1 SUBMITTED 21 DEC 22 1 REVISION REQ. 13 FEB & 26 APR 23; REVISION RECD. 19 MAR & 24 MAY 23 2 ACCEPTED 20 JUN 23 3 ONLINE-FIRST: JULY 2023 4 DOI: https://doi.org/10.18295/squmj.7.2023.044 5 6 Expression Patterns of ER, PR, Her-2/neu and p53 in Association with 7 Nottingham Tumor Grade 8 A retrospective hospital-based study 9 *Kamoru A. Adedokun,1 Waheed A. Oluogun,2 Musiliu A. Oyenike,3 Sikiru O. 10 Imodoye,4 Lukman A. Yunus,2 Ismaila A. Lasisi,5 Ibrahim O. Bello,6 Ramat T. 11 Kamorudeen,7 Saheed A. Adekola8 12 13 1Department of Immunology, Roswell Park Comprehensive Cancer Center, Buffalo, New York, 14 USA; 2Department of Morbid Anatomy and Histopathology Osun State University Teaching 15 Hospital (UNIOSUNTH), Osogbo, Nigeria; 3Department of Medical Laboratory Science, Ladoke 16 Akintola University of Technology, Ogbomosho, Oyo State, Nigeria; 4Department of Oncological 17 Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah, USA; 5Laboratory 18 Unit, Health Centre, Osun State Polytechnic Iree, Osun State, Nigeria; 6Department of Biological 19 Sciences, Southern Illinois University at Edwardsville, Edwardsville, Illinois, USA; 7Department 20 of Public Health, University of South Wales, Pontypridd, UK; 8MBM Molecular Laboratory, 21 Dubai, United Arab Emirate. 22 *Corresponding Author’s e-mail: adeolokun@yahoo.com 23 24 Abstract 25 Objectives: Histological grading has been an integral part of cancer diagnosis for a long time. Recent 26 molecular studies show that breast cancer is a heterogeneous disease, and several molecular changes 27 may accumulate over time to influence treatment response. As a result, employing reliable molecular 28 biomarkers to monitor these modifications may help deliver personalized treatment. However, this 29 may be unrealistic in the resource-limited parts of the world. Thus, we studied the expression pattern 30 of hormone receptors and p53 tumor suppressor using immunohistochemistry (IHC) in breast cancer 31 2 (BC) compared to the traditional tumor grade. Methods: Two hundred and five (n =205) cases were 32 investigated. The Modified Bloom-Richardson score system was adopted in grading the tumors. 33 Tissue sections of the cases were stained with specific primary antibodies (at dilutions of 1:60 for 34 estrogen (ER) and progesterone receptors (PR), 1:350 for human epidermal growth factor (Her-35 2/neu), and 1:50 for p53. The Chi-square test was used to determine the association between the 36 tumor grade and IHC markers. Results: Invasive ductal carcinoma of no-specific type (190 37 cases;92.7%) was predominant. Grade II tumor (n =146; 71.22%) was the most frequent. Hormone 38 receptors (ER+; n =227 and PR+; n =145) had 62.0% and 70.7% positive cases; 34.2% (n =70) were 39 positive for Her-2/neu, while 76.1% (n =156) were positive for p53. We observed strong associations 40 between Nottingham grade and expression patterns of ER (P ˂ 0.01), PR (P ˂ 0.001), Her-2/neu (P 41 ˂ 0.001), and p53 (P =0.001). Conclusion: Nottingham grade has a high degree of concordance with 42 the patterns of expression of hormone receptors, Her-2/neu, and p53, suggesting that it may play an 43 important role in connection with the predictive and prognostic biomarkers for BC. 44 Keywords: Breast cancer, Her-2/neu, hormone receptor, Nottingham grade, p53 mutation. 45 46 Advances in Knowledge 47 • Grade II tumors displayed higher levels of ER and PR expression than grade III tumors, 48 indicating that as the disease progresses, the proportion of cells expressing ER and/or PR steadily 49 declines. 50 • Similarly, we observed higher HR positivity than in many black populations, including Guinea, 51 Ghana, South Africa, and Mali emphasizing potential identifiable intra-racial factors influencing 52 the diverse variation. 