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Overexpression/amplification of Her-2/neu in malignant tumors-A Short Review

Overexpression/amplification of Her-2/neu in malignant tumors-A Short Review

*Kabir E

Abstract

Her-2/neu overexpression has been shown to play a significant role in development and progression of many malignant tumors of body, mostly affects tumors of epithelial origin. Her-2 is a protein involved in normal cell growth. However, HER2/neu may be made in larger than normal amounts by some types of cancer cells.This may cause cancer cells to grow more quickly and spread to other parts of the body. Checking the amount of HER2/neu on some types of cancer cells may help plan of treatment.  Amplification and/or overexpression of HER-2/neu in human tumor tissue has been found  in  breast, ovarian, endometrial, colon, gastric or gastroesophageal junction, urothelial, bladder, salivary duct and cervix cancer. The degree of overexpression correlates with tumor progression, resistance to chemotherapy and a poor prognosis. Testing for this overexpression/amplification in tumor and its recurrence is very important. Immunohistochemistry and FISH are two important modern techniques which can identify overexpression of this protein in formalin fixed paraffin embedded tissue.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):145-150]

Keywords: Her-2/neu overexpression, Immunohistochemistry of Her-2/neu, FISH, parrafin embedded tissue

*Professor Enamul Kabir, Professor of Pathology, Sir Salimullah Medical College, Dhaka, Bangladesh. enamulkabir1213@gmail.com

Introduction

EGFR was the first discovered epidermal growth factor receptor.1 HER2 is so named because it has a similar structure to human epidermal growth factor receptor, or HER1. Neu is so named because it was derived from a rodent glioblastoma cell line, a type of neural tumor. ErbB-2 was named for its similarity to ErbB (avian erythroblastosis oncogene B), the oncogene later found to code for EGFR.2 ERBB2, a known proto-oncogene, is located at the long arm of human chromosome 17 (17q12).2

Growth factor binding results in receptor dimerization, subsequent tyrosine kinase activity and autophosphorylation of specific tyrosine residues. After that event downstream activation of signal transduction cascades occur and  MAPK, Akt and JNK pathways become activated. It  leads to DNA synthesis, cell proliferation, and differentiation. EGFR, also known as ErbB-1, and the three related receptors of the ErbB family: ErbB-2, ErbB-3, and ErbB-4. ErbB-2 is also known as HER2 in humans and neu in rodents. The HER-2/neu oncogene was first identified as a dominant transforming gene in chemically induced adrenal neuroblastomas of neonatal mice and was referred to as neu.3,4 Subsequently, three groups independently identified the human homologue of this gene.5,6 Sequence analysis of the gene demonstrated a close relation ship to the human epidermal growth factor receptor (HER-i) or c-erbB oncogene.5,6 Because of the similarities with HER-i, this gene was considered to code for a membrane receptor.5,6

Pathophysiology

Normal cells express 40,000 to 100,000 EGFR receptors, cancer cells may express up to 2,000,000 receptors.1 Stimulation of overexpressed EGFR receptors may cause  cancer by inducing cancer-cell proliferation while simultaneously blocking apoptosis. Cancer cells cause  invasion and metastasis by activating invasion and metastasis of hyperproliferative cells and by stimulating tumor-induced neovascularization.1

Amplification and/or overexpression of HER-2/neu in human tumor tissue has been associated with a poor prognosis in   cancers of breast,7 ovary,8 endometrium,9 colon,10 gastric or gastroesophageal junction,11 urothelial,12 bladder,12 salivary duct13 and cervix.14 Amplification, also known as the over-expression of the ERBB2 gene, occurs in approximately 15-30% of breast cancers, 7-34% of patients with gastric cancer and in 30% of salivary duct carcinomas.2 The degree of overexpression correlates with tumor progression, resistance to chemotherapy and a poor prognosis.1

However, regarding involvement of  HER2 protein 3+ expression in nonepithelial malignancies, it was very rare, often non-existent, in malignancies of non-epithelial origin. Out of 965  malignant melanoma cases, only one showed HER2 3+ expression. In 1,211 sarcomas of soft tissues and 1,136 neuroendocrine tumors, none exhibited 3+ HER2 protein expression. No HER2 3+ expression was detected in gastrointestinal stromal tumors (GIST), small cell lung cancers (SCLC), kidney cancers, and glioblastomas.15,16

Tests for Her-2

Immunohistochemistry and in situ hybridization (ISH, FISH) are the recommended methods for determining Her2 status for treatment with Her-2-targeted therapy. Neither method is 100% sensitive or specific.Updated ASCO-CAP (2013) guidelines have resulted in increased proportion of patients being eligible for Her2-targeted therapy. Her2-positive cases are not a homogeneous group – borderline positive cases may not be as responsive to Her-2-targeted therapy. Challenges in Her-2 laboratory testing include polysomy / co-amplification, and genetic heterogeneity.17,18

Immunohistochemistry (IHC) and HER-2 in situ hybridization are the most commonly used techniques for Her-2 expression. IHC can be done on formalin-fixed, paraffin-embedded tissue . Tests are usually performed on biopsy samples obtained by either fine-needle aspiration, core needle biopsy, vacuum-assisted breast biopsy, or surgical excision. Immunohistochemistry is used to measure the amount of HER2 protein present in the sample. The sample is given a score based on the cell membrane staining pattern. Specimens with equivocal IHC results should then be validated using fluorescence in situ hybridisation(FISH).2 HER-2 scoring was reported per American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines published in 2007 and updated in 2013.   IHC test was considered positive (IHC+) when IHC3+ was obtained above the guidelines defined thresholds; an IHC test was considered negative (IHC-) when IHC 2+ (equivocal), IHC 1+, or IHC 0 was obtained.15,17,18,19

HER2 in situ hybridization technique

In a study 37,992 samples  were analyzed by IHC and 21,642 samples were also examined with ISH. FISH was used for evaluation of the HER2 amplification status. FISH was performed with a probe specific for HER2 (17q11.2-q12 region) and a probe for the pericentromeric region of chromosome 17. Interphase nuclei were examined and the ratio of HER2 signals to chromosome 17 centromere signals were evaluated to indicate amplification status of this gene.15. HER2/CEP17 ratio higher than 2.2 was considered amplified (ISH+), and HER2/CEP17 ratio between 1.8 and 2.2 (equivocal) in FISH or HER2/CEP17 ratio <1.8 in FISH was considered non-amplified (ISH-). 15,17

 

HER2 amplification was also evaluated by CISH. The HER2 and chromosome 17 probes are detected using two color ISH in formalin-fixed, paraffin-embedded human cancer tissue specimens following staining on automated slide stainer, and visualized by light microscopy. Consistent with the CISH package insert, HER2/CEP17 ratio higher than 2.0 was considered amplified (ISH+); HER2/CEP17 ratio <2.0 in CISH was considered non-amplified (ISH-). 11,670 patients had IHC and FISH; 9972 patient, IHC and CISH. IHC test was considered positive (IHC+) when IHC 3+ was obtained. ISH test was considered positive (ISH+) when the HER2/CEP17 ratio was >2.2 (by FISH) or 2.0 (by CISH).15, 17,18,19

The extracellular domain of HER2 can be shed from the surface of tumour cells and enter the circulation. Measurement of serum HER2 by enzyme-linked immunosorbent assay (ELISA) offers a far less invasive method of determining HER2 status than a biopsy and consequently has been extensively investigated. Results so far have suggested that changes in serum HER2 concentrations may be useful in predicting response to trastuzumab therapy.2

Clinical Significance

The ErbB family consists of four plasma membrane-bound receptor tyrosine kinases. One of which is erbB-2, and the other members being epidermal growth factor receptor, erbB-3 (neuregulin-binding; lacks kinase domain), and erbB-4. All four contain an extracellular ligand binding domain, a transmembrane domain, and an intracellular domain that can interact with a multitude of signaling molecules and exhibit both ligand-dependent and ligand-independent activity. Notably, no ligands for HER2 have yet been identified.2

HER2 has been firmly established in preclinical and clinical settings. Among all four HER family proteins, HER2 has the strongest catalytic kinase activity and functions as the most active signaling complex of the HER family after dimerization with other HER family members. 20,21 Overexpression of HER2 in breast cancer leads to increased homodimerization (HER2:HER2) and heterodimerization (e.g., HER2:HER3), which initiates a strong pro-tumorigenic signaling cascade.22 Overexpression of HER2 protein drives malignant transformation in cell culture and transgenic mouse models.23,24 The anti-HER2 antibody trastuzumab represents an effective, targeted therapy with significant efficacy in treatment of HER2-positive breast and gastric cancer.25,26

