Immunohistochemical Expression Of Cytokeratin 8 And 18 In Breast Carcinoma.
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1 ISPUB.COM The Internet Journal of Pathology Volume 13 Number 3 Immunohistochemical Expression Of Cytokeratin 8 And 18 In Breast Carcinoma. B Rattan, A Baghla, M Manjari, P Kakkar, S Kahlon, S Paul Citation B Rattan, A Baghla, M Manjari, P Kakkar, S Kahlon, S Paul. Immunohistochemical Expression Of Cytokeratin 8 And 18 In Breast Carcinoma.. The Internet Journal of Pathology Volume 13 Number 3. Abstract Immunohistochemical study was done in 40 cases of invasive ductal carcinoma including 16 cases with metastatic lymph nodes. The cases were evaluated for expression of cytokeratin (CK) 8/18 and to correlate it with histological grade of tumour for predicting prognosis and therapeutic approach. Immounohistochemical score of 0 4 (negative) was seen in 6 (15%) cases and 5 12 (positive) was seen in 34 cases (85%) cases. Out of negative cases, one (1) case was of grade II and five (5) cases were of grade III. While 2 cases of grade I, 9 cases of grade II and 23 cases of grade III were positive CK 8/18. Secondary deposits in lymph nodes expressed CK 8/18 expression in similar manner to a primary tumour. Thus, it showed that a high proportion of IDC cases express luminal epithelial cell cytokeratin 8/18 indicating a differentiated glandular luminal phenotype, a finding associated with good prognosis. INTRODUCTION Carcinoma of the breast is one of the commonest malignancies in women.over the past few years, considerable attention has been focused on keratins as potential markers of differentiation and neoplastic progression in human breast epithelial cells1. Expression of intermediate filaments particularly cytokeratins (CKs) a component of cytoskeleton of cell, reflects epithelial cell type, state of tissue growth and differentiation in addition to the functional status of tissue.in normal breast CKs 7, 8, 18 and 19 are expressed in the luminal cells, while smooth muscle actin (SMA) and CKs 5/6,14 and 17 are found in myoepithelial/basal cells2.keratins have a number of distinct 3 advantages as marker proteins.it would thus seem worthwhile to characterize and subdivide this type of cancer with appropriate markers for prognosis and therapeutic approach. AIMS AND OBJECTIVES The aim of the study was to find CK 8/18 expression in breast carcinoma, to correlate its expression with histological grade of tumour and to see CK 8/18 expression in lymph node if metastases present. MATERIAL AND METHODS The study group comprised of group A carcinoma breast (Lumpectomy specimens) 13 cases and group B carcinoma breast (Mastectomy specimens) with lymph nodes, 27 cases. Group B was further divided into subgroup B I with lymph node showing reactive pathology and subgroup B II with metastatic deposits. The cases were analyzed for CK 8/18 expression by immunohistochemistry on four micron thick paraffin embedded tissue sections with standard peroxidase, antiperoxidase method by using prediluted liquid mouse monoclonal antibody specific for human CK 8/18 intermediate filament protein, clone : Rtu - 5D3 ( novocastra ). OBSERVATION AND RESULTS Histologically, 33 (82.5%) cases were of invasive ductal carcinoma (IDC) no special type NST, IDC with intraductal element 5 (12.5%), invasive carcinoma mucinous 1 (2.5%) and IDC with areas of squamous differentiation 1 (2.5%) Table 1.Age of these patients varied from 20 to 80 years, youngest patients was 23 years and oldest was 75 years, with 32.5% cases in 3 rd to 4 th decade. All the patients were females. Left side was involved in 60% cases. Size of growth varied from 2cm to 15 cm with ulceration of skin in 3 cases and retraction of nipple in 3 cases. Lymph node status of 27 cases in group B was, 11 cases (40.74%) had reactive pathology (subgroup B I) and 16 (59.25%) cases had metastatic deports (subgroup B II).Out of 40 cases, majority of 28 (70%) cases were of grade III. While 10 (25%) cases were of grade II and remaining 2 (5%) cases 1 of 7
