its dependency on the
time of sera collection. e picture illustrates: Highly variable
[EGF] with higher mean values at 4 h, mean values ± standard
mean errors are represented in gray (A); [EGF]4h>[EGF]1h in the
majority of patients, excluding some exceptions at To (3/25 (12%))
and T1 (1/18 (6%)), examples are indicated by arrows (B and C).
Figure 2: EGF and EGF-combined variables: HC
vs.
NSCLC
patients before/aer chemoradiotherapy (NSCLC1/NSCLC2). e
picture shows that: patients and controls have on average the same
stock of EGF (A); patients have the EGF more accessible to
circulation at diagnosis (B); and dierences in EGF accessibility are
more evident when combined variables are compared (C and D).
Mean values ± standard mean errors are represented in gray.
Serum EGF levels and platelets count in NSCLC patients
Figure 3A shows extremely signicant dierences between the count
of platelets in controls and patients at diagnosis, with thrombocytosis1
present in 36% (9/25) of cases, which is in agreement with other
reported incidences [36-38]. Moreover, Figure 3A also shows that
chemoradiotherapy reduced platelets count (p=0.0072) and
thrombocytosis (11% (2/18)) although not signicantly (Chi-Square
p=0.0650, odds ratio=4.5). Such decreases were expected, as
thrombocytopenia is a well-known complication of chemotherapy [39]
and can also be induced by radiation [40].
A positive linear correlation between [EGF] and platelets count has
been reported in humans [14,41], evidencing that platelets are the
main source of EGF in human blood. erefore, we also studied this
correlation in our patients (Figures 3B and 3C). Surprisingly, at
diagnosis (To) there was no signicant correlation between [EGF]1h or
[EGF]4h and platelets count (Figure 3B). Aer rst-line therapy (T1),
however, there was no correlation for [EGF]1h, but a weak correlation
was observed for [EGF]4h (Figure 3C). Altogether, these results show
that in NSCLC patients exists a dierent relationship between EGF and
platelets, as compared to that observed in healthy controls.
Interestingly, rst-line chemoradiotherapy reduces serum EGF
concentrations and platelets counts, but also tends to partially recover
the correlation between them.
To further study the relation between [EGF] and platelets counts in
NSCLC patients, some normalized variables were evaluated (Figures
3D-3F). Figure 3D shows that the average total stocks of EGF per
platelet ([EGF]4h/platelets/L) were equal in healthy controls and
patients before/aer chemoradiotherapy. Conversely, the EGF per
platelet accessible to circulation ([EGF]1h/platelets/L) was signicantly
higher in patients, also before/aer chemoradiotherapy (Figure 3E),
despite the reduction of [EGF] by therapy. Consequently, the EGF
stored per platelet (d/platelets/L) was on average signicantly lower in
patients at diagnosis
vs.
healthy controls, and slightly lower aer
therapy (p>0.05, Figure 3F). erefore, the comparison of cohorts
through the normalized variables makes more evident the dierences
between them, further suggesting an altered relationship between EGF
and platelets in NSCLC patients, as contrasted with healthy controls.
rombocytosis is a hallmark in NSCLC at diagnosis, therefore, we
attempted to understand the relationship between EGF and platelets in
this specic context. For this aim, we divided naive patients into two
strata, with and without thrombocytosis (platelets counts above 400 ×
109 platelets/L). We compared in these subgroups the average values of
the study variables [EGF]1h, [EGF]1h/platelets/L and d/platelets/L
(Figures 3G-3I). Interestingly, the average EGF stored per platelet (d/
platelet/L) and the concentration of free EGF ([EGF]1h) were the same
in patients with/without thrombocytosis. However, the EGF in
circulation per platelet ([EGF]1h/platelets/L) was on average reduced in
thrombocytotic patients (p=0.0239, Figure 3H). Moreover, when the
stratication cut-o was established at 350 × 109 platelets/L, the most
common used threshold of thrombocytosis [21], this dierence was
extremely signicant (p=0.0005, data not shown). Overall, these results
further suggest the existence of an aberrant relationship between EGF
and platelets in NSCLC patients. In thrombocytotic patients there is an
increase in total platelets count. ese platelets store an amount of EGF
similar to that found in patients without thrombocytosis. But, the free
1
(Supplementary Table S2). Nevertheless, the dierences are expected not to be signicant, given the report of Biino et al. [24], declaring
a 9 × 109/L decrease in platelets count per each 10-years of increase in age. is drop is about two orders lower than the dierence we
found between the compared cohorts. Additionally, gender-related dierences in adults are much lower than those related to aging.