Regarding the mechanism of activation of EGFR, It starts by ligand binding at the extracellular domain which results in homo and heterodimerization, leading to phosphorylation, activation of downstream signaling pathways which upregulate expression of genes, proliferation and angiogenesis. Abnormalities in the expression of EGFR play an essential role in the development of different types of cancer. HER2 is the preferred heterodimerization partner for EGFR.; this biological characteristic together with structural homology has played a key role in the development of dual synthetic inhibitors against EGFR/HER2. 27

Overactivation of the ErbB protein family, which is comprised of 4 receptor tyrosine kinase members, can drive the development and progression of a wide variety of malignancies, including colorectal, head and neck, and certain non-small cell lung cancers (NSCLCs). As a result, agents that target a specific member of the ErbB family have been developed for the treatment of cancer.28

Her2 targeted therapy include Herceptin (trastuzumab) and Others: pertuzumab (Perjeta), T-DM1 (Kadcyla), and lapatinib (Tykerb). Recent data shows that a combination of pertuzumab, trastuzumab, and docetaxel (PTD) improved progression free survival compared to patients who had only trastuzumab and docetaxel (TD).29,30

Recent development  of HER2 mutation

Apart from gene amplification ,somatic HER2 (encoded by ERBB2) mutations, , have been reported recurrently in the literature. Mutations in HER2 are clustered in the extracellular, transmembrane and kinase domains. HER2 mutations have been found in non-small-cell lung cancers (NSCLC) and can direct treatment31. Also, HER-2 mutations are infrequent in a wide variety of cancers  but targetable. As for example In breast cancers, activating mutations were identified as follows: G309A, D769H/Y, V777L, P780ins, V842I, and R896C,32 L755S was associated with lapatinib resistance. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. Recently, phase II SUMMIT trial, which is a HER2 mutant basket trial, showed mutation status can contribute to response to neratinib regardless of tumor type 33.

Conclusion

Checking the amount of HER2/neu on some types of cancer cells may help plan treatment. It’s overexpression is mostly confined in malignant tumors of epithelial origin. Immunohistochemistry and FISH are two preferred techniques for identification of its overexpression/amplification. Targetting of Her-2/neu gene with proper drug is important for both treatment and recurrence of many cancer.

 

References

  1. Murphrey MB, Bhimji SS. Biochemistry, Epidermal Growth Factor Receptor. [Updated 2018 Jan 19]. In: Stat Pearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2018 Jan. Available from: https://www.ncbi.nlm.nih.gov/books/NBK482459/
  2. Wikipedia contributors. “HER2/neu.” Wikipedia, The Free Encyclopedia. Wikipedia, The Free Encyclopedia, 3 Feb. 2018. Web. 11 Feb. 2018.
  3. Shih C, Padhy LC, Murray M, and Weinberg R A. Transforming genes ofcarcinomas and neuroblastomas introduced into mouse fibroblasts. Nature (Land.), 1981;290: 261-264.
  4. Schechter AL, Stern DF, Vaidyanathan L, Decker SJ, Drebin JA, Greene MI and Weinberg RA. The neu oncogene: an erb-B-related gene encoding a185,000Mr tumour antigen. Nature (Land.), 312: 513(5161,984).
  5. Semba K, Kamata N, Toyoshima K, and YamamotoT. A v-erbB-related protooncogene, c-erbB-2, is distinct from the c-erbB-1/epidermal growth factor-receptor gene and is amplified in a human salivary gland adenocarcinoma. Natl. Acad. Sci. USA, 82: 6497-6501, 1985.
  6. Coussens L, Yang-Feng TL, Chen YC LE, Gray A, McGrath J, Seeburg PH, Libermann T A, Schlessinger J, Francke U, Levinson, and Ullrich A. Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science (Washington DC), 1985;230: 1132-1139.
  7. Slamon D J, Aark GM, Wong SG, Levis WJ, Ullrich A, and McGuire WL. Human breast cancer:correlation of relapse and survival with amplification of the HER-2/neu Science (Washington DC), 235: 177-18129,87.
  8. Slamon D3, Godoiphin W, Jones LA, Holt JA,Wong SG, Keith DE, Ievin WJ, Stuart SG, Udove J, Ullrich A, and Press MF. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science (Washington DC), 244: 707-712,1989.
  9. Hetzel DJ, Wilson T0, Keeney GL, Roche PC, Cha SS, and Podratz KC. HER-2/nexepuressaimona: joprrognofasctitcoinrendomectarinacler. Gynecol. Oncol, 1992; 47: 179-185.
  10. Nathason DR, Culliford AT, Shia J, Chen B, D’Alessio MD, Zeng Z, Nash G, Gerald W, Barany F, Paty P: HER-2/neu expression and gene amplification in colon cancer. Int J Cancer 2003.
  11. Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, Sawaki A, Lordick F, Ohtsu A, Omuro Y, Satoh T, Aprile G, Kulikov E, Hill J, Lehle M, Rüschoff J, Kang YK, ToGA Trial Investigators. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. 2010 Aug 28; 376(9742):687-97.
  12. Gandour-Edwards R, Lara PN Jr, Folkins AK, LaSalle JM, Beckett L, Li Y, Meyers FJ, DeVere-White R. Does HER2/neu expression provide prognostic information in patients with advanced urothelial carcinoma? 2002 Sep 1;95(5):1009-15.
  13. Williams MD, Roberts DB, Kies MS, Mao L, Weber RS, El-Naggar AK. Genetic and expression analysis of HER-2 and EGFR genes in salivary duct carcinoma: empirical and therapeutic significance.Clin Cancer Res. 2010 Apr 15; 16(8):2266-74.
  14. Bajpai S, Awasthi S, Dutta S, Mittal A, Kumar A, Ahmad F.Role of HYPERLINK “https://www.ncbi.nlm.nih.gov/pubmed/29207712″HER-2/neuHYPERLINK “https://www.ncbi.nlm.nih.gov/pubmed/29207712″in Premalignant and Malignant Lesions of Uterine Cervix. J Clin Diagn Res. 2017 Sep;11(9):EC01-EC04. doi: 10.7860/JCDR/2017/26583.10547. Epub 2017 Sep 1.
  15. Min Yan, Maria Schwaederle,David Arguello, Sherri Z. Millis, Zoran Gatalica, and Razelle Kurzr. HER2 expression status in diverse cancers: review of results from 37,992 patients. Cancer Metastasis Rev. 2015; 34: 157–164. Published online 2015 Feb 25. doi:  [10.1007/s10555-015-9552-6
  16. Lynne Eldridge, MD  | Reviewed by Doru Paul, MD. HER2 Positive vs. HER2 Negative Breast Cancer: Important Differences:Is It Good or Bad to Have HER2 Positive Breast Cancer? Caan, B., Sternfeld, B., Gunderson, E. et al. Cancer Causes Control (2005) 16: 545. https://doi.org/10.1007/s10552-004-8340-3 Updated October 23, 2018.
  17. Wolff AC, Hammond MEH, Schwartz JN, Hagerty KL, Allred DC, Cote RJ. College of American Pathologists. (2007). American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology, 25(1), 118–145. doi:10.1200/JCO.2006.09.2775 .
  18. Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, Allred DC, Bartlett JM, Bilous M, Fitzgibbons P, Hanna W, Jenkins RB, Mangu PB, Paik S, Perez EA, Press MF, Spears PA, Vance GH, Viale G, Hayes DF, American Society of Clinical Oncology., College of American Pathologists.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 Clin Oncol. 2013 Nov 1; 31(31):3997-4013.
  19. Rüschoff J, Dietel M, Baretton G, Arbogast S, Walch A, Monges G, Chenard MP, Penault-Llorca F, Nagelmeier I, Schlake W, Höfler H, Kreipe HH. HER2 diagnostics in gastric cancer-guideline validation and development of standardized immunohistochemical testing.Virchows Arch. 2010 Sep; 457(3):299-307.
  20. Garrett TP, McKern NM, Lou M, Elleman TC, Adams TE, Lovrecz GO, Kofler M, Jorissen RN, Nice EC, Burgess AW, Ward CW. The crystal structure of a truncated ErbB2 ectodomain reveals an active conformation, poised to interact with other ErbB receptors. Mol Cell. 2003 Feb; 11(2):495-50.
  21. Graus-Porta D, Beerli RR, Daly JM, Hynes NE. ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J. 1997 Apr 1; 16(7):1647-55.
  22. Holbro T, Beerli RR, Maurer F, Koziczak M, Barbas CF 3rd, Hynes NE. The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. Proc Natl Acad Sci U S A. 2003 Jul 22; 100(15):8933-8.
  23. Di Fiore PP, Pierce JH, Kraus MH, Segatto O, King CR, Aaronson SA. erbB-2 is a potent oncogene when overexpressed in NIH/3T3 cells. 1987 Jul 10; 237(4811):178-82.
  24. Ursini-Siegel J, Schade B, Cardiff RD, Muller WJ. Insights from transgenic mouse models of ERBB2-induced breast cancer. Nat Rev Cancer. 2007 May; 7(5):389-97.
  25. Spector NL, Blackwell KL. Understanding the mechanisms behind trastuzumab therapy for human epidermal growth factor receptor 2-positive breast cancer. J Clin Oncol.2009 Dec 1;27(34):5838-47. doi: 10.1200/JCO.2009.22.1507. Epub 2009 Nov 2.
  26. Romond EH, Perez EA, Bryant J, Suman VJ, Geyer CE Jr, Davidson NE, Tan-Chiu E, Martino S, Paik S, Kaufman PA, Swain SM, Pisansky TM, Fehrenbacher L, Kutteh LA, Vogel VG, Visscher DW, Yothers G, Jenkins RB, Brown AM, Dakhil SR, Mamounas EP, Lingle WL, Klein PM, Ingle JN, Wolmark N.Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med. 2005 Oct 20; 353(16):1673-84.
  27. Bello M, Saldaña-Rivero L, Correa-Basurto J, García B, Sánchez-Espinosa VA. Structural and energetic basis for the molecular recognition of dual synthetic vs. natural inhibitors of EGFR/HER2. Int J Biol Macromol. 2018 May;111:569-586. doi: 10.1016/j.ijbiomac.2017.12.162. Epub 2018 Jan 9.
  28. Roskoski R Jr. The ErbB/HER family of protein-tyrosine kinases and cancer. Pharmacol Res. 2014 Jan;79:34-74. doi: 10.1016/j.phrs.2013.11.002. Epub 2013 Nov 20.
  29. CLEOPATRA trial. Most recent: Swain et al, NEJM 2015 Feb 19;372(8):724-34.
  30. NeoSphere trial. Gianni et al, Lancet Oncol 2012 Jan;13(1):25-32.
  31. Mazières J, Peters S, Lepage B, Cortot AB, Barlesi F, Beau-Faller M, et al. (June 2013). Lung cancer that harbors an HER2 mutation: epidemiologic characteristics and therapeutic perspectives. Journal of Clinical Oncology. 31 (16): 1997–2003. doi:1200/JCO.2012.45.6095. PMID2361010
  32. Bose R, Kavuri SM, Searleman AC, et al. Activating HER2 mutations in HER2 gene amplification negative breast cancer. Cancer Discov 2013;3:224-37.
  33. Hyman DM, Piha-Paul SA, Won H, et al. HER kinase inhibition in patients with HER2- and HER3-mutant cancers. Nature 2018;554:189-94.
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Role of Cytopathology in Eyelid Growth with Histopathological Correlation

