Epidermal growth factor receptor (EGFR) overexpression in triple-negative breast cancer: association with clinicopathologic features and prognostic parameters
Surgical and Experimental Pathologyvolume 2, Article number: 6 (2019)
Triple-negative breast cancers are a poor prognostic group of breast cancers that don’t respond to conventional hormonal and her2neu targeted therapy. A subset of triple-negative breast cancer is known to overexpress epidermal growth factor receptor (EGFR); however prognostic significance of this biomarker has not been widely studied in our population. Therefore, we aimed to evaluate the frequency of EGFR overexpression in triple-negative breast cancer in our setup and its association with prognostic and predictive factors.
We performed EGFR immunohistochemistry on 150 cases of triple-negative breast cancers. Intensity and percentage of EGFR expression were combined to formulate an EGFR score, that was compared with prognostic features of breast cancer and recurrence status of patients.
Positive EGFR expression was noted in 18.7% ( 28 cases); out of which 16% (24 cases) showed low EGFR expression, whereas high EGFR expression was seen in 2.7% ( 4 cases). No significant association of EGFR expression was noted when compared with various clinicopathological parameters and recurrence status of the patients.
We found EGFR protein expression in 18.7% of cases while high expression was seen in only 2.7 % cases of triple-negative breast cancer which may harbor underlying genetic alterations like altered EGFR gene copy number, chromosome 7 copy number or average EGFR gene: chromosome 7 ratio; therefore we suggest that molecular tests like FISH to evaluate these EGFR molecular alterations should be performed in EGFR over expressing triple negative breast cancers in our setup to identify patients that can benefit from anti-EGFR targeted therapy. Moreover, regional difference in EGFR expression (high expression in chinese population compared to our population) are may be due to different underlying genetic alterations in triple-negative breast cancers, further necessitating a need of devising personalized therapeutic protocols for locoregional population.
Triple-negative Breast Cancers (TNBC) are a poor prognostic group of breast cancers and accounts for 20% of human breast cancers (Swain, 2008; Nakai et al., 2016; Kim et al., 2006; Hashmi et al., 2014a; Hashmi et al., 2014b; Hashmi et al., 2018a). TNBC are diagnosed pathologically by < 1% expression of Estrogen Receptor (ER) and Progesterone Receptor (PR) and Human Epidermal Receptor 2 (Her2neu) 0/1+ or 2+ (FISH negative) as per CAP/ ASCO recommendation (Hammond et al., 2010; Wolff et al., 2013). Due to lack of expression of hormonal and Her2neu receptors; conventional targeted therapies (hormonal and anti-her2neu) are ineffective in TNBC. Human Epidermal Growth Factor Receptor (EGFR) consists of several transmembrane glycoproteins. Activating mutations of EGFR results in uncontrolled cell proliferation and survival (Masuda et al., 2012). Such mutations of EGFR have been reported in many human cancers especially lung adenocarcinoma, head and neck squamous cell carcinoma and colorectal carcinoma (Scagliotti et al., 2004; Chung et al., 2005; Hashmi et al., 2018b; Hashmi et al., 2018c). Therefore EGFR represents a useful therapeutic target for EGFR inhibitor therapy. At present anti-EGFR therapy has not been approved in the treatment of TNBC and therefore routine EGFR testing is not recommended. Moreover, there are no set guidelines for the immunohistochemical interpretation of EGFR overexpression. It has been noted that EGFR abnormalities exhibit geographic and ethnic variations and its expression varies in different regions of the world (Nakajima et al., 2014; Grob et al., 2012; Secq et al., 2014; Tilch et al., 2014; Jacot et al., 2011).EGFR overexpression has not been evaluated in breast cancers of our population. Therefore in this study we aimed to evaluate the EGFR overexpression in TNBC in our population and its prognostic significance.
Total 150 cases of TNBC were retrieved from records of pathology department. The surgical specimens were those of modified radical mastectomies, simple mastectomies and wide local excisions with sentinel lymph node dissection. All of these patients had surgeries at Liaquat National hospital, Karachi from January 2008 till December 2013. Hematoxylin and eosin stained slides of all of these cases were reviewed independently by two senior histopathologists and pathologic characteristics were recorded. Clinical records of all patients were also retrieved and reviewed from institutional files to evaluated patients age and recurrence status. Moreover, representative blocks of each case were selected for immunohistochemistry. ER, PR and Her2neu immunohistochemistry were done to reconfirm triple-negative status. CK5/6 immunohistochemistry was done to subcategorize TNBC cases into basal and non-basal subtypes.
