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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2022  |  Volume : 65  |  Issue : 4  |  Page : 791-795
Role of CTLA4 immunohistochemistry in the diagnosis of colon cancers


1 Department of Pathology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India
2 Associate Professor, Departments of Pathology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India
3 Department of General Surgery, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India

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Date of Submission30-Jan-2021
Date of Decision30-Jul-2021
Date of Acceptance01-Sep-2021
Date of Web Publication21-Oct-2022
 

   Abstract 


Background: There are a wide range of diagnostic markers for colorectal cancers like detection of mutated KRAS, TP53, and APC genes. However, genetic and immunological factors have also been attributed to the cancer prognosis. The present study was carried out to evaluate the expression of CTLA-4 in colorectal cancers. Methods: This cross-sectional study was carried out among 30 resected specimens of colorectal cancer. Paraffin blocks were made on samples from tumor areas along with adjacent normal areas. Immunohistochemistry for CTLA-4 was done on the sections along with controls. Gross findings were recorded from the blocks. Blocks with section containing normal epithelium and tumor were chosen for immunohistochemistry. Results: Overexpression of CTLA-4 was observed in 43.3% of the tumors. There was a significantly high tumor infiltration among those specimens showing overexpression of CTLA-4. The observed difference was statistically significant (P < 0.05). On comparing the grade of the tumor with intensity of CTLA4 uptake, it was observed that majority of the well-differentiated tumors (66.7%) had an intensity of 1+ whereas majority of the poorly differentiated tumors had an intensity of 3+ (66.7%). Conclusion: The present study has demonstrated overexpression of CTLA-4 in colorectal cancer specimens, and also highlighted the potential scope for anti-CTLA-4 agents like Ipilimumab in cancer therapy. The need for further evaluation to examine five-year survival with such immunotherapies is essential to document candid therapeutic recommendations for colorectal cancers.

Keywords: Colorectal cancers, CTLA-4, immunohistochemistry, T lymphocytes

How to cite this article:
Narayanan V, Pavithra V, Dhanapal D, Sundaram S, Narayanan C D. Role of CTLA4 immunohistochemistry in the diagnosis of colon cancers. Indian J Pathol Microbiol 2022;65:791-5

How to cite this URL:
Narayanan V, Pavithra V, Dhanapal D, Sundaram S, Narayanan C D. Role of CTLA4 immunohistochemistry in the diagnosis of colon cancers. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Dec 7];65:791-5. Available from: https://www.ijpmonline.org/text.asp?2022/65/4/791/359306





   Introduction Top


Colorectal cancers are invasive cancers, which predominantly occur in the developed countries. According to the Global Burden of Disease estimates in 2017, there were 1.8 million cases worldwide, and the cancer incidence has witnessed an increase of 9.5% between 1990 and 2017.[1] In developing countries like India, the incidence is fairly low, with an age-standardized incidence of 7.2 per 100,000 population in 2017. However, being an invasive cancer, the disease is associated with increased mortality and low survival, with five-year survival rates being < 40%.[2] Furthermore, the age distribution of these cancers in India has demonstrated alarming incidences in the age group of 15-59 years (62.5%), thereby increasing the vulnerability among the economically productive population.[3]

The etiopathogenesis of colorectal cancers relate to various lifestyle factors like smoking, dietary factors, and excessive alcohol consumption.[4] Several factors have been attributed to the low survival, of which challenges in diagnosis and management are crucial. As a majority of these tumors are asymptomatic, early diagnosis is often missed, especially in Indian settings. There are a wide range of genetic and epigenetic tumor markers, including detection of mutated KRAS, TP53, and APC genes.[5] In addition, genetic and immunological factors have also been attributed to the cancer incidence and, immunohistochemical markers have been recently used for diagnosis and assessment of prognosis. Some of these markers include Epidermal Growth Factor Receptor (EGFR), Ki-67 proliferative index, matrix metalloproteinase-9, cytokeratin 7 (CK7), and CDX2. One such factor is the Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) gene.[6] CTLA-4 is a polymorphic gene located on chromosome 2q33. It binds to B7 molecules and inhibits cytokine production thereby providing negative feedback to the T lymphocytes.[7] As the presence of CTLA-4 can significantly alter the immune response, its role in susceptibility to certain cancers have long been explored. Detection of CTLA-4 has been considered as an immunosurveillance marker for colorectal cancers and certain studies have demonstrated the immunomodulatory and immunotherapeutic strategies using anti-CTLA-4 antibody in colorectal carcinoma.