2 were of grade I (Table II). Figure 4 Figure 1 TABLE IV: STAINING INTENSITY OF CK 8/18 IN GROUP A AND B TABLE I: HISTOLOGICAL DIAGNOSIS OF GROUPA AND B IHC SCORE IN GROUP A AND B IHC score of 0-4 (negative) was seen in 6(15%) cases and 5-12 (positive) was seen in 34(85%) cases (Table V). Figure 2 Figure 5 TABLE II: HISTOLOGICAL GRADING TABLE V: IHC SCORE GROUP A AND B IHC STAINING OF CYTOKERATIN 8/18 CORRELATION OF HISTOLOGICAL GRADE WITH PERCENTAGE OF POSITIVE STAINED CELLS IN GROUP A AND B PERCENTAGE POSITIVITY OF MALIGNANT CELLS IN GROUP A AND B. Out of 40 cases, majority of 27(67.5%) cases had more than 80% (>80%) stained cells. While 7(17.5%) cases had 50-80% stained cells and 2(5%) cases had 10-50% stained cells. Remaining 2 cases (5%) had <10% stained cells and 2(5%) cases had no positive stained cell (Table III). Figure 3 TABLE III: PERCENTAGE FOR POSITIVE STAINED CELLS OF CK 8/18 IN GROUP A AND B A majority of 28(70%) cases out of 40 cases of carcinoma breast were of grade III. While 10(25%) cases were of grade II and 2(5%) cases were of grade I. Out of 28 cases of grade III, 18 cases showed >80% and 5 cases showed 50-80% positive stained cells for CK 8/18. While 2 cases showed 10-50% and 1 case <10% positive stained cells for cytokeratin 8/18. Remaining 2 cases showed 0(0%) positivity. Out of 10(25%) cases of grade II, 7 cases showed >80% and 2 cases showed 50-80% positive stained cells for cytokeratin 8/18. While 1 case showed <10% positive stained for CK 8/18. Two (5%) cases of grade I showed more than 80% positive stained cells for cytokeratin 8/18 (Table VI). STAINING INTENSITY IN GROUP A AND B Figure 6 Out of 40 cases of invasive ductal carcinoma majority 21(52.5%) cases had strong staining intensity and 15(37.5%) cases had moderate staining intensity. Remaining 2(5%) cases had weak staining intensity and 2(5%) cases had no staining (Table IV). TABLE VI: CORRELATION OF HISTOLOGICAL GRADE WITH PERCENTAGE OF POSITIVE STAINED CELLS IN GROUP A AND B 2 of 7
3 CORRELATION OF HISTOLOGICAL GRADE WITH STAINING INTENSITY IN GROUP A AND B Out of 40 cases of carcinoma breast, 28(70%) cases were of grade III. Among 28 cases of grade III, majority 14 cases showed strong and 10 cases showed moderate staining intensity. Remaining 2 cases had weak staining intensity and 2 cases showed no staining intensity. Figure 8 TABLE VIII: PERCENTAGE POSITIVITY OF MALIGNANT CELLS, STAINING INTENSITY OF CYTOKERATIN 8/18 AND GRADE OF CASES WITH IHC SCORE 0-4 (NEGATIVE) IN GROUP A AND B (N=6) Out of 10(25%) cases of grade II, 5 cases showed moderate and 5 cases showed strong staining intensity. While remaining 2 cases of grade I had strong staining intensity (TableVII) Figure 7 TABLE VII: CORRELATION OF HISTOLOGICAL GRADE WITH STAINING INTSNITY IN GROUP A AND B In subgroup B II, 16 cases had metastatic deposits in lymph nodes. All cases showed 100% positivity with IHC score 5-12(Table IX). Figure 9 TABLE IX: IHC SCORE OF METASTATIC DEPOSITS IN LYMPH NODES IN SUBGROUP B II PERCENTAGE OF MALIGNANT STAINED CELLS AND STAINING INTENSITY OF CYTOKERATIN 8/18 AND GRADE OF CASES WITH IHC SCORE 0-4 (NEGATIVE) IN GROUP A AND B) In group A and B, 6(15%) cases had IHC score 0-4. Out of 3 cases in group A with IHC score 0-4, 2 cases were of grade III and one case was of grade II. One case of grade II showed <10% positive stained cells with moderate expression. While one case of grade III showed <10% positive stained cells with weak expression. One case of grade III had no positive stained cell and no staining intensity. In group B with IHC score 0-4 all the 3 cases were of grade III. One case showed 10-50% positive stained cells with weak expression and one case showed 10-50% positive stained cells with moderate expression. One case showed no positive stained cell and no staining intensity (Table VIII). 3 of 7 IHC STAINING OF CK 8/18 IN INTRADUCTAL ELEMENT ASSOCIATED WITH INVASIVE CARCINOMA Intraductal element was seen in 5(12.5%) cases of invasive ductal carcinoma (NST). No significant difference was seen in percentage positivity of cells, staining intensity or IHC score observed in intraductal element and invasive component.
4 Figure 10 Figure 12 Fig 1: Carcinoma breast, Grade III, Negative for Cytokeratin 8/18 (IHC 40x). Fig 3: Carcinoma Breast, Grade III, showing 50-80% CK8/18 positivity with moderate staining intensity(ihc 10x). Figure 11 Fig 2: Carcinoma breast with squamous differentiation grade III showing 10 50% cytokeratin 8/ 18 positivity with weak staining intensity(ihc 10x). 4 of 7 Figure 13 Fig 4: Carcinoma breast mucinous grade II, showing >80% CK 8/18 positivity with moderate staining intensity(ihc 10x).