Role of Cytopathology in Eyelid Growth with Histopathological Correlation

*Paul R,1 Kundu UK,2 Kabir E,3 Islam MN4

Abstract
The purpose of this study was to evaluate diagnostic accuracy of cytopathology in  different eyelid lesions using fine needle aspiration cytology (FNAC) & scrape cytology with histopathological correlation. Accurate diagnosis of eyelid tumors is necessary to guide ophthalmologists to design optimal management. Fine needle aspiration from 85 eyelid growth and histopathological correlation were studied. Immunohistochemical analysis were done in few cases.  A majority of the patients (43 out of 85) were in the 26-50 age group (53.49% male vs 46.51% female). Mean age was 43.22±17.42 (range 19 – 90 years) years. Of the malignant lesions, basal cell carcinoma were highest (12 in number, 36.36%) followed by sebaceous gland carcinoma and squamous cell carcinoma. Less common  malignant tumor were Non-Hodgkin’s lymphoma. Among benign neoplastic lesions, nevi were most common (14 in number, 43.75%) followed by haemangioma and squamous papilloma. Other less common  benign tumors were fibroepithelial polyp, adenoma, lipoma and neurofibroma. Most common benign cystic lesions of eyelid are cyst (10 in number, 50%) of moll/Hydrocystoma/Sudoriferous cyst, followed by Dermoid cyst, Epidermal inclusion cyst and Sebaceous cyst. Present study revealed that accuracy of cytopathological diagnosis of malignant eyelid growths were 97.65%. Cytopathology had a high diagnostic accuracy rate. Aspiration cytology was cost effective and offers rapid diagnosis with minimal discomfort to the patient.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):134-144]

Key wards: Eye lid growth, Cytopathology, Histopathology, Correlation

Introduction
Eyelid growth is a common cause to be presented to ophthalmologists1 and many of them can be treated as day care service. At the same time some of the tumors demand emergency surgical intervention and thus early referral.2 Introduction of cytopathology prior to excision biopsy would contribute to early diagnosis and management plan.3 Pathologic conditions affecting the eyelid may be inflammatory or neoplastic. Neoplastic lesion may be benign or malignant.1 It becomes difficult to decide clinically either it is a true neoplasm or an inflammatory lesion. In all such cases, cytopathology (FNAC / scrape cytology) proved to be very useful in quickly determining the nature of the lesion and also deciding the mode of treatment.4 FNAC has a high diagnostic accuracy rate, if the aspirated material is sufficient for microscopical examination and if it is properly interpreted.5 Aspiration cytology is also cost effective and offers rapid diagnosis, with minimal discomfort to the patient..6

  1. *Dr. Rita Paul, Assistant Professor, Department of Pathology, Ibrahim Medical College, Dhaka. ritapaul16@gmail.com
  2. Utpal Kumar Kundu, Assistant Professor (Eye), Mugda Medical College, Dhaka.
  3. Professor Enamul Kabir, Professor, Department of Pathology, Sir Salimullah Medical College, Dhaka.
  4. Professor Md. Nasimul Islam, Professor and Head, Department of Pathology, Sir Salimullah Medical College, Dhaka.

*For correspondence

Method

This was a cross sectional study conducted at the Departmentof Pathology of Sir Salimullah Medical College, Dhaka and National Institute of Ophthalmology and Hospital, Dhaka, Bangladesh from January 2012 to December 2013. 85 adult patients with eye lid growths of both sexes were recruited. Purposive sampling technique was used. FNAC was done using 22 Guage needle without anaesthesia and smears were stained with Papanicolaou’s stain. Biopsy was taken by clinician and diagnosis was confirmed by histopathological examination. Inclusion criteria were adult patients with eyelid growth. Exclusion criteria included patients who were clinically diagnosed to  have inflammatory eyelid lesions, patients belonging to less than 18 years of age, and very tiny growth <0.5cm in diameter.

FNA Features of different Eye-lid lesions

Smears from nevus showed single and small clusters of cells with rounded or oval nuclei and indistinct cytoplasm.7 Smears from haemangioma showed only blood, with a few cases showing an occasional cluster of endothelial cells. Smears from squamous papilloma showed degenerated squamous epithelial cells along with mature superficial squamous cells. Smears from neurofibroma showed cohesive spindle-shaped cells within fibrillary mesenchymal background material. Smears from hidrocystoma / Sudoriferous cyst / Cyst of Moll showed foamy macrophages in the background of proteinecious material. Smears from epidermal inclusion cyst showed high cellularity with numerous nucleated squamous cells and anucleated squames in a background of keratinous debris.8 Smears from dermoid Cyst showed anucleated and nucleated squamous epithelium and keratin debris.9 Smears from chalazion showed a polymorphic picture with neutrophils, plasma cells and macrophages. The granulomas are more of histiocytic cells with abundant vacuolated cytoplasm; the backround is generally dirty with nuclear debris and fat spaces.10 Smears from molluscum contagiosum showed Molluscum bodies, in the enlarged superficial cells of the epidermis.11 Molluscum bodies, also called Henderson-Patterson bodies, were large, round cytoplasmic inclusions (within the enlarged cells of epidermis), which push the nucleus to the periphery.12  Smears from Rhinosporidiosis showed many scattered basophilic rhinosporidial endospores and rhinosporidial spores in a background of amorphous eosinophilic material.13 Smears from basal cell carcinoma showed tightly cohesive small clusters of uniform hyper­chromatic basaloid cells with high nuclear-cytoplasmic ratio and absence of cytoplasmic vacuolation. Peripheral palisading of nuclei may be evident in some clusters. Squamous, sebaceous and adenoid differentiation may be seen and pigmented variant may be seen.14 Smears from sebaceous gland carcinoma showed large pale cells and vacuolated cytoplasm however another type is poorly differentiated cells with dark and irregular nuclei. The smears of squamous cell carcinoma showed markedly enlarged hyper-chromatic nuclei of variable size and keratinization.10 Smear from cutaneous melanoma showed atypical dispersed population of cells with abundant cytoplasm, eccentric uniform hyperchromatic nuclei, internuclear inclusions in the background of melanin pigment.15 In Non-Hodgkin’s lymphoma, cytology smears showed a monotonous population of lymphocytes with round nuclei having coarse granular chromatin. Histopathological examination of biopsied tissue confirmed the diagnosis16 and in a few cases by the help of immuno-histochemical analysis using specific antibodies.

