Intrinsic Molecular Subtyping of Breast Cancer In Low Resource Setting

Immunohistochemistry is an invaluable technique used clinically in the characterisation of breast cancer in various intrinsic subtypes. Such characterisation into the intrinsic subtypes is of great prognostic value in the management of breast cancer. Methodology Two hundred and seventy-six cases of formalin-xed paran-embedded (FFPE) tissue blocks were selected from 2012–2016 cases from Korle Bu Teaching Hospital (KBTH). The hormonal markers Estrogen Receptor (ER), Progesterone Receptor (PR), HER 2 and Ki67 were determined for cases using a semi-automated immunohistochemical method with commercially prepared antibodies from BioSB. Results There is The while TNBC was unfavourable for 65.5% of the cases with 21.8% favourable and 12.7% being borderline. The various subtypes are signicantly associated with vascular invasion.


Introduction
Immunohistochemistry is a valuable technique used clinically in the characterisation of breast cancers into various intrinsic subtypes. These include Luminal A (Estrogen Receptor positive (ER+) and/Progesterone Receptor positive/negative (PR±) and Human epidermal receptor negative (Her2-), Luminal B (ER+, PR + and Her2+), Her2 (ER-, PR-and Her2+) and Triple-negative (ER-, PR-and Her2-) breast cancers. Such characterisation into these intrinsic subtypes is of great prognostic value in the management of breast cancer. Luminal A generally has good prognosis compared to the other subtypes followed by luminal B [1][2][3]. Her2 and Triple-negative subtypes however generally have poor prognosis [1][2][3][4][5][6]. Molecular phenotype pro ling of breast cancer into these histologic subtypes has become crucial in the current era of molecular targeted therapy, and personalised treatment of breast cancer. In low resource settings plagued with nancial constraints and lack of facilities, studies into the pattern of occurrence of these molecular subtypes with various demographic characteristics and the clinicopathological signi cance are of immense importance in setting up realistic goals of breast cancer management.
Racial differences in molecular subtypes have been reported. For example, the triple-negative subtype appears to be more common in African-American populations, especially among younger African-American women, compared with European-ancestry populations. In this work we used immunohistochemical antibodies of Estrogen, progesterone, human epidermal factor to subtype breast cancer among Ghanaian patients and correlate it with the clinicopathological features.

Methodology
Two hundred and seventy-seven (277) cases of formalin-xed para n-embedded tissue blocks were selected out of the cases received from 2012-2016. The Estrogen Receptor, Progesterone Receptor, Human Epidermal Receptor2 and Ki 67 were determined using immunohistochemistry. Sections of 3 µm were taken from the FFPE blocks of the various cases using the microtome and having the ribbons transferred on the silane coated slides. The tissue was depara nised using xylene, ethanol and then washed in water followed by the immunohistochemistry process.

Depara nization
The depara nization process was done to remove the para n wax. This was done by putting tissue in three washes of xylene for 5 minutes each. Tissue was then placed into descending grades of alcohol thus 100%, 95%, 70%, and 50% for 10miutes for two washes each. Slides were then placed in distilled water for two wash for 5 minutes each. The tissues were transferred to heat retrieval stage using digital water bath at 97 C for 45 min with initial pre-warming at 85 C and followed by antibody treatment in the following stepwise method.

Heat retrieval and immunohistochemistry
A dedicated water bath 1.5L of distilled water and warm it to a pre-boiling temperature of 97 C was used.
Slides were placed in a pre-warmed staining dish containing the ImmunoDNA retrieval in the steamer, covered and steamed for 60 minutes. After heat treatment, slides were transferred in ImmunoDNA retriever with citrate to room temperature for 20 minutes and washed with changes of IHC wash buffer. Slides were placed in PolyDetector Peroxidase Blocker for 5 minutes. Wash with 3 changes of IHC wash buffer. Tissue was covered with Primary Antibody using prediluted antibodies from BioSB (ER, PR, HER 2 and Ki 67) for 60 minutes. (This was done separately for each of the cases in consideration).Wash with 3 changes of IHC buffer. Tissue was then covered with PolyDetector Plus Link, incubated for 15 minutes and washed with three changes of Immunohistochemistry buffer.
Tissue was covered with PolyDetector Horseradish peroxidase (HRP) label, incubate for 15 minutes and washed with 3 changes of IHC wash buffer. Diaminobenzidine (DAB) was prepared by adding PolyDetector DAB Chromogen per ml of PolyDetector DAB Buffer and mixed. Tissue was covered with prepared DAB substrate-chromogen solution, incubate for 5 minutes. Rinse with 3 changes of IHC wash buffer. Counterstain Meyer's haematoxylin was used and then dehydrated and coverslip. The slides were dehydrated, cleared and mounted using the following stepwise method. For each of the markers Dehydration and mounting of slides Tissue( slides ) were dehydrated in increasing order of alcohol thus two wash of 95% alcohol for 10 minutes each and also in 100% alcohol for two wash for 10minutes each. Slides were then placed in three wash of xylene for 5 minutes each. Slides were mounted with Distyrene (DPX) and coverslip.

Reporting of the slides
The slides were reported using the Allred scoring system as shown in Table 1 below for the oestrogen and progesterone receptors. Cases for HER 2 were reported using the algorithm Fig. 1. were calculated for quantitative variables. Chi-square was applied to determine associations. Student ttest was applied to compare the differences in means between groups. P-value of ≤ 0.05 as signi cant.