Moreover, despite the mentioned imbalance, HC3 does not show dierences by [EGF], as compared to the properly balanced controls
HC1 and HC2 (Supplementary Table S2).
Citation: Idania GP, Hassiul CLH, Camilo RP, Adriana CP, Kalet LM (2018) Normalized Serum EGF Levels as a Potential Biomarker in Non-
Small Cell Lung Cancer: The Role of Platelets. J Mol Biomark Diagn 9: 402. doi:10.4172/2155-9929.1000402
Page 5 of 11
J Mol Biomark Diagn, an open access journal
ISSN: 2155-9929
Volume 9 • Issue 5 • 1000402
Note that HC3 cohort, employed as control in comparisons by platelets count, could not be properly balanced by age and gender

EGF is slightly lowered in thrombocytotic
vs.
non-thrombocytotic
patients, although is still higher than that in healthy controls.
Figure 3: Platelets counts and its relation with EGF levels in NSCLC
patients: e eect of chemoradiotherapy. e picture shows that:
thrombocytosis is reduced by chemoradiotherapy (A); there is no
correlation between [EGF] and platelets count in NSCLC patients at
To (B), but rst-line therapy restores a weak correlation for [EGF]4h
(C); the normalization of [EGF] by platelets count highlights the
higher accessibility of EGF in patients (E and F); the concentration
of free EGF ([EGF]1h) and the average EGF stored per platelet (d/
platelet/L) are the same in patients with and without
thrombocytosis (G and I) but the average EGF contributed to
circulation per platelet ([EGF]1h/platelets/L) is signicantly reduced
in thrombocytotic patients (H). Mean values ± standard mean
errors are represented in gray.
Discriminatory capacity of dierent variables
Table 2 summarizes the ROC analysis for discrimination between
NSCLC patients and healthy controls, for all variables that showed
some relevant dierences between these cohorts (Figures 2 and 3).
With the obtained sample sizes and the ratios healthy/patients in ROC
analysis, statistical powers of 0.8-0.9 were achieved in the detection of
clinically important dierences, at α=0.05, according to Obuchowski
[42]. Several variables achieved a successful discrimination at
diagnosis (To) and aer chemoradiotherapy (T1). At To the variables
d=[EGF]4h-[EGF]1h and [EGF]1h/platelets/L discriminated just fairly
(AUC 0.70-0.80), [EGF]1h, r and platelets/L had good discriminatory
capacities (AUC 0.80-0.90), while the variable d/platelets/L reached the
best discrimination with an AUC=0.8875. At T1, however, only the
normalized variables had enough accuracy for a fair discrimination.
Figure 4 shows the ROC curves for those variables with higher
discriminatory capacities at To and T1, respectively. e curves cross
each other and none of pairwise comparisons were statistically
signicant. is suggests that all selected variables have a roughly
equivalent discriminatory capacity (at least with this relatively small
sample size). However, the simultaneous discrimination obtained at To
and T1 with the normalized variables ([EGF]1h/platelets/L, d/
platelets/L) might favor them as potential candidates for patient’s
stratication. Actually, d/platelets/L had the highest pAUC in the range
of clinical interest (Se ≥ 0.7, Sp ≥ 0.8), among all variables, at To and
T1. is result, along with the absence of bias for this variable under
thrombocytosis, might support its choice for stratication purposes
before/aer rst-line therapy.
