Quantitative expression analysis of the apoptosis-related gene, BCL2L12, in head and neck squamous cell carcinoma

Article (PDF Available)inJournal of Oral Pathology and Medicine 42(2) · July 2012with43 Reads
DOI: 10.1111/j.1600-0714.2012.01190.x · Source: PubMed
Abstract
J Oral Pathol Med (2013)42: 154–161 Background: BCL2L12 is a recently identified gene belonging to the BCL2 family, members of which are implicated in head and neck squamous cell carcinoma (HNSCC). We have recently shown that BCL2L12 mRNA expression is an unfavorable prognostic indicator in nasopharyngeal carcinoma (NPC) and that BCL2L12 can be regarded as a novel, useful tissue biomarker for the prediction of NPC patients’ short-term relapse. The aim of this study was to analyze the mRNA expression of the novel apoptosis-related gene BCL2L12 in patients with HNSCC. Methods: Total RNA was isolated from 53 malignant tumors originating in larynx, pharynx, tongue, buccal mucosa, parotid glands, and nasal cavity, as well as from 34 adjacent non-cancerous tissue specimens, resected from patients with HNSCC. A highly sensitive real-time PCR method for BCL2L12 mRNA quantification in head and neck tissues was developed using the SYBR® Green chemistry. After preparing cDNA by reverse transcription, relative quantification was performed using the comparative CT () method. Results: BCL2L12 mRNA levels were lower in laryngeal tumors of advanced tumor, node, metastasis (TNM) stage or bigger size and in well-differentiated malignant tongue neoplasms, compared with early-stage laryngeal tumors or poorly differentiated tongue tumors. Interestingly, the BCL2L12 expression showed significant discriminatory value, distinguishing efficiently patients with tongue squamous cell carcinoma (SCC) from non-cancerous population. Conclusions: This is the first study examining the BCL2L12 mRNA expression in HNSCC. Our results suggest that BCL2L12 mRNA expression may serve as a potential prognostic biomarker in tongue and/or larynx SCC, which principally constitute the great majority of HNSCC cases worldwide.
Quantitative expression analysis of the apoptosis-related
gene, BCL2L12, in head and neck squamous cell carcinoma
Panagiota-Aikaterini Geomela
1
, Christos K. Kontos
1
, Ioannis Yiotakis
2
, Andreas Scorilas
1
1
Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Athens, Athens, Greece;
2
First Ear, Nose and
Throat Clinics, Faculty of Medicine, University of Athens, Athens General Hospital Hippokration’, Athens, Greece
BACKGROUND: BCL2L12 is a recently identified gene
belonging to the BCL2 family, members of which are
implicated in head and neck squamous cell carcinoma
(HNSCC). We have recently shown that BCL2L12 mRNA
expression is an unfavorable prognostic indicator in
nasopharyngeal carcinoma (NPC) and that BCL2L12 can
be regarded as a novel, useful tissue biomarker for the
prediction of NPC patients’ short-term relapse. The aim
of this study was to analyze the mRNA expression of the
novel apoptosis-related gene BCL2L12 in patients with
HNSCC.
METHODS: Total RNA was isolated from 53 malignant
tumors originating in larynx, pharynx, tongue, buccal
mucosa, parotid glands, and nasal cavity, as well as from
34 adjacent non-cancerous tissue specimens, resected
from patients with HNSCC. A highly sensitive real-time
PCR method for BCL2L12 mRNA quantification in head
and neck tissues was developed using the SYBR
Green
chemistry. After preparing cDNA by reverse transcrip-
tion, relative quantification was performed using the
comparative C
T
(2
DDC
T
) method.
RESULTS: BCL2L12 mRNA levels were lower in laryngeal
tumors of advanced tumor, node, metastasis (TNM)
stage or bigger size and in well-differentiated malignant
tongue neoplasms, compared with early-stage laryngeal
tumors or poorly differentiated tongue tumors. Inter-
estingly, the BCL2L12 expression showed significant dis-
criminatory value, distinguishing efficiently patients with
tongue squamous cell carcinoma (SCC) from non-can-
cerous population.
CONCLUSIONS: This is the first study examining the
BCL2L12 mRNA expression in HNSCC. Our results sug-
gest that BCL2L12 mRNA expression may serve as a
potential prognostic biomarker in tongue and
or larynx
SCC, which principally constitute the great majority of
HNSCC cases worldwide.
J Oral Pathol Med (2012)
Keywords: BCL2 family; larynx; oral cancer; tongue; tumor
biomarkers
Introduction
Head and neck carcinomas constitute the sixth most
commonly diagnosed cancer worldwide, and the over-
whelming majority of them (90%) are characterized
as squamous cell carcinomas (SCCs) (1). Despite the
recent advances in the field of cancer prevention and
treatment, head and neck squamous cell carcinoma
(HNSCC) represents a significant cause of cancer-
related deaths in the developed world (2). However,
HNSCC takes its toll especially on populations of
Southern China and Southeast Asian countries, where
the disease is endemic (3).
