Journal of Global Pharma Technology

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1 ISSN Journal of Global Pharma Technology Available Online at Research Paper The Degree of Agreement between Manual and Digital Histological Evaluation of Immunohistochemical Expression of Estrogen and Progesterone Receptors in Breast Carcinoma Hameda Abd-Al Mahdi Ghazi College of medicine, University of Babylon- Hilla-Iraq. Abstract Background Malignant neoplasm involving the breast is by far one of the most frequent cancers that affect mankind. Globally, it is estimated that breast cancer accounts for about 25% of all cancers affecting women. Carcinoma of the breast is heterogeneous; however, they all share the common origin from the terminal duct lobular unit. They are generally classified into ducal and lobular. The three gold standard immunohistochemical makers, nowadays, are progesterone and estrogen receptors (PR and ER) and her2neu. Other markers like p53 and bcle2 expression are also important. The study of PR and ER receptors permits the decision of adding and adjunct mode of treatment, hormonal treatment. Aim of the study the objective of this study was to assess automated digital study of immunohistochemical markers as a substitute for the original manual method. Materials and methods: The present study included 35paraffin embedded, formalin fixed histological blocks of breast carcinoma. The duration of the study extended from January 2017 to June 2017 and was conducted in the teaching laboratories of College of Medicine, Al-Qadissiyah Univerist, Al-Diwaniayah province, Iraq. The paraffin blocks were retrieved from the teaching laboratories of Al-Diwaniyah teaching hospital, Al-Diwaniayah province, Iraq. Results: Following conversion into positive and negative cases, matching was assessed using kappa agreement statistic and the results were as following: number of positive ER cases were 14 and 16 according to manual and digital scoring, respectively; whereas the number of positive PR cases were 15 and 17 according to manual and digital scoring, respectively, as shown in table 3 and 4. The degree of agreement between manual and digital scoring was 0.87, considered good, in both ER and PR expression status. Conclusion Digital scoring appears to be faster than and as accurate as manual scoring; however it needs to be correlated with clinical prognosis of breast carcinoma. Introduction Malignant neoplasm involving the breast is by far one of the most frequent cancers that affect mankind. Globally, it is estimated that breast cancer accounts for about 25% of all cancers affecting women. Although, it is well known disease in women, it can affect men; nevertheless, its incidence in male patients is less than 1: 100 when compared to female patients [1]. According to [2] 2014, breast cancer in Iraqi women ranks the first among other forms of cancers and accounts for about 33.8%. Anatomically speaking, the breast or mammary glands are specialized accessory glands that are located in the pectoral region and present in both sexes; however, they are strictly functional in women and secrete milk. The breast harbors fifteen to twenty five lactiferous ductss that start at the nipples then divide into smaller ducts and terminates in the terminal ductt lobular unite (lobule) that is formed of a terminal duct and a number of small acini. The ducts and ductless are lined by a layer of cubical to columnar epithelial cells and an external my epithelial cell layer. The connective tissue within the lobule is made up of fibroblasts in a matrix of collagen and acid mucins, together with histiocytes and little number of lymphocytes. The interlobular stroma is hypocellularr and formed of fibroadipose tissue. The lining epithelium and the stroma are responsive to hormones. Throughout pregnancy there is a pronounced proliferation of acini, leading to enlargement of lobules, and the epithelial cells possess abundant cytoplasm rich in secretary vacuoles [3] , JGPT. All Rights Reserved 201