Inference of EGF-dependency in NSCLC
EGF levels in circulation, EGFR mutation status and platelets counts
have documented implications in prognosis of NSCLC and its response
to therapy. e latter fact supports the existence of NSCLC variants
with dierent underlying biology of the EGF/EGFR system.
Particularly, the seminal results of Rodriguez et al. [10] suggest the
existence of NSCLC patients with dierent levels of dependency on the
availability of EGF in serum. NSCLC patients with high [EGF] have a
poor prognosis and respond better to therapy with CIMAvax-EGF®
vaccine, which induces a deprivation of free EGF in the blood of
treated patients. Inspired in these ndings, we tried to further infer the
dependency on EGF in dierent NSCLC patients, using the studied
above EGF-related variables, for stratication purposes. We reasoned
that those variables with a higher capacity for discrimination between
patients and healthy controls might better capture the aberrant EGF
biology in cancer patients. erefore, these variables might be better
for the identication of those patients probably more sensitive to
therapies attempting to normalize EGF/EGFR interactions.
Stratication of patients with study variables
Patients were stratied using for each study variable the optimal
cut-o value C(>/<), obtained in ROC analysis as explained in Materials
and Methods. Patients were predicted as highly EGF-dependent for
values of the study variable above C>/below C<, or vice versa, in each
specic case. To compare alternative stratications, its percentages of
overlapping in predictions were calculated by pairs of variables (Table
3). In this analysis each equal classication of a given patient (either as
dependent or as independent of EGF) by two dierent study variables,
directly increases its overlapping percentage.
For the sake of comparison, we also included the stratication
method reported by Rodriguez et al. [10], for the identication of
patients more benetted from CIMAvax-EGF® vaccine. In Rodriguez´s
method, patients with [EGF] above the median of the studied
population appear to carry tumors apparently more EGF-dependent.
To apply this stratication method to our data, the cut-o values (C>)
were set to the medians of either the [EGF] at 1 h and 4 h. Note that in
Rodriguez´s report the time of sera separation was not controlled,
although it was likely close to our 4 h processing, since the median
[EGF] reported (873 pg/ml) is close to our value for [EGF]4h (829
pg/ml) in the T1 sample.
Table 3 summarizes the comparison of stratications achieved with
the dierent variables in our study. At the time of diagnosis (To) the
variable [EGF]1h/platelets/L appears to classify patients quite similarly
to variables d/platelets/L and [EGF]1h, with 83% and 92% of
coincidence between the overall selections, respectively. Interestingly,
the classications by the medians of [EGF] ([EGF]1h,4hb in Table 3)
were quite dierent to those obtained with the normalized variables at
To, with the maximal overlapping of about 70% between [EGF]1hb and
[EGF]1h/platelets/L.
Table 3 also shows a low overlapping between the classications
achieved by the EGF-related variables and the variable platelets/L. is
Citation: Idania GP, Hassiul CLH, Camilo RP, Adriana CP, Kalet LM (2018) Normalized Serum EGF Levels as a Potential Biomarker in Non-
Small Cell Lung Cancer: The Role of Platelets. J Mol Biomark Diagn 9: 402. doi:10.4172/2155-9929.1000402
Page 6 of 11
J Mol Biomark Diagn, an open access journal
ISSN: 2155-9929
Volume 9 • Issue 5 • 1000402

result suggests that both could determine the bad prognosis in patients
through dierent pathways. is is not contradictory, considering that,
in addition to EGF, platelets possess an armory of other pro-angiogenic
proteins, which can be released under its activation [43]. Moreover,
platelets also contain several anti-angiogenic proteins, which are
delivered following specic stimuli [44]. erefore, the classication of
patients by platelets might go no via EGF pathway, but through other
of these several factors and/or its interactions.
Aer rst-line chemoradiotherapy (T1), the normalized variables
showed a remarkably high (94%) coincidence in patient´s
classication, and moderate overlappings with [EGF]1hb (82%-88%).