Results
In Table 1, of the various hormonal status, luminal A, Luminal B, HER-2(+ ve), and triple-negative were found to be more predominant in patients who were 40 years and above (87.9%, 81.8%, 86.2% and 84.4% respectively) whereas these hormonal status were less or not predominant in patients below the age of 40(12.1%,18.2%,13.8%,and 15.6% respectively). There was no signi cant association between the hormonal status and age since p value 0.05 In Table 2 In Table 3 Luminal A and Triple Negative signi cantly presented with tumour sizes above 5 cm in their widest dimension whereas tumour sizes of below 5 cm were predominantly associated with Her2 + and Luminal B hormonal receptor subtypes. There was a signi cant association between size range of tumor and HER-2(+ ve) since p value 0.05   (Table 7). However, high tumour grades had the highest frequency for favourable (6), unfavourable (29) and borderline (5) Ki67 phenotype while these phenotypes had least expression in low grade tumours for favourable (4), unfavourable (2) and borderline (0) phenotypes respectively (Table 9).       In Fig. 1, a total of 277 patients whose hormonal status were identi ed using the immunohistochemistry method. There were 117(42.2%) luminal A, 34(12.3%) luminal B, 29(10.5%) Her2 + and 97(35.0%) Triple Negative. In Fig. 2, 65.5% of patients presenting with malignant breast conditions generally presented with an unfavourable ki67. Only 21.8% presented with a favourable Ki67 and 12.7% presented as borderline.

Discussion
The mean age of presentation of breast cancer was 52.4 ± 12.7 a value higher than some studies conducted in Africa [8] with most women presenting in the post-menopausal years of 50 years and above (54.9%).In conformity with a Galukande et al's study in Uganda [8], a 2-3 fold increase in TNBC (35%) was realised when compared to studies from other Caucasian populations with a prevalence of 12-17% [9][10][11]. However, a 21% Triple-negative was recorded in a study in Soweto, South Africa [12]. Luminal A was the most occurring subtype representing 42.2% compared to the Uganda study recording 38% but on the contrary to that study, the least occurring subtype was Her2 + representing 10.5% as opposed to 22%. 12.3% had Luminal B subtype as opposed to 5% in the Uganda study [8].

Triple Negatives
One of the major clinical challenges in breast cancer management is triple-negative breast cancer receptor status usually associated with poor prognosis and refractory to endocrine therapy or other available targeted therapy [13][14][15]. The intrinsic poor prognosis compared with other receptor subtypes stems from its high metastatic potential, high recurrence rate and poor overall survival [16].
This study recorded a high prevalence of triple-negative breast cancer associated with poorer prognosis including higher tumour grade, size and unfavourable ki67 phenotype. This further con rms the high prevalence of triple-negative breast cancer in Africans [17][18][19]. The study contrary to some studies [11,20] but in keeping with others (Carey, 2006) realised triple-negative tumours to be more predominant in patients who were 40 years and above. Metastatic TNBC is associated with a high proliferation index evidenced by the high ki67 score, correlating with visceral and CNS metastases [21] and poor outcome in spite of treatment creating a major clinical challenge [16,22]. The average survival of advanced TNBC is 12 months, much shorter than the duration of survival observed in other subtypes of advanced Breast Cancer.
In conformity with literature, this study a rmed the relative better prognosis of Luminal A tumours compared with the other subtypes. Luminal B, Her2 + and Triple-negative were all associated with highergrade tumours whereas Luminal A was not. It was also evident in the study that luminal A and B were predominantly associated with smaller tumour sizes compared with Her2 and Triple-negative also a rming the aggressiveness and relatively poorer prognosis of the later in keeping with Xue et al's review in 2012. The study also identi ed a non-association of triple-negative tumours with vascular invasion although all other histologic subtypes had a signi cant vascular invasion. This may be indicative of the fact that the aggressiveness of triple-negative tumours may not be as a result of vascular invasiveness.
This may explain the reason why anti-angiogenic agents such as Bevacizumab and sorafenib which are anti-VEGF on TNBC have not yielded positive outcome [23].
The identi cation of this receptor subtype is valuable in selecting high and low-risk subsets of patients for other personalised and targeted treatment. The high prevalence of triple-negative breast cancer in this population together with the low socioeconomic status of most patients and late reporting to health facilities is indicative of a greater challenge in breast cancer management in Ghana. The major breast cancer treatment modalities available in Ghana include Chemotherapy, radiotherapy and endocrine therapy. Limited treatment options available for patients also compounds the already worse situation.
Although several promising therapeutic options are being developed for triple-negative breast cancer targeting pathways such as the Notch signalling [24], Wnt/β-catenin [25] and Hedgehog pathways [26], in addition to EGFR [27], PARP1 [28], mTOR [29], and TGF-β [29,30] due to lack of facilities and nancial constraints our patients do not bene t from either research or therapy. Early detection of cases is, therefore, a key in the management of breast cancer in Ghana since between 3-4 out of 10 patients presenting with breast cancer present with TNBC. Heightened efforts are to be made for the public to be breast aware and to ensure early reporting of cases to appropriate health facilities for prompt management. Through public health awareness creation, the general public is to be educated on the need for patients to report early to health care facilities. A national policy of screening of patients 40 years and above will be valuable in ensuring we are a step ahead of the disease as a country. Self-breast examinations and regular breast screening exercises should be encouraged. Further characterisation of tumours to elucidate the aggressiveness of tumours of African origin is highly warranted. It is also recommended that hormonal receptor status should be part of routine investigations for all histology con rmed breast cancer cases to inform the decision on the best modality of management to employ. Active inclusion of breast cancer management on the national health insurance scheme.
In conclusion, greater percentage of breast cancer among Ghanaian patients are hormonal positive and should have done well on hormonal treatment but because of the late presentation and tumour characteristics.