ER, PR, Her2neu and Ki67 IHC were performed using DAKO antibodies as shown below, with EnVisionTM FLEX, high pH DAKO kit according to manufacturer’s protocol.
FLEX Monoclonal Rabbit Anti-human Estrogen Receptor alpha, Clone EP1.
FLEX Monoclonal Mouse Anti-human Progesterone receptor clone PgR 636
Polyclonal Rabbit Anti-human c-erbB-2 oncoprotein
FLEX Monoclonal mouse Anti-human Ki67 Antigen clone MIB-1
For ER and PR, nuclear staining in >1% of cancer cells was taken as positive. For her2neu, cases were scored 1+, 2+ and 3+ according to CAP guidelines. For equivocal cases (2+) fluorescent in situ hybridization (FISH) was done using Path Vysion Her2neu probe kit according to manufacturer’s recommendations. Paraffin embedded tissues were cut at 5 microns and mounted on positively charged glass slides. Four slides were prepared, with one slide stained with hematoxylin and eosin. Well preserved invasive carcinoma was marked on the H & E slide as the target area. Target area was etched with a diamond tipped scriber on the back of the unstained slide to be assayed. Glass slides with paraffin embedded tissue sections were baked at 56 degree centigrade overnight. Tissue was then processed in xylene, ethanol, purified water and wash buffer. Probe mixture was applied to the target area of the tissue on slide, sealed with rubber cement and then incubated in thermobrite for hybridization. After post-hybridization buffering, DAPI (counter stain) was applied. Slides were interpreted by two pathologists, evaluating 60 interphase nuclei using fluorescence microscope equipped with appropriate excitation and emission filters allowing the visualization of the orange and green fluorescent signals (Hashmi et al., 2014b). Results were expressed as ratio of her2neu signals as compared to CEP17 signals according to ASCO/CAP guidelines (Wolff et al., 2014).
For Ki67 immunostaining, proportion of positive stained cancer cells were recorded (Jalava et al., 2006). Furthermore on the basis of Ki67 index, cases were categorized into <15%, 16-24%, 25-44% and >44% ki67 index categories.
CK5/6 IHC was performed by using FLEX Monoclonal Mouse Anti-human Cytokeratin 5/6, clone D5/16 B4 by using DAKO envision kit. Intermediate to strong cytoplasmic and membranous staining in more than 10% cells was considered positive CK5/6 expression.
EGFR immunohistochemistry was performed using DAKO EnVision method using DAKO Monoclonal Mouse Anti-human Epidermal growth factor Receptor (EGFR), clone H11 according to manufacturers protocol.
IHC staining was performed manually in batches of 10-15 slides. Positive and negative controls were run along with each batch. For IHC staining, 3-4 micrometer sections were cut on DAKO IHC coated slides. After fixation of slides in oven at 70-80 degree centigrade for 20 mins, sections were de-waxed in xylene and then processed through decreasing concentrations of alcohol after which antigen retrieval was performed. For antigen retrieval, Slides were placed in antigen retrieval solution in water bath at 99-100 degree centigrade for 40 mins. After being kept at room temperature, slides were washed with wash buffer solution 2-3 times, followed by blocking peroxidase for 10 mins in humidity chamber. Subsequently, slides were washed 2-3 times with washing buffer. Primary antibody (EGFR) was applied with dilution of 1:200 for 20 mins, then washed 2-3 times. After application of secondary antibody, slides were incubated for 20mins and then again washed with wash buffer, followed by application of DAB chromogen solution (Wen et al., 2013). Both membranous and cytoplasmic staining for EGFR were evaluated. Intensity of staining was categorized into no staining (0), weak (1+), intermediate (2+), strong (3+) as shown in Fig. 1 as follows
Weak intensity 1+: Faint, incomplete staining of membranes and focal weak positivity in cytoplasm which is only evident on 100X magnification.
Moderate intensity 2+: Complete and moderate staining of membranes and weak to moderate staining in cytoplasm easily appreciable on 40X magnification.
Strong intensity 3+: Complete and strong staining of membranes and moderate to strong staining of cytoplasm appreciable on 40X magnification.
