Association of tumor budding and tumor infiltrating lymphocytes with clinicopathological parameters in gallbladder carcinoma

Sana Ahuja; Adil Aziz Khan; Pooja Verma; Sufian Zaheer

doi:10.14216/kjco.24001

ABSTRACT

Purpose

Gallbladder carcinoma (GBC) poses significant challenges in oncology due to its aggressive nature and limited treatment options. The lack of effective biomarkers for early detection and prognosis exacerbates the prognosis for GBC patients. Tumor budding (TB) and tumor infiltrating lymphocytes (TILs) have emerged as potential prognostic indicators in various cancers, reflecting tumor-host immune interactions and tumor aggressiveness. The study of TB and TILs in GBC is particularly important due to the limited literature available.

Methods

This retrospective observational study aimed to evaluate the association of TB and TILs with clinicopathological parameters in GBC patients. Clinicopathological data were collected from patients with histologically confirmed GBC who underwent surgical resection. The sections were evaluated for TB and TILs using standardized methods. Statistical analysis was performed to assess associations between these parameters and clinicopathological variables.

Results

Tumor stage and grade showed significant associations with TB and TILs, indicating their potential as prognostic markers. High TB correlated with advanced tumor stage and higher grade, while high TIL infiltration was associated with early tumor stage and lower grade. Additionally, TILs exhibited a significant association with lymphovascular invasion. Interestingly, an inverse association was observed between TB and TILs, highlighting the dynamic interplay between tumor aggressiveness and host immune response.

Conclusion

TB and TILs hold prognostic significance in GBC, offering insights into its pathogenesis and potential therapeutic targets. Future research exploring the mechanistic underpinnings of tumor-host immune interactions in GBC is crucial for translating these findings into clinical applications and improving outcomes for patients.

Keywords: Gall bladder cancer, Tumor infiltrating lymphocytes, Adenocarcinoma, Malignancy

INTRODUCTION

Gallbladder carcinoma (GBC) is a highly aggressive malignancy associated with poor prognosis and limited therapeutic options. According to GLOBOCAN, although gall bladder carcinoma accounts for only 1.2% of the total cases, it is responsible for almost 1.7% of cancer-related deaths [1]. Despite advancements in diagnostic and therapeutic modalities, the prognosis for GBC remains dismal, largely due to the lack of effective biomarkers for early detection and prognostic stratification.

Tumor budding and tumor infiltrating lymphocytes (TILs) have emerged as promising prognostic indicators in various solid tumors, reflecting the tumor-host immune interactions and tumor aggressiveness. Tumor budding, characterized by the presence of single cells or small clusters of tumor cells at the invasive front, has been linked to tumor progression, metastasis, and poor clinical outcomes in several cancers [2]. Conversely, TILs, comprising various immune cell populations infiltrating the tumor microenvironment, have been associated with favorable prognosis and improved response to immunotherapy in various malignancies [3].

However, the significance of tumor budding and TILs in GBC remains poorly understood. Investigating their association with clinicopathological parameters could provide valuable insights into the underlying biological mechanisms driving GBC progression and aid in identifying potential prognostic biomarkers and therapeutic targets.

METHODS

Patients

This retrospective observational study aimed to assess the association of tumor budding and TILs with clinicopathological parameters in patients diagnosed with GBC. The study was conducted in accordance with the principles outlined in the Declaration of Helsinki and after approval by the Institutional Ethics Committee of Safdarjung Hospital (IEC/VMMC/SJH/2019–09–11/41). Written informed consent was taken from all patients included in the study.

All patients diagnosed with histologically confirmed GBC, who underwent surgical resection at our institution, from 2021 to 2023 were included in the study. Patients with a prior history of neoadjuvant chemotherapy or radiotherapy were excluded.

Clinicopathological data including age, gender, tumor grade, T stage, nodal status, perineural invasion, and lymphovascular invasion were collected from the medical records.

Methods

Formalin-fixed paraffin-embedded tissue sections from GBC specimens were evaluated by (SA, AAK, SZ) blinded to clinical data. The study analyzed 30 cases of gall bladder carcinomas. Each case underwent routine histopathological processing, which included staining with hematoxylin and eosin, as well as immunohistochemistry for CD3, CD20, and CD68 markers.

Tumor budding was assessed using hematoxylin and eosin-stained slides according to the criteria proposed by the International Tumor Budding Consensus Conference [3]. It was categorized as 0–4 buds, low budding; 5–9 buds, intermediate budding; 10 or more buds-high budding. For the assessment of TILs, a standardized method recommended by the International Immuno-Oncology Biomarkers Working Group was employed. This involved scanning the entire slide at low magnification using a ×5 or ×10 objective lens, followed by higher magnification with a ×20 objective lens.

The assessment focused on the stromal area and excluded polymorphonuclear leucocytes, areas of necrosis, fibrosis, crush artifact, abscess formation, and stromal areas not adjacent to the tumor. Mononuclear immune cells (lymphocytes and plasma cells) were scored, and the average number of TILs was determined across multiple stromal areas [4]. TILs were quantified as a continuous score, ranging from 5% to 100%. Based on this score, cases were categorized into low TILs (less than 20%) and high TILs (20% or more).

Statistical analysis

The statistical analysis was done using Statistical Package for Social Sciences software, version 25.0 (IBM Corp.). The chi-square test and the Fischer exact test were done to determine the association between clinicopathological variables with tumor budding and TILs. A P-value of less than 0.05 was considered to be statistically significant.

