CD326 EVALUATION IN HEPATOCELLULAR CARCINOMA PATIENTS WITH AND WITHOUT PORTAL VEIN TUMOR THROMBOSIS

CD326 EVALUATION IN HEPATOCELLULAR
CARCINOMA PATIENTS WITH AND WITHOUT
PORTAL VEIN TUMOR THROMBOSIS
Mohamed Elshal1 ; Mohamed Y. Nasr 2; Mahmoud Gadallah 3
and Aya Nasser*4
1Professor of molecular biology, Department of biochemistry and molecular biology,
Genetic Engineering and Biotechnology Research Institute, Sadat University. Egypt.
2 Lecturer of Molecular Diagnosis, Molecular Diagnosis Department, Genetic
Engineering and Biotechnology Research Institute, Sadat City University, Egypt.
3 Lecturer of Molecular Diagnosis, Molecular Diagnosis Department, Genetic
Engineering and Biotechnology Research Institute, Sadat City University, Egypt.
4 Master degree in Department of biochemistry and molecular biology, Genetic
Engineering and Biotechnology Research Institute, Sadat City University, Egypt
*01111710212 aya@gmail.com
Key Words: cluster of differentiation 326, circulating tumor cell, patient
management, hepatocellular carcinoma, flow cytometry
ABSTRACT.
Aim : to evaluated the presence and clinical relevance of a cluster of
differentiation (CD)326+subset of circulating tumor cells (CTCs) in
blood samples of hepatocellular carcinoma patients with portal vein
tumor thrombus and without portal vein tumor thrombosis , who had
undergone curative or palliative intervention, in order to find a novel
prognostic factor for patient management and follow-up.
Method: In total, 47 HCC patients, along with 21 with PVTT and 26
without PVTT were included. The easily transferable methodology of
flow cytometry, along with multiparametric antibody staining were used
to selectively evaluate CD326+CTCs in the peripheral blood samples of
HCC patients. The multiparametric selection allowed any enrichment
methods to be avoided thus rendering the whole procedure suitable for
clinical routine.
Result: The presence of CD326+ cells was strongly correlated with poor
survival and high metastasis rates in this novel patient population .as well
as PVTT probability for CD 326 > or < 0.45 was 88.0%. Several factors
independently correlated with CD326 using univariate multiple logistic
regression of CD 326 with laboratory parameters Correlation was found
between EPCAM activity and AST, ALT, AFP at P Value less than 0.01
In conclusion: CD326+ CTCs are an independent prognostic factor for
tumor aggressiveness rate in multivariate analysis, suggesting that their
evaluation could be an additional factor for liver cancer progression risk
evaluation in patient management
Egypt. J. of Appl. Sci., 35 (11) 2020 188-199
INTRODUCTION
Hepatocellular carcinoma (HCC) is responsible for significant
morbidity and mortality in cirrhosis and also accounts for between 85%
and 90% of primary liver cancer[1-2] . Most of HCCs in the world occur
in the setting of cirrhosis and over half-million of people develop liver
cancer every year and an almost equal number die of it[3] . The most
important causes leading to HCC are the HBV and HCV infections,
heavy alcohol consumption, aflatoxin B1, age and gender (males are
more susceptible than females), tobacco consumption, obesity associated
with nonalcoholic fatty liver disease, and the increase of the Diabetes II
mellitus (that rises the risk factor between 2 and 3), genetic
hemochromatosis, primary biliary cirrhosis, and alpha1-antitrypsin
deficiency and autoimmune hepatitis [4] .
Usually HCC develop during long process of inflammation and
fibrosis eventually leading to cirrhosis [5], Portal vein tumour thrombosis
is common complications of HCC that occur in approximately 10_40 /of
HCC patient at time of diagnosis .although great improvement have been
made in diagnosis of HCC with PVTT and treatment during the past few
decades the long term survival of these patients remain unfavourable, due
to high mortality. conventional prognostic marker for HCC alpha feto
protein and tumor node metastasis, but their value varies between
patients with this diseases[6],therefore new method are needed to provide
predictive information about existing metastasis and probability of early
recurrence.