53 • Some patients had higher grades in the Her-2/neu+ expression group than in the Her-2/neu- 54 expression group but lower in ER+ and PR+ expressions compared to ER- and PR- of the highest 55 grade III. 56 • We found a higher Her-2+ than the majority of previous studies, but most of these cases co-57 expressed HR+ with Her-2/neu- rather than Her-2+ tumors, indicating that the cancer cells are 58 responsive to hormone treatment, have a better prognosis, and are less aggressive, contrary to 59 the common opinion that black population has aggressive breast cancer presentation. 60 • The proportion of TNBC patients was rather low, implying that hormone therapy or targeted 61 therapies targeting at Her-2 would benefit the majority of our cancer patients. 62 63 3 Application to Patient Care: 64 • Our study shows that cancer phenotype can exhibit location-dependent variations due to several 65 factors including genetic predisposition, lifestyle, and environmental influences. 66 • We observed that the underlying factors for regional, ethnic or racial variation can impact the 67 expression patterns of various biomarkers. 68 • As a result, this study implies that understanding regional variations in cancer phenotype and 69 biomarker expression patterns, as well as tumor grade, can help guide personalized treatment 70 decisions, optimize therapy selection, and perhaps improve patient outcomes. 71 72 Introduction 73 Cancer continues to be one of the deadliest noncommunicable diseases worldwide.1 Although the 74 literature shows that BC is more common in developed countries, a recent GLOBOCAN estimate 75 shows that Africa constitutes a nerve-racking proportion of BC deaths, possibly due to poorer 76 prognosis and limited access to appropriate diagnosis and treatment.2 Before the advent of molecular 77 diagnosis, most cases of BC were solely diagnosed using histological methods. Yet, the histological 78 method is still commonly and exclusively used, especially in low-resource settings in many African 79 countries.3 80 81 In this new genomic era, molecular markers are gaining wide acceptance as sensitive and inclusive 82 methods to understand the behavior of advanced cancers. Specifically, hormone receptors, p53, 83 Ki67, and human epidermal growth factor receptor 2 (Her-2/neu) are used for the diagnosis, 84 classification, prognosis, and prediction of response to therapy in BC; even so, histological 85 assessment is used primarily.4 Each of these biomarkers is important in diagnosing BC and may 86 sometimes correlate with other disease diagnostic indicators. Overexpression of Her-2/neu has been 87 linked to a higher histological grade, increased tumor size, the number of affected lymph nodes, p53 88 mutation, and lower ER expression (or even ER expression in some cases).5 Similarly, ER and PR 89 patterns have been linked to BC grade, potentially influencing treatment options.6,7 Furthermore, a 90 mutation in the p53 gene, a tumor suppressor gene, represents a genetic predisposition to cancers 8 91 and has been associated with tumor aggressiveness 9, making them a possible indicator of 92 histological grade. 93 94 4 Meanwhile, histological grade enables a description of a tumor's level of aggressiveness and is 95 regarded as a forerunner for morphological evaluation of tumor biological characteristics.10 96 According to a study on gene expression, histological grade reveals information about the molecular 97 makeup of BC in addition to tumor size or lymph node involvement.11 Furthermore, evidence from 98 genome-wide microarray-based expression profiling elucidates many characteristics of tumor 99 biology in BC, adding to the evidence that the biological features revealed by histological grade are 100 critical in determining tumor behavior. 