Percentage of positively stained cells was also measured and scored as a continuous variable. Intensity score and percentage scored were multiplied to formulate an H-score (0-300). Cases with EGFR expression were further categorized into low and high EGFR expression with a cut-off H-score of 200.
Follow-up and recurrence
Recurrence status and follow-up were evaluated by reviewing patient’s hospital record. Overall survival was defined as the time from surgical excision till death or last follow-up. Disease free survival was considered as the time between surgical excision and local recurrence or distant metastasis, last follow-up or patient’s death.
Statistical package for social sciences (SPSS 21) was used for data compilation and analysis. Mean and standard deviation were calculated for quantitative variables. For qualitative variables, frequency and percentage were calculated. Chi-square test was performed to determine the association. Student t test or Mann witney test were applied to compare difference in means among groups. Survival curves were plotted using Kaplan- Meier method and the significance of difference between survival curves were determined using log-rank ratio. P-value ≤0.05 was taken as significant.
Mean age of the patients was 48.9 years. Median follow up time was 28.4+8.3 months. Most common presentation was at stage T2 (52.7%). Most tumors were high grade (grade III), 86.7%. Similarly, 53.35 of cases showed high ki67 index (>44%). Nodal metastasis was seen in 42.7% cases. Although 84% cases had invasive ductal carcinoma, NST; a significant proportion of cases were of metaplastic histology (9.3%). Lymphovascular invasion was present in 24% cases, while 10.7% cases were of basal phenotype based on CK5/6 expression as shown in Table 1.
Positive EGFR expression was noted in 18.7% ( 28 cases); out of which 16% (24 cases) showed low EGFR expression, whereas high EGFR expression was seen in 2.7% ( 4 cases). No significant association of EGFR expression was noted when compared with various clinicopathological parameters of TNBC (Table 1). Similarly no significant association of EGFR expression was noted with recurrence status of the patients (Fig. 2). Association of clinicopathologic parameters of TNBC with EGFR was also evaluated using different H-score cutoffs, however no significant association was seen except for histologic subtype as shown in Table 2.
In the present study, we found that a small subset of TNBC of our population was found to have EGFR overexpression by immunohistochemistry (IHC) while no significant association was found between EGFR over expression and various prognostic parameter of TNBC.
EGFR overexpression in TNBC markedly varies in different populations. A study conducted in chinese population revealed as high as 42% EGFR expression in TNBC cases (Wang et al., 2015). On other hand, in another study consisting of 567 TNBC cases in Korean population revealed; 1+ EGFR expression in 11.8 % , 2+ in 8.5 % & 3+ in only 3.2 % cases of TNBC & except for lymphovascular invasion , no significant association was found between EGFR expression & various prognostic parameters in TNBC (Kim et al., 2017). These findings were concordant with the results of our study. Similarly Park et. al, reported EGFR over expression associated with tumor stage , while association with other prognostic parameter was not significant (Park et al., 2014a). In the current study we used H-score incorporationg both the intensity and percentage of cells to evaluate EGFR expression. In most of the previous only positive EGFR expression was correlated with clinicopathologic variables without categorizing EGFR expression into low or high expression. In most of these studies only intensity of EGFR expression was taken into account and categorized into weak, moderete and strong expression neglecting the percentage of positivity stained cells (Changavi et al., 2015). Due to heterogenity of tumor cells, there is usually variable expression of IHC markers and therfere its always good to score IHC expression, but unfortunately as no standard protocol of interpreting EGFR IHC expression is there, therefore we used different cutoffs.
One of the limitations of our study was that mutation analysis of EGFR gene was not performed as positive over expression does not necessarily correlates with underlying gene mutation in TNBC; and it is necessary to document gene amplification or activating mutations of EGFR in order to benefit from anti-EGFR targeted therapy. Moreover, mechanism of protein over expression in the absence of activating gene mutations is not well understood. Overall EGFR gene mutation is relatively low in TNBC. Studies failed to reveal such mutations in European , Australian & Japanese patients, while 3.4% and 1-2% of cases showed EGFR gene mutations in a subset of American & Korean populations respectively (Reis-Filho et al., 2006; Santarpia et al., 2012; Kim et al., 2013). A frequency of 10% & 11.4% EGFR mutations were reported in Chinese population (Lv et al., 2011a). These findings reveal marked geographic differences in EGFR amplifying gene mutations which may result in protein overexpression in TNBC . High IHC Expression of EGFR may be due to various underlying genetic alterations involving EGFR gene or chromosome 7, including EGFR gene amplification and chromosomal alterations like disomy, low trisomy, high trisomy, low polysomy and high polysomy (Lee et al., 2015).