2 Neoplasms are uncontrolled cellular proliferation that affect body tissues and result in districted structure and function. Neoplasms affecting breast tissue are diverse and can originate from epithelium, connective tissues, lymphoid cells, vascular tissue and melanocytes. Tumors originating from epithelial tissues are by far the most common and usually present in the form of adenoma or carcinoma. The most common benign breast tumor is fibro adenoma, whereas the most frequent malignant breast tumor is carcinoma [4]. Carcinoma of the breast is heterogeneous; however, they all share the common origin from the terminal duct lobular unit. They are generally classified into ductal and lobular. Ductal carcinoma are characterized by formation of duct like structures with stromal invasion, while, lobular type is characterized by acinar formation and single cell infiltration of the stroma. Further classification is determined by the presence or nascence of basement membrane breaching and stromal invasion by malignant cells [5]. The general scheme will be in the form of one the following major groups: insituductall carcinoma, invasiveductall carcinoma, insituu lobularr carcinoma, invasive lobular carcinoma. Invasive ductal carcinoma is further sub-classified into: tubular, papillary, come do, medullar and mutinous according to specific morphologic features, however, invasive ductal carcinoma of no special type or in other words not otherwise specified (NOS) is by far the most frequent and accounts for about 75-80% [6]. Management of carcinoma of breast is dependent up on the stage and grade of the disease and other characteristics. Stage means the degree of spread of the lesion while grade means the degree of differentiation or resemblance of the malignant lesion to the original breast tissue. Surgical removal is the standard way of treatment together with chemotherapy and or radiotherapy [7]. Another important determinant of the way of treatment is the expression of immunohistochemical markers. The three gold stand ardimmunohistochemical makers, nowadays, are progesterone and estrogen receptors (PR and ER) and her2neu [8]. Other markers like p 53 and bcle 2 expression are also important. The study of ER and PR receptors permits the decision of adding and adjunct mode of treatment, hormonal treatment. The study of these two immmunohistochemical markers, is nowadays, totally manual and subjective and great inter-observer and intra-observer variations exist. So the objective of this study was to assess automated digital study of immunohistochemical markers as a substitute for the original manual method [8]. Materials and Methods The present study included 35 paraffin embedded, formalin fixed histological blocks of breast carcinoma. The duration of the study extended from January 2017 to June 2017 and was conducted in the teaching laboratories of College of Medicine, Al- Qadissiyah Univerist, Al-Diwaniayah province, Iraq. The paraffin clocks were retrieved from the teaching laboratories of Al-Diwaniyah teaching hospital, Al- Diwaniayah province, Iraq. The first step was to review the biopsy reports available at the laboratories from January 2015 through the present time of the study in order to get the most recent convenient sample of paraffin blocks belonging to patients already diagnosed with breast carcinoma. Next, from each paraffin block, thin sections (5 μm widths) were prepared, 3 in number: one for revaluation of H and E stained sections and two for immunohistochemical staining with ER and PR markers. Standard immunostaining was carried out according to the providing company and included the following steps: deparafinization and attaching tissues to positively charged slides followed by antigen retrieval and addition of the primary antibody after pap pen labeling and then incubation at 37 C for 30 minutes. The next step involved the addition of the conjugated secondary antibody and then the addition of avid in horse radish peroxides complex and then the addition of the chromogen substrate. Each step was followed by proper washing using standard washing solution. The results of staining procedures are shown in figures (1and 2) , JGPT. All Rights Reserved 202