10 101 102 The investigation of the connection between histological grade and molecular biomarker expression 103 patterns is thought to add to the body of diagnostic knowledge, particularly in the areas where 104 molecular testing is currently lacking. Even though they are complementary, more research is needed 105 to determine the magnitude of the relationship between traditional tumor grading and the more 106 contemporary IHC methodologies, particularly regarding expression patterns. This attempt may 107 highlight the importance of histological grade in low-resource settings as a low-cost, easy, accurate, 108 and validated approach to diagnosing BC. In the present study, we investigated the frequency and 109 patterns of expression of some clinically significant molecular markers in patients with BC. We 110 explored the link between the biomarkers' expression patterns and histological tumor grade to 111 determine their role in disease diagnosis. 112 113 Methods 114 Study design and patients 115 This investigation was a hospital-based retrospective study. It involved archival tissue blocks and 116 records of female patients older than 18 years referred to LAUTECH Hospitals in Osogbo and 117 Ogbomosho, Osun and Oyo States, respectively (at the time of the investigation). The study included 118 patients on record between 2005 and 2014 for breast biopsy or surgery diagnosed with BC in their 119 pathology reports. 120 121 Slide preparation 122 Tissue blocks were retrieved and new thin sections of about 3µm were made using rotary microtome 123 from formalin-fixed paraffin-embedded blocks following a previous method.3 124 125 5 Clinicopathological features 126 Data vis-à-vis; age, histological grade, nuclear grade, tumor size, and lymph node involvement were 127 extracted from patients’ records. 128 129 Tumor classification 130 Histological classification of the breast tumor was made following World Health Organization 131 (WHO) guidelines. Tumor grading was done using Nottingham modification of the Scarff-Bloom-132 Richardson (SBR) grading system. Tumor staging was done using the TNM system adopted by 133 International Union against Cancer (UICC) and the American Joint Committee on Cancer and End 134 Results Reporting (AJC)12. 135 136 Immunohistochemical assessment 137 All samples were evaluated by immunohistochemical (IHC) staining under the direct supervision of 138 a Chief Histopathology Scientist and reported by two different Consultant Pathologists, which were 139 then compared in a blinded fashion. The procedures for IHC staining were performed using the 140 primary antibody specific for ER (ER6F11) (Dako), PR (Dako), Her-2/neuis (ERBB2) (Dako), and 141 p53, Do-7 (Santacruz) at the Breast Cancer Laboratory Medical Genetic and Bioethics Research 142 Unit, Institute for Advanced Medical Research and Training (IMRAT), University College Hospital, 143 Ibadan. The sections were exposed to the primary antibody (dilutions of 1:60 for ER and PR, 1:350 144 for Her-2/neu, and 1:50 for p53 for one hour). Negative and positive controls were performed by 145 including the control tissues specified by the antibody vendors, respectively. 146 147 Scoring of ER and PR status 148 The scoring was performed using the modified immunohistochemical score (“Quickscore”), a 149 modified semi-quantitative assessment method by Allred 13. Nuclear staining intensity was scored 150 from 0 to 3+ in combination with the proportion of cells involved to get a range of 0–7 as the final 151 score for ER and PR positivity [Figure 1]. 152 153 The criteria used are explicitly described as follows: "Quickscore" determines the percentage or 154 range of stained cells from 1 to 4 and overall intensity from 1 to 3. The scores are added to give a 155 total maximum score of 7 (Table 1). Chances of benefit from Hormonal Therapy were classified as 156 6 follows: 0–1 = No effect; 2–3 = Small (20%) chance of benefit; 4–6 = Moderate (50%) chance of 157 benefit; 7 = Good (75%) chance of benefit. 158 159 Scoring of Her-2/neu status 160 For Her-2/neu expression, the only membrane staining pattern was scored from 0 to 3+, where 0/1+ 161 indicates negative, 2+ stands for equivocal, and 3+ means positive following the standards outlined 162 by Ellis et al.14. 