HEPARANASE EXPRESSION IN TISSUE OF EARLY STAGE CERVIX CANCER

PATIENT RECEIVING PACLITAXEL – CARBOPLATIN NEOADJUVANT

CHEMOTHERAPY

Avicena Muhammad Iqbal, Heru Priyanto, Supriyadi Hari Respati, Sri Sulistyowati,
Uki Retno
Fakultas Kedokteran, Universitas Sebelas Maret
ABSTRACT
Introduction: Cervical cancer is number two cancer causing death in women under 45 years
old in the world. In Indonesia, cervical cancer is number one malignancy occurred in women
(17,8%). Neoadjuvant chemotherapy is chemotherapy which is given before main therapy
(radiotherapy and radical hysterectomy). One of the prognostic factors in patients with
invasive cervical cancer is heparanase expression. Heparanase is a endoglucoronidase that
has a role in extracellular matrix degradation, and may be important in cancer invasion and
metastasis. This study aim to analyze the difference of heparanase expression in tissue
before and after the administration of neoadjuvant chemotherapy.
Method: Pre and post test experimental study, consist of 30 early stage cervix cancer patients
(Ib2 dan IIA2 stadium) with bulky lesions in Obstetry and Gynecology Department in Dr.
Moewardi Hospital, which were given neoadjuvant chemotherapy using 3 series Paclitaxel
– Carboplatin on Desember 2017 – June 2018. Before and after neoadjuvant chemotherapy,
a biopsy and immunohistochemical examination was performed to assess heparase
expression by the Anatomy Pathology Department. The difference in the biomarker’s mean
value of pretest and posttest was analyzed using T-test.
Result: About 80% of patients (24 people) are aged 40-60 years, with 2-4 parity in 21 people
(70.0%). Most of the patients are in the Stage IIA2 (27 people or 90.0%) compared to stage
IB2 with only 3 patients (10.0%). The type of most cells were Squamous Cell Carcinoma
(83.4%), others were Adenocarcinoma cells (13.3%) and Small Cells (3.3%). In most stage
IIA2 cervical cancer patients (46.7%) there was a decrease in heparanase expression after
being given Paclitaxel-Carboplatin neoadjuvant chemotherapy. The mean of Heparanase
expression level before the administration of Paclitaxel-Carboplatin neoadjuvant
chemotherapy (5.37) is higher compared with after the administration of Paclitaxel –
Carboplatin neoadjuvant chemotherapy (4.53) with a Z-score = -2.212; p = 0.027 (p <0.05).
This shows a significant difference between Heparanase expression levels before and after
Paclitaxel – Carboplatin neoadjuvant chemotherapy.
Conclusion: The expression of heparanase in early stage cervical cancer patients is
decreased after the administration of Paclitaxel – Carboplatin neoadjuvant chemotherapy.
Keywords: Heparanase, Neoadjuvant Chemotherapy, Cervical Cancer
INTRODUCTION

Cervical cancer still becomes a health problem for women in developing countries
including in Indonesia due to the high incidence and mortality rate. 1Cervical cancer is
number two cancer causing death in women under 45 years old in the world, and is currently
the third most common reason for cancer death amongst women worldwide after breast and
lung cancer. 2In Indonesia, cervical cancer is the first malignancy in women (17.8%). 3 One
of the major problems in the treatment of cervical cancer is symptoms that are not typical at
an early stage so that most patients come to the hospital at an advanced stage.
Neoadjuvant chemotherapy is a chemotherapy which is given before primary therapy
such as radiotherapy or radical hysterectomy. One of the goals of neoadjuvant chemotherapy
is to reduce the size of cervical lesions and cancer invasion in the vaginal wall, so that the
radicality of surgery increases.4Neoadjuvant chemotherapy is given to early stage cervical
cancer before radical hysterectomy is performed, especially in patients with lesions more
than 4 cm or called bulky lesion. 5 Carboplatin / Paclitaxel is an adjuvant chemotherapy
regimen that improves the progress of free survival and quality of life, and it can be used
easily with lower resistancy.6,7
Heparanase is endo-ß-D-glucoronidase which catalyzes the hydrolysis of ß-1,4-
glycosidic bonds between D-glucoronate and D-glucosamine in heparan sulfate. Heparan
sulfate and heparan sulfate proteoglycans (HSPGs) are found in extracellular matrix,
basement membrane and cell surface. The expression and secretion of heparanase is related
to the degradation of extracellular matrix and cancer cell metastasis and also increases
angiogenesis. Increased expression of heparanase is found in various types of tumors and
correlates with poor prognosis.8,9 Examination of biomolecular markers related to the
developing and metastatic cancer cells ability is expected to be used as a prognostic factor
to evaluate the response of neoadjuvant chemotherapy.