Although various immunohistochemical markers have been evaluated, very few studies in India have explored the significance of CTLA-4 in colorectal cancers. Apart from being a primary source of malignancy, colorectal cancers also manifest as a consequence of various other forms of cancers including ovarian carcinoma. Therefore, the scope of utilizing this marker for immunosurveillance and immunotherapies are greater and wider, and can potentially influence the prognosis and survival outcomes of colorectal carcinoma in Indian setting. The present study was taken up to evaluate the role of CTLA-4 immunohistochemical marker in colorectal cancer in a single tertiary institute setting.


   Methodology Top


Study setting and participants

The present study was carried out as a cross-sectional study among the resected specimens of colorectal cancer received in the Department of Pathology of our tertiary teaching institution for a period of 1 year between February 2019 and January 2020. All the specimens received during the study period were taken up for the study. A total of 30 specimens were evaluated.

Ethical approval

Approval was obtained from the Institutional Ethics Committee prior to the commencement of the study.

Data collection

A structured proforma was used to obtain clinical information regarding the participant's demographics and medical history from the hospital medical records. Paraffin blocks were made on samples from tumor areas along with adjacent normal areas from colectomy specimens received in the department. IHC for CTLA-4 was done on the sections along with controls. Gross findings were recorded from the blocks. Blocks with section containing normal epithelium and tumor were chosen for immunohistochemistry.

Hematoxylin and Eosin staining was done for microscopic analysis, for resected lymph nodes and surgical margins. Other histopathological features observed were intratumoral and peritumoral inflammatory response.

The proportion of positivity in tumor cells and tumor infiltrating lymphocytes was also tabulated as percentage [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9].
Figure 1: Intensity of CTLA-4 uptake by tumor cells

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Figure 2: H&E poorly differentiated adenocarcinoma (Signet ring cell carcinoma)- colon ×400

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Figure 3: H&E moderately differentiated adenocarcinoma -colon ×200

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Figure 4: H&E well-differentiated adenocarcinoma – colon ×200

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Figure 5: CTLA-4 immunohistochemistry: Mild cytoplasmic staining (Score + 1) ×100

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Figure 6: CTLA-4 immunohistochemistry: Moderate cytoplasmic staining (Score + 3) ×100

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Figure 7: CTLA-4 immunohistochemistry: Strong cytoplasmic staining (Score + 4) ×200

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Figure 8: CTLA-4 immunohistochemistry: tumor infiltrating lymphocytes – no staining ×100

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Figure 9: CTLA-4 immunohistochemistry: tumor infiltrating lymphocytes – positive staining ×200

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Data analysis

Data was entered and analyzed using SPSS ver. 15 software. The CTLA-4 positivity was expressed as percentages. Chi-square test was used to evaluate the association between immunohistochemical findings and tumor characteristics. A P value < 0.05 was considered statistically significant.


   Results Top


The present study was carried out among 30 resected specimens. The mean age of the participants was 55.7 ± 15.1 years. Majority of the participants belonged to the age group of 50-65 years (40%) followed by >65 years (30%). Hemicolectomy was the most common procedure performed (56.7%). Both the genders equally constituted the study population [Table 1].
Table 1: Background characteristics of the study participants

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With regards to the tumor characteristics, majority of the tumors were moderately differentiated (80%) and belonged to the TNM stage III (60%) of the AJCC staging of cancers. Histopathologically, adenocarcinoma was the most common diagnosis (53.3%). Lymphovascular and perineural invasion was present in 36.7% and 16.7% of the specimens respectively [Table 2].
Table 2: Tumor characteristics among the study specimens

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Immunostaining of CTLA4 in colon carcinoma[8]

Immunostaining of CTLA4 was done using Lyophilized Mouse Monoclonal Antibody CTLA4 (IgG1) at a dilution of 1:50. Section from appendix was taken as a positive control, where distinct brown colored cytoplasmic staining was regarded as positive staining. A negative control (without addition of primary antibody) was included in every batch of immunostaining. CTLA4 stain intensity scoring was defined as +1 = Negative (Tumour cells with no staining) +2 = Positive (Tumour cells with weak staining ) +3 = Positive (Tumour cells with moderate staining ) +4 = Positive (Tumour cells with strong staining ). All scoring was done without any bias with the clinical correlation.

