Expression of inflammatory proteins STAT & NFĸB for phenotypic diagnosis of gall bladder cancer: A pilot study

Materials & Methods

This study was undertaken at the department of Biochemistry, All India Institute of Medical Sciences, Rishikesh between March 2020 to July 2023 and included histologically confirmed cases of GBC (n=13); patients with cholelithiasis (n=23) were recruited as controls. This study was approved by the Institute’s Ethics Committee (IEC), and informed consent was in accordance with the Declaration of Helsinki. Participants were recruited after obtaining informed consent. Individuals with metachronous and synchronous malignancies were excluded from the study. Clinical and demographic characteristics of all the study participants were recorded. Histological staging was carried as per the AJCC (American Joint Committee on Cancer) 8^th edition¹².

Results & Discussion

In the present study, the mean age of participants with GBC and cholelithiasis was 50.84±14.5 yr and 44.0±11.9 yr, respectively. GBC was more common in females than males (69.23% female and 31% male). Similarly, in cholelithiasis, the percentage of male vs. female was 26.09 and 73.91 per cent. In several studies, gallstones and GBC have been found to be more prevalent in women globally as well as in the Indian population^13-16, which supports the present study showing a 2.23 times higher incidence rate in the case of females compared to males.

In the present study, stages II, III, and IV of GBC were observed in 3 (23.08%), 8 (61.54%) and 2 (15.38%) participants, respectively. Liver infiltration was observed in 61 per cent of the GBC patients, whereas 69 per cent of the patients presented with lymph node involvement and metastasis. The baseline demographic and clinico-pathological parameters of the study participants have been summarised in supplementary table I.

Around 75 per cent of GBC patients in the current study presented at an advanced stage. This corroborates the large body of evidence that demonstrates, owing to the insidious onset of GBC and lack of typical clinical manifestations in the early stages, most patients presented at the advanced stage when diagnosed, resulting in a loss of opportunity for radical surgery^17-19. This also explains the small sample size of the GBC patients in the present study.

The tissue expression of STAT3, STAT6, and NFĸB was significantly higher in GBC as compared to the cholelithiasis group. The mean ±SD scores for STAT3, STAT6, and NFĸB in GBC vs. cholelithiasis were 4 ±1.5, 1.3 ±1.2 (P<0.01); 3.8±2.4, 2.0±1.4; P<0.01 and 3.8±2.1, 2.4±2.0; P<0.05, respectively. Representative IHC images for both GBC and cholelithiasis have been shown in figure 1. ROC analysis showed a high area under the curve (AUC) of 0.9 and 0.73 for STAT3 and STAT6. While AUC for NFĸB was 0.69 (Fig. 2). The sensitivity and specificity for STAT3 and STAT6 were 91, 47 and 75, 39 per cent with optimal cut-off of 1.5 and 2.5, respectively. NFĸB showed a sensitivity of 58 per cent and specificity of 30 per cent at an optimal cut-off of 3.5.

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Fig. 1.

Immunohistochemistry of STAT and NFĸB in GBC and cholelithiasis. Representative immunohistochemistry (IHC) staining images illustrating the expression of STAT3, STAT6, and NFĸB in cholelithiasis and gallbladder adenocarcinoma. Panels (A), (C), and (E) show nuclear and cytoplasmic expression of STAT3, STAT6, and NFĸB in cholelithiasis, while panels (B), (D), and (F) depict the expression in gallbladder adenocarcinoma. Images (A), (C), (D), (E), and (F) are captured at 20× magnification, whereas image (B) is captured at 40× magnification. Additionally, panels (G) and (H) present representative images of STAT6 expression in gallbladder adenocarcinoma at 40× and 100× magnification, respectively, highlighting tumor cells exhibiting predominantly cytoplasmic and focal nuclear immunopositivity for STAT6.

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Fig. 2.

Receiver operating characteristics curve for STAT and NFĸB. ROC curve for STAT3, STAT6 and NFĸB with corresponding AUC (area under curve) value.

