Immunohistochemical evaluation of tumor hypoxia and angiogenesis: Pathological significance and prognostic role in head and neck squamous cell carcinomas

INTRODUCTION

Head-and-neck squamous cell carcinoma (HNSCC) is an important cause of carcinoma related morbidity and mortality worldwide. Notably, in India oral cavity carcinoma is a leading cause of cancer among men due to higher prevalence of tobacco chewing and alcohol consumption.^[1,2] The mainstay of treatment in HNSCC is surgery with or without radiation and chemotherapy.

Hypoxia and angiogenesis are key tumor microenvironmental factors that have been implicated in conferring radiation resistance and an overall more aggressive biological phenotype in solid malignant tumors.^[3,4]

Hypoxia-inducible factor (HIF) is a family of heterodimeric transcription factors that are upregulated in response to hypoxia and regulate hypoxia-driven changes in tumor cells. HIF-1 α is a member of this family.^[5] Under normoxic conditions, HIF-1 α subunits are degraded in the cytoplasm, whereas in hypoxia, the degradation is inhibited, as enzymes that modify HIF become inactive. HIF-1 α, then, binds to hypoxia response elements (HREs) present in the nuclear deoxyribonucleic acid and regulates changes in expression of downstream target genes. Vascular endothelial growth factor (VEGF) a hypoxia-responsive gene is a key downstream target of HIF-1 α activation, and induces tumor angiogenesis.^[6] Tumor angiogenesis is commonly assessed by intratumoral microvessel density (MVD). Cluster of Differentiation (CD31) is a marker that can selectively detect a glycoprotein of 130kDa in vascular endothelial cells and can aid in assessment of MVD.^[7]

Biomarkers that can detect tumor hypoxia and angiogenesis early enough in HNSCCs and predict tumor aggressiveness and its sensitivity to treatment have been a key area of research in recent years. HIF-1 α and VEGF have shown promise in this respect. The immunohistochemical evaluation of these markers associated with clinicopathological data may be a cost-effective method to guide treatment. This is particularly in resource-limited set ups of low- and middle-income group countries.^[3,8]

The present study was thus undertaken to study the immunohistochemical expression patterns of these markers and to evaluate their association with known prognostic clinical and histopathological parameters of HNSCC.

MATERIALS AND METHODS

This cross-sectional study conducted by the Departments of Pathology, Otorhinolaryngology, and Radiotherapy, of a tertiary care center in Central India included 44 patients. These patients were operated for HNSCC between January 2020 and December 2021. The clinical details and demographic profile of each patient were recorded. Tumor focality, size, tumor thickness, dimensions of metastatic nodes, any bony, or salivary gland infiltration were noted. Hematoxylin and eosin (H&E) stained slides were studied for tumor histology, grade, depth of invasion, lymphovascular invasion (LVI), perineural invasion (PNI), a worst pattern of invasion (WPOI), tumor stroma ratio, presence of metastatic nodes, extranodal extension, and status of resection margins. Tumor grading and pathological TNM staging were done according to AJCC 8^th edition.^[9] Surgically resected margins were considered free when tumor was a distance of ≥5 mm involved when <1 mm and close when the distance was 1–5 mm.^[10]

Patients recruited for the study were followed up till April 2022. This study was approved by the Institutional Human Ethics Committee for Postgraduate Research (2020/PG/Jan/19).

RESULTS

A total of 44 participants were included in this study. The median age was 45 years (age range: 26–73 years) with 75% (n = 33) of study patients being <50 years. Male patients predominated, comprising 36 cases (81.8%), with a male-to-female ratio of 4.5:1.

Buccal mucosa was the most frequent site (63.6%), followed by the tongue (15.9%), alveolus (11.4%), floor of the mouth (4.5%), lower lip (2.3%), and larynx (2.3%). Wide local excision of buccal mucosa (34.09%) was the most common resection procedure received.

On histopathological examination, 61.4% of cases were moderately differentiated (grade-2), 22.7% were well-differentiated (grade-1), and 15.9% were poorly differentiated (grade-3). More than half of the cases had resection margins close to the tumor (56.80%).

LVI was detected in 13 cases (29.5%), PNI in 15 cases (34.1%), and WPOI-5 in 15 cases (34.1%). Majority (34, 77.3%) were stroma-poor tumors.