In fact, HNSCC includes a large variety of tumors
arising from different sites of the head and neck region.
Particularly, malignancies are developed in nasal cavity
and paranasa l sinuses, nasopharynx, hypolarynx, larynx
and trachea, oral cavity and oropharynx, salivary
glands, and ear, while miscellaneous tumors such as
neurogenic neoplasms and paragangliomas can also
arise (4).
Undoubtedly, the novel therapeutic procedures have
contributed to improvement of the 5-year survival rates
for advanced laryng eal and pharyngeal carcinomas.
Nevertheless, it is common knowledge that the overall
survival (OS) rate for patients suffering from HNSCC
remains among the lowest. Treatment strategies often
fail or procure rather modest improvement and conse-
quently, locoregional recurrence, distant metastases,
and second primary tumors usually occur. In particu-
lar, the presence of lymph node metastases is consid-
ered as the most adverse independent prognostic factor
in HNSCC (5–7). The poor prognosis for advanced
cases of HNSCC and the high morbidity rate for
this malignancy can be largely attributed to lack of
established biomarkers for early detection, treatment
response failure, and inadequate understanding of the
molecular mechanisms involved in this disease (8).
Correspondence: Andreas Scorilas, PhD, Department of Biochemis-
try and Molecular Biology, Faculty of Biology, University of Athens,
Panepistimiopolis, 15701 Athens, Greece. Tel: +30 210 727 4360,
Fax: +30 210 727 4158, E-mail: ascorilas@biol.uoa.gr
Accepted for publication May 24, 2012
doi: 10.1111/j.1600-0714.2012.01190.x
J Oral Pathol Med
ª 2012 John Wiley & Sons A/S Æ All rights reserved
wileyonlinelibrary.com/journal/jop
Moreover, it is wi dely accepted that HNSCC is a
heterogeneous disease characterized by variations in its
biological behavior and complex molecular abnormal-
ities. As a result, despite the fact that the stage and site
of the primary tumor as well as the presence or absence
of distal metastases determine the selection of an
appropriate treatment, these clinicopathological fea-
tures of the tumor often prove to be insufficient,
because they are not able to predict treatment response
(2, 5). Taking all this information into consideration,
the identification of novel, reliable molecular biomar-
kers for early diagnosis, reliable prognosis and or
effective treatment response monito ring, which will
contribute utmost to clinical decision making, remains
an important research topic.
The BCL2L12 gene, a member of the BCL2 family,
was discovered and cloned by members of our group
in 2001. The classical BCL2L12 protein isoform
(334 aa) contains a highly conserved BH2 domain, a
BH3-like motif and a proline-rich region (9). Alterna-
tive splicing of this gene has very recently been shown
to give birth to several alternatively spliced variants
(10). The prognostic potential of BCL2L12 mRNA
expression has already been assessed in several solid
tumors (11–17) as well as in hematological malignan-
cies such as chronic lymphocytic leukemia (CLL) (18)
and acute myeloid leukemia (AML) (19). In addition,
the discriminatory power of BCL2 mRNA expression
in CLL was improved when this latter was combined
with BCL2L12 mRNA expression (18).
BCL2L12 mRNA expression analysis demonstrated
the overexpression of this gene in colon cancer samples
as compared to their paired normal mucosa (11, 12).
Furthermore, the expression of BCL2L12-A, an alter-
native transcript of BCL2L12, is associated with Dukes’
stage and lymph node status (11), while higher expres-
sion of the full-length BCL2L12 variant is linked with
less aggressive forms of intestinal cancer (12). Patients
with colon cancer showing BCL2L12 overexpression
have significantly longer disease-free survival (DFS) and
OS, suggesting a favorable prognostic role for BCL2L12
mRNA expression (12). Similarly, BCL2L12 mRNA
seems to constitute a novel, independent favorable tissue
biomarker in breast cancer as well, because BCL2L12
mRNA-positive patients have a lower probability of
relapse and or death than BCL2L12 mRNA-negative
patients (13, 14). Moreover, an association between
BCL2L12 and BCL2, mRNA expression was shown in
breast tumors (14). Increased expression of BCL 2L12
has also been associated with favorable outcome in
patients with gastric cancer (15). On the other hand,
mRNA upregulation and robust protein expression of
this apoptosis-related gene has been noticed in primary
glioblastoma, compared with surrounding normal brain
tissue (16). Furthermore, BCL2L12 mRNA expression
has recently been linked to unfavorable prognosis in
nasopharyngeal carcinoma (NPC) and has been sug-
gested as a novel, useful tissue biomarker for the
prediction of NPC patients’ short-term relapse. Inter-
estingly, BCL2L12 overexpression may also account for
the resistance of NPC patients with advanced-stage
disease to chemotherapeutic and irradiation treatment
(17).