Majority of the tumors showed an intensity of 3+ uptake of CTLA-4 whereas 36.7% of the tumors showed an intensity of 1+. There was a significantly high tumor infiltration among those with an uptake of 3+, with a mean difference of 19.9. The observed difference was statistically significant (P < 0.05). Similarly, there was a significant increase in the tumor quantity among those with uptake of 3+ with a mean difference of 19.75. The observed difference was statistically significant (P < 0.05) [Table 3].
Table 3: Uptake of CTLA-4 (with intensity >3+) among tumor cells

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On comparing the grade of the tumor with intensity of CTLA4 uptake, it was observed that majority of the well-differentiated tumors (66.7%) had an intensity of 1+ whereas majority of the poorly differentiated tumors had an intensity of 3+ (66.7%). Among the moderately differentiated tumors, 45.8% of the tumors had an intensity of 3+. However, the association was not statistically significant [Table 4].
Table 4: Association between grade of the tumor and CTLA4 intensity of uptake

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   Discussions Top


Although the incidence of colon cancers is considerably low in India, poor survival outcomes and high mortality rates warrant the need for effective strategies for early diagnosis and treatment. Further, the existing treatment options with chemotherapy have provided moderate results in terms of efficacy and remissions, warranting novel therapeutic options. In recent times, immunotherapy has provided significant hope in cancer therapy, especially for solid tumors like bronchogenic carcinoma and melanoma. The immunologic role of certain cancers, involving T-cell response has been explored in the lines of adoptive cell therapy in cancer treatment. The present study evaluated the role of CTLA-4 in tumor cell infiltration of colon cancers. The mean age of the participants was 55.7 years and adenocarcinoma was the most common histopathological diagnosis (53.3%). Majority of the specimens showed overexpression of CTLA-4 (43.3%). The intensity of CTLA-4 expression varied between 1+ and 3+ among the study specimens. Specimens showing intensity greater than 2+ was found to be associated with increased infiltration of immune cells in comparison with lower intensity of expression (1+), and this correlated significantly with tumor quantification with a statistical significance (P < 0.05). Very few studies have been carried out to evaluate the uptake of CTLA-4 specific to colorectal cancers. In a study done by Kitsou M, et al.[9] elevated CTLA-4 was observed in both colon and rectal adenocarcinoma and the study also established improved survival outcomes in the presence of high expression of CTLA-4, indicating effective immune response.

Human CTLA-4 consists of two isoforms – membrane bound mCTLA-4 and soluble sCTLA-4. Both these isoforms act as inhibitory molecules, downregulating T-cell signaling pathways and reducing T-cell activation to maintain immune tolerance. Studies have also explored the engagement of CD28 by CTLA-4 on cell surfaces to prevent cell death.[10] However, CTLA-4 isoforms are similar in expression with CD4 cells, and CD8 cells express 2.5 times more in comparison with CTLA-4.[11]

Among the various immunomodulators, Ipilimumab and Tremelimumab function as a human CTLA-4 blocking antibody, effective in the management of nonresectable cancers. They are said to trigger anti-tumor immune response by competitively binding CTLA-4, thereby preventing T-cell activation. Furthermore, they are said to reduce the accumulation of Tregs, thereby improving the outcome of colorectal cancers. The present study findings distinctly correlate the validity of these immunomodulators, given the strong statistical significance between overexpression of CTLA-4 with the extent of tumor infiltration. Therefore, the presence of intensity >2+ may be used as a valid benchmark for initiation of immunotherapy, and also a potent predictor for assessment of survival and prognosis. In a systematic review and meta-analysis done by Hu P, et al.[11] significant correlation was observed between CTLA-4 in subset of Single Nucleotide Polymorphism (SNP) and overall survival. Some studies have demonstrated co-expression of CTLA-4 on CD4+ Foxp T cells rendered enhanced immunosuppressive cytokines like IL-10 and TGF-β, thereby providing a more potent immune checkpoint in cancer therapeutics for colorectal cancers.[12]