Results

Age distribution of the patients presented with eyelid growths showed almost half (50.59%) of the patients comprised of middle age group (26-50 years) with a little more than one-fourth (30.59%) above 50 years of age. Gender distribution of the patients presented with eyelid growths showed slightly male preponderance (50.59%) and female constituted 49.41%. Mean age was 43.22±17.42 (range 19 – 90 years) years.

Among benign neoplastic lesions, nevus was most common, followed by vascular lesion and squamous papilloma. Others were adenoma, lipoma and hamartoma. Correlation with cytopathological and histopathological examination were done. Cytopathologically diagnosed fourteen (14) cases of Nevus were confirmed by histopathological examination. Cytologically diagnosed Vascular Lesion in eight (8) cases were histologically confirmed in seven (7). The other case was histologically diagnosed as Hamartoma. Five (5) cases of squamous papilloma corresponded cytologically and histologically. One (1) case of cytologically diagnosed lipoma was also confirmed histologically. Both (2) cases of histologically diagnosed Fibroepithelial Polyp were cytologically diagnosed as benign Mesenchymal lesion. One (1) case of histologically diagnosed Neurofibroma was cytologically diagnosed as a malignant peripheral nerve sheeth tumor (False positive; Table III).

Cytologically diagnosed Cyst of Moll/ Hydrocystoma/ Sudoriferous Cyst corresponded histologically in all ten (10) cases. Five (5) cases of cytologically diagnosed Dermoid Cyst were confirmed histologically. Cytologically diagnosed three (3) cases of Epidermal Inclusion Cyst corresponded histologically. Two (2) cases of cytologically diagnosed Sebaceous Cyst were later on confirmed histologically (Table IV). Among the malignant lesions encountered during the present study, Basal cell carcinoma was the most common malignancy, followed by Sebaceous Gland Carcinoma and Squamous Cell Carcinoma. Non-Hodgkin’s lymphoma (NHL) was found to be less common. Cytological diagnosis was confirmed by histopathological examination in the present study. Eleven (11) cases of cytologically diagnosed Basal Cell Carcinoma were confirmed histologically. One (1) case of cytologically diagnosed Nevus (False negative) was histologically diagnosed as Basal Cell Carcinoma. Sebaceous Gland Carcinoma corresponded cytologically and histologically in all nine (9) cases. Nine (9) cases of cytologically diagnosed Squamous Cell Carcinoma were also confirmed histologically. Lymphoma corresponded cytologically and histologically in both (2) cases, which was later on confirmed by immuno-histochemical study as Non-Hodgkin’s lymphoma of ‘B’ cell origin. One (1) case of cytologically diagnosed Small Cell Tumor was confirmed histologically (Table V).

Comparison of diagnosis between cytopathology with histopathology among the malignant eyelid growths

Among 85 eyelid growths, 32 cases were cytopathologically and histopathologically true positive for malignant lesions. The comparison between cytopathology and histopathology were statistically highly significant (p < 0.0001; Table VI). Out of 85 (100%) patients of eyelid growths 32 (37.64%) were positive for malignancy, 51(60.00%) were negative for malignancy, 01(1.18%) was false positive(1.18%) and 1 (1.18%) was false negative (Table VII). The validity of cytopathology to diagnose malignant eyelid growths, Sensitivity, Specificity, PPV, NPV and Accuracy were 96.97%, 98.08%, 96.97%, 98.08% and 97.65% respectively (Table VIII).

Out of a total 33 histologically confirmed cases of malignant tumor, thirteen (13) cases were ulcerated. From these ulcerated lesions, samples were collected by scraping. Out of these, nine (9) cases were Basal Cell Carcinoma, three (3) cases were Squamous Cell Carcinoma and one (1) case was found to be Sebaceous (Meibomian) Gland Carcinoma.

Table I: Distribution of different eyelid lesions with Cytological and Histological Diagnosis (n=85)

Eyelid Growth Cytological Diagnosis Histological Diagnosis
Nevus 15 (17.65) 14 (16.47)
Vascular Lesion 8 (9.41) 7 (8.23)
Squamous Papilloma 5 (5.88) 5 (5.88)
Adenoma 1 (1.18) 1 (1.18)
Lipoma 1 (1.18) 1 (1.18)
Hamartoma 0 (0.00) 1 (1.18)
Benign Mesenchymal Lesion 2 (2.36) 0 (0.00)
Fibroepithelial Polyp 0 (0.00) 2 (2.36)
Neurofibroma 0 (0.00) 1 (1.18)
Cyst Of Moll/ Hydrocystoma/ Sudoriferous Cyst 10 (11.76) 10 (11.76)
Dermoid Cyst 5 (5.88) 5 (5.88)
Epidermal Inclusion Cyst 3 (3.52) 3 (3.52)
Sebaceous Cyst 2 (2.36) 2 (2.36)
Basal Cell Carcinoma 11 (12.94) 12 (14.12)
Sebaceous Gland Carcinoma 9 (10.58) 9 (10.58)
Squamous Cell Carcinoma 9 (10.58) 9 (10.58)
Lymphoma 2 (2.36) 2 (2.36)
Small Cell Tumor 1 (1.18) 1 (1.18)
Malignant Peripheral Nerve Sheath Tumor (MPNST) 1(1.18) 0 (0.00)
Total 85 (100) 85 (100)

Table II: Age distribution of the patients of all types of eyelid lesions with percentage

(n=85)

Age Frequency Percentage
Up to 25 16 18.82
26 – 50 43 50.59
Above 50 26 30.59
Total 85 100.0

Table III: Distribution of different benign neoplastic eyelid growths

Final Histological Diagnosis  

Cytological Diagnosis
Total (Final Histology) Nevus Haemangioma Squamous Papilloma Adenoma Lipoma Hamartoma Benign Mesenchymal Lesion Fibroepithelial Polyp Neurofibroma

 

 MPNST
Nevus 14 14 0 0 0 0 0 0 0 0 0
Haemangioma 7 0 7 0 0 0 0 0 0 0 0
Squamous Papilloma 5 0 0 5 0 0 0 0 0 0 0
Adenoma 1 0 0 0 1 0 0 0 0 0 0
Lipoma 1 0 0 0 0 1 0 0 0 0 0
Hamartoma 1 0 1 0 0 0 0 0 0 0 0
Fibroepithelial Polyp

 

 2 0 0 0 0 0 0 2 0 0 0
Neurofibroma 1 0 0 0 0 0 0 0 0 0 1
Total = 32 Total= 32

Table IV: Distribution of different benign cystic eyelid lesions

Final Histological

Diagnosis

 Cytological Diagnosis
Total Final Histology Cyst of Moll Dermoid Cyst Epidermal Inclusion Cyst Sebaceous Cyst
Cyst of Moll 10 10 0 0 0
Dermoid Cyst

 

 5 0 5 0 0
Epidermal Inclusion Cyst

 

 3 0 0 3 0
Sebaceous Cyst 2 0 0 0 2
Tolal 20 Total = 20

 

Table V: Distribution of malignant eyelid lesions (total of 85 cases each)

Final

Histological

Diagnosis

 Cytological Diagnosis
Total Final Histology Basal Cell Carcinoma Sebaceous Gland Carcinoma Squamous Cell Carcinoma Lymphoma Small Cell Tumor Nevus
Basal Cell Carcinoma

 

 12 11 0 0 0 0 1
Sebaceous Gland Carcinoma

 

 9 0 9 0 0 0 0
Squamous Cell Carcinoma

 

 9 0 0 9 0 0 0
Lymphoma 2 0 0 0 2 0 0
Small Cell Tumor 1 0 0 0 0 1 0
Total = 33 Total = 33

 

TableVI: Comparison of diagnosis between Cytopathology with Histopathology among the Malignant Eyelid Growths

 

Cytopathology Histopathology Total
Malignant Benign
Malignant 32 (37.64) 1 (1.18%) 33 (100.0%)
Benign 1 (1.18%)  51 (60.00%) 52 (100.0%)
Total 33 (38.82%)   52 (61.18%)  85 (100.0%)