The above data encouraged us to analyze BCL2L12
mRNA expression in HNSCC and adjacent non-can-
cerous tissue specimens from patients bearing malignant
tumors in larynx, pharynx, tongue, buccal mucosa,
parotid glands, or nasal cavity, by an ultrasensitive
quantitative real-time polymerase chain reaction (PCR)
(qRT-PCR) method, and to evaluate its clinical signif-
icance and application as a novel tissue biomarker for
HNSCC.
Materials and methods
Tissue specimens and RNA extraction
Included in this study were 53 malignant tumors and 34
adjacent non-cancerous tissue specimens from patients
having undergon e surgical treatment for primary
HNSCC at Athens General Hospital Hippokration’
(Athens, Greece), between 2005 and 2007. These tumors
originated in larynx (20 cases), pharynx (five cases),
tongue (14 cases), buccal mucosa (five cases), parotid
glands (five cases), and nasal cavity (four cases). Patient
age ranged from 34.0 up to 90.0 years with a
mean ± SE of 63.1 ± 1.6. The selection criteria for
the specimens included the availability of sufficient
tissue mass for RNA extraction and assay. All tissue
specimens had been frozen in liquid nitrogen immedi-
ately after the resection of the specimen.
This study was performed in accordance with the
ethical standards of the World Medical Association
Declaration of Helsinki (version: 2008) and was
approved by the institutional review board of Athens
General Hospital Hippokration’ (Athens, Greece).
First-strand cDNA synthesis
Tissue specimens were pulverized and then dissolved in
TRI
Reagent (Ambion Europe Ltd., Huntingdon,
UK). Following the manufacturer’s instructions, total
RNA was extracted and diluted in a RNA Storage
Solution (Ambion Europe Ltd.) and stored at )80C
until use. First-strand cDNA was then synthesized using
the M-MuLV Reverse Transcriptase, RNase H
)
(Finn-
zymes Oy, Vantaa, Finland), RNaseOUT RNase
inhibitor (Invitrogen, Carlsbad, CA, USA) and oli-
go(dT)
12–18
as primer, according to the manufacturer’s
instructions.
Quantitative real-time PCR
Based on the information of the BCL2L12 and GAPDH
cDNA sequences (GenBank Accession Numbers:
NM_138639.1 and NM_002046.3, respectively), two
pairs of gene-specific primers were designed. The
sequences of the BCL2L12 real-time PCR primers were:
5¢-CCCTCGGCCTTGCTCTCT-3¢ and 5¢-TCCGCAG-
TATGGCTTCCTTC-3¢, producing a 182-bp PCR
amplicon with a T
m
of 84.5C, while the sequences of
the GAPDH real-time PCR primers were the following:
5¢-ATGGGGAAGGTGAAGGTCG-3¢ and 5¢-GGGT
CATTGATGGCAACAATATC-3¢, resulting in a 107-
bp PCR amplicon with a T
m
of 79.4C. Quantitative
BCL2L12 mRNAexpressioninHNSCC
Geomela et al.
2
JOralPatholMed
real-time PCR was performed in a 7500 Real-Time PCR
System (Applied Biosystems, Foster City, CA, USA)
using the SYBR
Green chemistry (Applied Biosys-
tems), and dissociation curves of the PCR products were
generated after amplification, as previously described
(17), to distinguish between the main PCR products and
primer-dimers or other non-specific products. Each real-
time PCR was performed in duplicate, to evaluate data
reproducibility.
Calculations were made with the use of the compar-
ative C
T
(2
DDC
T
) method, as previously described (20).
In our study, GAPDH was used as an internal control
gene to normalize the PCRs for the amount of RNA
added to the reverse transcription reactions, while the
Caco-2 cell line was used as a calibrator. Normalized
results were expressed as arb itrary units (a.u.), which
stand for the ratio of BCL2L12 mRNA copies to 1000
GAPDH mRNA copies, calculated for each specimen,
in relation to the same ratio, calculated for Caco-2
cells.
Statistical analysis
Extensive biostatistical analysis was performed only for
the laryngeal squamous cell carcinoma (LSCC) and
tongue squamous cell carcinoma (TSCC) specimens, as
the small number of cases in the rest Patients with
HNSCC groups did not allow an advanced analysis to
be undertaken. Analyses of the differences in BCL2L12
profiles between cancerous and non-cancerous tissues
were performed with the non-parametric Mann–Whit-
ney U-test. To assess the diagnostic value of BCL2L12
mRNA expression in TSCC, receiver operating charac-
teristic (ROC) curves were constructed for BCL2L12
expression levels and the areas under the ROC curves
(AUC) were analyzed by Hanley and McNeil method.
Patients with LSCC and TSCC were also classified in
subgroups according to the size, TNM stage and
histological grade of their tumors, and analysis of the
differences of BCL2L12 mRNA expression levels in
these subgroups was performed with the non-parametric
Mann–Whitney U or Kruska l–Wallis test, where appro-
priate.