3 Manual reading was carried out by two blinded pathologist using Olympus light microscope. Nuclearr staining was assessed for ER and PR. A score for the proportion of stained cells (zero = no nuclear staining, one = <1percent nuclear staining, two = 1-10percent nuclear staining, three = 11-33percent nuclear staining, four = 34-66percent nuclear staining and five = percent nuclear staining) and the intensity of staining (0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining) were assigned to each tumor. The score for the proportion of cells stained and the score for the intensity of staining were added to get the total score, which ranged from 0 to 8, A score of 0-2 was regarded as negative while 3-8 as positive. [9].Digital studying was done using Scan scope Leika (Germany) fully automated system Digital scoring was made to correspond to manual scoring so that we depend on two parameters, the proportion of cellss stained and intensity of color. The results were then transferred into Microsoft Excel sheet for further analysis. Data were statistically analyzed using SPSS. version 22. Mann Whitney U tool was utilized to compare mean scores, whereas, Chi-square was used to evaluate ratios. P- value was considered significant at a point equal to or less than Results The results of manual quick score are shown in table 1 and 2. Regarding estrogen receptor (ER) immunohistochemical staining score the following results were obtained: the number of cases were 16, 1, 0, 4, 4, 2, 2, 1 and 1 for as retrieved from rater 1 reports; the number of cases were 15, 2, 1, 3, 4, 2, 2, 1 and 1 for as retrieved from rater 2 reports. Regarding progesterone receptor (PR) immunohistochemical staining score the following results were obtained: the number of cases were 14, 2, 0, 0, 4, 3, 4, 3 and 1 for as retrieved from rater 1 reports; the number of cases were 13, 3, 0, 1, 3, 4, 3, 2 and 2 for as retrieved from rater 2 reports. Table 1: results of manual scoring for ER immunohistochemical expression ER PR Score Rater 1 Rater 2 Rater 1 Rater (51.6) 15 (48.4) 14 (45.2) 13(41.9) 1 1 (3.2) 2 (6.5) 2(6.5) 3(9.7) 2 0 (0.0) 1 (3.2) 0(0.0) 0(0.0) 3 4 (12.9) 3 (9.7) 0(0.0) 1(3.2) 4 4 (12.9) 4 (12.9) 4(12.9) 3(9.7) 5 2 (6.5) 2 (6.5) 3(9.7) 4(12.9) 6 2 (6.5) 2 (6.5) 4(12.9) 3(9.7) 7 1 (3.2) 1 (3.2) 3(9.7) 2(6.5) 8 1 (3.2) 1 (3.2) 1(3.2) 2(6.5) ER: estrogen receptors; PR: progesterone receptors Results of digital scoring are shown in table 2 and they were as following: the number of cases, concerning ER status, were 14, 1, 0, 4, 4, 2, 2, 2 and 2 for scores 0, 1, 2, 3, 4, 5, 6, 7 and 8, respectively. In PR status, the number of cases were 13, 1, 0, 0, 4, 3, 4, 4 and 2 for scores 0, 1, 2, 3, 4, 5, 6, 7 and 8, respectively. Table 2: results of digital scoring for ER immunohistochemical expression Score ER PR 0 14 (45.2) 13 (41.9) 1 1 (3.2) 1 (3.2) 2 0 (0.0) 0(0.0) 3 4 (12.9) 0(0.0) 4 4 (12.9) 4(12.9) 5 2 (6.5) 3(9.7) 6 2 (6.5) 4(12.9) 7 2 (6.5) 4 (12.9) 8 2 (6.5) 2 (6.5) ER: estrogen receptors; PR: progesterone receptors , JGPT. All Rights Reserved 203

4 Following conversion into positive and negative cases, matching was assessed using kappa agreement statistic and the results were as following: number of positive ER cases were 14 and 16 according to manual and digital scoring, respectively; whereas the number of positive PR cases were 15 and 17 according to manual and digital scoring, respectively, as shown in table 3 and 4. The degree of agreement between manual and digital scoring was 0.87, considered good, in both ER and PR expression status. Table 3: Rate of positive ER cases according to manual and digital scoring Kappa =0.87 Manual scoring Positive Negative Total Positive Digital scoring Negative Total Table 4: Rate of positive PR cases according to manual and digital scoring Kappa = 0.87 Manual scoring Positive Negative Total Positive Digital scoring Negative Total A B C D Figure 1: Immunohistochemical expression of estrogen receptors (ER) in breast carcinoma. A: Technical negative control. B: Negative. C: weak positive. D: Strong positive A B C D Figure 2: Immunohistochemical expression of progesterone receptors (PR) in breast carcinoma. A: Technical negative control. B: Negative. C: weak positive. D: Strong positive , JGPT. All Rights Reserved 204