RESULTS

Clinicopathological parameters

The majority of the cases (n=17, 56.7%) belonged to the age group 41–50 years followed by 61–70 years (n=8, 26.7%) with a mean age of 46.6±5.8 years. The study included 63.3% female (19 cases) and 36.7% male (11 cases) patients. All cases were gall bladder adenocarcinomas which were categorized based on their histological grade. Most of the cases were moderately differentiated (n=15, 50%) followed by well (n=8, 26.7%) and poorly differentiated (n=7, 23.3%) respectively.

Based on tumor stage, seven (23.3%), 14 (46.7%), and nine (30%) cases belonged to T1, T2, and T3 respectively. None of the cases belonged to T4. According to the nodal status, three cases (10%) showed N1 stage while 27 cases (90%) had no nodal involvement. None of the cases had N2 or N3 stage. Out of 30 cases, lymphovascular and perineural invasion was observed in five (16.7%) and four (13.3%) cases respectively (Fig. 1). Based on tumor budding, the cases were categorized–11 (36.7%), 13 (43.3%), and six cases (20%) into low, intermediate, and high budding respectively (Fig. 2). Based on the categorization of TILs into low high, 13 cases (43.3%) exhibited a low infiltration while the remaining 17 cases (56.7%) showed a high infiltration of TILs (Fig. 3).

Table 1 depicts the association of TILs and tumor budding with clinicopathological parameters. Tumor budding and stromal TILs had a significant association with tumor stage (P=0.0003, P=0.03) and grade (P=0.01, P=0.008). Stromal TILs in addition exhibited a significant association with lymphovascular invasion (P=0.02) also. However, no significant association was found with age, gender, nodal status and perineural invasion. Further, tumor budding, and TILS also showed a significant association (P=0.02), the tumors with high tumor budding low TILs and vice versa (Table 2). Immunohistochemistry showed an almost similar proportion of CD3, CD20, and CD68 positive cells in all the cases and showed no significant association with clinicopathological parameters.

DISCUSSION

GBC presents a formidable challenge in oncology, characterized by its aggressive nature and limited therapeutic avenues. Despite advancements in diagnosis and treatment, the prognosis for GBC remains bleak, primarily due to the absence of effective biomarkers for early detection and prognostic stratification. In this context, exploring novel prognostic indicators such as tumor budding and TILs in GBC is imperative to unraveling its underlying biological mechanisms and identifying potential therapeutic targets.

Tumor budding, characterized by the presence of single cells or small clusters of tumor cells at the invasive front, has garnered attention as a marker of tumor progression and metastasis in various cancers [2]. Conversely, TILs, representing the immune cell populations infiltrating the tumor microenvironment, have shown promise as indicators of favorable prognosis and improved response to immunotherapy in different malignancies [3,5]. However, their significance in GBC remains poorly understood.

In this retrospective observational study, we endeavored to shed light on the association of tumor budding and TILs with clinicopathological parameters in patients diagnosed with GBC. Our findings offer valuable insights into the intricate interplay between tumor biology and host immune response in GBC progression.

Our study revealed a significant association between tumor budding and clinicopathological parameters such as tumor stage and grade. Specifically, high levels of tumor budding were correlated with advanced tumor stage and higher histological grade, indicative of tumor aggressiveness and poor clinical outcomes. This underscores the potential of tumor budding as a prognostic marker in GBC, aiding in risk stratification and treatment planning.

Similarly, we observed a significant association between TILs and clinicopathological parameters, particularly tumor stage, grade, and lymphovascular invasion. High levels of TILs were associated with early tumor stage, lower histological grade, and absence of lymphovascular invasion, suggesting a potential role of the host immune response in restraining tumor progression and invasion in GBC. Notably, the association of TILs with lymphovascular invasion underscores their importance in modulating tumor microenvironment and inhibiting tumor dissemination.

Furthermore, our study elucidated an intriguing relationship between tumor budding and TILs, wherein tumors with high tumor budding exhibited low TIL infiltration and vice versa. This inverse association underscores the dynamic interplay between tumor aggressiveness and host immune response in shaping the GBC microenvironment. Moreover, the lack of significant association between immunohistochemical markers (CD3, CD20, CD68) and clinicopathological parameters highlights the unique immunological landscape of GBC, necessitating further exploration of immune-related biomarkers.

The study of tumor budding and TILs in GBC is particularly important due to the limited literature available on this subject [6–9]. As of now, there has been only one study in the literature that evaluated the association of both TILs and tumor budding in GBC. Secinti et al. [6] conducted this study, assessing the prognostic role of these biomarkers. Their study revealed significant associations between high tumor budding and adverse clinicopathological features, including higher tumor grade, advanced tumor stage, nodal and distant metastasis, as well as lymphovascular invasion. Conversely, the high TILs group showed associations with favorable histological grade, reduced lymphovascular invasion, and decreased distant metastasis.

Moreover, other studies by Kim et al. [7] and Oz et al. [8] corroborated the adverse prognostic implications of higher tumor budding in GBC. They found associations between elevated tumor budding and poor tumor differentiation, advanced tumor stage, perineural and lymphatic invasion, and nodal metastasis. However, Rivera-Moncada and Lino-Silva [9] only found a significant association between high tumor budding and tumor stage.

In conclusion, the present study underscores the prognostic significance of tumor budding and TILs in GBC, offering valuable insights into its pathogenesis and potential therapeutic targets. Future research endeavors focusing on unraveling the mechanistic underpinnings of tumor-host immune interactions in GBC are warranted to translate these findings into clinical applications, ultimately improving outcomes for patients battling this aggressive malignancy.