Cervix cancer is the most common cancer in females, particularly in developing countries like India. The incidence of the disease increases in 30 – 34 years and summits at 55 – 65 years with a median age of 38 years. Cervical cancer is a preventable and most frequent carcinoma in India. It accounts for 1/3 of global cervical carcinoma.1In India every year around 1,22,844 women are diagnosed with cervical carcinoma and around 67,000 die from the disease.2 EGFR-1 has been suggested as a biomarker of aggressiveness in multiple cancer types like lung, colon and pancreas.3 The study is intended to see expression of EGFR-1 and Her2neu on samples of Squamous cell carcinoma of cervix and correlate it with patient’s age and histological grading. Data would help in planning the prognostic and therapeutic approach in patients with cervical carcinoma and also identify prognostic markers and diagnostic markers. It will be beneficial to mark Her2 neu and EGFR as therapeutic and diagnostic marker in case of Cervix carcinoma.

H&E Slides of sixty patients diagnosed as cervix carcinoma, proved on histopathology were taken and EGFR and Her2 neu expression by IHC were observed, followed by FISH. Data analysis: Data is presented in the form of percentage and proportions, using Chi-square test for statistical analysis with SPS Software version 23. Inclusion criteria – All cases of squamous cell carcinoma of cervix proved histopathologically. Exclusion criteria – 1.Cases where there is extensive tumor necrosis without sufficient viable tumor cells for accurate evaluation of the immunohistochemical results. 2.Cases of adenocarcinoma and other tumors of cervix. 3. Follow up cases of squamous cell carcinoma received and on radiotherapy /chemotherapy. Surgical specimens from patients operated for cervical carcinoma were received, formalin fixed, paraffin embedded, Cut at Four to five micron and stained by haematoxylin and eosin (H & E) for histopathological study. Procedure for H & E staining for paraffin section: 1. Deparaffinize sections, hydrate through graded alcohol to water. 2. Remove Fixation pigments if needed. 3. Stain in an alum Haematoxylin for suitable time. 4. Wash in running tap water until sections blue for 5 minutes or less. 5. Differentiated in1% acid alcohol for 5-10 seconds. 6. Wash well in tap water until sections again blue (10-15 minutes) or. 7. Blue by dipping in an alkaline solution followed by a 5-minute tap water wash. 8. Stain in 1% eosin Y for 10 minutes. 9. Wash in running tap water for 1-5 minute. 10. Dehydrate through alcohols, clear, dry and mount in DPX. Cervical carcinoma was classified according to WHO histological classification of invasive carcinoma of cervix. Squamous cell carcinoma was sub typed into Keratinizing and Non Keratinizing types. All the cases diagnosed histologically as Squamous cell carcinoma were subjected to immuno histochemical study for EGFR-1 expression. Control used for expression of EGFR-1 in normal (nondysplastic) cervical epithelium which was helpful in expression of EGFR-1 to interpret sections as Weak, Moderate & Strong expression in Squamous cell carcinoma of cervix. 3 micron thickness tissue sections were taken for immuno histochemical analysis to determine EGFR-1 expression pattern. The super sensitive polymer – HRP based immunohistochemistry Kit of BIOGenex [EP22] was used and procedure followed as per manual provided/Standard Protocol. Evaluation of immunohistochemistry (IHC) results Epidermal growth factor receptor (EGFR) EGFR expression was evaluated using the same scoring criterion as for HER2. Scores 0 and 1+ were considered negative, 2+ was considered equivocal, and 3+ was considered positive 4. HER2neu HER2 neu was scored as positive if there was strong and diffuse nuclear and cytoplasmic staining present in greater than 70% of the malignant cells. 5. Fluorescence in situ hybridization (FISH) analysis The manufacturer’s instructions are followed. Three micron sections from the tumor paraffin block are prepared and mounted on a positively charged slide. Slides are initially pretreated after being incubated at 70˚c for 10 min. Pretreatment is done through immersion of the slide in xylene, graded alcohol concentrations for 5 min each, washing in distilled water, immersing in prewarmed citric solution for 15 min and then allowing for pepsin digestion for 10 min. Serial washes followed by dehydration are applied. After drying, denaturation at 75˚C is done. 10 μl of dual color probe EGFR LSI spectrum green/ CEP7 spectrum orange (Zytovision, CE marked) is applied on the slide. The slide is then transferred to a hybridization chamber for the next day. After serial washing followed by ethanol solutions, 10 μl of DAPI is applied on the slide. The slide is then incubated in the dark for 15 min before visualization. Fluorescence microscope evaluation The slides are visualized using a Zeiss axioscope fluorescent microscope using orange, green, DAPI, and dual orange and green filters. Zeiss imaging software system is used. We evaluated the EGFR gene copy number by counting green signals and the CEP7 copy number by counting orange signals. Representative images of each image were acquired with a CCD camera in monochromatic layers, which were merged by the Zeiss software. At least 100 nonoverlapping interphase nuclei from whole samples were scored. The number of copies of EGFR probes and that of chromosome 7 were assessed.6 Patients were divided into six groups with ascending number of copies of the EGFR gene per cell according to the frequency of tumor cells with a specific number of copies of the EGFR gene and chromosome 7 centromere. In this study, we classified the patients into two groups for more simplification; amplified and not amplified. The unamplified group included balanced disomy and balanced trisomy. The amplified group included amplification of EGFR with or without polysomy. The case was considered as balanced disomy (not amplified) if the EGFR copy number is 2 and the chromosome copy number is 2. If chromosome 7 copy number is 3 and EGFR gene copy number is also 3 in most nuclei, this is defined as balanced trisomy and the case is considered not amplified. If the EGFR copy number is 4 or more, the case is considered amplified whether it is associated with polysomy or not 6.