   Conclusions Top


The management of colorectal cancers in India is a clinical challenge, owing to latent manifestation and poor survival outcomes. There is a need for a novel immunomodulator, which not only helps in detecting the prognosis, but also helps in providing immunotherapeutic benefit, thereby prolonging the survival outcome. The present study has demonstrated overexpression of CTLA-4 in colorectal cancer specimens, and also highlighted the potential scope for anti-CTLA-4 agents like Ipilimumab in cancer therapy. There is, however, limited evidence in support of the same among Indian subset of population. The need for further evaluation in terms of prospective studies to examine 5-year survival with such immunotherapies is essential to document candid recommendations for incorporating these immunomodulators in the treatment regimen of colorectal cancers.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
World Health Organization. Cancer. Key facts. Available from: https://www.who.int/news-room/fact-sheets/detail/cancer.  Back to cited text no. 1
    
2.
Patil PS, Saklani A, Gambhire P, Mehta S, Engineer R, De'Souza A, et al. Colorectal cancer in India: An audit from a tertiary center in a low prevalence area. Indian J Surg Oncol 2017;8:484-90.  Back to cited text no. 2
    
3.
SRS: Chapter—2 population composition.” Available from: http://www.censusindia.gov.in/vital_statistics/srs_report/9chap2-2011.pdf.  Back to cited text no. 3
    
4.
Rossi M, Anwar MJ, Usman A, Keshavarzian A, Bishehsari F. Colorectal cancer and alcohol consumption-Population to molecules. Cancers (Basel) 2018;10:38.  Back to cited text no. 4
    
5.
Hong SN. Genetic and epigenetic alterations in colorectal cancer. Intest Res 2018;16:327-37.  Back to cited text no. 5
    
6.
Reimers MS, Zeestraten ECM, Kuppen PJK, Leifers GJ, van de Velde CJH. Biomarkers in precision therapy in colorectal cancers. Gastroenterol Rep (Oxf) 2013;1:166-83.  Back to cited text no. 6
    
7.
Schildberg FA, Klein SR, Freeman GJ, Sharpe AH. Coinhibitory pathways in the B7-CD28 ligand receptor family. Immunity 2016;44:955-72.  Back to cited text no. 7
    
8.
Paulsen EE, Kilvaer TK, Rakee M, Richardsen E, Hald SM, Andersen S, et al. CTLA-4 expression in the non-small cell lung cancer patient tumor microenvironment: Diverging prognostic impact in primary tumors and lymph node metastases. Cancer Immunol Immunother 2017;66:1449-61.  Back to cited text no. 8
    
9.
Kitsou M, Aiyomamitis GD, Zaravinos A. High expression of immune checkpoints is associated with the TIL load, mutation rate and patient survival in colorectal cancer. Int J Oncol 2020;57:237-48.  Back to cited text no. 9
    
10.
Yu H, Yang J, Jiao S, Li Y, Zhang W, Wang J. Cytotoxic T lymphocyte antigen 4 expression in human breast cancer: Implications for prognosis. Cancer Immunol Immunother 2015;64:853-60.  Back to cited text no. 10
    
11.
Hu P, Liu Q, Deng G, Zhang J, Liang N, Xie J, et al. The prognostic value of cytotoxic T lymphocyte antigen 4 in cancers: A systematic review and meta-analysis. Sci Rep 2017;7:42913.  Back to cited text no. 11
    
12.
Jacobs J, Smits E, Lardon F, Pauwels P, Deschoolmeester V. Immune checkpoint modulation in colorectal cancer: What's new and what to expect. J Immunol Res 2015;2015:158038.  Back to cited text no. 12
    

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Correspondence Address:
V Pavithra
Department of Pathology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpm.ijpm_112_21

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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