Abstract

Background: Breast carcinoma (BC) is one of the most common malignancies in women, affecting 1 in 8.Interleukin 6 (IL6) is a proinflammatory cytokine. The role of IL6 pathways in breast cancer motivated the development of anti-IL6 agents or monoclonal antibodies, which inhibit the IL6/signal transducer and activator of transcription 3 (STAT3) pathway. This study aimed to determine the proportion and intensity of immunohistochemical (IHC) IL6 expression in invasive ductal carcinoma (IDC) breast tissue sections and estimate plasma IL6 levels using an enzyme-linked immunosorbent assay (ELISA) in the same patients to evaluate the association between IHC and plasma IL6 levels.

Methods: This laboratory observational cross-sectional study examined new primary BC cases between January 2021 and June 2022. IL6 IHC was performed on tissue sections and analyzed using the histochemical (H)-score system. Plasma samples were taken from the same cases to estimate IL6 levels using an ELISA. The data were analyzed for an association between IHC and plasma IL6 levels in paired samples using SPSS software (version 22).

Results: Among 50 IDC cases, the mean IHC-based IL6 H-score was 201.6 88.4, and the mean ELISA-based plasma IL6 level was 68.13 89.98 pg/mL. A slight positive correlation existed between IHC-based IL6 H-scores and ELISA-based plasma IL6 levels (p = 0.217). IHC-based IL6 H-scores were not associated with clinicopathological parameters. ELISA-based plasma IL6 levels were significantly associated with premenopausal status (p = 0.010), positive erb-b2 receptor tyrosine kinase 2 (ERBB2/HER2/NEU) expression (p = 0.040), low marker of proliferation Ki-67 (MKI67) index (p = 0.040), and the ERBB2/HER2/NEU enriched molecular category (p = 0.040).

Conclusion: IHC-based IL6 H-scores increased with ELISA-based plasma IL6 levels among the cases. ELISA-based plasma IL6 levels were significantly associated with premenopausal status, positive ERBB2/HER2/NEU expression, low Ki-67 proliferative index, and the ERBB2/HER2/NEU enriched molecular category. Therefore, plasma IL6 could be a potential marker to assess prognosis in patients with IDC.

Introduction

Breast cancer (BC) is the most common malignancy among women, with an estimated global incidence of 22,61,419 (11.7%) and mortality of 6,84,996 (6.9%) in 2020. The increased incidence of BC is one of the leading causes of cancer-related deaths worldwide1. The GLOBOCAN data for 2020 indicate that BC accounted for 13.5% (n = 178,361) of all cancer cases and 10.6% (n = 90,408) of all cancer-related deaths in India, with a cumulative risk of 2.811. In India, BC has a reported age-adjusted incidence among females of 25.8 per 100,000 population and mortality of 12.7 per 100,000 population. BC has a reported burden of 34.4% in Bangalore2 and a reported prevalence of 6.41% of all cancers in females in Kolar3.

Cytokines are a large group of proteins, glycoproteins, or peptides secreted by specific immune system cells. Cytokines include interleukins, colony-stimulating factors, interferons, and growth factors. Cytokines are primarily synthesized by leukocytes and primarily act on other leukocytes, leading them to be called interleukins (ILs)4, 5. ILs represent a large group of cytokines (IL-1 to IL-17) produced mainly by T cells, although some are also produced by mononuclear phagocytes and tissue cells (including adipocytes). Each IL acts on a specific group of cells with its corresponding receptors. IL6 is produced by macrophages and fibroblasts. Two types of IL6 receptors exist: transmembrane IL6 receptors expressed on the cell surface and soluble IL6 receptors in the circulation. They perform various functions, and their main role is directing other cells to divide and differentiate6, 7, 8.

Adipokines are molecules secreted by adipocytes and have an endocrine function. Cancer-associated adipocytes (adjacent to invasive BC tissue) contribute to BC progression and metastasis and secrete adipokines, including the cytokine IL67, 8. Cytokines form oligomeric protein complexes, bind with high affinity to transmembrane receptors, and induce IL6 cytokine family signal transducer (IL6ST/GP130) homo- or hetero-dimers, triggering intracellular signaling9, 10. IL6ST/GP130 dimerization induces the signal transduction and activation of various pathways, resulting in carcinogenesis. Therefore, anti-IL6 monoclonal antibodies are used as an adjuvant targeted therapy in BC and inhibit the IL6/signal transducer and activator of transcription 3 (STAT3) pathway11.