TABLE -1- Types of Specimen – 90 % -Biopsies ,10 % - Hysterectomies Type of Specimen Number Of cases Percentage (%) Hysterectomies 08 13.33% Cervical biopsies 52 86.6% Total 60 100% TABLE -2 Expression Score of EGFR-1 in SCC EGFR – 1 expression And Score SCC (n=60) 0(Negative) - 2-3 (Weak) 10 (66.6%) 4-5 (Moderate) 05(33.3%) 6-7 (Strong) - • Out of 60 cases of Squamous cell carcinoma of cervix 15 cases were well differentiated in which 66.6% cases showed weak expression of EGFR-1 which 57% cases showed moderate Expression of EGFR-1 while 1 case showed strong expression of EGFR-1. Expression of EGFR -1 In different stages – In all stages correlation with EGFR-1 expression is significant (p value =0.009).

Cervical cancer is the fourth most common cancer worldwide 3. Globally, the average age at diagnosis of cervical cancer was 53 years 3,7. In this study, the mean age of the patient was ranging from 32 to 67 years. Squamous cell carcinoma was the most common histological type constituting 80% of cases followed by adenocarcinoma (18%) and neuroendocrine carcinoma (2%) This is similar to what is reported by Meng et al, where squamous cell carcinoma comprises 74.5% of cases followed by adenocarcinoma (21.4%) 8. Epidermal growth factor receptor (EGFR) has been an attractive target for anticancer therapy due to its involvement in multiple cellular processes, thus contributing to the initiation and development of cancer 9. HER2 NEU The Her2 neu positivity was detected in 60% of cases in the form of nuclear and cytoplasmic staining. This percentage is much lower than that reported by Suppulaxmi N, et al. (17.% of invasive cervical cancer cases are associated with Her2 neu positivity) and in the same study, 95% of cervical cancer is associated with HPV infection 11. Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Figure 1(a,b) depicts Gross images of Cervix carcinoma. Figure 1c and Figure 3 depicts H&E image of Cervix carcinoma. Figure 2 depicts IHC marker EGFR membranous positivity in Cervix Carcinoma Moderately differentiated. Figure 4 depicts and 5 depicts Her2 neu membranous positivity in Cervix Carcinoma MD and Poorly differentiated. Figure 6 (a,b,c,d) depicts FISH analysis for cervix carcinoma. 4 A dual color probe for EGFR LSI spectrum green and CEP7 spectrum orange is used. (A) Balanced disomy. Each nucleus contains two green EGFR signals and two orange CEP7. (B) Balanced trisomy. Some nuclei contain three orange CEP7 signals and three green EGFR signals which appear to overlap in most nuclei giving three yellow signals (arrow). (C) Combined EGFR amplification and CEP7 polysomy. Some nuclei (arrow) contain up to four orange CEP7 signals and four green EGFR. (D) Clusters of green signals denoting amplification of the EGFR gene associated with 2-3 red signals of CEP-7. Her2 neu Positivity was detected in 60% of cases. Positive reaction to EGFR (score 3+) was detected in 18% of cases. The EGFR gene was not amplified in 81% of cases. Statistical significance was found between histological type, Her2 neu, and EGFR expression. A fair agreement was detected between EGFR expression and EGFR amplification. EGFR is a prognostic marker and Her2 neu is a diagnostic marker as EGFR is expressed in only poorly differentiated carcinoma and Her2 neu is expressed in all variants.