163 The criteria used are explicitly described as follows: Negative (0 scores): Membrane staining <10% 164 of the tumor cells, or no staining detected. Negative (1+ score): Membrane staining detected in >10% 165 of the tumor cells or faint staining detected. The stain was observed only in some parts of the 166 membrane. Equivocal (2+ score): A weak to moderate complete membrane staining was detected in 167 >10% of the tumor cells. Positive (3+score): A strong complete membrane staining was detected in 168 >10% of the tumor cells. The molecular classification was based on the positivity and negativity of 169 ER, PR, and Her-2/neu [Figure 1]. 170 171 Scoring of p53 status 172 For p53 expression, the nuclear staining pattern was scored from 0, 1+, 2+ to 3+, the numbers 0, 1+, 173 2+, and 3+ were used to describe the intensity of the staining of the p53 protein in the cells (reported 174 by Bergh)15. The degree staining was used to determine whether the p53 protein is overexpressed or 175 not. The numbers 0 and 1+ indicated negative staining, while the numbers 2+ and 3+ indicated 176 positive staining, as depicted in Figure 1c. The p53 protein is considered negative if it is not 177 overexpressed or mutated. 178 179 Statistical analysis 180 Data obtained were reported in percentage and proportion using descriptive statistics. No calculation 181 of sample size was done, and all cases with complete information were entered into the study. The 182 Chi-square test was used to determine the association between histological tumor grades (I, II, and 183 III) against the expression patterns of individual selected molecular markers (for ER/PR expression, 184 Her-2/neu overexpression, and p53 mutation). A value of P <0.05 was considered statistically 185 significant. 186 187 7 Ethical approval and consent 188 Ethical approval was obtained from the LAUTECH Health Research Ethics Committee. This study 189 posed no risk to the participants and the community at large. Data generated were made confidential, 190 and no patients' names were recorded. 191 192 Results 193 This was a hospital-based retrospective study involving biopsy/surgical cases of BC recorded over 194 10 years. Two hundred and five (n = 205) cases were investigated for IHC markers—hormone 195 receptors (estrogen receptor, ER and progesterone receptor, PR), human epidermal growth factor 196 receptor (Her-2/neu), and p53 immunomarkers. 197 198 Age distribution 199 The age range was 21 and 87 years (mean = 49.30 years) of the total cases. The peak age of this 200 incidence was 50–59 years. 201 202 Laterality 203 By laterality, the records showed that BC occurred at nearly the same rate between the left (n = 103 204 cases; 50.2%) and the right (n = 102 cases; 49.8%) breast sides among those with complete records. 205 206 Histological type 207 The most frequent histological phenotype of female BC recorded was infiltrating ductal carcinoma 208 (IDC) (190 cases: 92.7%). Other less frequent types were invasive lobular carcinoma (ILC) (8 cases; 209 3.9%) and medullary carcinoma (3 cases; 1.5%), while the rare frequent phenotypes were mucinous 210 carcinoma, carcinosarcoma, metaplastic carcinoma, and poorly differentiated carcinoma had 1 case 211 each (0.49%), respectively. 212 213 Tumor grade 214 Using Nottingham modification of the Bloom-Richardson system, the frequency distribution by 215 tumor grade was recorded [Table 2]. 216 217 8 Tumor size 218 All the cases had specified tumor sizes ranging between 1–22 cm in the widest diameter (mean = 219 5.8 cm). The frequency distribution is shown in Table 2. 220 221 Lymph node metastasis 222 Table 2 also illustrates the degree of lymph node (LN) involvement. LN biopsy was reviewed in the 223 record for a possible note of metastasis in individual cases. The frequency distribution is shown in 224 Table 2. 