AIM
1. To determine the differences of Heparanase expression in tissue of cervical cancer
patients who had been given Paclitaxel-Carboplatin neoadjuvant chemotherapy.
2. To determine whether Heparanase expression in tissue of early stage cervical cancer can
be used as a parameter to evaluate chemotherapy responses.

METHODS
This study was a pre and post test experimental study, consist of 30 early stage cervical
cancer patients (IB2 and IIA2) with bulky lesions (diameter of cervical lesions> 4 cm) and
had never received treatment for cancer in the Obstetrics and Gynecology Department Dr.
Moewardi Hospital. All patients who fitted in the inclusion criteria were received 3 series
of neoadjuvant chemotherapy Paclitaxel - Carboplatin at intervals between chemotherapy
for 3 weeks in December 2017 - June 2018.
Before and after neoadjuvant chemotherapy, a biopsy and immunohistochemical
examination was performed to assess liver expression by the Anatomy Pathology
Department. The heparanase expression in the tissue used heparanase antibody from Abcam
Cambridge Science Park, UK, 2012. Staining reagent used Avidin Biotin Complex from
Biocare Medical, LLC, Concord, USA. The unit of measurement was the addition of the
colored field percentage and the percentage of its intensity. To analyze the difference in the
biomarker’s mean value of pretest and posttest was analyzed using T-test.

RESULTS
The results of this study are demographic data includes age, education, parity, stage
and cell carcinoma types. Based on age, there were <40 years old (3 people or 10.0), 40-60
years old (24 people or 80.0%), and >60 years old (3 people or 10.0%) with an average age
was 47.93 years old.
From the education data, there were patients who graduated from elementary school
(15 people or 50.0%), junior high school (4 people or 13.33%), senior high school (8 people
or 26.7%), diploma (1 people or 3.3%), and undergraduate education (2 people or 6.7%).
Based on pregnancy status obtained by patients with 0-2 parity (8 people or 26.7%), 2-4
parity (21 people or 70.0%), and >4 parity (1 person or 3.3%).
In this study, most patients were in stage IIA 2 (27 people or 90.0%) compared to
stage IB2 which only 3 patients (10.0%). The type of most cells were Squamous Cell
Carcinoma (25 people or 83.4%), others were Adenocarcinoma cell types (4 people or
13.3%), and Small Cell types (1 person or 3.3% )
Table 1. Research subjects demographic data based on cell type (histology)

Histology Amount Percentage

Squamous Cell Carcinoma 25 83,4%

Adenocarcinoma 4 13,3%

Small Cell 1 3,3%

Source: Primary Data (2018)

Table 2 shows that in most stage IIA2 cervical cancer patients (46.7%) there was a
decrease in heparanase expression after being given Paclitaxel-Carboplatin neoadjuvant
chemotherapy.
 Tabel 2. Clinic Pathological Relationship with Hheparinase Expression Level
Amount Heparanase
Variable
of cases (-) 0 (+)
Stadium
I B2 3 1 (3.3%) 1 (3.3%) 1 (3.3%)
14 9 (30.0%) 4 (13.3%)
II A2 27
(46.7%)

Table 3. Difference mean test of Heparanase Expression Level in tissue of early stage cervical
cancer before (pre-test) and after (post-test) Paclitaxel-Carboplantin Neoadjuvant
Chemotherapy
Heparanase
Z
Group N Expression SD P
Score
Mean Level
Before Paclitaxel-
Carboplantin 1,1
30 5,37
Neoadjuvant 89
Chemotherapy - 0,0
After Paclitaxel- 2,212 27*
Carboplantin 1,4
30 4,53
Neoadjuvant 08
Chemotherapy
Notes: * p < 0,05 was statistically significant