However, the classication by the median of [EGF]4hb showed a very
low overlapping with the selections of any other study variable,
including [EGF]1hb (53%).
Overall, our results suggest that normalized variables [EGF]1h/
platelets/L and d/platelets/L are quite complementary and therefore
will classify patients similarly. However, they could provide
classications dierent to those obtained using the medians of
[EGF]1h,4hb, as proposed by Rodriguez et al. [10], especially when
using [EGF]4h. is result was expected, as we know from our data that
[EGF]4h are unable to discriminate cases from controls.
Variable To T1
AUC p Ca (>/<) Se (%)bSp (%)bAUC p Ca (>/<) Se (%)bSp (%)b
Platelets/L 0.8181 0.0009553 302 (>) 75 87 -- -- -- -- --
[EGF]1h 0.8250 0.0007371 291 (>) 83 73 -- -- -- -- --
r 0.8278 0.0006635 0.68 (>) 54 100 -- -- -- -- --
d 0.7056 0.0178 239 (<) 58 100 -- -- -- -- --
[EGF]1h/platelets/L 0.7389 0.01308 0.63 (>) 83 53 0.7431 0.0071 1.62 (>) 65 80
d/platelets/L 0.8875 <0.0001 1.80 (<) 92 80 0.7059 0.0349 1.84 (<) 71 80
AUC: Area Under Curve; C(>/<): Optimized cut-off points (cases are above/below C>/C<, respectively); Se: Sensitivity; Sp: Specificity; aExpressed in the standard units
of each variable; bRounded values
Table 2: ROC analysis of discriminatory variables.
Figure 4: ROC curves for the evaluation of the discriminatory capacity of the study variables. e picture shows the curves of variables with
higher AUCs at To (HC3
vs.
NSCLC1) (A) and T1 (HC3
vs.
NSCLC2) (B).
Discussion
Overall, EGF concentrations in NSCLC patients were highly
variable, just as reported before in healthy people. is natural
variability could be mainly attributed to the single nucleotide
polymorphism (SNP) at the promoter region of the EGF gene [45,46],
which is functional and determines dierent total stocks of EGF in
individuals. Environmental factors could additionally contribute
according to Pantsulaia et al. [35].
e higher accessibility of EGF to circulation in NSCLC patients, as
compared to healthy controls, suggests that this increase in free/
accessible EGF in blood circulation might be relevant to the biology of
NSCLC, most likely because it reects as well a higher accessibility to
this tumoral growth factor by the tumor. Moreover, it is known that
platelets are oen activated in patients [47,48], which elicits the release
of several anti- and pro-angiogenic proteins by them [44], including
EGF. Additionally, the circulating tumor cells can induce the
degranulation of platelets [49], also provoking the EGF release in
Citation: Idania GP, Hassiul CLH, Camilo RP, Adriana CP, Kalet LM (2018) Normalized Serum EGF Levels as a Potential Biomarker in Non-
Small Cell Lung Cancer: The Role of Platelets. J Mol Biomark Diagn 9: 402. doi:10.4172/2155-9929.1000402
Page 7 of 11
J Mol Biomark Diagn, an open access journal
ISSN: 2155-9929
Volume 9 • Issue 5 • 1000402

patients, and its higher accessibility to serum as suggest our results.
Previous ndings, altogether with the proven ecacy of the EGF-
targeted-immunotherapy CIMAvax-EGF®, support the involvement of
EGF in the biology of cancer.
Our results further suggest the existence of an aberrant relationship
between EGF and platelets in NSCLC patients. e correlation [EGF]-
platelets/L, previously reported for normal individuals [14], is lost in
naive patients, probably due to the signicant reduction of circulating
EGF levels per platelet under thrombocytosis. us, thrombocytosis,
an independent indicator of bad prognosis [36,38,48], and low survival
and response to therapy in LC [50,51], modies this correlation.
Moreover, the better discrimination achieved by the normalized
variables, also conrms the altered relationship EGF-platelets in
patients. Finally, the good accuracy of the variable platelets/L in ROC
analysis reveals the platelets count as a good surrogate marker of the
tumor.