* p value < 0.0001

 

Table VII: Assessment of diagnostic accuracy of cytopathology of eyelid growths

 

Cytopathological Diagnosis No. of cases Percentage
Positive for Malignancy 32 37.64%
Negative for Malignancy 51 60.00%
False Positive 1 1.18%
False Negative 1 1.18%
Total 85 100%

 

Table VIII: Cytopathological validity of different malignant eyelid growths

Sensitivity Specificity Positive Predictive Negative Predictive Value Accuracy
96.97% 98.08% 96.97% 98.08% 97.65%

Figure 1. (A) Case 1. Basal cell carcinoma, (B) Photomicrograph of cytology smear of basal cell carcinoma showing tightly cohesive small clusters of uniform hyper­chromatic basaloid cells (Pap’s, x200), (C) Photomicrograph of Basal cell carcinoma showing atypical basaloid cell with retraction artifact (H&E, x400)

Figure 2. (A) Case 2.  Squamous cell carcinoma, (B) Photomicrograph of cytology smear of Squamous cell carcinoma showing enlarged hyper-chromatic nuclei of variable size and keratinization (Pap’s, x400), (C) Photomicrograph of Squamous cell carcinoma (Grade-I) showing atypical squamous cell invading deeply into the dermis. It also shows squamous pearl. (H&E, x200)

Figure 3. (A) Case 3. Sebaceous (meibomian) gland carcinoma, (B) Photomicrograph of cytology smear of sebaceous (meibomian) gland carcinoma showing atypical tumor cells arranged in clusters and singly with foamy eosinophilic cytoplasm (Pap’s, x400), (C) Photomicrograph of sebaceous (meibomian) gland carcinoma showing atypical tumor cells and necrosis (H&E, X200).

Figure 4. A. Case 4. Non-Hodgkin’s Lymphoma, B. Photomicrograph of cytology smear of Non-Hodgkin’s Lymphoma showing monomorphous population of atypical lymphoid cells, scanty cytoplasm with clumped chromatin. (Pap’s x200), C. Photomicrograph of Non-Hodgkin’s Lymphoma showing  hypercellular proliferations. Most of the tumor cells are monotonous in appearance, having large nuclei with condensed chromatin. (H&E, x400), D. Photomicrograph of IHC study showing lymphoid cells with positive staining for LCA. (IHC, x400), E. Photomicrograph of IHC study showing scattered lymphoid cells with positive staining for CD3. (IHC, x400), F. Photomicrograph of IHC study showing  majority of atypical  lymphoid cells with positive staining for CD20 (IHC, x400) Conclusion: Immunostaining results favor the diagnosis of Non-Hodgkin’s Lymphoma of “ B” cell origin.

Figure 5. A. Case 5.  Hydrocystoma. B. Photomicrograph of cytology smear of benign cystic lesion showing foamy macrophages in the background of proteinecious material (Pap’s, x200), C. Photomicrograph of hydrocystoma showing cyst wall lined by a double layer of columnar cells with eosinophilic cytoplasm and prominent papillary projections. (H&E x400)

Figure 6. A. Case. Nevus, B. Photomicrograph of cytology smear of nevus showing single and small clusters cells with rounded or oval nuclei and indistinct cytoplasm (Pap’s x200). C. Photomicrograph of nevus showing nests of round cells in the underlining dermis. (H&E, x200)

Discussion

The present study was conducted with an aim to assess the cytopathological and histopathological correlation of different types of eyelid growths. It was a hospital based cross sectional study which enrolled 85 clinically suspected eyelid growths. Out of them 52 (61.18%) were benign and 33 (38.82%) were malignant. A recent study by Mondal and Dutta,8 Fine needle aspirates from 80 eyelid swellings were studied.  Forty eight cases of benign and 32 cases of malignant lesions were diagnosed by FNAC.

Mean age in the present study was 43.22 years (SD ±17.42) (range 19 – 90 years). Pombejara et al.17 reported mean age of presentation 52.4 years ± SD 21.8 years in Thailand. Most benign growths were within the age group 26-50 years and malignant eyelid lesions were in patients above 51 years of age.Mondal and Dutta8 studied 80 eyelid lesions by FNAC, in which 32 cases were malignant. In that study most common malignant lesion was basal cell carcinoma (12 cases, 15%) followed by sebaceous gland carcinoma (nine cases, 11.25%) and squamous cell carcinoma (eight cases, 10%). In the present study, malignancy were 38.82% (33 out of 85), and the most frequent malignant tumor was basal cell carcinoma (12 out of 33, 36.37%) followed by sebaceous gland carcinoma (09, 27.27%), squamous cell carcinoma (09, 27.27%), Non-Hodgkin’s lymphoma (2, 6.06%) and small cell carcinoma (1,3.03%).

Among benign lesions, in the present study, nevus was most common in 14 cases (43.75%), followed by haemangioma 7 cases (21.88%) and squamous papilloma in 5 cases (15.64%). Other less common lesions were fibroepithelial polyp, adenoma, lipoma, neurofibroma and hamartoma. In the present study, among benign cystic lesions (20 cases) of eyelid, sudoriferous cyst was most common in 10 cases (50.00%) followed by dermoid cyst in 5 cases (25.00%), epidermal inclusion cysts in 3 cases (15.00%), and sebaceous cysts in 2 cases (10.00%). In a recent study by Toshida et al.,18 the most frequent diagnosis among 106 benign lesions were nevus in 23 cases (21.7%). The second common was squamous cell papilloma in 18 cases (17.0%), followed by seborrheic keratosis in 14 cases (13.2%). Less common causes were epidermal cyst in 10 cases (9.4%) and dermoid cyst in 7 cases (6.6%).

 

Ulcerated skin of eye-lid can be scraped safely and it is recommended to combine FNAC with scrape cytology for any ulcerated lesions of eyelid skin and conjunctiva (Rai, 2007). In the present study, out of a total 33 histologically confirmed malignant tumors, thirteen (13) ulcerated cases were taken by scraping. Of these, nine (09) were Basal Cell Carcinoma, three (3) were Squamous Cell Carcinoma and one (1) was found to be Sebaceous (Meibomian) Gland Carcinoma.

In recent studies by Mondal and Dutta 8 and  Arora et al.5 showed accuracy of cytological diagnosis of eyelid growths were 83.87% and 89.4% respectively. In the present study, accuracy of cytological diagnosis of malignant eyelid growths was 97.65%. These comparisons are clearly emphasizing need for cytopathology and histopathology of all surgically removed specimens.19,20 The present study also compared cytopathological and histopathological diagnosis of all specimens.When comparing cytopathology with histopathology of the clinically suspected malignant eyelid growths, the comparison between cytopathology and histopathology was statistically highly significant (p<0.0001).In the present study, Sensitivity, Specificity and Accuracy of cytopathology to diagnose malignant eyelid growths were 96.97%, 98.08% and 97.65% respectively.