Results
Validation of the comparative C
T
(2
DDC
T
) method for
BCL2L12 mRNA quantification
The application of the comparative C
T
(2
DDC
T
) method
is based on the assumptions that the PCR amplification
efficiencies of the target and the reference genes are quite
equal and close to 100% (21). In our study, the
requirements for the application of the 2
DDC
T
method
were checked in a validation experiment, in which C
T
values of BCL2L12 and GAP DH amplification were
measured in a dilution seri es of control cDNA over a
10
4
-fold range and then plotted against log cDNA
dilution. The C
T
values for both genes corresponded to
the number of cycles at which the fluorescence emission
monitored in real-time reached a threshold of 10 times
the standard deviation of the mean baseline emission
from cycles 3 to 15 (Fig. 1A). All PCR products were
gene-specific, as depicted by the respective dissociation
curves (Fig. 1B).
As illustrated in Fig. 1C, the slopes of BCL2L12 and
GAPDH amplification plots are similar ()3.334 and
)3.394, respectively). On the basis of the formula E
(%) = [)1+10
()1 a)
]
.
100, where E (%) is the real-time
PCR efficiency for amplification of each gene and a is
the slope of the corresponding amplification plot, the
calculated efficiencies for BCL2L12 and GAPDH ampli-
cons were 99.5 and 97.1%, respectively, thus fulfilling
both prerequisites for the application of the 2
DDC
T
method for the relative BCL2L12 mRNA quantification
in head and neck specimens.
BCL2L12 mRNA expression analysis in cancerous and
non-cancerous specimens of the head and neck
BCL2L12 mRNA expression was significantly higher in
TSCC specimens than in non-malignant counterparts
(P = 0.003; Fig. 2), ranging from 1.6 to 7356.4 a.u.
with a mean ± S.E. of 1282.1 ± 509.1 a.u. in the
former, while varying between 1.6 and 648.0 a.u. with
a mean ± S.E. of 237.1 ± 87.7 a.u. in the latter
(Table 1). On the other hand, BCL2L 12 mRNA
expression levels presented a slight–though not sta-
tistically significant–decrease in LSCC specimens, in
comparison with their non-malignant counterparts.
Therefore, BCL2L12 mRNA levels in laryngeal
tumors fluctuated between 3.3 and 1639.0 a.u. with a
mean ± SE of 459.8 ± 104.7, whereas in non-malig-
nant laryngeal tissue specimens, they ranged from 10.9
to 8310.8 a.u. with a mean ± SE of 971.9 ± 575.1
(Table 1).
Table 2 shows features of the distribution of
BCL2L12 mRNA expression in tumors resected from
pharynx, buccal mucosa, parotid glands, and nasal
cavity. Because of the small number of cases of each
group, only descriptive statistical analysis was per-
formed for these results. Hence, BCL2L12 mRNA
levels seem to be higher in tumors of the pharynx than
in non-malignant pharyngeal tissue specimens. In con-
trast, lower levels of BCL2L12 transcripts were detected
in malignant neoplasms of parotid glands and nasal
cavity, compared with their non-cancerous counter-
parts.
Diagnostic value of BCL2L12 mRNA expression in
TSCC
To evaluate the potential of BCL2L12 mRNA expres-
sion as a diagnostic biomarker in tongue SCC, we
performed ROC analysis. As illustrated by the ROC
curve in Fig. 3, BCL2L12 mRNA expression was found
to distinguish efficiently patients with TSCC from
healthy controls (area under the curve [AUC] = 0.69,
95% confidence interval [95% CI] = 0.59–0.78,
P < 0.001).
BCL2L12 mRNA expression
a
analysis in subgroups of
patients with LSCC and TSCC
Patients within LSCC and TSCC groups were further
classified into subgroups, according to classical clinico-
pathological parameters, such as tumor size, TNM
BCL2L12 mRNA expression in HNSCC
Geomela et al.
3
JOralPatholMed
stage, and histological grade. BCL2L12 mRNA expres-
sion analysis was then performed for each subgroup,
and BCL2L12 mRNA levels were compared among
distinct patients’ subgroups using the non- parametric
Mann–Whitney U or Kruskal–Wallis test, as described
in Materials and methods’.
Regarding patients with LSCC, those bearing small
tumors displayed significantly increased levels of
BCL2L12 mRNA, in contrast to patients with bigger
tumors (P = 0.043). In particular, in patients with
tumor size £ 2 cm, BCL2L12 expression showed a
mean ± SE of 740.7 ± 189.6 a.u., whereas in patients
with tumor size >2 cm the respective mean ± SE was
235.1 ± 48.1 (Table 3). Similarly, patients with LSCC
being at an early disease stage (TNM stage I or II)
displayed elevated BCL2L12 expression, in comparison
with patients diagnosed at an advanced-stage (TNM
stage III) (P = 0.027).
