Immunohistochemical evaluation of glutaminase-1 enzyme in oral squamous cell carcinoma

Methods

The study was conducted in the department of Oral Pathology, Vasantdada Patil Dental College and Hospital, Kavalapur, Sangli, from August 2023 to Feb 2025. This study was approved by the Institutional Ethical Committee (IEC) of the Institute. A total of 98 histologically proven OSCC patients were considered for this retrospective study based on sample size estimation (Supplementary Material). Demographic details such as age, gender, site, and TNM staging were recorded from the departmental archives. The patients were grouped based on the World Health Organization classification¹² as follows:

• Group A (n=48): Well-differentiated squamous cell carcinoma (WDSCC).

• Group B (n=34): Moderately differentiated squamous cell carcinoma (MDSCC).

• Group C (n=16): Poorly differentiated squamous cell carcinoma (PDSCC).

Diagnosed cases of primary, conventional OSCC were included. Histopathological variants of OSCC, patients with incomplete clinical history, paraffin blocks with insufficient tissue, patients with recurrence and patients with second primary tumour were excluded from the study.

Results

The demographic details of the patients are depicted in Table I.

GLS1 immunoexpression was positive in 90 (91.83%) out of 98 OSCC patients. Correlation of GLS 1 immuno expression in patients of various grades of OSCC, in term of staining intensity score, staining extent score, and immunoreactive score is shown in Table II. This is also depicted graphically in Figures 1 and 2.

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Fig. 1. Graphs showing co-relation for Glutaminase1 staining (A) intensity, (B) extent, (C) Glutaminase1 immunoreactive score (IRS) with oral squamous cell carcinoma cases in various grades, (D) Percentage of oral squamous cell carcinoma cases with varying Glutaminase1 immunoreactive score with Tumour Node Metastasis (TNM) staging, and (E) Glutaminase1 immunoreactive score with level of Tumour Infiltrating Lymphocytes.

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Fig. 2. Photomicrograph shows Glutaminase1 staining in OSCC (10X) H and E- section of (A) Well differentiated squamous cell carcinoma; (B) Corresponding Glutaminase1 stained section; (C) H and E stained section of Moderately differentiated squamous cell carcinoma (MDSCC); (D) Corresponding Glutaminase1 stained section of MDSCC; (E) H and E stained section of Poorly differentiated squamous cell carcinoma.(PDSCC); (F) Corresponding GLS1 immunostained section of PDSCC. Inset: Strong GLS 1 expression in tumour strands (40X).

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Tumour infiltrating lymphocytes (TIL)

38.8% and 30.61% of OSCC patients showed moderate and high level of TIL, respectively; 50% of OSCC with moderate level of TIL (score-2) and 53.33% patients with higher TIL; showed a weak IRS in tumour cells. No statistically significant relation was noted. (P=0.30; Table III, Figs. 1E and 3).

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Fig. 3. Photomicrograph (10X) (A) H and E-stained section showing high level TIL, (B) Corresponding to IHC section. Inset: GLS 1+ ++TIL, (C) H and E-stained section showing moderate level TIL, (D) Corresponding IHC section, (E) H and E-stained section showing low level TIL, (F) Corresponding IHC section.

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Discussion

We noted a highly significant increase in the intensity and the extent of GLS1 expression with worsening grades of OSCC; indicating a strong correlation between increasing GLS1 expression and worsening tumour grade.

Similar results were noted by Cetindis et al¹³ and Kamarajan et al⁴, who reported GLS1 expression to be high in primary and metastatic OSCC tissues compared to controls. Yang et al¹ hypothesised that glutamine supplementation promotes OSCC growth. The overexpression of functional glutaminolysis is crucial for the bioenergetic and biosynthetic homeostasis especially in proliferative and invasive cancer cells.¹⁴ Serum glutamate levels are also known to be associated with tumour aggressiveness⁹. The GLS transcript has a pH-responsive element, which results in increased expression at acidic pH. Intratumoural hypoxia increases the accumulation of HIF-1α (hypoxia inducible factor), which binds to the GLS1 gene’s hypoxia-response element, increasing its transcription, which regulates multiple downstream target genes associated with cancer migration, invasion, metastasis, and relapse.⁶ Kamarajan et al⁴ concluded that glucose metabolism may be more important for survival and proliferation, whereas glutamine/glutamate metabolism may be critical for subsequent aggressive transitions, such as acquiring stemness properties and metastatic potential. Thus, increased GLS1 expression may suggest that the tumour is of an aggressive phenotype with a probability of nodal metastasis, loco-regional recurrence, and second primary tumour in the future.^4,6,9 A previous study however, found that glutamine may not be the dominant energy source and noted that OSCC are glycolytic tumours. However, this study was done on cell culture.¹⁵