Table 3 shows the heparanase expression mean level before the administration of
Paclitaxel–Carboplatin neoadjuvant chemotherapy (5.37) was higher than after the
administration of Paclitaxel – Carboplatin neoadjuvant chemotherapy (4.53) with Z-score =
-2.212; p = 0.027 (p <0.05). This shows a significant difference between the heparanase
expression mean level before and after the administration of Paclitaxel – Carboplatin
neoadjuvant chemotherapy. Therefore Paclitaxel – Carboplatin neoadjuvant chemotherapy
was effective in heparanase expression mean level in early stage cervical cancer lesion
tissue.
 5.6
Heparanase Expression Mean 5.37
5.4 Pretest rerata
Pre-test of Heparanase
kadar
Expression Mean
ekspresi Heparanase
5.2 Level
5
Level

4.8 Post testpertama

Post-test of
Heparanase
rerata Expression
kadar ekspresi
4.6 4.53
Mean Level
Heparanase
4.4
4.2
4

Figure 1. Histogram of Heparanase expression levels in early stage cervical cancer tissue
lesion before and after Paclitaxel – Carboplatin neoadjuvant chemotherapy.

Figure 2. Microfotographic description of Heparanase expression in tissue. Figure A was

an expression of Heparanase in tissue before chemotherapy. Figure B was an image of
Heparanase expression in tissue after chemotherapy at 10x magnification. Pictures C and D
at 400x magnification. In figure C, the black arrow shows a strong expression of the
cytoplasm with dark brown color. Yellow arrow shows strong expression of intercellular
(dark brown color). Figure D, black arrow shows expression of Heparanase in cytoplasm,
and yellow arrow shows Heparanase expression in intercellular after neoadjuvan
chemotherapy (light brown color). Brownish color was a staining reaction of Heparanase
antibody with hematoxylin eosin. Examination used Cx 21 Olympus microscope at 400 x
magnification.

Table 4 shows that the heparanase expression variable in tissue of early stage cervical
cancer before the administration of Paclitaxel-Carboplatin neoadjuvan did not have a
significant relationship with the heparanase expression variable in tissue of early stage
cervical cancer after the administration Paclitaxel-Carboplatin neoadjuvant (p = 0.925; p >
0.05). The relationship between the expression of heparanase in tissue of early stage cervical
cancer before and after the administration of Paclitaxel-Carboplatin neoadjuvant was r = -
0.018 (very weak: 0.00-0.199). The correlation or relationship was negative, it can be
concluded that the higher of heparanase expression level in tissue of early cervical cancer
before the administration of Paclitaxel-Carboplatin neoadjuvant, the lower of heparanase
tissue expression level in early cervical cancer after administration of Paclitaxel Carboplatin
neoadjuvant. And otherwise the lower of heparanase expression level in tissue of early
cervical cancer before the administration of Paclitaxel-Carboplatin neoadjuvant, the higher
of heparanase expression level in tissue of early-stage cervical cancer after administration
of Paclitaxel-Carboplatin neoadjuvant.
The results of the correlation or relationship analysis also shows that if there was no
significant correlation with the heparanase expression level in tissue of early cervical cancer
before and after administration of Paclitaxel-Carboplatin neoadjuvan, then the modification
in heparanase expression in tissue of early cervical cancer were truly because of the
administration of Paclitaxel-Carboplatin neoadjuvant and not because of other factors.

Table 4. Relationship between heparanase expression in tissue of early stage cervical

cancer before and after the administration of Paclitaxel-Carboplatin neoadjuvant.
Paired Samples Correlations
N Correlati Sig.
on (r)
Pair Pre_Heparanase & 30 -.018 .925
1 Post_Heparanase
Notes: * p <0.05 was statistically significant.

CONCLUSION
The expression of heparanase in early stage cervical cancer patients is decreased after the
administration of Paclitaxel-Carboplatin neoadjuvant chemotherapy.
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