5 Figure 14 Fig 5: Primary tumour, carcinoma breast (NST), grade III, showing >80% CK 8/18 positivity with strong staining intensity(ihc 10x). Figure 15 Fig 6: Lymph node with metastatic deposits showing >80% CK 8/18 postivity with strong staining intensity(ihc 10x). DISCUSSION Keratins have a number of distinct advantages as marker 3 proteins. It has been shown that primary carcinomas and their metastases have virtually the same cytokeratins as 2 normal breast epithelium. Tsubura et al showed percentage positivity of 83% for cytokeratins 8 and 18 in invasive breast carcinoma3.dm Abd E I Rehim et al found percentage positivity of 98.3% and 88% for cytokeratin 8 and cytokeratin 18 respectively4. Rajthar et al (2000) showed immunohistochemical positivity for CK 8 in (100/100)(100%). and for CK 18 in (100/100) (100%) cases 5 of invasive ductal carcinoma). The percentage positivity given by different studies varied 5 of 7 from 78%-100% for CK 8 and 18 expression. The present study showing positivity in 85% cases. This could be due to sensitivity of different monoclonal antibodies used (antibody clones). Five cases in the present study showing the intraductal component showed no difference from invasive component in cytokeratin positivity and staining intensity. Sorensan et al (1987) demonstrated that essentially all tumour cells in infiltrating ductal carcinomas containing a prominent intraductal component showed a homogenous, moderate to strong reactivity with the two antibodies AEI and AE31. In the present study solitary case of invasive mucinous carcinoma showed moderate staining intensity thereby showing 100% positivity which is very well 6 comparable with finding of Heatley et al (1995). In the present study 1 case of invasive ductal carcinoma with areas of squamous differentiation showed weak staining intensity for cytokeratin 8/18 because areas of squamous differentiation express epidermal type of keratins only. In a study done by DM abd E1 Rehimet et al (2004) demonstrated that 5/5 cases of grade 3 carcinoma NST (no special type) with squamous differentiation were positive for positive for at least one of the basal marker, high molecular 4 weight epidermal type keratin.in the present study 5/16 (31.25%) cases of secondary metastatic deposits in lymph nodes showed moderate staining intensity and 11/16(68.75%) cases showed strong staining intensity and thereby 100% positivity. Higher results in the present study can be attributed to the fact that present study comprised of small number of cases and currently, high quality antikeratin monoclonal antibodies to all of the 20 keratins are commercially available from many biomedical reagent vendors. Moll et al and Jarach et al found that metastatic lymph nodes from patients with breast cancer reacted with antibodies in the same way as normal breast epithelium and primary tumour respectively2,7. CONCLUSION High proportion of IDC cases express luminal epithelium cell cytokeratins 8 and 18 indicating a differentiated glandular luminal phonotype, a finding associated with good prognosis. IDC of any grade if negative for cytokeratins 8 and 18 are presumed to be carrying poor prognosis as these could be positive for basal phenotype. Hence cytokeratins have role in subclassification, for origin of carcinoma and to identify the tumour at the metastatic site and can be used as a marker for evaluation as for as prognosis and treatment are concerned.
6 References 1. Sorensen SC, Bonnie BA, Jomes LC, Burstein NA, ASch HL. Structural distinctions among human breast epithelial cells revelaed by the monoclonal antikeratin antibodies AE1 and AE3. Journal of pathology 1987; Moll R, Krepler R, Franke WW. Complex cytokeratin Complex cytokeratin polypeptide patterns observed in certain human carcinomas. Differentiation 1983; 23: Tsubura A, Okada H, Senzaki H, Hatano T, Morii S. Keratin expression in normal breast and in breast carcinoma. Histopathology 1991; 18: AbdEI-REhim DM, Pinder SE, Paish CE, Bell J, Blamey 6 of 7 RW, Robertson JF et al. Expression of luminal and basal cytokeratins in human breast carcinoma. Journal of Pathology 2004; 203(2): Rejthar A, Nenutil R. The intermediate filaments and prognostically oriented morphological classification in ductal breast carcinoma. Neoplasma 1997; 44: Heatley M, Maxwell P, Whiteside C, Toner P. Cytokeratin intermediate filament expression in benign and malignant breast disease. J ClinPathol 1995; 48: Jarasch ED, Naegle RB, Kaufmann M, Maurer C, Bocker WJ. Differential diagnosis of benign epithelial proliferations and carcinomas of the breast using antibodies to cytokeratins. Hum Pathol 1988; 19:
7 Author Information Bharti Rattan Senior Resident(Pathology), Government Medical College Aditi Baghla Assistant Professor(Pathology), Maharaja Agarsen Medical College Mridu Manjari Professor and Head(Pathology), Shri Guru Ramdas Medical College Parbodh Kakkar Ex. Professor(Pathology), Government Medical College S.K Kahlon Ex. Professor and Head(Pathology), Government Medical College Surinder Paul Associate Professor(Pathology), Government Medical College 7 of 7
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