In the field of biology of tumors, some expressions have been coined
for the different types of circulating cellular elements. The term circulating
tumor cells (CTC) defines a small population of cancer cells that have
escaped from the primary tumor into the body’s circulatory system, and
establish metastases in distant organs, The presence of CTC reflects the
aggressiveness nature of a solid tumor. Many attempts have been made to
develop assays that reliably detect and enumerate these cells. The clinical
results obtained with such assays suggest that in some tumor types, CTC
detection and identification can be used to estimate prognosis and may serve
as an early marker to assess antitumor activity of treatment.
In addition, CTC can be used to predict progression-free survival
and overall survival. CTC are an interesting source of biological
information in order to understand dissemination, drug resistance and
treatment-induced cell death[7-8] .
In HCC animal models showed that 10 to 10 000 CTC are capable
to initiate new metastasis . Even after curative resection, the tumor
recurrence rate remains high. [9]
Although CTC detection has been applied and well documented in
different types of cancer, CTC detection is not routinely performed in
HCC follow-up and remains in the experimental field. However ,HCC
189 Egypt. J. of Appl. Sci., 35 (11) 2020
CTC detection might bring new interesting information of metastatic
process might be used as diagnostic tool of early recurrence and may
allow a better patient selection for liver transplantation. [10] but there
are a number of problems affect CTC identification and enumeration;
each method used for their detection has several drawbacks[11] .The
scarcity of CTCs in peripheral blood samples means that an enrichment
step is often required prior to the analysis[12] , however, all methods
used to enrich CTCs from blood samples (i.e., filtration, density gradient
separation and magnetic isolation) exhibit a low purity grade[13]
Also , major techniques used to identify CTCs (i.e., quantitative
polymerase chain reaction and CellSearch system) are too expensive and
time consuming to be predominantly used in the clinical routine[14] . In
addition, nucleic acid-based methods are markedly affected by the
presence of a huge number of contaminant cells inside peripheral blood
samples[15] while intact cell analysis by the use of specific markers is
defective due to the lack of unambiguous specificities[16] ; in fact
several of the markers expressed by cancer cells are shared by
leucocytes. Flow cytometry is a suitable technique to analyze CTCs , as it
allows single cell analysis and permits the researcher to include or
exclude doubtful origin populations and suspect objects from the analysis
at any time following sample acquisition. The universally recognized
markers of CTCs are the epithelial specificities, CD326 (EpCAM) and
the cytokeratins , [17] however, recent findings have highlighted the
complex nature of cancer cell dissemination, which involves deep cell
changes, including the epithelial-to-mesenchymal (EMT) transition . The
types of modifications that occur in the cell in such transitions are not
univocally clarified; it has been demonstrated that cells reduce the
epithelial characteristics as mesenchymal features appear , this transition
appears to promote cancer cell dissemination[18]
In addition, it has been reported that intermediate phenotypes are
observable in CTCs with the presence of epithelial and mesenchymal
markers[19] . The assessment of biologically significant markers is
likely to provide more clinically relevant information than simple
enumeration[20] ,so Recent work suggest using Flow cytometry to
identify expression of CD326 as tumor marker in CTCs of
hepatocellular carcinoma with PVTT compared to patient without PVTT
Egypt. J. of Appl. Sci., 35 (11) 2020 190
for improvement standard cancer staging criteria and assessment risk at
early stage.
Recruitment of individuals
Patients collected between January 2020 and March 2020 from
HCC CLINIC, NATIONAL LIVER INSTITUTE, Menoufia university.
Patients with diabetes were excluded from these studies. In total, 47
patients, consisting of 26 without portal vein tumor thrombus and 21 with
portal vein tumor thrombus patients, were included. According to
Magnetic resonance imaging and computerized tomography scan 9 HCC
patients with patent portal vein and 17 HCC patients with distal portal
vein. Furthermore, 21patients, including 16 patients with partial portal
vein tumor thrombus In the remaining five patients with complete portal
vein tumor thrombus.