References

  1. Abdi U, Tyagi N, Maheshwari V, Gogi R and Tyagi SP. Tumors of eyelid: A clinicopathologic study. J Indian Med Assoc, 1996;94: 405-9.
  2. Cook BE Jr. and Bartley GB. Epidemiologic characteristics and clinical course of patients with malignant eyelid tumors in an incidence cohort in Olmsted county, Minnesota. Ophthalmology, 1999; 106: 46-50.
  3. Hughes MO. A Pictorial Anatomy of the Human Eye/Anophthalmic Socket: A Review for Ocularists. The Journal of Ophthalmic Prosthetics, 2004; 51-53.
  4. Jakobiec FA, Bonanno PA and Sigelman J. Conjunctival adnexal cysts and dermoids. Arch Ophthalmol, 1978;96: 1404-1409.
  5. Arora R, Rewari R and Betheri SM. Fine needle aspiration cytology of eyelid tumors. Acta cytol, 1990; 34(2): 227-32.
  6. Dey P, Radhika S, Rajwanshi A, Ray R, Nijhawan R and Das A. Fine needle aspiration biopsy of orbital and eyelid lesions. Acta Cytol, 1993; 37: 903-7.
  7. Brooks Christine, Scope, Alon, Braun, Ralph, P,Marghoob and Ashfaq A. Dermoscopy of nevi and melanoma in childhood. Expert Review of Dermatology, 2011; 6 (1): 19–34.
  8. Mondal SK and Dutta Cytohistological study of eyelid lesions and pitfalls in fine needle aspiration cytology. J Cytol, 2008; 25(4):133-7.
  9. Baschinsky D, Hameed A, Keyhani-Rofagha S. Fine-needle aspiration cytological features of dermoid cyst of the parotid gland: a report of two cases.diagncytopathol, 1999; 20(6): 387-8.
  10. Vemuganti GK and Rai NN. Neoplastic lesions of eyelids, eyeball and orbit. J Cytol, 2007; 24: 30-36.
  11. Brown ST, Nalley JF, Kraus SJ. Molluscum contagiosum. Sex Transm Dis. Jul-Sep, 1981; 8(3), pp227-34.
  12. Stulberg DL, Hutchinson AG.Molluscum contagiosum and warts. Am Fam Physician.  2003; 67(6): 1233-40
  13. Pathak D and Neelaiah S. Disseminated cutaneous rhinosporidiosis: diagnosis by fine needle aspiration cytology. Acta Cytol, 2006; 50: 111-2.
  14. Baron K, Curling OM, Paridaens AD and Hungerford JL. The role of cytology in the diagnosis of peri-ocular basal cell carcinomas.OphthalPlastReconstr Surg, 1996; 12: 190-4.
  15. Saqi A, McGrath CM, Skovronsky D and Yu GH. Cytomorphologic features of fine-needle aspiration of metastatic and recurrent melanoma.Diagn Cytopathol, 2002; 27: 286-290.
  16. Pugh WC, Manning JT and Butller JJ. Paraimmunoblastic variant of small lymphocytic lymphoma/leukemia. Am J Surg Pathol, 1988; 12: 907-917.
  17. Pombejara FN, Tulvatana W and Pungpapong K. Malignant tumors of the eye and ocular adnexa in Thailand: A six-year review at King Chulalongkorn Memorial Hospital. Asian Biomed, 2009; 3: 551-5.
  18. Toshida H, Mamada N, Fujimaki T, Funaki T, Ebihara N, Murakami A andOkisaka S. Incidence of Benign and Malignant Eyelid Tumors in Japan, Int J Ophthalmic Patho, 2012; l1(2): 112-14.
  19. Kerstern R, Ewing-Chow D, Kulwin DR. and Gallon M. Accuracy of clinical diagnosis of cutaneous eyelid lesions. Ophthalmology, 1997; 104: 479-484.
  20. Margo CE and Waltz K. Basal cell carcinoma of the eyelid and periocular skin, Survey of ophthalmol, 1999; 38(2):169-192.
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Correlation of Ki-67 Proliferating Index with Histological Stage and Grade in Colorectal Carcinoma

Correlation of Ki-67 Proliferating Index with Histological Stage and Grade  in Colorectal  Carcinoma

 *Sultana S,1 Islam N,2 Kabir E,3 Akhter S,4 Paul R,5 Shirin A,6  Khan AA,7 Jahan N8

Abstract
Colorectal carcinoma is the most  common cancer of gastrointestinal tract. It is the 3rd most  commonly  diagnosed  cancer  and  the  3rd  leading  cause  of  cancer  death. The  growth  of  tumor  in  colorectal   carcinoma  is  highly  variable  and  its  histological grading and  staging  has  important  role  in  diagnosis, treatment  and  overall  prognosis. To observe the Ki-67 expression in colorectal carcinoma and find out the possible correlation of Ki-67 proliferating index with histological grading and Duke’s staging. This  cross sectional  study  was  conducted  at  Sir Salimullah Medical College  from  July 2014  to June 2016. 98  patients  with colorectal carcinoma  enrolled  in  this  study  by  purposive  sampling. The H&E staining was done on paraffin embedded tissue sample. Ki-67 expression by IHC method. Ki-67 is a proliferation associated  nuclear  antigen  which  can be recognize by  MIB-1 monoclonal antibody, correlate with histological staging and grading in  colorectal carcinoma. Then tumours were graded according to WHO grading criteria and pathological staging was done according to Duke’s staging system and immunohistochemical staining for Ki-67 antigen expression. Results were subjected to statistical analysis. The results were considered to be significant when the P< 0.05. Ki-67 proliferative index was high in well and moderately differentiated adenocarcinoma but low in poorly differentiated which is statistically significant (p <0.05).  Ki-67 expression was high in early Duke’s stage A and B but low expression  in advanced Duke’s stage C (p>0.05). The result of  this  study will enlighten the clinician regarding  the need for  doing Ki-67 in  colorectal carcinoma which   would  contribute to better understanding  of the  treatment as well as prognosis.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):125-133]

Key words: Colorectal carcinoma, Immunohistochemistry, Ki-67

  1. *Dr. Sahela Sultana, Assistant Professor, Department of Pathology, Dr. Sirajul Islam Medical College, Dhaka. sultana.sahela83@gmail.com
  2. Professor Dr. Nasimul Islam, Professor and Head, Department of Pathology, Sir Salimullah Medical College, Dhaka.
  3. Professor Dr. Enamul Kabir, Professor, Department of Pathology, Sir Salimullah Medical College, Dhaka.
  4. Salma Akhter, Assistant Professor, Department of Pathology, Universal Medical College, Dhaka.
  5. Rita Paul, Assistant Professor, Department of Pathology, Ibrahim Medical College, Dhaka.
  6. Afroz Shirin, Assistant Professor, Department of Pathology, Enam Medical College Savar, Dhaka.
  7. Abu Anis Khan, Assistant Professor, Department of Pathology, International Medical College, Dhaka.
  8. Nusrat Jahan, Lecturer, Department of Pathology, Sir Salimullah Medical College, Dhaka.

 

*For correspondence

Introduction

Colorectal carcinoma is the most common malignancy of gastrointesinal (GI) tract and is a major cause of morbidity and mortality worldwide.1 Colorectal cancer accounts for 10% of all cancers and it is the 2nd leading cause of death from malignancy in the industrialized world.2 There are nearly one million new cases of colorectal cancer diagnosed worldwide each year and half a million death.3 In 2013, there were an estimated 1,177,556 people living with colon and rectal cancer in the United States and the number of new cases of colon and rectal cancer was 41.0 per 100,000 men and women per year.4 Regarding age  incidence of colorectal carcinoma, recent reports show that in the USA it was the most frequent form of cancer among the person aged between 60-70 years and fewer than 20% of cases occurs before the age 50.5 The incidence of  colorectal cancer in Bangladesh is exactly not known but estimated population are approximately 15,10.1%.6 The distribution of colorectal carcinoma worldwide seems to be related to industrialization and socioeconomic standard and the incidence rate is higher in industrialized countries including Western Europe, Scandinavia and North America, whereas in the developing countries (sub-Saharan, Africa and Asia) the incidences appear to be lower.7

There are several staging system for colorectal carcinoma among these TNM and Duke’s staging systems are the most common way of staging and grading of colorectal carcinoma.8 The American Joint Committee (AJC) and the Union  for International  Cancer control (UICC) joined to produce the TNM system, which attempt to record clinical and pathological data, guide therapy and forecast prognosis, all in one.9 Whereas  the  classification   into  Duke’s stage A,B,C  cases   is  the  measurement  of  the  boundaries  reached and both   methods   permit the grouping  of  cases into favorable and unfavorable outcome.10 Histologically  the tumor is graded according to WHO grading criteria as well differentiated, moderately differentiated and poorly differentiated and the histological appearance of colorectal carcinoma may vary considerably with its major importance being related to prognosis.10

The use of monoclonal antibodies raised against specific antigens associated with the cell proliferation.11  Ki-67 is a proliferation associated nuclear antigen expressed in all cycling cell except resting cell in the G0 phase and it reflects cell in the S/G2+M phases in particularly.12  MIB-1 is a monoclonal antibody and it recognizes the Ki67 nuclear antigen in the formalin fixed paraffin embedded tissue section.13 Ki-67 expression is estimated as the percentage of tumors cells positively stained by the antibody with nuclear staining.12 The importance of Ki-67 as an indicator of tumor behavior and in colorectal cancer this index may be used as a marker of prognosis.12

The proliferative activity as measured by Ki-67 antibody is closely associated with histological  grade and stage.2 In 2008 Uzma Nabi, Nagi A H and Waqas Sami, Department of Pathology, University of Health Sciences, Lahore, Pakistan conducted a study on Ki-67 proliferating index and histological stage and grade  of colorectal carcinoma and observed that proliferative index is high in well and moderately differentiated  adenocarcinoma and in an early Duke’s stage (A or B).2 But Ki-67 proliferating activity is low in poorly differentiated tumour and  in an advanced Duke’s stage C.13,8 But there are some studies of Lanza, Cavazzinil  in 1990 and  Yokoyama N, Okomoto H in Japan in 2005 contradicting the above mentioned  association of Ki-67 versus grading and staging of colorectal carcinoma, they concluded that proliferating index of Ki-67 was increasing with increasing grade, stage.14, 15

Methods

This  cross sectional study was conducted among the 98 histopathologically diagnosed patients having colorectal carcinoma  over a period of two years in the department of surgery, Sir Salimullah Medical College. Study population were the patients having colorectal cancer underwent surgical  treatment in the department of surgery of Sir Salimullah Medical College. The proliferative activity as measured by Ki-67 antibody is closely associated with histological  grading  and staging of colorectal carcinoma. The representative sections were submitted for Immunohistochemical staining. The Ki-67 immunostaining were performed according to manufacture’s recommendation, using the MIB-1 clone (DAKO, Carpenteria, CA & Ventena Medical System, Tucson, AZ). Ki-67 immunostainined slides were examined via light microsccopy. Positive Ki-67 staining was observed brown granular nuclear staning. For Ki-67 scoring the most positive area of the tumor was selected avoiding foci of inflammation. The number of positive nuclei were counted in 500 tumor cells in a high power field. The average of the counts over the same slides was taken and expressed as the percentage of Ki-67 positive cells  in the tumor.