We in our study included 60 positive SCC Cervix cases, Her2 neu was block positive in 70% cases and EGFR 1 strong expression was found in Stage III and Stage IV. EGFR and Her2 neu have an independent role in the development of cervical cancer in South Asian females. EGFR overexpression and EGFR amplification represent two different genetic events in other words, an increase in gene copy number does not mean that there will be an increase in the function of the gene as expressed by an increase in protein expression. Therefore association of Her2 neu as diagnostic marker and EGFR 1 as prognostic marker will be very useful in diagnosis and treatment of patient.

1.T.soonthornthum,H.Aris- pulido –Epidermal growth factor receptor as a biomarker for cervical carcinoma Department of internal medicine university of new Mexico cancer research and treatment centre , Annals of oncology 2011; volume 22 :2166-2178. 2. ICO Information centre on HPV and Cancer (summary Report 2014 :08-22. Human papilloma virus and Related Disease in India 2014. 3.Arbyn M, Weiderpass E, Bruni L, Saraiya M, Ferlay J, Bray F (2020). Estimates of incidence and mortality of cervical cancer in 2018: a worldwide analysis. Lancet Glob Health 8:191–203. 4. Shen L, Shui Y, Wang X, Sheng L, Yang Z, Xue D, Wei Q (2008). EGFR and HER2 expression in primary cervical cancers and corresponding lymph node metastases: implications for targeted radiotherapy. BMC Cancer 8:1. p.1.) 5. Schache AG, Liloglou T, Risk JM, Filia A, Jones TM, Sheard J, et al. Evaluation of human papilloma virus diagnostic testing in oropharyngeal squamous cell carcinoma: sensitivity, specificity, and prognostic discrimination. Clin Cancer Res 2011;17:6262–6271. 6.Cappuzzo F, Hirsc FR, Rossi E, Bartolini S, Ceresoli GL, Bemis L, et al. (2005). Epidermal growth factor receptor gene and protein and gefi tinib sensitivity in non – small-cell lung cancer. J Natl Cancer Inst 97:643–55. 8.Chen Q, Huang Y, Shao L, Han-Zhang H, Yang F, Wang Y, et al. (2020).An EGFR-amplified cervical squamous cell carcinoma patient with pulmonary metastasis benefits from Afatinib: A case report. Onco Targets Ther 13:1845–1849. 7. Mokhtar N, Salama A, Badawy O, Khorshed E, Mohamed G, Ibrahim M, Abd elAzim H (2016). Cancer Pathology registry 2000-2011. Egypt: Cairo Press; 85–88. 8. Meng Y, Chu T, Lin S, Wu P, Zhi W, Peng T, et al. (2021). Clinicopathological characteristics and prognosis of cervical cancer with different histological types: A population-based cohort study. Gynecol Oncol 163:545–551. 9. Kato S, Okamura R, Macendonia M, Leee S, Goodman A, Patel SP, et al. (2019). Revisiting epidermal growth factor receptor (EGFR) amplification as a target for anti-EGFR therapy: analysis of cell-free circulating tumor DNA in patients with advanced malignancies. JCO Precis Oncol 3:1–14. 10. Da Mata S, Ferreira J, Nicolás I, Esteves S, Esteves G, Lérias S, et al. (2021). P16 and HPV Genotype significance in HPV-associated cervical cancer. A large cohort of two teritiary referral centers. Int J Mol Sci 22:2294. 11. Suppulaxmi N, Bhuvneshwari G et at al. (2023). Evaluation of her2/neu expression in carcinoma cervix JAMP DOI: 10.47009/jamp.2023.5.2.142