Variable Platelets/La[EGF]1harada[EGF]1h/platelets/Lad/platelets/La[EGF]1hb[EGF]4hb
T0 Platelets/La100 54 33 38 58 63 42 58
[EGF]1ha-- 100 71 67 92 79 67 63
ra-- -- 100 88 71 63 92 50
da-- -- -- 100 63 67 92 46
[EGF]1h/platelets/La-- -- -- -- 100 83 71 29
d/platelets/La-- -- -- -- -- 100 63 21
[EGF]1hb-- -- -- -- -- -- 100 58
[EGF]4hb-- -- -- -- -- -- 100
T1 [EGF]1h/platelets/La-- -- -- 100 94 88 41
d/platelets/La-- -- -- -- 100 82 47
[EGF]1hb-- -- -- -- -- -- 100 53
[EGF]4hb-- -- -- -- -- -- -- 100
aThe selected cut-off points were C>/C< from ROC analysis; bThe selected cut-off points were the medians of [EGF]; a,bThe intersections row-column show the
percentages of patients that were equally classified by both variables, in rounded values
Table 3: Percentages of agreement between dierent classications of patients.
Our study has also an added value to the eorts of nding an
ecacy biomarker for CIMAvax-EGF® vaccine. is vaccine is
approved as a second-line therapy, so the selection of patients for this
treatment occurs aer chemoradiotherapy. Overall, our results suggest
that normalized variables [EGF]1h/platelets/L and d/platelets/L are
quite complementary and therefore will provide similar selections of
patients for this treatment. However, they could provide a classication
dierent to that obtained using the median [EGF] as cut-o, especially
when using [EGF]4h, a variable which was not able to discriminate
cases from controls. erefore, although in Rodriguez´s approach
[EGF]4h could explain in some measure the prognosis of patients and
the vaccine´s ecacy aer chemoradiotherapy [10], we believe that the
normalized variables, discriminatory at T1, might be more valuable for
these purposes than [EGF]4h.
Finding´s scope
ere are enough proofs supporting the role of EGF/EGFR axis in
tumor progression and metastasis in several cancer types. In NSCLC
specically, the EGFR is overexpressed in 40-85% of cases [52,53].
Moreover, this overexpression has been implicated in the process of
malignant transformation by promoting cell proliferation, cell survival
and motility [54]. e EGF, a potent growth factor measured in
numerous tumors [55-62] including LC [12,57], has been implicated in
the process of invasion and metastasis [63] and correlated with disease
stage, course and prognosis [64]. Additionally, some authors have
evidenced the mutual interaction and regulation established between
EGF and EGFR. Clark et al. reported that EGF regulates its own
receptor [65] and an association between EGFR overexpression and an
increased expression of its ligands has also been reported [66].
Consequently, EGF and EGFR are frequently synchronously expressed
in gastric [56], lung [52] and other carcinomas. Similarly, minimally
invasive colorectal resection is associated with a signicant decrease in
EGF levels early post operation [57]. All these facts sustain the
usefulness of EGF/EGFR as potential biomarkers for several
carcinomas.