225 226 Nottingham prognostic index 227 The Nottingham Prognostic Index (NPI) traditionally involves a combination of the assessments of 228 nodal status, tumor size, and histological grade for its potential survival outcome. It is based on a 229 recent prognostic scoring, namely, NPI-I (excellent) ≤2.4; NPI-II (good) >2.4 but ≤3.4; NPI-III 230 (moderate) >3.4 but ≤5.4; and NPI-IV (poor) >5.4 16. Our data showed that out of 205 cases, 63 231 cases (30.7%) indicated a good prognosis, 100 cases (48.7%) signified a moderate prognosis, and 232 42 cases (20.5%) showed a poor prognosis. 233 234 Immunohistochemical profile 235 Two hundred and five female (n = 205) BC cases were processed and stained for ER, PR, Her-2/neu 236 antigen, and p53 positivity. 237 Two hundred and five female (n = 205) BC cases were immunostained for ER and PR. One hundred 238 and twenty-seven cases (n = 127; 62.0%) were positive, ER+, while 78 cases (38.0%) were ER-. 239 One hundred and forty-five cases (n = 145; 70.7%) were PR+, while 60 cases (29.3%) were PR-. 240 The intensity and its score are shown in Table 3A and Figure 1. 241 242 Two hundred and five cases (n = 205) were immunostained for Her-2/neu. Seventy cases (n = 70) 243 34.2%) were Her-2/neu+, while eighty-one cases (n = 81; 39.5%) were Her-2/neu-. Fifty-four cases 244 (n = 54; 26.3%) were equivocal. For the equivocal result, the stains were not furthered with 245 fluorescent in situ hybridization (FISH) due to limited funding but considered Her-2/neu-. 246 247 The staining intensity and the score for Her-2/neu are shown in Table 4 and Figure 1b. 248 9 Two hundred and five cases (n = 205) were analyzed for p53 immunostain. One hundred and fifty-249 six cases (n = 156; 76.1%) were p53+, while 49 cases (23.9%) were p53-. The staining intensity and 250 the score for p53 mutation are shown in Table 4 and Figure 1c. 251 252 Immunohistochemical profile and Nottingham tumor grade 253 We observed associations between the expression profile of hormone receptors (ER and PR), Her-254 2/neu, and p53 compared to the Nottingham tumor grade. The pattern of expression in ER (positivity) 255 showed a significant difference (P ˂ 0.01) compared to the distribution of patients according to 256 tumor grades, in the same way as PR positivity (P ˂ 0.001). Likewise, the pattern of Her-2/neu 257 expression (connecting positive, negative, and equivocal staining distribution among the incident 258 cases) showed a significant difference (P ˂ 0.001) compared to the Nottingham tumor grade pattern. 259 Also, the association (P = 0.001) between the p53 expression pattern and the Nottingham tumor 260 grade pattern was observed. 261 262 Based on the results provided above, we classified the breast cancer subtypes along with their 263 proportions in this study into the following groups: 264 265 ER/PR positive, Her-2/neu negative cases were 110 (53.6%); This subtype was characterized by the 266 presence of estrogen and progesterone receptors but the absence of Her-2 overexpression through 267 ER+/PR+, Her-2-; ER-/PR+, Her-2- and ER+/PR-, Her2-. 268 269 ER/PR positive, Her-2/neu positive cases were 48 (23.4%); This subtype was defined by the 270 presence of both estrogen and progesterone receptors, as well as Her-2 overexpression through 271 ER+/PR+, Her-2+: ER-/PR+, Her-2+ and ER+/PR-, Her-2+. 272 273 ER/PR negative, Her-2/neu positive cases 22 (10.7%); This subtype was identified by the absence 274 of estrogen and progesterone receptors but the presence of Her-2 overexpression through ER-/PR-, 275 Her-2+. 276 277 Triple-negative cases were 25 (12.2%): This subtype was specified by the absence of estrogen and 278 progesterone receptors, as well as Her-2 overexpression through ER-/PR-, Her-2- and ER-/PR-, Her-279 2-. 280 10 281 Discussion 282 In this study, we retrospectively investigated 205 BC cases in western Nigeria for hormone receptor 283 (HR) expression (HR: estrogen receptor [ER] and progesterone receptor [PR]), human epidermal 284 growth factor receptor (Her-2/neu), and p53 expression profile in terms of pattern and frequency. 