Immunohistochemical Expression of Hypoxia-Inducible Factor-1? in Triple Negative Breast Cancer

Mahdi Jassim Muhammad Ali,Haider Abdul Ridha AL-Khafaji,Abbas Fadhil Hassoon
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Keywords : Triple negative-breast cancer, HIF-1? and prognostic factors.
Medical Journal of Babylon  12:4 , 2016 doi:1812-156X-12-4
Published :22 January 2016

Abstract

Triple-negative breast cancer is a malignant neoplasm characterized by absent expression of estrogen, progesterone and human epidermal growth factor-2 receptors. Hypoxia-inducible factor-1 alpha is up-regulated under hypoxia and associated with induction of angiogenesis resulting in proliferation, aggressive tumor phenotype and metastasis. Determination of the potential role of Hypoxia-inducible factor-1 alpha in the development and conventional clinicopathologic factors, and to demonstrate their prognostic relevance in Triple-negative breast cancer. A pro and retrospective study of 281 female patients with breast cancer. The fifty four triple-negative breast cancer formalin-fixed, paraffin-embedded Specimenspresented from all cases were immuno-stained by Hypoxia-inducible factor-1 alpha Immunohistochemical marker. The age groups distributed from 23-85 years old, mean age 51.23±11.26 years. Overall cases, triple negative breast cancer presented in 19.2%; most frequently younger,grade III, tumor size ? 2 cm, and positive lymph node metastasis or recurrent more than other subtypes. Hypoxia-inducible factor-1 alpha expression was strongly associated and significantly differentwith grade (p=0.001), lymph node metastasis (p<0.001) and Ki-67 expression (p=0.003). Detected Hypoxia-inducible factor-1 alpha expression has an important role in pathogenesis, drug resistance and in uncontrolled proliferation cells or as important conductor of the worst prognosis in triple negative-breast cancer.

Introduction

Out of the 26 different types of cancer, breast cancer is the most common in women with approximately 1.38 million new cases worldwide, which corresponds to 23% of the total cases and 14% of cancer death. In Iraq it constitutes 19.59% of the total cancer cases and alone is accounted for 31% of all new cancer cases among females [1, 2, 3, 4, 5]. Breast cancer is heterogeneous with a variable prognosis depending on different histological and molecular characteristics, which influence patient prognosis and tumor behavior [6, 7].The mortality rates of breast cancer were decreasedbyearly diagnosisand hostile different approachesof treatment protocols. The treatment approachesaredetermined by using prognostic and predictive parameters like the patient’s age, pathological tumor grade, menstrual status, status of hormone receptors and human epidermal growth factor receptor 2 (HER2) [8]. Among all molecular subtypes, triple negative breast cancer (TNBC) has generated the greatest interest. Due to lack of expression of estrogen, progesterone and HER-2 receptor,specific targeted therapies are not effective, and chemotherapy is currently the only modality of the available systemic therapy [7, 9].TNBC tumors typically express of genes associated with proliferation, inhibition of apoptosis and tumor invasion [10].Therefore the documentation of new therapeutic goals and treatment tactics for TNBC is desperately needed [11]. It represents an important clinical subtype and overlap with basal-like group[9, 12, 13, 14]. Moreover, from a clinical point of view they are characterized by aggressive behavior and poorer prognosis. Standard therapy is associated with high relapse rates mainly localized to the lungs, central nervous system and lymph nodes and death within 5 years of diagnosis[15, 16, 17, 18].Numerous studies carried vision into risk factors related to TNBC were more likely to arise among women with a younger age, premenopausal, family history of BRCA1 (Breastcancergene1) mutation carriers and African American women than among women of other ethnicities[9, 19, 20,21]. Tumor hypoxia, in the last few years, molecular and in vivo studies leads to a series of biological changes that can promote enhanced malignancy growth primarily, metastatic behavior by the expression of a large number of metastasis related genes, maintenance of cancer stem cell, angiogenesis, glycolysis and other processes involved in cell proliferation and survival through the action of Hypoxia-inducible factor (HIF-1/HIF-2) driving several oxygen-sensitive signaling pathways[22, 23, 24,25]. It is related to poor response to therapy in various cancer types[26]. In addition, very low levels of oxygen induce the unfolded protein response to reduce metabolic demand under condition of severe energy "starvation"[27]. HIF-1 is a heterodimer transcription protein composed of an oxygen-labile ?- subunit (HIF1-?) and a constitutively expressed HIF1- ? subunit, also documented as aryl hydrocarbon receptor nuclear translocator (ARNT). While HIF-1? is constitutively expressed, HIF-1? levels are tightly regulated by rapid upregulation and degradation [25, 28]. HIF-1? is found almost in every tissue and highly expressed in many different tumors, including primary breast cancers, but infrequent in most normal tissues [29, 30]. HIF-1? stimulates the expression of many enzymes and other transcriptional genes[31]. The indicators of tumor hypoxia inTNBC are central fibrosis and necrosis [18]. The association between HIF-1? and the frequently triple negative familial breast cancer brings to special molecular features of TNBC Response to endoplasmic reticulum stress, activation of unfolded protein response pathway, a factor that promotes degradation of HIF and blunts HIF-induced malignant cell behavior as (Sharp1) andhypoxia[32]. In addition, also associated with novel targeted therapy for TNBC, namely anti-HIF-1? chemotherapy and related agents. This is especially reasonable for given the frequent link of TNBC with central necrosis, a surrogate morphological marker for hypoxia [18]. This study was directed in the aim of finding out the percentage of TNBC in Iraqi breast cancer women and if there is any association between clinico-pathological parameters of the TNBC and other breast cancer subtypes also to detect any association between several clinical and pathological factorsof TNBC and overexpression of HIF-1?.