Eight out of 98 OSCC patients showed no GLS1 expression. About 26.47% of MDSCC and 6.25% of PDSCC patients showed weak GLS1 expression. The probable reason for the weak staining could be attributed firstly to the fact that samples were collected from various cancer centres, wherein there could be variability in tissue preservation and fixation, which could have affected the immunohistochemical staining. However, we ensured that the processing protocol was nearly uniform at the centres. Secondly, solid tumours contain significant heterogeneity of perfusion, such that many tumour cells reside in nutrient- and oxygen-poor environments; hence, areas within the same tumour can be driven by different oncogenes and can be metabolically divergent.^16,17 Considering glutaminase expression to be a marker of aggressiveness; patients with strong expression should be kept on a keen follow up to see for the treatment outcomes. In a clinical scenario, it is noted that some exceptional cases of early OSCC with better differentiation; succumb to cancer. This provides a scope to evaluate GLS1 in OSCC patients and correlate it with survival in future prospective studies.

To date, the TNM classification scheme is still the most practical method to predict prognosis in OSCC. Therefore, we evaluated for correlation between GLS1 expression and TNM staging and found no association. Pan et al² noted that GLS1 expression was higher in T3/T4 prostate cancer, and in tumours with higher Gleason scores. Similar results were noted in colorectal carcinoma.¹⁸ GLS1 expression in OSCC is a direct effect of the genotype, which decides the metabolic phenotype of the tumour. TNM staging is based on anatomic characteristics of the tumour, such as the tumour size, lymph node metastasis, which is decided by the metabolic phenotype. Hence, the lack of correlation indicates that studying the tumour metabolomic is more important.

There are many supplementary histopathologic prognosticators. Prominent among them is, TIL. TILs are part of the immune response that plays a critical role in recognising and eliminating tumours and is also assumed to influence the clinical behavior of tumours.^19,20 Tumours of the same clinical stage and/or same histopathologic grade may have extremely variable immune responses.²¹ TIL is associated with determining survival and local recurrence.^19-22 Thus, the study further evaluated the correlation between GLS1 IRS and TIL and found no significant association. Kim et al⁵ observed that higher glutaminase expression in tumour cells correlated with a low level of TILs in triple negative breast cancer patients. They concluded that a metabolic competition exists in the tumour microenvironment; a high level of GLS1 expression in tumour cells might result in glutamine deprivation in TILs, thereby hindering the protective role of T cells in cancer progression. There is a link between metabolic reprogramming and immune evasion, as changes in the tumour cell metabolism can influence the components and function of the inflammatory infiltrates.²⁰ When glutamine is deficient in the cell growth environment, T cell activation is completely inhibited.²³

Our study shows that with worsening tumour grade the GLS1 expression increases significantly, indicating that GLS1 expression marks tumour aggressiveness. Literature search revealed no study wherein a comparison was done between GLS1 expression with differentiation grades, TNM staging, and TIL in OSCC. There are few lacunae in our study. This was a retrospective study, it did not address the association of GLS1 expression with many of the possible variables associated with survival or recurrence, such as tumour margin status, pattern of invasion, the expression in various anatomical sites treatment regimen or concomitant medical problems; due to time and financial constraints.

The dependence of tumour on glutamine metabolism has formed the basis for use of anti-glutamine metabolites in cancer therapeutics. Anti GLS1 drugs like telaglenastat has shown promising results in Phase I and II clinical trials in OSCC.²⁴ Glutamine is also used in cancer imaging in brain tumours and has been successfully used for tumour detection in glioma patients validating it as a diagnostic tool.⁸ Accumulating evidence shows that the predictive capability of 8th AJCC staging system still need to be improved in order to obtain a more robust prognostic stratification.²¹ Hence, determining the GLS1 expression in OSCC patients carries more weightage and can play an important role in risk stratification in oral cancer patients. More research needs to be carried out on OSCC metabolism in the Indian population to determine glutamine dependence of tumours, whether such tumours are associated with treatment failure, drug resistance, and their role in modifying the tumour immune microenvironment.