Statistical analysis were performed in SPSS statistical software program, Version 17.0. To correlate histological grading and staging of colorectal carcinoma with Ki-67 proliferating index were performed with  Mann- Whitney U test. The result were consider to be  significant when P<0.05. One way ANOVA followed by Bonferroni test was performed to compare between groups.

Results

98 cases were included in the present study.  Age incidence ranged from 28-78 years and their mean ± SD 47.38 ± 10.37. Maximum patients (30.6 %) were found in 41-50 years age group where M: F was 1.72: 1 (Table I). Regarding site of tumor more than 50% of patient had tumors in the left side of colon and in the rest of the cases tumors were present in caecum (26.5%; Table-II).  98 cases having different sizes of tumors and in most of the cases fifty nine cases (60.2%) tumors size were 3-4 cm and  their mean ± SD 4.8 ± 1.8 (Table-III). Different morphological types of tumor were observed in present study. In maximum forty cases (40.8%) the morphological types of tumors were ulcerative and only five cases (5.3%) tumors were infiltrative type. Rests were annular (33.5%) and polypoid (20.3%; Table IV).

In the present study, tumors were graded according to WHO grading system into well differentiated, moderately differentiated, poorly differentiated and grouped into A, B, C accordingly. The maximum cases 69 (70.4%) of colorectal carcinoma were well differentiated and their mean Ki-67 proliferating index was 47.83 ± 15.23. There were significant differences among the groups (A vs B vs C), when mean proliferating index of Ki-67 were compared among the three groups. The result was found statistically significant (P<0.05). However when compared in between groups only A vs C (between well differentiate & poorly differentiate) groups were found also statistically significant (P<0.05). In the present study, it was also observed that with increasing grade, Ki-67 proliferating index decreases (Table V) .

Regarding staging of the tumor (Duke’s staging) where  maximum  cases (44.8%) were in stage B1 and their mean Ki-67 proliferating index was 46.25±14.06. There was no significant differences among the stages and the result was not statistically significant (P>0.05). In the present study it was also observed that with increasing stage of the tumors, there was decreasing Ki-67 proliferating index (Table VI).

Table I: Distribution of patients according to age group with male female ratio (n=98)

Age groups         Frequency            M: F      Percentage
Total  Male Female
≤30

 

 10    7    3           2.3:1       10.2
31-40 23   13   10           1.3: 1       23.5
 

41-50

  

30

  

19

  

11

  

1.72: 1

  

30.6
 

51-60

  

26

  

17

  

9

  

1.88: 1

  

26.5
 

≥60

  

9

  

9

  

0

  

9.00: 0

  

9.2
 

Total

  

98
 

Mean ± SD

 

  

47.01± 10.99
Range (Min-Max)                                              28 – 78

 

 

Table II: Distribution of tumors according to site (n=98)

Site of tumor Frequency (%)
Left side of colon
Sigmoid colon 34 (34.7)
Transverse colon 24 (24.6)
Rectum 14 (14.7)
Right side of colon
Caecum 26 (26.5)
Total 98 (100)

Table III: Distribution of patients according to tumor size (n=98)

Tumor size (cm) Frequency (%)
1 – 2 14 (14.3)
3 – 4 59 (60.2)
5 – 6 19 (19.4)
Mean ± SD 4.8 ± 1.8
Total 98 (100)

 

Table IV: Distribution of patients according to morphological types of tumor (n=98)

Morphology Frequency (%)
Ulcerative 40 (40.8)
Annular 33 (33.5)
Polypoid 20 (20.3)
Infiltrating 5 (5.3)
Total 98 (100)

 

Table V: Relation of Ki-67 proliferating index with histological grading (n=98)

Grading Frequency

n(%)

 Ki-67 expression

(Mean ± SD)

 P
Well differentiated (A) 69 (70.4) 47.83± 15.23
Moderate differentiate
(B)		 15(15.3) 46.33 ± 18.07
Poor differentiated (C)  14(14.3) 35.35 ± 11.17
Statistical analysis
A vs B vs C 0.023*
A vs B 1.000ns
A vs C 0.019*
B vs C 0.162 ns

ANOVA followed by Bonferroni test was performed to compare between groups

Table VI: Relation of Ki-67 proliferating index with histological stage (n=98)

 

Duke’s staging

 

 Frequency

n (%)

 Ki-67 expression

(Mean ± SD)

 p
Stage A 3 (3.3%) 53.33 ± 20.81 0.727ns
Stage B1 44 (44.8) 46.25 ± 14.06
Stage B2 20(20.5) 43.25 ± 14.06
Stage C1 14(14.8) 44.64 ± 21.07
Stage C2 17(17.5) 42.05 ± 13.69

 

ANOVA test was done to measure the level of significance.

 

 

 

 

 

Figure 1. Photomicrograph of histopathological section of well differentiated adenocarcinoma of colon (H&E method x100)

 

 

 

 

 

Figure 2. Photomicrograph of   well differentiated adenocarcinoma stained with Ki-67 immunostain showing high proliferative Index(x100).

 

 

 

 

Figure 3. Photomicrograph of histopathological section of poorly differentiated adenocarcinoma of colon ( H& E method x400).

 

 

 

 

 

Figure 4. Photomicrograph of   poorly differentiated adenocarcinoma stained with Ki-67 immunostain showing low Proliferative Index (x400).

Discussion

Colorectal cancer (CRC) is one of the most common malignancies and a leading cause of cancer death worldwide.16 The incidence of cancer colon and rectum was 41.0 per 100,000 men and women per year and the number of deaths was 15.1 per 100,000 men and women per year in Bangladesh.17 Management of  colorectal carcinoma depends on a number of  morphological and biological factors which include the pathological tumor stage (including involvement of lymph nodes, breach of serosa, distant spread etc.), primary tumor characteristics (including depth of tumor penetration in the bowel wall, histological subtype, histological grade and differentiation, venous and lymphatic invasion, perineural invasion and lymphocytic infiltration), status of surgical resection margins (free or involved).18 With assessment of tumor cell proliferation may predict tumor behavior.19 The aim of this study was to evaluate the proliferating index (PI) in formalin fixed, paraffin embedded tissue section of colorectal carcinoma, using monoclonal MIB-1 antibody (Ki-67) and to assess the relationship between proliferative index (PI) and various pathological findings in colorectal carcinoma including histological grade, and stage.