This study examined the immunohistochemical (IHC) expression of IL6 in invasive ductal carcinoma (IDC) breast tissue sections and estimated plasma IL6 levels in blood samples using an enzyme-linked immunosorbent assay (ELISA) from the same cases (paired samples). It also assessed the association between IL6 IHC expression and plasma ELISA levels, a liquid biopsy concept.

Methods

This laboratory observational cross-sectional study was conducted in a tertiary health care center of the Department of Pathology in collaboration with the Department of Surgery attached to Sri Devaraj Urs Medical College, Kolar, Karnataka, India, between January 2021 and June 2022. Its sample size was statistically estimated as 50 based on the study by Ahmad N et al.12. Its inclusion criteria were new cases of IDC of the breast diagnosed by fine needle aspiration cytology (FNAC)/trucut biopsy. Its exclusion criteria were BC cases already given chemotherapy or radiotherapy or with metastatic deposits in the breast, BC recurrence, any other malignancy, and chronic inflammatory disorders.

This study received ethical approval from the Institutional Ethics Committee (approval no.: DMC/KLR/IEC/688/2022-23) before being conducted. Informed consent was obtained from the study participants before starting the study. Case details were collected from the case files and interactions with the patient, including their age; clinical presentation; relevant laboratory and radiological investigations; lesion site; tumor size; surrounding structure involvement; nipple and areola involvement, including skin changes; and the number of palpable lymph nodes. Cases were classified as normal, overweight, obese, severely obese, morbidly obese, and super obese according to Asian body mass index (BMI) criteria. The tumor’s clinical stage was noted. The breast tissue, either trucut or mastectomy specimens, was fixed in 10% neutral buffered formalin overnight and then grossed per the laboratory’s standard operating procedure, and representative portions were provided. The tissue portions were processed per the laboratory’s standard protocol. Tissue sections were stained with hematoxylin and eosin and analyzed for histomorphological features, including histopathological type, tumor-infiltrating lymphocytes (TILs), lymphovascular invasion (LVI), Nottingham prognostic index (NPI), and tumor grade according to the modified Scarff–Bloom–Richardson (SBR) grading system. The tumor’s estrogen receptor 1 (ESR1/ER), progesterone receptor (PGR/PR), erb-b2 receptor tyrosine kinase 2 (ERBB2/HER2/NEU), and marker of proliferation Ki-67 (MKI67) status were captured from pathology department records.

× Figure 1 . Microphotograph showing expression of IHC IL6 (IHC IL6, 100X) . Figure 1 . Microphotograph showing expression of IHC IL6 (IHC IL6, 100X) .

Tissue sections were subjected to IHC for IL6 using a rabbit polyclonal immunoglobulin G primary antibody (cat. no.: GTX109204; GeneTex) according to the manufacturer’s protocol. The test was run with positive (tonsil) and negative controls. Cytoplasmic staining was considered positive (Figure 1). IHC was scored using the histochemical (H) intensity grading score calculated with the following formula: (% of weakly-stained cells × 1) + (% of moderately-stained cells × 2) + (% of strongly-stained cells × 3)12, 13. The H-score ranged from 0 to 300.

A 6 mL blood sample was taken from the patient in a K2EDTA vacutainer after confirmation of IDC diagnosis by either FNAC or trucut biopsy and before the patient underwent a mastectomy. The blood sample was centrifuged at 1500 rpm for 10 minutes to separate the plasma. The plasma IL6 concentration was estimated using the Human IL6 ELISA Kit (Diaclone) according to the manufacturer’s protocol and expressed as pg/mL. IHC-based IL6 H-scores were correlated with ELISA-based plasma IL6 levels.

The data were entered in an MS Excel sheet. Statistical analyses were performed using IBM SPSS software (version 22). Continuous data are expressed as means and standard deviations with confidence intervals. Categorical data were expressed as frequencies and percentages. The significance of differences was assessed using the chi-square test. The correlation between IHC-based IL6 H-scores and ELISA-based plasma IL6 levels was assessed using Pearson’s correlation coefficient. Results with a p-value of

Table 1.

Basic data of the cases in the present study

Basic characteristics Frequency Percentage (%) Age category 35 - 45 6 12.0 46 - 55 18 36.0 56 - 65 20 40.0 66 – 75 6 12.0 Menopausal Status Premenopausal 13 26.0 Postmenopausal 37 74.0 Parity Multipara 47 96 Primipara 3 4 BMI Underweight 14 28.0 Normal 32 64.0 Overweight 4 8.0 Tumor Infiltrating lymphocytes (TIL) Absent 33 66 Present 17 34 Lymphovascular Invasion (LVI) Absent 49 98 Present 1 2 pT (Tumor size) T1 13 26 T2