5 Discussion Although, the estimation of malignant cell estrogen receptor (ER) expression status by immunohistochemical method has gained routine practice, the reproducibility of the assay has not been measured sufficiently. The issue of inter laboratory mismatch in the immunohistochemicall estimation of estrogen receptor status using a breast cancer tissue has been raised and investigated by Parker et al [10]. Their work involved a breast tumor tissue microarray, and they examined inter laboratory mismatch in ER reading. A 2-fold tissue microarray block was synthesized from twenty nine malignant breast blocks. Unstained slides were distributed to 5 laboratories and each laboratory immunostained and scored 1 slide for ER. Agreement among laboratories ranged from moderate to high (overall. kappa = 0.54 for zero-three+ grading; overall. kappa = 0.84 for negative vs positive assessment of ER status). When 1 observer scored each of the 5 slides, inter laboratory agreement was slightly better (kappa = 0.63 for 0-3+ scoring; kappa = 0.96 for positive versus negative scoring). When looking to the work of Parker et al., one can conclude that although the experiment used tissue microarray, the level of concordance was not always excellent among different labs and observers. In our opinion, the use of well-equipped and optimally standardized fully automated digital method in assessing immunohistochemical status of ER and PR may not result in such wide range of disagreement among different labs. The progesteron receptor and estrogen receptor status of human breast malignancy is regarded as an important marker from prognostic and predictive points of view of human breast cancer that affect therapeutic measures but their subjective way of interpretation result in intra observer, interaserver and fatigue variability. Inter observer agreement for estrogen receptor immunohistochemical analysis in breast cancer was also assessed by Diaz et al.[11]. In this study a series of seventy consecutive invasive breast malignant tumors, ER was determined using two methods: manual stating method and digital method. The comparison between manual and digital methods in the study of Diaz et al [11] reveled strong agreement (kappa =0.84); this results supports our findings and also they described high level of inter observer agreement related to manual method; also in agreement with the results of our study. Probably the availability of well-trained expert lab workers in the study performed by Diaz et al. and us is the cause behind good level of inter observer agreement and this may not always available and perhaps the use of digital scoring may reduce the need for well-trained lab workers and that minimal manipulation for well-established digital machine may give excellent results. Prasad et al. [12] carry out a comparison of manual and digital assessment immunostaining of 60 breast cancer tissue specimens. They performed comparison among three labs regarding manual method as well comparison of manual with digital scoring. The level of correlation between two pathologist was 0.46 and 0.66 for two pathologist regarding ER and PR expression respectively. This low level of correlation, in the study of Prasad et al., clearly demonstrated the effect of subjectivity of the manual determination of PR and ER status in breast carcinoma. The results of the present showed that digital scoring is characterized by overestimation of positive cases, ER and PR, when compared to manual scoring. This overestimation may be the direct results of variation in intensity measurement between manual and digital methods; however, the kappa agreement statistics gave a good result of A lower level of agreement was recorded by Hwatet al., in [13] when measuring the expression of ER in breast cancer both manually and digitally, and the level was (kappa =0.76). Similar rate of agreement was reported by Krecsáket al., in [14] of about 86% for both ER and PR. The overestimation of positive results, together with less than 100% agreement may raise the question of which method is more accurate in defining ER and PR status for breast carcinoma. For answering such a question, one should perform a large cohort study to relate overall survival with digital and manual scoring. Unfortunately this aim is out of scope of our study; nevertheless, such study was carried out by Stålhammar et al. [15] and stated that the digital scoring system was in major , JGPT. All Rights Reserved 205