A pathological tumor stage of pT3 was present for 18 cases (40.91%) followed by 13 cases (29.55%) in stage pT2, 8 cases (18.18%) in stage pT4a, and 5 cases (11.36%) of pT1. Metastatic nodes were present in 20 cases (45.45%). On pathologic prognostic stage grouping, the largest subset of tumors (16, 36.36%) fell into group Stage IV A.

HIF-1 α immunohistochemical expression

HIF-1 α was localized in both the nucleus and cytoplasm. Patchy to diffuse positivity in tumor cells with variable staining intensity was noted. A strong intensity nuclear staining was noted in a majority (36, 81.88%). Cytoplasmic staining was fine, granular to coarse, with strong cytoplasmic staining noted in (9, 20.5%). Well-differentiated squamous cell carcinomas exhibited strong HIF-1 α nuclear and cytoplasmic expression, particularly within the keratin pearls [Figure 1].

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Figure 1:

Hypoxia inducible factor 1 alpha (HIF-1 α) in tumor cells: (a) Cytoplasmic expression (DAB, ×40 objective lens), (b) nuclear expression (DAB, ×40 objective lens), (c) both cytoplasmic and nuclear expression of HIF-1 α (DAB, ×40 objective lens), and (d) strong HIF-1 α immunostaining around keratin pearls in well differentiated tumor (hematoxylin and eosin, ×10 objective lens, inset of DAB). DAB: Diaminobenzidine.

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Tumor cells adjacent to blood vessels were negative for HIF-1 α, whereas tumor cells away from blood vessels were strongly nuclear positive [Figure 2].

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Figure 2:

(a) Image showing tumor hypoxia in relation to distance from blood vessel (Figure made with BioRender online software), (b) photomicrograph showing positive expression of hypoxia inducible factor (HIF)-1α in tumor cells ~150 µm away from blood vessel (DAB, ×10 objective lens; Red arrow head: Blood vessel; Black arrow head: Tumor cells with no expression for HIF-1α; Yellow arrow heads: Nuclear expression of HIF. DAB: Diaminobenzidine.

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Tumor adjacent nondysplastic mucosal epithelium showed nuclear expression of HIF-1 α, in the lower half of the epithelium, except in the basal layer., While the dysplastic mucosal epithelium showed nuclear expression in the lower half of the epithelium,including the basal layer.

HIF-1 α was negative in non-tumoral cells of the stroma except in inflammatory cells, mainly lymphocytes which exhibited strong nuclear staining for HIF-1 α serving as an internal control.

The median H-score for cytoplasmic expression of HIF-1 α was 80 (Range 2–300), with 25 cases (56.8%) of high H-score (>80) and 19 cases (43.2%) with low H-score (<80). The median H-score for nuclear expression of HIF-1 α was 20, with 23 cases (52.2%) having a high H-score and 21 cases (47.7%) with low H-score.

VEGF immunohistochemical expression

Cellular localization of VEGF was observed mainly in the cytoplasm (n = 40, 90.09%), with occasional cases showing additional membranous staining. Staining intensity was variable. Majority (26, 59.1%) showed a weak VEGF expression. Nuclear expression,too was observed, although in only four cases (9.1%), all with higher histologic grades, lymph node metastasis, and belonging to an advanced stage group (stage IVA) [Figure 3]. Tumor adjacent to normal mucosal epithelium was negative for VEGF.

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Figure 3:

(a) Strong cytoplasmic expression of vascular endothelial growth factor (VEGF) in tumor cells (DAB, ×40 objective lens), (b) nuclear expression of VEGF in tumor cells (DAB, ×20 objective lens), (c) high microvessel density (MVD) with numerous dilated, tortuous and many slit like vessels highlighted by CD31 (DAB, ×10 objective lens), and (d) low MVD with small slit like vessels highlighted by CD31 (DAB, ×10 objective lens). DAB: Diaminobenzidine.

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For quantification of VEGF expression, H-score was calculated and ranged from 0 to 175, with a median value of 20. Of the 44 cases, 25 (56.8%) had high H-scores, while 19 (43.2%) had low H-scores for immunohistochemical expression of VEGF in tumor cells.

DISCUSSION

Previous studies have shown tumor hypoxia and angiogenesis to have a bearing on therapeutic resistance, locoregional recurrence, and the metastatic potential of HNSCC.^[18] In India, a high burden of cancer cases is attributed to squamous cell cancers of the head and neck region, particularly in the oral cavity, due to the higher prevalence of tobacco chewing. This was noted in the study cohort too, where oral cavity,particularly the buccal mucosa was the most common site of malignancy. Furthermore, there was a rise in the incidence of HNSCC in the young that was also noted in the present study cohort.