On the other hand, BCL2L12 mRNA expression was
lower in well-differentiated tongue tumors than in
tumors of high histological grade (P = 0.032). Thus,
in TSCC patients with histological tumor grade III,
BCL2L12 mRNA presented a mean ± SE of
AB
C
40
Cτ vs log cDNA dilution
35
30
y = –3.334 log(x) + 21.726
R
2
= 0.9976
25
20
Thershold cycle (C
T
)
y = –3.394 log(x) + 18.654
R
2
= 0.9958
15
GAPDH
BCL2L12
0.00001 0.0001 0.001 0.01 0.1 1
cDNA dilution
Figure 1 Real-time PCR quantification of BCL2L12 mRNA expression in a tongue cancer tissue specimen. Amplification plots of BCL2L12 and
GAPDH cDNAs (A), dissociation curves of the respective amplicons, showing DR
n
plotted against cycle number (B), and validation of the
comparative C
T
(2
DDC
T
) method, to assess the efficiency of amplification of BCL2L12 and GAPDH (C).
50 000
10 000
5000
1000
500
100
50
10
5
1
BCL2L12 mRNA expression (a.u.)
Non-cancer Cancer
P = 0.003
N = 14
N = 9
Figure 2 Comparison of the distribution of BCL2L12 mRNA
expression in tongue cancer specimens and non-cancerous counter-
parts. The bold lanes indicate the median value (50th percentile) of
each group.
BCL2L12 mRNAexpressioninHNSCC
Geomela et al.
4
JOralPatholMed
3796.1 ± 1921.1 a.u., while expression of this gene was
514.1 ± 46.2 a.u. and 661.2 ± 43.6 a.u. in malignant
tongue neoplasms of histological grade I and II,
respectively (Table 4); yet, no statistically significant
differences wer e observed between subgroups of patients
with TSCC classified according to the TNM stage or size
of the tumor.
Discussion
Head and neck cancer represents a noticeable threat to a
large group of population in both developed and
developing countries of the world. Although the inci-
dence of the disease is lower in the former than in the
latter, its consequences should not be disregarded or
underestimated. Although extensive research efforts
have been devoted to unravel the pathobiology of the
disease and to assist those who have already been struck
by a type of HNSCC, its complexity makes it seem as a
difficult puzzle (22).
Recently, scientists have focused on the discovery of
new biomarkers which will contribute to early diagnosis,
prognosis, treatment decision making, and prediction of
therapeutic response of patients with HNSCC. Several
oncogenes have been implicated so far in HNSCC
carcinogenesis, including members of the RAS, MYC,
and BCL2 gene family (5, 23). Furthermore, overex-
pression of EGFR mRNA has been noticed in HNSCC
cell lines and primary tumors (24), while genetic
alterations and enhanced transcription of the EGFR
gene have been shown to predict poor prognosis in
patients with LSCC (25, 26). EGFR and CCND1 gene
amplification, also, constitute poor prognostic factors in
HNSCC, in terms of OS (27–29). Moreover, the
CCND1 CDK4 complex, CDK2, and CDKN1B are
related to the proliferative activity of cancerous lar-
yngeal squamous cells and to patients’ prognosis (30–
32). In patients with locally advanced LSCC, CDKN2A
Table 1 BCL2L12 mRNA expression
a
analysis in patients with laryngeal or tongue tumors and their healthy counterparts
Variable Mean ± SE
b
Range
Percentiles
10 25 50 75 90
Median
BCL2L12 in tongue tumors (N = 14) 1282.1 ± 509.1 1.6–7356.4 176.3 373.5 731.5 915.5 5311.7
BCL2L12 in non-cancerous specimens (N = 9) 237.1 ± 87.7 1.6–648.0 1.63 1.63 136.5 516.4 595.3
P = 0.003
c
BCL2L12 in laryngeal tumors (N = 18) 459.8 ± 104.7 3.3–1639.0 80.3 201.6 270.5 639.8 1479.7
BCL2L12 in non-cancerous specimens (N = 14) 971.9 ± 575.1 10.9 8310.8 41.1 174.3 342.8 523.2 4977.9
P = 0.86
c
a
a.u., arbitrary units.
b
Standard error of the mean.
c
Calculated using the Mann–Whitney U-test.
Table 2 Distribution of BCL2L12 mRNA expression
a
in tumors resected from pharynx, buccal mucosa, parotid glands, and nasal cavity, and in
respective normal tissue specimens
Tissue
Cancerous samples Non-cancerous samples
Mean ± SE
b
Median Range Mean ± SE
b
Median Range
Pharynx 651.7 ± 152.0 660.7 257.7–1158.3 256.0 ± 46.4 300.8 116.9–305.5
Buccal mucosa 397.5 ± 180.4 216.9 1.6–861.6
Parotid glands 435.3 ± 151.9 274.0 179.9–982.1 651.2 ± 42.4 642.9 570.0–749.0
Nasal cavity 272.4 ± 84.1 207.7 154.4–519.8 1424.1 ± 804.7 622.0 616.9–3033.5
a
a.u., arbitrary units.
b
Standard error of the mean.