285 We explored the expression patterns of these biomarkers in connection with the tumor's 286 aggressiveness using the conventional Nottingham grade. 287 288 From our findings, the molecular characteristics of the tumor showed that ER and PR were positive 289 in 62% and 70.7% of the total recorded cases, respectively. There were associations between ER and 290 PR's expression patterns and the tumor grades' frequency. This is following the report on Polish 291 women, which showed an association between tumor grades and HR positivity.17 The present study 292 showed that grade II tumors had a higher ER and PR positive frequency than grade III. Meanwhile, 293 a previous report 18 indicated that the number of cells expressing ER and/or PR gradually decreases 294 with disease progression. This was substantiated by the report of Badowska-Kozakiewicz et al. 17, 295 which showed an inverse correlation between ER expression and the size of the primary tumor. In 296 specific terms, in addition to positively predicting therapeutic outcomes, estrogen receptor α (ERα) 297 is believed to inhibit epithelial-mesenchymal transition by promoting epithelial phenotype and 298 preventing tumor invasion in breast cancer.19 We observed higher HR positivity than in many 299 African populations, including Guinea 20, Ghana 21, South Africa 22, and Mali 23. Although there is 300 no specific identifiable factor influencing the diverse variation from one population to another, a 301 previous study suggested that small sample sizes recruited for studies across African countries could 302 be a possible reason.20 Even though our study showed higher HR positivity compared to a study of 303 a considerably similar population in Nigeria, where a multicentric study involving 507 patients was 304 previously carried out.24 Conversely, our data are in tandem with reports involving BC patients in 305 Western countries 6 and the Saudi population 7, where high HR is also documented. Potemski and 306 coworkers 25 reported related results and revealed that the higher the level of receptor expression, 307 the lesser the mortality. In line with their observations, our study also showed that the majority of 308 our incident cases had a moderate prognosis with high HR positivity and lower tumor grades, 309 indicating a possible association between HR expression and tumor grade. 310 311 11 In addition, regarding the Her-2/neu expression pattern in this study, some (39.5%) of the cases were 312 negative and were more than the positive (34.2%) outcome, with an unexpected increase in Her-2+ 313 proportion than many reported cases. Equally, patients were classified histologically as having 314 higher grades in the Her-2/neu+ expression group than in the Her-2/neu- expression group but lower 315 in ER+ and PR+ expressions compared to ER- and PR- of the highest grade III [Table 4]. In 316 agreement with our study, Arafah 7 reported that the histologic grade of BC was significantly 317 associated with both ER and PR expressions but, in turn, found a negative correlation between HR 318 and Her-2/neu stains. Also, Aman et al. 26 recently associated overexpression of Her-2/neu with 319 higher Nottingham grade in an Ivorian population. Again, in the literature, concurring with the 320 present study, a study involving the Chinese population reported a link between Her-2/neu 321 overexpression and a higher histological grade with a higher incidence rate of infiltrating ductal 322 carcinoma, among many other factors.8 Although the majority (92.7%) of the incident cases in this 323 study were infiltrating ductal carcinoma, which is in line with the study of Ding and his colleagues9, 324 our analysis also showed a strong association between histological grading and the pattern of 325 expression of Her-2/neu. However, our observations indicated that Her-2/neu overexpression is 326 linked to the aggressive forms of