These clinical and demographic factors have a peculiar bearing on the treatment outcome of HNSCC patients treated in resource-limited settings of low- and middle-income group countries. The radiation therapy facilities in these countries are often unevenly distributed. Biomarker studies that can either predict treatment responses or guide treatment decisions are, thereby, specifically relevant.

A noteworthy finding in this study was the presence of both cytoplasmic and nuclear expression of HIF-1alpha in all tumors. The cytoplasmic expression was significantly associated with the histological grade (P = 0.030*). Hence, even though a few previous studies have considered only nuclear expression for a positive interpretation that we recommend taking into account both cytoplasmic and nuclear expression.^[19-24]

This can be further substantiated by the inherent biology of cancer cells,where a dip in the oxygen concentration in the tumor microenvironment initiates increased intracellular accumulation of HIF-α subunits; Thereby, this translates into cytoplasmic overexpression of HIF-1 α on IHC. On the other hand, nuclear localization of the HIF-α subunit occurs during hypoxia, where the HIF-1 α/ARNT dimer binds to HREs of target genes in the nucleus required for adaptive cellular responses. Tumor cells showing nuclear overexpression for HIF-1 α on IHC may thus be interpreted as frankly hypoxic.

Furthermore, HIF-1 α nuclear overexpression was noted in >60% cases (n = 26) of advanced T stages (T3/T4) of tumor in this study cohort.^[25]

The delivery of cancer chemotherapeutics is crucial for radiotherapy and chemotherapy response. Poorly vascularized tumors may hinder drug delivery, while highly vascularized tumors may not fully benefit from hypoxia sensitizers in radiation therapy. Tumor angiogenesis enables growth but may lead to leaky vessels,hindering drug diffusion to tumor cells. VEGF is a key pro-angiogenic factor that regulates tumor angiogenesis.^[14] Likewise erstwhile studies, immunohistochemical expression of VEGF was observed in the cytoplasm of tumor cells.^[6,13,26-33] However, nuclear staining in addition to cytoplasmic staining, was noted in four cases that belonged to the stage group IVA with metastatic tumor deposits in the lymph nodes. A couple of preceding studies have noted nuclear localization of VEGF (messenger RNA) postulating a nuclear function,especially during hypoxia.^[34,35]

Statistically significant associations were not recorded for VEGF with HIF-1 α expression or MVD, similar to a few previous studies.^{[14,26,30,36-38]} To increase the specificity and nonspecific background staining, we used a rabbit monoclonal antibody. This may explain the weak to moderate intensity cytoplasmic staining in a restricted pattern in most of our study cases.

In our study, MVD showed a statistically significant association with the nuclear expression of HIF-1 α. We observed a higher nuclear expression of HIF-1 α cases with high MVD compared to those with low MVD (OR = 6.9, 95% CI = 0.49–3.1 P = 0.057). Although, cytoplasmic staining was noted, it was the nuclear expression of HIF-1 α and not cytoplasmic expression that was associated with angiogenesis. This supports the concept of hypoxia- inducing angiogenesis through nuclear localization and upregulation of the HIF-1 α in tumor cells.^{[13,30,31,39,40]}

Our study is also one of the first to report a significant association between LVI and MVD in HNSCC. Regression analysis also o performed revealed that high MVD and the presence of LVI were associated statistically (P = 0.015*).

We quantified angiogenesis and found MVD significantly associated with the stage of disease (P = 0.042*). On logistic regression analysis, an advanced-stage of tumor showed higher odds of showing high MVD compared to those in the early stage (OR: 0.23, 95% CI: 0.04–0.93, P = 0.051).

CONCLUSIONS

The findings of this study indicate immunohistochemical expression analysis of hypoxia and angiogenesis markers; Notably, HIF-1 α coupled with MVD quantification, may be promising and cost-effective biomarkers that can be utilized for guiding escalation or de-escalation of treatment in HNSCC patients. This will be of great significance particularly in low-to-middle-income group countries, where radiotherapy centers are unevenly distributed and limited resources are present. For positive interpretation of HIF-1 α, we recommend taking into account both nuclear and cytoplasmic staining. Nuclear expression of VEGF was also a unique finding in a few cases of advanced-stage HNSCC with lymph node metastasis. Nuclear localization of VEGF in existing literature has been linked to chronic hypoxia. This is an aspect of VEGF expression on IHC that may be taken up in future studies for further validation.