1 - Specificity
1007550250
Sensitivity
100
75
50
25
0
AUC (BCL2L12) = 0.69
95% CI = 0.59 – 0.78; P < 0.001*
*Null hypothesis: true area = 0.5
Figure 3 Receiver operating characteristic analysis for BCL2L12
mRNA expression. BCL2L12 expression was found to distinguish
successfully patients with tongue cancer from healthy controls.
BCL2L12 mRNAexpressioninHNSCC
Geomela et al.
5
JOralPatholMed
mutations have prognostic significance in predicting
adverse outcome (33).
Regarding BCL2, the founder member of the BCL2
apoptosis-related gene family, its overexpression is asso-
ciated with poor treatment outcome and shorter OS in
patients with early-stage HNSCC (34, 35). BCL2 is also
associated with aggressive disease, neck lymph node
metastasis, and poor prognosis of patients with TSCC
(36). Similarly, BCL2 mRNA expression constitutes an
unfavorable and independent prognostic marker in NP C
(37), and its protein levels represent an important
predictor in advanced-stage NPC (38). Furthermore,
BCL2 modulates lymph node meta stasis of NPC cells
(39). Not surprisingly, pre-treatment BCL2 expression
predicts an increased risk of treatment failure in patien ts
with oropharyngeal squamous cell carcinoma (OPSCC),
particularly through distant metastasis, after concurrent
chemoradiation (40, 41). However, the necessity of
discovering novel, reliable molecular tissue biomarkers
in HNSCC remains high, because no such marker has
been established and used in clinical practice so far.
BCL2L12 is a novel member of the BCL2 family,
members of which are involved in head and neck cancer.
Although it is clear that BCL2L12 is involved in
apoptosis, it remains unclear or even controversial
whether it exerts a pro- or anti-apoptotic role. Interest-
ingly, in vitro experimen ts have revealed that BCL2L12
can interact with the apoptotic BCLX
L
protein, most
likely through its BH3-like domain (42). Unlike typical
BCL2 family members, cytoplasmic BCL2L12 does not
affect cytochrome C release or apoptosome-driven
caspase-9 activation, but most likely impedes effector
caspase activation, at least in murine cortical astrocytes
and human glioma cell lines (16, 43, 44). On the other
hand, nuclear BCL2L12 interacts with the tumor
suppressor protein p53 and inhibits its binding to some
of its target gene promoters. Thus, BCL2L12 attenuates
endogenous p53-directed transcriptomic alterations fol-
lowing genotoxic stress and obstructs p53-dependent
DNA damage-induced apoptosis (45). Expression anal-
ysis of BCL2L12 uncovered its prognostic potential in
various malignancies, including breast (13, 14), colon
(11, 12), and gastric cancer (15). According to our
recently published data, BCL2L12 can be considered as
a novel biomarker for the prediction of short-term
relapse in NPC, as its mRNA expression is an unfavor-
able and independent prognostic indicator of DFS in
NPC patients (17).
In the current study, we investigated the mRNA
expression of the novel apoptosis-related gene BCL2L12
in SCCs of the larynx, pharynx, tongue, buccal mucosa,
parotid glands, and nasal cavity, as well as in adjacent
non-cancerous counterparts. Our results revealed that
BCL2L12 mRNA expression differs significantly in
patients with LSCC, when classified according to the
TNM stage and or size of malignant tumors. Con-
cretely, patients with LSCC bearing tumors of advanced
TNM stage and or bigger than 2 cm displayed lower
BCL2L12 mRNA expression than patients with less
advanced tumors. Despite the fact that clinical comor-
bidities have been demonstrated to significantly affect
survival over TNM prognosticators (46), the TNM
tumor classification consistently correlates, on multivar-
iate survival analyses, with DFS and OS (47). Taking
into acco unt that the TNM staging of LSCC remains the
most significant predictor of survival and that BCL2L12
mRNA levels are high in early-stage patients, BCL2L12
could constitute an unfavorable prognostic indicator in
LSCC. In this framework, further investigation of the
potential correlation between BCL2L12 mRNA expres-
sion and DFS and or OS of patients with LSCC should
be worthy. Additionally, as aforementioned, BCL2L12
impedes the capacity of p53 to bind some of its target
gene promoters; hence, BCL2L12 expression could add
to the prognostic value of p53, the expression status of
which is, on its own, inconclus ive for LSCC (48–50).
Data about the prognostic value of HNSCC grading
are controversial. Although a number of older studies
had shown that the histological grade of HNSCC
correlates poorly with patient outcome (51, 52), more
recent studies support the notion that the prognostic
value of grading improves when the deeply invasi ve
Table 3 BCL2L12 mRNA expression
a
analysis in subgroups of
patients with laryngeal cancer, classified according to classical clini-
copathological parameters
Variable Mean ± SE
b
Median P-value
Tumor size
£2 cm 740.7 ± 189.6 635.7 0.043
c
>2 cm 235.1 ± 48.1 223.2
TNM stage
I 868.2 ± 286.9 625.8 0.027
c
II III 302.7 ± 59.6 231.8
Histological
grade
I 356.9 ± 83.2 381.3 0.64
d
II 656.9 ± 269.3 625.9
III 415.5 ± 210.9 223.6
a
a.u., arbitrary units.
b
Standard error of the mean.
c
Calculated using the Mann–Whitney U-test.
d
Calculated using the Kruskal–Wallis test.