In the present study, the mean age of the patients was 47.01± 10.99 years and the highest number of malignant cases were seen in the 4th and 5th decades. Male female ratio was 1.72:1 and 1.88:1 in 4th and 5th decades accordingly. So, in present study, male were predominant than female. These findings were similar to other studies.20, 21

In 34.7% patients the tumors were located in sigmoid colon followed by caecum (26.5%), transverse colon (24.5%) and rectum (14.3%).  As per gross morphological type of cancer, maximum 40.8% were ulcerative and 33.5% were annular type and rest were polypoid (20.3%) and infiltrating type (5.3%). In one study, colon was more commonly affected site compared to rectum and most of the lesions were ulcerative.22 In another study rectum was the most common affected site and predominant lesions were annular.23

In present study, 60.2% of malignant cases had tumors size between 3-4 cm and 19.4% malignant cases had tumors size 5-6 cm. In a study in 2011 by Kornprat showed that maximum size of the tumor in colon cancer were in between 4.5 to 6.5 cm in majority of cases.24 In 2013 ASCO annual meeting showed a report where a study conducted on tumor size in colorectal cancer found that in majority of the cases the tumors sizes were in between 4-6 cm in colorectal carcinoma.25

In this present study it was observed that  the Ki-67 labelling index was high in grade I and grade II  compared to grade III. These results showed that proliferating index was low in poorly differentiated tumor compared to well and moderately differentiated tumor. When Ki-67 proliferating index was compared among three groups a statistically significant correlation was found in present study (p<0.05). These findings are similar to studies in Japan2 Pakistan12 and Finland.26 On the other hand, some of the studies showed contradictory result that Ki-67 proliferating index increased with increasing histological grade.26,27

In the present study, it was observed that Ki 67 labeling index was high in Duke’s stage A and B  and tumor in advanced stage (Duke’s C) have a low proliferating index  compared to tumors in  an early invasive stage (Duke’s A and B). Regarding the correlation of Ki-67 LI with staging, no statistically significant correlation was found. This result was similar with other studies in which they concluded that Ki-67 proliferating index was significantly lower in carcinoma in subserosa or deeper invasion compared to carcinoma with submucosa or muscularis mucosa invasion.2,8,26 In another study  contradictory results have been reported on associations of Ki-67 with prognosis and survival of colorectal tumors with a low proliferation index in Duke’s A and B tumors to be associated with survival impairment compared to those with high values.28 Tumors with high proliferative activity are known to be most responsive to radiotherapy and Willett and collaborators showed that radiation eradicates preferentially rapidly dividing cells in rectal cancer, whereas populations with slow proliferation show greater radioresistance.29

Conclusion

It is concluded that Ki-67 labeling index is high in well to moderately differentiated adenocarcinomas in an early Duke’s stage A or B compared to poorly differentiated adenocarcinomas, and  in an advanced Duke’s stage C. Thus Ki-67 proliferating index can be useful in a patient with colorectal carcinoma as a ancillary diagnostic support. Moreover, it may help in the prognostic evaluation of patient, survival as well as in considering them for post surgical treatment.

References

  1. Kumar V, Abul K Abbas, Nelson F, Jon C Aster. The Gastrointestinal Tract, Robbins and Cotran Pathologic Basis of Disease, 8th edition,Saunders, 2010;17: 822-826.
  2. Uzma N, Nagi A.H, Waqas S. Correlation of Ki 67 with histological grade, stage in colorectal carcinoma. Dept of pathology,University of health science, Lahore  Pakistan, J Ayub Med Coll, 2008;20(4):44-8.
  3. Bisgaard ML, Jager AC, Myrhoj T, Bernstein I, Nielsen FC. Hereditary non-polyposis colorectal cancer (HNPCC): phenotype-genotype correlation between patients with and without identified mutation. 2002; 20(1):20-7.
  4. Andrew RH, Hongmei N. Epidemiology of colorectal cancer. Int Journal of Molecular Epidemiology and  Genetics, 2016;7(3):105–114.
  5. Soreide K, Janssen MA, Soiland H, H, Baak APJ. Microsatellite  instability in colorectal cancer. Br J Surg, 2006; 93(4):395-406.
  6. Sayed A H, Richard S. Cancer control in Bangladesh. Jpn J Clin Oncol, 2013; 43(12):1159-1169.
  7. Alice G, Davies A, Liz G, Evropi T, Harry Cl. Estimating the incidence of colorectal cancer in  Sub-Saharan Africa: A systematic analysis. J Glob Health, 2012;2(2):020404.
  8. Ishida H, Sadahiro S, Suzuki T, Ishikawa K, Kamijo A, Tajima T. Proliferative, infiltrative, and metastatic activitie s in colorectal tumors assessed by MIB-1 anti-body. 2003;10(6):1741-5.
  9. Sobin LH, Godspodarowicz MK, Wittekind C. UICC TNM Classification of Malignant Tumours. 7th edition. Wiley-Blackwell, 2009;12(4):100-105.
  10. Gordon PH, Nivatvongs S. Missouri: Karen Berger, Principles and practice of surgery for the colon, rectum  and anus.1992;23(3):503–520.
  11. Mokrý J, Nĕmecek S. Immunohistochemical detection of proliferative cells’,Sb Ved Pr Lek Fak Karlovy Univerzity Hradci Kralove.1995;38(3):107-.130.
  12. Oshima FTC, Iriya K, Forones MN. Ki-67 as a prognostic marker in colorectal cancer but not in gastric cancer. Neoplasm, 2005;5(4):420-4.
  13. Parkin D, Bray F, Ferlay. Global cancer statistics. CA Cancer J Clinic, 2005;55(2):74-108.
  14. Lanza G Jr, Cavazzini L, Borghi L, Ferretti S, Buccoliero F and Rubbini M. Immunohistochemical assessment of growth fraction in colorectal adenocarcinomas with monoclonal antibody Ki-67. 1990;186 (1):608-18.
  15. Yokoyama N, Okamoto,Valentina V, Walter B,  Suda HT, Hatakeyama K. Clinical significance of Ki-67 proliferation index in disease progression and prognosis of patients with resected colorectal  carcinoma Version of   Record British Journal of Surgery Society  2005; 10:4858-15.
  16. World Health Organization Cancer Incidence in Five Continents. Lyon: The World Health Organization and The International Agency for Research on Cancer; WHO, Geneva, 2002. http://www.iarc.fr/en/publications/pdfs-online/epi/index.php.
  17. National Institutes of Health and Human service. What You Need To Know About Cancer of the Colon and Rectum. Bethesda, MD: U.S. Department of Health and Human Services & National Institutes of Health.2010. http://www.cancer.gov/cancertopics/types/colon-and-rectal.
  18. Ola Marzouk and John Schofield. Review of Histopathological and Molecular Prognostic Features in Colorectal Cancer Cancers (Basel). 2011;3(2):2767–2810.
  19. Valera V, Yokoyama N, Walter B, Okamoto H, Suda.T, Hatakeyama K. Clinical significance of Ki-67 proliferation index in disease progression and prognosis of patients with resected colorectal carcinoma’. Br J Surg 2005;92:1002-7.
  20. Bhagyalakshmi, Sreelekha A. Kasi Babu SV, Kumar P. Muralikrishnm Ki-67 proliferation index and clinicopathological patterns in colorectal carcinomas. Departments of   Pathology, Biochemistry, Surgery, Gastroenterology, Andhra Medical College, Visakhapatnam. 2015;4:119-28.
  21. El-Bolkainy TN, Sakr MA, Nouh AA, El-Din NH. A comparative study of rectal and colonic carcinoma: demographic, pathologic and TNM staging analysis. J Egypt Natl Canc Inst, 2006;18: 258-263.
  22. Missaouia N, Jaidaine L, Abdelkader AB, Beizig N, Anjorin A, Yaacoubi MT. . Clinicopathological patterns of colorectal cancer in Tunisia. Asian Pacific J Cancer Prev 2010; 11:1719-1722.
  23. Laishram RS, Kaiho N, Shimray R, Sorokhaibam BD, Pukhrambam P, Durlav CS. Histopathological evaluation of colorectal carcinomas status in Manipur, India. 2010; 8(1):5-8.
  24. Kornprat P, Pollheimer MJ, Lindtner RA, Schlemmer A, Rehak P, Langner C. Value of tumor size as a prognostic variable in colorectal cancer: a critical reappraisal. Am J Clin Oncol 2011; 34(1):43-9.
  25. ASCO 2013 Annual Meeting, Gastrointestinal Colorectal Cancer,ASCO university. https://meetinglibrary.asco.org/results/SessionTitle:/22Gastrointestinal/20/28Colorectal /Meeting:/222013/20ASCO.
  26. Eeva S, Salla P, Tero V, Peter J. Roberts, Karl-Owe Söderström. Increased proliferation activity measured by immunoreactive Ki-67 is associated with survival improvement in rectal/recto sigmoid cancer. Department of Oncology and Radiotherapy, Turku University Hospital, Finland 2005;11(21):3245–3249.
  27. Saleh HA, Jackson H, Khatib G, Banerjee M. Correlation of bcl2 oncoprotein immunohistochemical expression with proliferation index and histopathologic parameters in  colorectal neoplasia. Appl mmunohistochem Mol Morphol, 2002;8:175-182.
  28. Palmqvist R, Sellberg P, Oberg A, Tavelin B, Rutegård JN. Low tumour cell proliferation at the invasive margin is associated with a poor prognosis in Dukes’ stage B colorectal cancers. Br J Cancer 1999;79:577-581.
  29. Willett CG, Warland G, Hagan MP, Daly WJ, Coen J, Shellito PC, Compton CC. Tumor proliferation in rectal cancer following preoperative irradiation. J Clin Oncol 1999;13:1417-1424.