The following clinicopathological parameters were recorded: Age,
gender, blood transfusion , surgery .focal lesion number , ALT,AST,
AFP
The following was done for each patient:
1-Flowcytometric technique
10 ml were withdrawn from 48 HCC patient to measure the
percentage of CD326+ cells using flowcytometric technique. These blood
samples were separated by RBCs lysing buffer Then, the mononuclear
cells were extracted. Using phosphate buffer solution (PBS), a process of
washing the mononuclear cells is done twice by incubation for 7 minutes
at room temperature and Centrifugation at 250 x g for 7 minutes. After
that, the staining process is done by adding (FITC which was conjugated
with anti-CD 45 antibody, PE which was conjugated with anticd326
antibody) into appropriate amount of sediment. This system was placed
in a dark place for 15 minutes at 37°C; after which the cells were washed
with PBS. Finally, the CD326+cells are measured by flowcytometry
(Miltenyi Biotec, Germany).
2- Liver enzyme ,were represented by measuring the level of AST, ALT,
AFP by automated biochemistry Huma Star 300 SR German.
3- To assess infection with hepatitis B virus and hepatitis C virus
were measured using a (Stat Fax-4200 USA) device, qualitatively and
quantitatively, depending on the technique of immune absorption linked
to the enzyme (ELISA).
4- Statistical analyses were performed by IBM® SPSS® Statistics
software version All data are expressed as mean ± standard deviation
(X±SD). Comparisons were analyzed by ANOVA one-way analysis A
value of P <0.05: significant, P < 0.001: more significant. that was
considered statically significant. ROC curve was done to determine the
cutoff point, area under curve (AUC), sensitivity (Sn), specificity (Sp),
191 Egypt. J. of Appl. Sci., 35 (11) 2020
positive predictive value (PPV) and negative predictive value (NPV) of
presences of portal vein thrombosis.
Measure the diagnostic power of indirect scores which came in modern
scientific journals according to the following equations:
1- AST: platelets ratio index (APRI) as calculated using
a s formula: (AST (upper limit of normal)/ALT (IU/L)
×100)/platelet count (platelets x 109/L) X 100.
2- Fibrosis index (FI) was calculated using this formula as:
8.0-0.01 x platelet count (x109/L) – serum albumin (g/dl) .
RESULT
Total of 50 patient were included in this study 3 patient were
omitted because of missing data leaving 47 patients in final analysis 26
patient were HCC without PVTT. median age in all studied group
(patent, dilated, partial and complete PVTT)(table 1)
(60.0 83.5±,8538. 83.5±, .3..±.1306, .3.5±.8351)
Majority were male n = 39, and 44.6 % had HCC with portal vein
tumor thrombus , etiology of underlying liver cancer included ( HCV n
=46, HBV n = 1).
Table 1: Comparison between studied groups as regard Age and
Gender
Groups
Variables
Group1
N=9
Group2
N=17
Group3
N=16
Group4
N=5
Gender
Male 9
100%
13
76.5%
14
87.5%
3
60%
Female
0
0.00%
4
23.5%
2
12.5%
2
40.0%
Age (mean ± sd ) 60.00±5.38 58.53±5.68 60.19±6.66 65.40±6.34
Mean blood level of CD326 in 4 studied group was (0.49+ 1300 , 03.6+
.31.,83.5 +.305 , .3.8 +8366 )(table 2), the enzyme activities of ALT ,AST in
partial portal vein tumor thrombus[ 94.63 + 3.12 , 160.06 + 8.06] and complete
PVTT patients [ 99.40 + 0.89 , 181.40 + 5.77] were significant higher than in
patient without PVTT. . In contrast, significant differences were found in AFP
between (patent, dilated) versus (partial, complete) (P = 0.000).
Table 1: Comparison between studied groups as regard Laboratory
data
Groups
Parameters
Group1 N=9
Mean ± SD
Group2(N=17)
Mean ± SD
Group3(N=16)
Mean ± SD
Group4(N=5)
Mean ± SD
p-value
ALT (U/L) 53.89 + 5.07 63.75 + 6.63 94.63 + 3.12 99.40 + 0.89 0.000
AST(U/L) 104.67 + 3.71 127.59 + 8.65 160.06 + 8.06 181.40 + 5.77 0.000
AFP(ug/ml) 11.89 + 3.09 99.53 + 123.38 503.69 + 103.97 964.80 + 145.52 0.000
Cd326 0.49 + 0.77 1.39 + 3.06 2.68 + 3.18 6.62 + 5.99 0.008
Egypt. J. of Appl. Sci., 35 (11) 2020 192
Correlation between CD326 and different variable are illustrated in
table 3
highly significant association was observed between EPCAM CD
326 and the AFP . furthermore liver enzyme ALT and AST were
significant associated P< 0.01 with CD326 in all HCC patient included
in this study .