Table 4 BCL2L12 mRNA expression
a
analysis in subgroups of
patients with tongue cancer, classified according to classical clinico-
pathological parameters
Variable Mean ± SE
b
Median P-value
Tumor size
£2 cm 481.6 ± 110.8 375.0 0.228
c
>2 cm 2082.7 ± 947.3 764.7
TNM stage
I 2964.8 ± 2196.3 852.6 0.187
d
II 550.1 ± 120.4 552.8
III 1551.7 ± 858.7 764.7
Histological
grade
I 514.1 ± 46.2 730.5 0.032
d
II 661.2 ± 43.6 623.3
III 3796.1 ± 1921.1 3267.1
a
a.u., arbitrary units.
b
Standard error of the mean.
c
Calculated using the Mann–Whitney U-test.
d
Calculated using the Kruskal–Wallis test.
BCL2L12 mRNAexpressioninHNSCC
Geomela et al.
6
JOralPatholMed
margins of the tongue tumor are evaluated (53, 54). The
current study showed that BCL2L12 mRNA expression
differs significantly in patients with TSCC, when clas-
sified according to the histological grade of their
malignant tumors. More specifically, patients with
TSCC bearing poorly differentiated tumors had signifi-
cantly higher BCL2L12 mRNA expression than patients
with well-differentiated malignant neoplasms. There-
fore, it is tempting to speculate that BCL2L12 acts anti-
apoptotically in TSCC, one of the most aggressive types
of HNSCC, resulting in the survi val of cancerous
squamous cells and the progression of the disease (55,
56). Similarly, recent oncogenomic studies uncovered
the anti-apoptotic role of the BCL2L12 oncoprotein in
primary glioblastoma multiforme (GBM), where it is
robustly expressed, at a significantly higher level than in
oligodendrogliomas and oligoastrocytomas (57), and or
surrounding normal brain tissue (16). Furthermore, the
BCL2L12 expression possesses significant discrimina-
tory value, distinguishing efficiently patients with TSCC
from non-cancerous population, as demonstrated by the
ROC analysis.
To the best of our knowledge, this is the first study
examining the BCL2L12 mRN A expression in HNSCC.
Our results suggest that BCL2L12 mRNA expression
might serve as a potential prognostic biomarker in
TSCC and or LSCC, which principally constitute the
great majority of HNSCC cases worldwide. Undoubt-
edly, further studies are needed to confi rm the present
findings and to examine in depth the prognostic value of
BCL212 expression in LSCC, TSCC, and other types of
HNSCC. Furthermore, it would be useful to try to
elucidate the molecular pathways of HNSCC in which
BCL2L12 is involved. Our future goals include com-
parison of BCL2L12 mRNA expression profile in a
large cohort of patients with LSCC and TSCC and in
healthy controls. Moreover, we will study the effect of
treatment for HNSCC cell lines with anticancer drugs,
commonly used in the chemotherapy of HNSCC.
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Acknowledgements
This work was financially supported by the Commission of the European
Community through the INsPiRE project (EU-FP7-REGPOT-2011-1,
proposal 284460).
Conflict of interest
The authors declare no conflict of interest.
BCL2L12 mRNAexpressioninHNSCC
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    • "High BCL2 protein expression is also associated with recurrent LSCC cases, primarily treated by radiation [40]. Additionally, loss of BCL2L12 mRNA expression was observed in LSCC of advanced TNM stage, in comparison with early-stage laryngeal malignancies [41]. To the best of our knowledge, this study is the first to examine the prognostic potential of the BAX/BCL2 ratio in LSCC. "
    [Show abstract] [Hide abstract] ABSTRACT: Objectives: Laryngeal squamous cell carcinoma (LSCC), a common type of head and neck cancer, is associated with high rates of metastasis and recurrence. Therefore, accurate prognostic stratification of LSCC patients based on molecular prognostic tumor biomarkers would definitely lead to a better clinical management of this malignancy. The aim of this study was the investigation of the potential combinatorial prognostic value of BCL2 and BAX mRNA expression in LSCC. Design and methods: Total RNA was isolated from 105 cancerous laryngeal tissue specimens obtained from patients having undergone surgical treatment for primary LSCC. After cDNA preparation, a low-cost, in-house developed, sensitive and accurate real-time quantitative PCR (qPCR) methodology was applied for the quantification of BCL2 and BAX mRNA levels. Then, we carried out a biostatistical analysis to assess the prognostic value of the BAX/BCL2 mRNA expression ratio. Results: High BAX/BCL2 mRNA expression constitutes a favorable prognosticator in LSCC, predicting significantly longer disease-free survival (P=0.011) and overall survival (P=0.014) of patients. More importantly, the significant prognostic value of the BAX/BCL2 mRNA expression appeared to be independent of the histological grade and size of the malignant laryngeal tumor as well as TNM stage, as revealed by the multivariate bootstrap Cox regression analysis. Kaplan-Meier survival analysis demonstrated also that the BAX/BCL2 ratio can stratify node-negative (N0) LSCC patients into two subgroups with significantly different DFS and OS (P=0.021 and P=0.009, respectively). Conclusions: The BAX/BCL2 mRNA ratio is a putative molecular tissue biomarker in CLL and hence deserves further validation in larger cohorts of LSCC patients.