We found EGFR protein expression in 18.7% of cases while high expression was seen in only 2.7 % cases of triple negative breast cancer which may harbor underlying genetic alterations like altered EGFR gene copy number, chromosome 7 copy number or average EGFR gene: chromosome 7 ratio; therefore we suggest that molecular tests like FISH to evaluate these EGFR molecular alterations should be performed in EGFR over expressing triple-negative breast cancers in our set up to identify patients that can benefits from anti-EGFR targeted therapy. Moreover, regional difference in EGFR expression (high expression in chinese population compared to our population) are may be due to different underlying genetic alterations in triple-negative breast cancers, further necessitating a need of devising personalized therapeutic protocols for locoregional population.
Triple negative breast cancer
Changavi AA, Shashikala A, Ramji AS (2015) Epidermal Growth Factor Receptor Expression in Triple Negative and Nontriple Negative Breast Carcinomas. J Lab Physicians 7(2):79–83
Chung KY, Shia J, Kemeny NE, Shah M, Schwartz GK, Tse A et al (2005) Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J ClinOncol 23:1803–1810
Grob TJ, Heilenkötter U, Geist S, Paluchowski P, Wilke C, Jaenicke F et al (2012) Rare oncogenic mutations of predictive markers for targeted therapy in triple-negative breast cancer. Breast Cancer Res Treat 134:561–567
Hammond ME, Hayes DF, Dowsett M et al (2010) American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version). Arch Pathol Lab Med 134:e48
Hashmi AA, Edhi MM, Naqvi H, Faridi N, Khurshid A, Khan M (2014b) Clinicopathologic features of triple negative breast cancers: an experience from Pakistan. Diagn Pathol 9:43
Hashmi AA, Edhi MM, Naqvi H, Khurshid A, Faridi N (2014a) Molecular subtypes of breast cancer in South Asian population by immunohistochemical profile and Her2neu gene amplification by FISH technique: association with other clinicopathologic parameters. Breast J 20(6):578–585
Hashmi AA, Hussain ZF, Aijaz S, Irfan M, Khan EY, Naz S, Faridi N, Khan A, Edhi MM (2018b) Immunohistochemical expression of epidermal growth factor receptor (EGFR) in South Asian head and neck squamous cell carcinoma: association with various risk factors and clinico-pathologic and prognostic parameters. World J Surg Oncol 16(1):118
Hashmi AA, Hussain ZF, Irfan M, Edhi MM, Kanwal S, Faridi N, Khan A (2018a) Cytokeratin 5/6 expression in bladder cancer: association with clinicopathologic parameters and prognosis. BMC Res Notes 11(1):207
Hashmi AA, Hussain ZF, Irfan M, Khan EY, Faridi N, Naqvi H, Khan A (2018c) Prognostic significance of epidermal growth factor receptor (EGFR) over expression in urothelial carcinoma of urinary bladder. Edhi MM. BMC Urol 18(1):59
Jacot W, Lopez-Crapez E, Thezenas S, Senal R, Fina F, Bibeau F et al (2011) Lack of EGFR-activating mutations in European patients with triple-negative breast cancer could emphasise geographic and ethnic variations in breast cancer mutation profiles. Breast Cancer Res 13:R133
Jalava P, Kuopio T, Juntti-Patinen L, Kotkansalo T, Kronqvist P, Collan Y (2006) Ki67 immunohistochemistry: a valuable marker in prognostication but with a risk of misclassification: proliferation subgroups formed based on Ki67 immunoreactivity and standardized mitotic index. Histopathology 48(6):674–682
Kim A, Jang MH, Lee SJ, Bae YK (2017) Mutations of the Epidermal Growth Factor Receptor Gene in Triple-Negative Breast Cancer. J Breast Cancer 20(2):150–159
Kim MJ, Ro JY, Ahn SH, Kim HH, Kim SB, Gong G (2006) Clinicopathologic significance of the basal-like subtype of breast cancer: a comparison with hormone receptor and HER2/neu-overexpressing phenotypes. Hum Pathol 37:1217–1226