Table 3: correlation between CD326 and liver function test
Variables
Cd326
patent PV
N=9
dilated PV
(N=17)
partial
PVTT
(N=16)
complete
PVTT
(N=5)
Total
(N =47)
ALT (U/L) 0.670* 0.657* 0.761** 0.936* 0.531**
AST(U/L) 0.779* 0.577* 0.926** 0.920* 0.606**
AFP(ng/ml) 0.770* 0.733** 0.880** 0.944* 0.687**
**: significant at p= 0..01 *: significant at p= 0..05
The diagnostic abilities of CD326 versus those of APRI and FIB4
illustrated in table 4
Unfortunately, there are not significant correlation between CD326
and (APRI or FIB 4) among 4 studied group
cytometric analysis for CD326
A multiparametric cytometric analysis was used to evaluate the
CD326+ CTCs present in the peripheral blood samples of the PVTT HCC
patients and without PVTT HCC . The cells were first gated on physical
parameters in a dot plot [forward scatter versus side scatter (SSC)] to
exclude debris. Subsequently, in a CD45 versus SSC dot plot, the CD45-
cell population position was identified by discarding all hematopoietic
contaminants. Finally, a CD326 dot plot, + CTCs were identified ,finally
we calculated the cut point using the partial and complete PVTT groups
together as positive HCC group ,patient without PVTT(n= 26) served as
negative group .ROC analysis showed the best cut point at a sensitivity of
about 81% , 88% for CD26 and CD 326 . The specificity and sensitivity
values are as follows in fig 1.
Table 4: correlation between CD326 and (APRI, FIB 4)
CD326
complete pv
(N=5)
patent pv
N=9
dilated pv
(N=17)
partial pv
(N=16)
Variables
APRI SCORE -0.41102 -0.15308 -0.18365 -0.25981
FIB 4 SCORE -0.20775 -0.12181 -0.15877 -0.45762
DISCUSSION
In the past few decades, many promising candidate biomarkers for HCC had
been found, but most of them were not applied to clinical diagnosis due to their
limited practicability and high cost
[21] . Nevertheless, these new markers have
potential to be applied in clinical diagnosis for their higher sensitivity and
specificity. So far, 𝛼-fetoprotein (AFP) and imaging technology (e.g., ultrasound or
computed tomography) are two primary methods to diagnose HCC in hospitals.
193 Egypt. J. of Appl. Sci., 35 (11) 2020
AFP has been used as a serum marker for HCC for many years, but its sensitivity
was only about 39%–65% [22]
Recently, we have identified epithelial cell adhesion molecule (EpCAM), to be
an early biomarker of HCC because its expression is highly elevated in
premalignant hepatic tissues
[23] . EpCAM (also known as CO17-1A, EGP, EGP40,
GA733-2, KSA, Ly74, M1S2, M4S1, MIC18, MK-1, TROP1, and hEGP-2) is
highly expressed in many human cancers with an epithelial origin.
The function of EpCAM and the regulation of its expression are largely
unknown[24] . In the adult liver, hepatocytes are negative and bile duct epithelium
is positive for EpCAM expression[25] . However, in the embryonic liver, the
majority of hepatocytes express EpCAM . In the cirrhotic liver, EpCAM is
expressed in proliferating bile ductulus that are thought to be derived from HPC[26]
. but its distribution pattern is altered in hepatocellular carcinoma according to
previous study published by Gi Hong Choi who examined mRNA levels for K19,
EpCAM, and CD44 in peripheral blood and HCC tissues before and after operation
using real-time RT-PCR. Study participants were divided into high and low ratio
groups, according to the ratio of postoperative to preoperative mRNA levels for
each marker.. . their Findings revealed that Preoperative peripheral levels of K19
and EpCAM mRNA were higher in liver transplantation patients than in resection
patients, and they were also significantly higher in cirrhotic patients of Child–Pugh
Class B or C than those of Child–Pugh Class A (p\0.05 for all).. Preoperative
peripheral levels of K19 and EpCAM mRNA were influenced by background liver
status and HCC. Additionally, the ratio of postoperative to preoperative mRNA
levels for CSC markers, especially K19 and EPCAM, was shown to be important to
predict HCC recurrence[27] .