    Full-text · Article · Apr 2016
    • "High BCL2L12 mRNA expression constitutes an unfavorable prognosticator in chronic lymphocytic leukemia [55,56], acute myelid leukemia [57], gastrointestinal cancer [58,59], and nasopharyngeal carcinoma [60]. On the other hand, BCL2L12 has been suggested as a potential molecular biomarker of favorable prognosis in breast cancer [61,62] as well as in head and neck squamous cell carcinoma [63] . Undoubtedly, research efforts investigating the diagnostic and/or prognostic utility of distinct BCL2L12 transcripts in human disease could become fruitful. "
    [Show abstract] [Hide abstract] ABSTRACT: The next-generation sequencing (NGS) technology has enabled genome-wide studies, providing massively parallel DNA sequencing. NGS applications constitute a revolution in molecular biology and genetics and have already paved new ways in cancer research. BCL2L12 is an apoptosis-related gene, previously cloned from members of our research group. Like most members of the BCL2 gene family, is highly implicated in various types of cancer and hematological malignancies. In the present study, we used NGS to discover novel alternatively spliced variants of the apoptosis-related BCL2L12 gene in many human cancer cell lines, after 3'-RACE nested PCR. Extensive computational analysis uncovered new alternative splicing events and patterns, resulting in novel alternative transcripts of the BCL2L12 gene. PCR was then performed to validate NGS data and identify the derived novel transcripts of the BCL2L12 gene. Therefore, 50 novel BCL2L12 splice variants were discovered. Since BCL2L12 is involved in the apoptotic machinery, the quantification of distinct BCL2L12 transcripts in human samples may have clinical applications in different types of cancer.
    Full-text · Article · Jan 2016
    • "In addition, cyclin D3 (CCND3) mRNA overexpression and CCND1 gene amplification were shown to independently predict poor OS in this malignancy (Bellacosa et al., 1996; Pruneri et al., 2005). Many other molecules have also been studied in LSCC, including markers of proliferation Ki-67 (MKI67) and proliferating cell nuclear antigen (PCNA) (Franchi et al., 1996; Liu et al., 2003), proapoptotic as well as antiapoptotic BCL2 family proteins (Condon et al., 2002; Geomela et al., 2013), members of the epidermal growth factor receptor (EGFR) family (Ganly et al., 2007; Almadori et al., 2010) and the vascular endothelial growth factor (VEGF) subfamily (Teknos et al., 2002; Sullu et al., 2010), E-cadherin (Franchi et al., 1996), and cathepsins B and D (Maurizi et al., 1996; Li et al., 2011). Furthermore, some microRNAs (miRNAs) appear as emerging tumor biomarkers in LSCC (Saito et al., 2013; Zhao et al., 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: Abstract Several members of the family of tissue kallikrein and kallikrein-related peptidases have been suggested as promising tumor biomarkers with important prognostic significance. However, only one (KLK11) has already been studied in laryngeal squamous cell carcinoma (LSCC) as potential biomarker for LSCC diagnosis and/or prognosis. Our study investigated the prognostic value of kallikrein-related peptidase-4 (KLK4) mRNA expression as a molecular tissue biomarker in LSCC. For this purpose, KLK4 mRNA expression analysis was performed in 116 cancerous and 74 paired non-cancerous laryngeal tissue specimens obtained from patients having undergone surgical treatment for primary LSCC. A remarkable downregulation of KLK4 mRNA expression was noticed in laryngeal tumors, compared to non-cancerous laryngeal tissue specimens. KLK4 mRNA expression was also shown to distinguish well LSCC from non-cancerous laryngeal tissues. Furthermore, low KLK4 mRNA expression was shown to predict poor disease-free survival, independently of the histological grade and size of the malignant tumor as well as patient TNM stage. According to Kaplan- Meier survival analysis, low KLK4 mRNA expression predicts short-term relapse even among patients with well-differentiated tumors or those at an early TNM stage. Thus, KLK4 mRNA positivity could be regarded as a novel independent indicator of favorable prognosis for the disease-free survival of LSCC patients.
    Full-text · Article · Apr 2014
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