6 aspects a better alternative to manual marker scoring and has the advantage to minimize time consumption for doctors, as many of the steps in the technique are either automatic or easy to manage without pathological expertise. The issue of time consumption and inter observer variability of manual method was raised by Vijayashree et al.[16] and they measured time needed to accomplish manual work in comparison with digital work and concluded that unlike routine manual techniques that are time consuming, a single run of digital way takes only a few minutes. When time is spent by pathologist to read a single slide of ER immunohistochemistry of breast tissue is equal to the same time needed to carry out examination of several H and E slides to assess histological diagnosis, References 1. Makki J (2015) Diversity of Breast Carcinoma: Histological Subtypes and Clinical Relevance. Clinical Medicine Insights Pathology.8: Al-Hashimi MM, Wang XJ (2014) Breast cancer in Iraq, incidence trends from Asian Pac J Cancer Prev.15: Standring S, Gray H (2015) Gray's anatomy: The anatomical basis of clinical practice. Fortieth edition. Edinburgh: Churchill Livingstone/Elsevier Shah R, Rosso K, Nathanson SD (2014) Pathogenesis, prevention, diagnosis and treatment of breast cancer.world Journal of Clinical Oncology.5(3): Sharma GN, Dave R, Sanadya J, Sharma P, Sharma KK (2010) Various Types and Management Of Breast Cancer: An Overview. Journal of Advanced Pharmaceutical Technology & Research. 1(2): Sinn H-P, Kreipe H A (2013) Brief Overview of the WHO Classification of Breast Tumors, 4th Edition, Focusing on Issues and Updates from the 3rd Edition. Breast Care. 8(2): Zhang BN, Cao XC, Chen JY (2012) et al Guidelines on the diagnosis and treatment of breast cancer (2011 edition).gland Surgery.1 (1): Mouttet D, Laé M, Caly M (2016) et al Estrogen- Receptor, Progesterone-Receptor and HER2 Status Determination in Invasive Breast Cancer. Concordance between Immuno-Histochemistry and Map Quant TM Microarray Based Assay. Sapino A, ed. PLoS ONE. 11(2):e Mudduwa LK (2009) Quick score of hormone receptor status of breast carcinoma: correlation with other clinicopathological prognostic parameters. Indian J PatholMicrobiol.52: this will of course make digital scoring superior to manual scoring of ER and PR status. It should be mentioned that scoring 31 cases for ER and PR status manually, took 2 weeks in order to be accomplished, whereas, it was carried out at a single run in one day for the digital procedure. This time consumption by manual method is coasty and can be reduced significantly by the use of digital method. Also there, was some inter-observer variation between rater 1 and 2, though it was minimal, but expert pathologist is always needed to perform such tedious work. In conclusion, digital scoring appears to be faster than and as accurate as manual scoring, however it needs to be correlated with clinical prognosis of breast carcinoma. 10. Parker RL, Huntsman DG, Lesack DW (2002) et al Assessment of interlaboratory variation in the immunohistochemical determination of estrogen receptor status using a breast cancer tissue microarray. Am J ClinPathol. 117(5): Diaz LK, Sahin A, Sneige N (2004) Interobserver agreement for estrogen receptor immunohistochemical analysis in breast cancer: a comparison of manual and computer-assisted scoring methods. Ann DiagnPathol. 8 (1): Prasad K, Tiwari A, Ilanthodi S, Prabhu G, Pai M (2011) Automation of immunohistochemical evaluation in breast cancer using image analysis. World Journal of Clinical Oncology. 2(4): Howat WJ, Blows FM, Provenzano E (2015) et al Performance of automated scoring of ER, PR, HER2, CK5/6 and EGFR in breast cancer tissue microarrays in the Breast Cancer Association Consortium. The Journal of Pathology: Clinical Research. 1(1): Krecsák L, Micsik T, Kiszler G (2011) et al Technical note on the validation of a semiautomated image analysis software application for estrogen and progesterone receptor detection in breast cancer. Diagnostic Pathology Stålhammar G (2016) et al Digital image analysis outperforms manual biomarker assessment in breast cancer. Mod. Pathol.doi: /modpathol Vijayashree R, Aruthra P, Rao KR (2015) A Comparison of Manual and Automated Methods of Quantitation of Oestrogen/Progesterone Receptor Expression in Breast Carcinoma. Journal of Clinical and Diagnostic Research : JCDR. 9 (3):EC01-EC , JGPT. All Rights Reserved 206