Materials and methods

This prospective and retrospective study was conducted in the Department of Pathology in the College of Medicine-Babylon Universityinwhich281 female patients with primary breast cancer wereselected from data obtained from archives in the laboratories in AL- Hilla Teaching Hospital, Al-Sadr Teaching Hospital in Najafand AL-Hussein Medical City and Al-Sajad Private laboratory in Karbala during the period from November 2013 through April 2015. The patient s specimens were classified along with their immunohisto-chemical staining into two groups as 54cases of triple negativebreast cancer and 227 cases of other subtypes.  Histological types and grade were divided according to the World Health Organization classification and modified Bloom-Richardson grade [33]. The clinic-pathological variables comprising age, menopausalstatus, tumor sizeand site, type of surgery, recurrence, histological subtype, TNM Staging and grading system were estimated.
Tissue specimens:
Fifty four specimens of TNBC were maintained in 10% formalin and preceded routinely then embedded in paraffin blocks and new sections were made from each of the paraffin embedded blocks which included 4-5 ?m thickness where one section made on a glass slide to be processed and stained with routine haematoxylin and eosin stain for the confirmed histopathological study and others made on positively charged slides to be subjected for the purpose of conducting IHC procedures to detect HIF-1? [34,36].
Immunohisto-chemical procedures:
Immunostaining was completed using the Avidin, Biotin Complex detection (ABC) system [34]. The paraffin sections fixed on positively charged slides were back in an oven at 60 ?C for overnight, then the sections were deparaffinized in pre-warmed xylene and Rehydrated through absolute, 90%, 80%, and 70% ethanol. The section then treated with retrieval solution low PH (6.0) (DakoK8005 Kit) by heating in water bath at 95oC for 30 minutes (until boiling) to expose antigens before advance treatment. The whole technique below was achieved at room temperature and according to the instruction of the manufacturer (Universal Detection Kit, Dako, Denmark). Once washed in fresh phosphate buffer saline (PBS) between each steps belowfor 3-5 minutes, make a circle around sections byPAP pen, then the section incubated in enough hydrogen peroxide block for 5 minutes (Dako K8023 Kit).
The enough amount of primary antibody applied to cover specimens and incubated for 60 minutes using mouse anti human HIF-1? (1/200, HIF-1? [1A3] ab113642, Abcam, Cambridge, UK) before incubated with enough amount of Mouse (linker)(DakoK8023 Kit) for 10-20 minutes.Furthermore, afterapplied enough amount of horseradish peroxidase (HRP) conjugate for 20 minutes, followed by 5-10 minutes Incubation with the DAB(3,3 -Diaminobenzidinetetra hydrochloride) -containing Substrate Working Solution(Dako K8023 Kit).
Finally the slides Immersed in a bath of Myers haematoxylin and dehydrated with graded alcohol 70%, 80%, 90%, absolute then cleared with xylene before the cover slip was put on slides mounted with Di-N-Butyl Phthalate in Xylene(DPX).
The negative control in which the Sections untreated with primary antibody and replaced by PBS were used. In addition, Non-TNBC sections as positive internal control and cervical lymph adenitis as positive external control were added in each run of TNBC sections to the precision and standardization of the elaborated IHC results.
Statistical analysis
   Data were represented as numbers and percentages. The database was examined the association between different variables and the differences were associated for statistical significance by chi – square (X2) test [35]. The difference was considered significant at P <0.05. The statistical analysis was performed using statistical package for the social sciences (SPSS) version 20.0 for Windows (SPSS Inc, Chicago, Illinois, USA).




Results

Clinico pathological parameters of patients: The total number of 281 cases of breast cancer has been included in the present study, 19.20% (54 cases) of cases were TNBC. The age group distribution of the patients ranged from (23 to 85) years. The overall mean and slandered deviation of ages was 51.23±11.26 years. The most common age group involved was fifth to sixth decade, both in (32.4%). 55.6% of TNBC cases were below or equal the age of fifty years, while 50.7% of non- TNBC cases where more than fifty years oldas shown in Figure -1.

Discussions

The current study revealed that the age group of the breast cancer patients were ranging from 23-85 years, with a mean age and or standard deviation of 51.23±11.26 years, with 55.6% were younger than or equal 50 years. The peak age group frequently in the fifth to sixth decade of life was reported in this study. Our results consistent with most previous Iraqi researchers reports in Bagdad Like Alwan NA [3],AL-Hashimi MMY & Wang XJ and AL-Khafaji HA et al, [4, 5]. In presented study compared TNBC rate with a proportion of15%–20% in worldwide studies [20] which included 19.2% of the breast cancer patients. There were variable values as shown in Table (3) in comparison with this study may be due to variability in race, geographical area and IHC study.

Conclusions

The percentage of TNBC in Iraqi women is accounted 19.2% of all breast cancer cases when existed as a bad prognostic type of breast cancer. HIF-1? is the hypoxia responsive transcription factor that involved in breast carcinogenesis. The expression of it in TNBC frequently high was within the universal range detected by others. The expression of HIF-1?in TNBC showed a significant direct association with the grade of tumor and lymph node metastasis with no relation to the age, menopausal status, site, histological type, and size, and distance metastasis, type of surgery and tumor recurrence. The study presented a significant direct association between The HIF-1?and Ki-67 IHC expression in TNBC. This may play important role in prognosis and in detection of new treatment protocol later.

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