Kim Y, Kim J, Lee HD, Jeong J, Lee W, Lee KA (2013) Spectrum of EGFR gene copy number changes and KRAS gene mutation status in Korean triplenegative breast cancer patients. PLoS One 8:e79014
Lee HJ, Seo AN, Kim EJ, Jang MH, Kim YJ, Kim JH, Kim SW, Ryu HS, Park IA, Im SA, Gong G, Jung KH, Kim HJ, Park SY (2015) Prognostic and predictive values of EGFR overexpression and EGFR copy number alteration in HER2-positive breast cancer. Br J Cancer 112(1):103–111
Lv N, Xie X, Ge Q, Lin S, Wang X, Kong Y et al (2011a) Epidermal growth factor receptor in breast carcinoma: association between gene copy number and mutations. Diagn Pathol 6:118
Masuda H, Zhang D, Bartholomeusz C, Doihara H, Hortobagyi GN, Ueno NT (2012) Role of epidermal growth factor receptor in breast cancer. Breast Cancer Res Treat 136:331–345
Nakai K, Hung MC, Yamaguchi H (2016) A perspective on anti-EGFR therapies targeting triple-negative breast cancer. Am J Cancer Res 6:1609–1623
Nakajima H, Ishikawa Y, Furuya M, Sano T, Ohno Y, Horiguchi J et al (2014) Protein expression, gene amplification, and mutational analysis of EGFR in triple-negative breast cancer. Breast Cancer 21:66–74
Park HS, Jang MH, Kim EJ, Kim HJ, Lee HJ, Kim YJ et al (2014a) High EGFR gene copy number predicts poor outcome in triple-negative breast cancer. Mod Pathol 27:1212–1222
Reis-Filho JS, Pinheiro C, Lambros MB, Milanezi F, Carvalho S, Savage K et al (2006) EGFR amplification and lack of activating mutations in metaplastic breast carcinomas. J Pathol 209:445–453
Santarpia L, Qi Y, Stemke-Hale K, Wang B, Young EJ, Booser DJ et al (2012) Mutation profiling identifies numerous rare drug targets and distinct mutation patterns in different clinical subtypes of breast cancers. Breast Cancer Res Treat 134:333–343
Scagliotti GV, Selvaggi G, Novello S, Hirsch FR (2004) The biology of epidermal growth factor receptor in lung cancer. Clin Cancer Res 10(12 2):4227s–4232s
Secq V, Villeret J, Fina F, Carmassi M, Carcopino X, Garcia S et al (2014) Triple negative breast carcinoma EGFR amplification is not associated with EGFR, Kras or ALK mutations. Br J Cancer 110:1045–1052
Swain S (2008) Triple-Negative Breast Cancer: Metastatic Risk and Role of Platinum Agents 2008 ASCO Clinical Science Symposium, 2008
Tilch E, Seidens T, Cocciardi S, Reid LE, Byrne D, Simpson PT et al (2014) Mutations in EGFR, BRAF and RAS are rare in triple-negative and basal-like breast cancers from Caucasian women. Breast Cancer Res Treat 143:385–392
Wang XZ, Liu Q, Sun JJ, Zuo WS, Hu DW, Ma SG, Mu DB (2015) Yu ZY.Correlation between p53 and epidermal growth factor receptor expression in breast cancer classification. Genet Mol Res 14(2):4282–4290
Wen YH, Brogi E, Hasanovic A, Ladanyi M, Soslow RA, Chitale D, Shia J, Moreira AL (2013) Immunohistochemical staining with EGFR mutation-specific antibodies: high specificity as a diagnostic marker for lung adenocarcinoma. Mod Pathol 26(9):1197–1203
Wolff AC, Hammond ME, Hicks DG et al (2013) Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J ClinOncol 31:3997
Wolff AC, Hammond ME, Hicks DG et al (2014) Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology - College of American Pathologists (ASCO/CAP) Clinical Practice Guideline Update (2013). Arch Pathol Lab Med 138(2):241–256
We gratefully acknowledge all staff members of Pathology, Liaquat National Hospital, Karachi, Pakistan for their help and cooperation.
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Ethics committee of Liaquat National Hospital, Karachi, Pakistan approved the study. Written informed consent was obtained from the patients for the participation.
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