Another study by Taro Yamashita who identified EpCAM-positive HCCs by
cDNA microarray in 40 HCC cases and validated by oligonucleotide microarray
analysis of 238 independent HCC cases, which was further confirmed by
immunohistochemical analysis of an additional 101 HCC cases. EpCAM-positive
HCC displayed a distinct molecular signature with features of hepatic progenitor cells
including the presence of known stem/progenitor markers such as cytokeratin 19, c-
Kit, EpCAM, and activated Wnt-B-catenin signaling, whereas EpCAM-negative
HCC displayed genes with features of mature hepatocytes. Moreover, EpCAMpositive
and EpCAM-negative HCC could be further subclassified into four groups
with prognostic implication by determining the level of A-fetoprotein (AFP). These
four subtypes displayed distinct gene expression patterns with features resembling
certain stages of hepatic lineages. Taken together, they proposed an easy classification
system defined by EpCAM and AFP to reveal HCC subtypes similar to hepatic cell
maturation lineages, which may enable prognostic stratification and assessment of
HCC patients with adjuvant therapy and provide new insights into the potential
cellular origin of HCC and its activated molecular pathways[28] .
These result once more underline the pathological role of CD326 in HCC
Therefore, the current study selected EPCAM to explore the CTC populations in the
Egypt. J. of Appl. Sci., 35 (11) 2020 194
peripheral blood of HCC patients by flow cytometry. Our goal was to validate the
usefulness of the CD326 as diagnostic marker in progression liver cancer
Our cohort revealed that Portal vein tumor thrombosis patients of HCC had the
highest number of CD326+ CTCs and Blood levels of CD326+CTCs correctly
predicted tumor prognosis in 85,7%of the cases, by contrast , negative patient (patent
, dilated) expressed decrease number of cd 326+ CTC in 84,4% of cases, with a test
specificity of 88 %. (Fig 1).
Our second goal was to examine possible correlation between CD 326 activity
and other laboratory parameters of liver function. We focused on 3 parameters that
were associated with prognosis in HCC patients. included were markers of liver
damage such as AST, ALT,. also included were AFP that were correlated with tumor
aggressiveness.
Consistent with previous published data by UC Okonkwo et al who analyzed
bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline
phosphatase (ALP) and albumin in 64 patients with HCC and 120 patients without
HCC they observed that . , AST and ALT were elevated in 30(46.9%) and 31(48.4%)
patients, respectively, while ALP was elevated in 33(52%). Hyper bilirubinaemia was
present in 34(53%) and hypo albuminaemia in 54(84.3%) of the patients. Except for
bilirubin, liver function test was more frequently abnormal in HCC than in non-HCC
cases[29] This seems logical and in agreement with Our study which revealed
highly significant difference among studied groups in liver enzyme (AST,ALT) at
P<0.001 and highly significant correlation with CD326 at P <0.001
In present study we showed highly significant correlation between Cd326 and
AFP, at p value< 0.01 in total 47 patient, similar to our result Leonardo do Prado
Lima, et al, (2018) concluded that. EpCAM+ expression was associated with AFP +
(OR = 12.5, 95% CI, 1.9-84.1, p<0.001) in child Pugh A HCC patient undergoing
curative surgical resection [30] .
As predicted in our study, HCC patients showed a positivity for CD326+ even
when the primary cancer had a low pathological grade, suggesting that CTCs can
identify high-risk HCC patients at early stages. Notably, the spread of CD326+ cancer
cells into the blood circulation is a common event and 81% of all analyzed patients
exhibited these cells in their peripheral blood (figure 1) we have also proven to be
useful in detecting minor subgroups of cells present in the primary tissue which might
eventually be the cause of treatment resistance or relapse of the disease.