BC, as previously reported by Arteaga and his colleagues.27 327 328 Moreover, to better understand the therapeutic benefits for the patients, we classified the patients 329 based on histological phenotypes of the hormone receptor and Her-2/neu expression patterns. Most 330 notably and in agreement with the report of Gago et al.28, the majority of our breast cancer patients 331 co-express HR+ with Her-2/neu- rather than Her-2+ tumours, indicating that the cancer cells are 332 responsive to hormones such as estrogen and progesterone, better prognosis and also preventing 333 tumour aggressiveness. On the other hand, among the Her-2+ category, a smaller number of ER/PR-334 Her-2/neu+ was observed representing breast cancer cases where both the ER and PR are negative, 335 while the Her-2/neu is overexpressed. This subtype is commonly known as hormone receptor-336 negative, Her-2/neu-positive breast cancer. It suggests that the cancer cells do not respond to 337 hormones and have an overexpression of the Her-2/neu gene. More importantly, triple-negative 338 breast cancer (TNBC) is a vastly diverse group of tumours, which represents 15-20% of all breast 339 cancer cases Kummel et al.29. The proportion of the TNBC in our study is relatively small suggesting 340 an advantage against the studied population. Meanwhile, TNBC is the most difficult to treat among 341 all breast cancer phenotypes because the common hormonal therapy used for the majority of breast 342 12 cancer subtypes is treatment-refractory for TNBC. On the hand, TNBC is often treated in its early 343 stages with surgery, radiation, and chemotherapy. 344 345 Furthermore, most of our investigated cases (70.1%) were p53 positive, and there was a strong 346 association between the p53 expression pattern and the Nottingham tumor grade. Consistent with 347 other studies 5, 30, our findings, therefore, implied that the p53 positivity may have a connection with 348 tumor grade in terms of the frequency of the incident cases. Patients in the p53+ expression group 349 were classified histologically as higher grades than those in the p53- expression group, similar to the 350 previous report 24 and corresponding to the Her-2/neu expression pattern in this investigation. 351 Shokouh et al.5 earlier demonstrated that p53 expression had a significant association with the grade 352 of BC. Various reports have outlined the functional role of p53 in the progression of BC. 353 Mechanistically, p53 activates protein transcriptions involved in the DNA repair mechanism. 354 However, if the mechanisms fail due to a defective p53, aberrant cells may proliferate 355 uncontrollably, leading to cancer 31. A report shows that tumors with p53 mutations are more likely 356 to be aggressive and resistant to chemotherapy and radiotherapy.29 In other words, p53 357 immunoreactivity is linked to histologic grade, particularly a tumor's high mitotic index.8 358 359 Limitations of the study 360 According to the Her-2 testing guidelines of the American Society of Clinical Oncology and the 361 College of American Pathologists (ASCO/CAP), breast cancer that is reported 2+ equivocal by 362 IHC should be followed up with in-situ hybridization (ISH) testing to confirm the cases for 363 possible gene amplification. However, the current study is limited by the inability to verify the 364 negative (2+ score) results with fluorescence in situ hybridization (FISH), and thus considered 365 negative. This could have an impact on the negative result value. 366 367 Conclusion 368 Our observations suggest that expression patterns of PR, ER, Her-2/neu, and p53 were influenced 369 by the tumor grade (level of aggressiveness). In other words, there is an association between the 370 tumor grade and expressions of PR, ER, Her-2/neu, and p53, which suggests that the Nottingham 371 grade is still relevant as a reliable prognostic marker for BC. 372 373 13 Funding 374 No funding was received for this study. 