e status of EGFR was not assessed in our study, which could be
considered as a limitation of this work. Several reports have described
the role of the expression level and mutational status of EGFR in
predicting the ecacy of anti-EGFR therapies [67-71], but the
association between the expression of EGFR mutations and response
to anti-EGFR treatments in patients is still controversial. is responds
to variations in the assessed expressions, caused by the use of dierent
cut-o values for EGFR immunostaining [72], the use of antibodies
that do not discriminate between the wild-type and mutated forms of
the EGFR [73] and discordances between the expression of EGFR in
the primary tumor and the metastatic sites [74,75]. Moreover, the
interactions between the EGFR´s ligand(s) and the receptor (wild type
or mutated), the molecular mechanism of EGFR activation and its
Citation: Idania GP, Hassiul CLH, Camilo RP, Adriana CP, Kalet LM (2018) Normalized Serum EGF Levels as a Potential Biomarker in Non-
Small Cell Lung Cancer: The Role of Platelets. J Mol Biomark Diagn 9: 402. doi:10.4172/2155-9929.1000402
Page 8 of 11
J Mol Biomark Diagn, an open access journal
ISSN: 2155-9929
Volume 9 • Issue 5 • 1000402

impact on patient´s clinical outcome, are not fully elucidated [73]. e
existence of multiple routes for EGFR activation, including a ligand-
independent untethering in wild type EGFR (wEGFR), sensitive to
dierential ligand concentrations, has also been suggested [73,76]. is
alternative activation might explain the response to small molecules
erlotinib and getinib [77], achieved in NSCLC patients with wEGFR
(not harboring EGFRvIII). It could also explain the interaction
between the overexpressed wEGFR [78,79] and the mAb806 antibody,
originally raised against the EGFRvIII mutation [80].
On the other hand, little is known about the mechanisms of action
of the anti-EGF antibodies elicited by vaccination with CIMAvax-EGF®
or its dierential activity in tumors with EGFR mutations or other
genetic alterations. Nevertheless, this immunotherapy has proven its
ecacy in a phase III clinical trial carried out on unselected NSCLC
patients with advanced disease [10]. Recently, Rosell et al. [81]
published that anti-EGF antibodies generated with this vaccine
suppress the EGF-induced cell proliferation, the cycle progression and
also inhibit downstream EGFR signaling, in EGFR-mutant NSCLC cell
lines sensitive to dierent generations of EGFR TKIs. Finally, he
concludes that patients with an EGFR-mutant can also derive benet
from immunization against EGF, particularly if combined with EGFR
TKIs. In summary, Rosell´s ndings prove the contribution of EGF to
NSCLC progression also in patients with a mutant EGFR and hence
the possible role of this ligand as a biomarker in this subset of patients
too. erefore, present work´s results, along with Rossell´s ndings,
heighten the usefulness of EGF-related variables as biomarkers of
ecacy for CIMAvax-EGF® treatment, independently of the EGFR
mutational status. Further studies including the assessment of
mutations and expression of EGFR in NSCLC patients, will validate
the applicability of these biomarkers in the context of other EGF/EGFR
directed therapies.
Conclusion
Concluding, our study revealed that what dierentiates NSCLC
patients from healthy individuals is not the total stock of EGF, but its
higher accessibility to serum. Additionally, a dierent relation between
[EGF] and platelets count was observed in patients. Moreover, several
EGF-related variables with enough accuracy for discrimination were
identied. Particularly, those normalized by platelets count make more
evident the dierences between patients and controls, and might be
potential biomarkers in NSCLC, and good candidate biomarkers of
ecacy for CIMAvax-EGF® treatment, independently of the patient´s
EGFR mutational status. Further studies are needed to evaluate the
usefulness of these normalized variables on its predictive value to
select good responders to treatment with therapies targeting the EGF/
EGFR system, and also in prognosis, monitoring of therapy and
evaluation of response, in NSCLC and other epithelial cancers.
Acknowledgements
e author IGP wants to thank Elizabeth Cuétara Lugo, from the
National Institute of Oncology and Radiobiology, in Havana, for the
valuable discussions and comments on the manuscript.
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Small Cell Lung Cancer: The Role of Platelets. J Mol Biomark Diagn 9: 402. doi:10.4172/2155-9929.1000402
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Citation: Idania GP, Hassiul CLH, Camilo RP, Adriana CP, Kalet LM (2018) Normalized Serum EGF Levels as a Potential Biomarker in Non-
Small Cell Lung Cancer: The Role of Platelets. J Mol Biomark Diagn 9: 402. doi:10.4172/2155-9929.1000402
Page 11 of 11
J Mol Biomark Diagn, an open access journal
ISSN: 2155-9929
Volume 9 • Issue 5 • 1000402