Fig 1: CD326
195 Egypt. J. of Appl. Sci., 35 (11) 2020
ROC curve of CD326 between Patients group
Cut off
Area
under
the
curve
Sensitivity specificity PPV NPV
Pvalue
CD326 >0.45 0.831 88.0 81.0 84.6 85.7 0.01
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لمرضي سرطان الکبدية مع او بدون الوريد البابي المتخثر CD تقييم 326
محمد الشال 1 ، محمد يونس نصر 2 ، محمود جاد الله 3 ، آية ناصر 4
-1 استاذ بقسم الکيمياء الحيوية والبيولوجيا الجزيئية ، معيد بحوث اليندسة الو ا رثية والتکنولوجيا الحيوية
، جامعة السادات، مصر
2 - مدرس بقسم الکيمياء الحيوية والبيولوجيا الجزيئية ، معيد بحوث اليندسة الو ا رثية والتکنولوجيا
الحيوية ، جامعة السادات، مصر
3 - مدرس بقسم الکيمياء الحيوية والبيولوجيا الجزيئية ، معيد بحوث اليندسة الو ا رثية والتکنولوجيا
الحيوية ، جامعة السادات، مصر
-4 ماجستير بقسم الکيمياء الحيوية والبيولوجيا الجزيئية ، معيد بحوث اليندسة الو ا رثية والتکنولوجيا
الحيوية ، جامعة السادات، مصر
يعد سرطان الکبد مسؤول عن الوفاة بنسبة 85 % الي 90 % و معظم الحالات بسبب تميف الکبد
و نصف مميون شخص حول العالم يموت بسبب ذلک و من اىم اسباب سرطان الکبد فيروس الکبد ب ،
فيروس الکبد س ، الاف ا رط في تناول الکحول و السمنة المفرطة و مرضي السکري النوع الثاني
عادة ، سرطان الکبد يتطور خلال عممية طويمة من الالتياب و التميف ، مما يؤدي في النياية
إلى تميف الکبد تم السرطان
في مجال بيولوجيا الاو ا رم وجدت العديد من التعبي ا رت المسؤلة عن الکشف عن الخلايا السرطانية
و في ىذا السياق يمکن تعريف الخلايا السرطانية بانيا خلايا ىربت من ورم ابتدائي و انتقمت في الدم
Egypt. J. of Appl. Sci., 35 (11) 2020 198
لکي تستوطن في عضو اخر وکونت مايسمي بالانبتاث و ىذه الخلايا مسؤلة عن تطور المرض وليذا
فان ىناک العديد من المحاولات لفحص ىذه الخلايا وعدىا و بالتاي تکمن اىميتيا في معرفة مدي تطور
المرض
Cell و PCR ىناک العديد من التقنيات التي استخدمت لعد الخلايا السرطانية مثل جياز
و لکن تکمفتيا عالية و ميدرة لموقت و لکن الابحاث الجديدة اثبتت فعالية جياز search system
التدفف الخموي فيو يقوم بعد خمية خمية و يقوم باستئصال الخمية المشکوکة فيو
CD وليذا فان بحثنا يقوم بعد الخلايا السرطانية الموجودة في سرطان الکبد عن طريق عد 326
الموجود عمي اسطح الخلايا EPCAM او
المرضى الذين استخدموا في الد ا رسة جمعت بين کانون الثاني / يناير 2020 آذار / مارس عام
2020 من معيد الکبد جامعة المنوفية. المرضى الذين يعانون من مرض السکري استبعدوا من ىذه
الد ا رسة. في المجموع 47 المرضى ، تتکون 26 بدون وريد بابي متخثر و 21 مع الوريد البابي المتخثر
تم اخد جميع بيانات المريض
و قمنا بالتالي لکل مريض
AST, ALT , AFP -1 تحميل وظائف الکيد
الم وجود عمي الخلايا السرطانية CD -2 قياس نسبة 326
APRI -3 قباس
FIBRO index -4 قياس
-5 التحاليل الاحصائية
اظيرنسبة ايجابية في مرضي الحالات المبکرة من سرطان الکبد و استطاع CD لقد و جدنا ان 326
و انزيمات الکبد ,و لم يظير AFP تشخيص المرض بنسبة % 80 کما انو اظير علاقة ايجابية مع
APRI , FIBRO INDEX اي علاقة مع
199 Egypt. J. of Appl. Sci., 35 (11) 2020