375 376 Conflicts of interest 377 The authors declare no conflicts of interest. 378 379 Authors’ Contribution 380 KAA, WAO, and MAO were involved in conceptualization and design of the study. WAO, MAO, 381 LAY, and RTK collected the data. KAA and SOI analyzed and interpreted the results. 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Nat Rev Mol Cell Biol 2019; 20:199–210. 483 https://doi.org/10.3389/fcell.2021.762651 484 485 https://doi.org/10.1186/s12907-018-0081-4 https://doi.org/10.3389/fcell.2021.762651 17 Table 1: Scoring Guideline (“Quickscore”) for ER and PR 486 Proportion score Observation Intensity score Observation Zero Zero staining Zero No staining of any nuclei even at high magnification 1 1 – 25% 2 26 – 50% 3 51 - 75% 4 76 – 100% 1 Weak staining (only visible at high magnification) 2 Moderate staining (Readily visible at low magnification) 3 Strong staining (strikingly positive even at low magnification) The score for intensity is then added to the score for proportion, giving a range of 0-7. 487 488 Table 2: Frequency distribution of tumor grade, size, and lymph node involvement in female 489 breast cancers 490 Tumor index Frequency(%) Tumor gradeα I (Low) 16 (7.80) II (Intermediate) 146(71.22) III (High) 43 (20.98) Tumor sizeβ pT1 18 (8.78) pT2 106(51.71) pT3 81(39.51) Lymph node statusɤ pN0 156(76.1) pN1 46(22.44) pN2 3(1.46) “ɤ”rep.tumor grade(Nottingham grade): Grade 1 =I; Grade 2 =II; Grade 3 =III 491 “β” rep. lesion size (cm): pT1= ≤ 2 cm; pT2 = 2-5 cm; pT3 = >5cm 492 “ɤ” rep. node positivity: pN0= 0 nodes;pN1 = 1-3 nodes;pN2 = >3 nodes. 493 494 18 Table 3: Frequency distribution according to ER and PR expression status 495 ER Score Frequency (%) Cumulati ve PR Score Frequency (%) Cumulati ve Interpretation Zero 8 (3.9) 78 (38.0) Zero 15 (7.3) 60 (29.3) Negative 2 70(34.1) 2 45(22.0) Negative 3 43 (20.9) 127 (62.0) 3 61 (29.8) 145 (70.7) Positive 4 27 (13.2) 4 13 (6.3) Positive 5 25 (12.2) 5 55(26.8) Positive 6 14(6.8) 6 12 (5.9) Positive 7 18(8.9) 7 4(1.9) Positive Total 205 (100) 205(100) Total 205 (100) 205 (100) ER -Oestrogen receptor; PR - Progesterone receptor 496 497 Table 4: Frequency distribution according to Her-2/neu and p53 expression status 498 Her- 2/neu Score Freq. (%) Cumulati ve Interpreta tion p53 Score Freq. (%) Cumulati ve Interpret ation Zero 21 (10.2) 81 (39.5) Negative Zero 13(6.3) 49 (23.9) Negative 1+ 60 (29.3) Negative 1+ 36(17.6) Negative 2+ 54(26.3) 54(26.3) Equivocal 2+ 85(41.5) 156 (70.1) Positive 3+ 70(34.2) 70(34.2) Positive 3+ 71(34.6) Positive Total 205(100) 205(100) Total 205(100) 205(100) Her-2/neu – human epidermal growth factor receptor-2 499 500 Table 5: Expression profile of hormone receptors, Her-2/neu and p53 compared to 501 Nottingham tumor grade 502 Immunohistochemi cal markers Nottingham grade χ2, P-value, df Grade I Grade II Grade III ER+ (%) 10 (7.9) 103(81.1) 14 (11.0) 10.458, ˂0.01, 2 ER- (%) 3 (3.85) 53 (67.95) 22 (28.21) PR+ (%) 8(5.6) 121 (83.4) 16 (11.0) 18.581, ˂0.001, 2 PR- (%) 3 (5.00) 35 (58.33) 22 (36.67) Her2/neu+ (%) 4 (5.7) 46(65.7) 20(28.6) 27.317, ˂0.001, 4 Her2/neu-ve (%) 31 (38.27) 42 (51.85) 8(9.88) Her2/neu-Eq (%) 11 (20.37) 28 (51.85) 15 (27.78) p53+ (%) 8 (5.2) 118(75.6) 30 (19.2) 13.381, 0.001, 2 p53- (%) 9 (18.37) 38 (77.55) 2 (4.08) ER: oestrogenreceptor; PR: progesterone receptor; Her2/neu: human epidermal growth factor 503 receptor 2; +: positive; -/-ve: negative; Eq.: equivocal 504 19 505 Figure 1: A: Invasive ductal carcinoma (ER-positive X40). Note that the tumor cells pick up the 506 stain in the nucleus. The score in this case was 7. B: Invasive ductal carcinoma. (Her-2/neu 507 positive x40). Note that the intensity score for this case was 3. Her-2/neu stains in the membrane 508 compared to ER/PR which stains in the nucleus. C: Invasive ductal carcinoma (p53 positive x40). 509 Note that the intensity score for this case is 3. p53 stains in the nucleus like ER/PR. 510