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Chronic Inflammation is long known to be a potential risk factor for GBC. NFĸB and STAT are key mediators of crosstalk between cancer and inflammation. There is a limited body of evidence elucidating the molecular mechanisms underlying GBC, particularly in relation to the involvement of the JAK/STAT signalling pathway. As per the evidence from several studies, IL-6/JAK/STAT3 signalling is believed as a key signalling pathway for tumour progression, and has been confirmed in many types of cancers, including hepatocellular cancer, breast cancer and lung adenocarcinomas^20,21. A recent preclinical study demonstrated that GBC-associated fibroblasts promote vascular mimicry formation and tumour progression in GBC by upregulating NOX4 expression. This effect was driven via paracrine IL-6 mediating IL-6/JAK/STAT3 signalling pathway as evidenced by elevated expression levels of IL-6, JAK1, JAK2, and STAT3 in both in vitro and in vivo models²¹. Furthermore, another study²² reported that an increased expression of STAT3 and vascular mimicry in GCB tissues correlate with lower differentiation, greater malignancy, and advanced clinical stage. This suggests that STAT3 may facilitate vascular mimicry formation during tumour initiation, progression, and metastasis. Therefore, targeting STAT3 could inhibit tumour cell proliferation and invasion while preventing vascular mimicry development, making it a promising candidate for anti-angiogenic cancer therapy. This clearly shows the involvement of STAT3 and STAT6 in the molecular pathogenesis of cancer, and as potential candidates to be explored as biomarkers and therapeutic targets.

However, in the current study, NFĸB showed low sensitivity and specificity for discriminating cancer from chronic inflammatory conditions. The plausible explanation for this could be that NFĸB expression is more specific to inflammation per se and not specifically associated with cancer progression. Moreover, literature suggests the dual role of NFĸB as both pro-inflammatory and anti-inflammatory mediator in different phases of the disease²³. Another reason for the low expression of NFĸB in the current study could be the involvement of a larger number of patients from an advanced stage of GBC.

In the present study, a significant association was observed between high expression of STAT6 and lymph node involvement (P<0.05). Likewise, liver infiltration was seen with a high proportion in individuals with high STAT3 and STAT6 expression compared to low expression (6:1). No association was observed with tumour stage or metastasis. NFĸB failed to show any association with clinico-pathological parameters. The association of clinico-pathological parameters with IHC expression of STAT3, STAT6 and NFĸB proteins in GBC has been depicted in supplementary table II. The association of STAT6 and STAT3 expressions with lymph node and liver infiltration indicate the role of STAT as a mediator of not only cancer-related inflammation but also as a promoter of tumour spread. This result is in agreement with the previous studies, which demonstrated a significant association of STAT6 expression with the histological tumour grade, node infiltration and distant metastasis in breast cancer^24,25. However, GBC is still unexplored in this respect. Thus, the findings of the present study may pave the way towards STAT as a potential sensitive and specific biomarker for GBC and a potential therapeutic target with the potential to curtail the spread of such highly aggressive malignancy.

IHC has long been successfully employed in breast cancer for directing the targeted therapy¹⁰. Anti-inflammatory agents like aspirin and celecoxib are being explored for their therapeutic potential in colorectal cancer²⁶. A randomised phase II clinical trial has also evaluated the efficacy of JAK2 inhibition in individuals with triple-negative inflammatory breast cancer²⁷. Similarly, a recent clinical trial has investigated STAT3 inhibition as a therapeutic strategy in glioblastoma²⁸ demonstrating target engagement and clinical efficacy. However, as per the authors’ knowledge and a researching Clinical Trial Registry (CTRI) and other internet sources no clinical trials have been conducted to date targeting the JAK/STAT pathway in GBC. The current proof-of-principle study may be an important step to direct further research and clinical trials for the utility of STAT proteins in otherwise difficult-to-manage GBC patients.

In conclusion, this study demonstrated STAT as a sensitive and specific molecular biomarker for diagnosis and assessing the aggressiveness of GBC. However, this is a pilot study where most of the patients were from an advanced stage and from a single geographical area. Therefore, a conclusive statement on the utility of STAT and NFĸB may not be possible. However, this can be a first step towards the targeted approach in reducing the disease burden of otherwise difficult-to-manage and aggressive diseases like GBC. It is recommended to conduct further research with a larger sample size.