Head and neck squamous cell carcinoma (HNSCC) ranks sixth among the most common neoplasms in the world and is responsible for more than 350,000 cancer deaths worldwide every year,123. In India, where tobacco is the most common etiologic factor, HNSCC is the most common cancer in men and the third most common in women, with an increasing incidence reported in young adults1.

Despite an aggressive multidisciplinary approach and advances in treatment, the 5-year survival rate for HNSCC has not improved much4. Although a number of factors such as TNM stage56, histologic grade of tumour78, perineural invasion89, depth of tumour8, and extracapsular spread1011 have been used as prognostic markers in HNSCC, regional lymph node (LN) metastasis has been considered one of the most important factors determining treatment approach and thus long-term survival8101112. Even with advances in diagnostic imaging the sensitivity to detect occult LN metastasis remains limited, especially in Stages I and II1314. Furthermore, it has been observed that even among patients with same stage and site of HNSCC, some patients do better than others1. The past two decades saw innovative research in a number of molecular studies on the identification of occult LN metastasis. These studies have shown that development of HNSCC is driven by multistep accumulation of genetic alterations involving various oncogenes, tumour suppressor genes, and cell adhesion molecules, resulting in a heterogeneous cell population with probable differences in invasive and metastatic behaviour and clonal proliferation of these cells11314151617. This has led to increased interest in assessing cancer progression in HNSCC and predicting its course using different molecular markers, which has in turn fuelled the search for markers that can act as definitive prognostic/predictive markers. Furthermore, tumour heterogeneity has led to the use of multiple selected markers together with histopathology in prognostic assessment of HNSCC151819.

E-cadherin is a 120-kDa calcium-dependent transmembrane glycoprotein encoded by the CDH1 tumour suppressor gene located on chromosome 16q21. It is a cell adhesion molecule that connects epithelial cells via homotypic interaction. Disruption of this interaction promotes detachment of cancer cells from their primary sites, the first step in the tumour invasion process4. Reduced or aberrant E-cadherin expression appears to be associated with various parameters such as aggressiveness of tumour, enhanced invasion, and metastatic potential in a number of malignancies including HNSCC20212223. The cyclin D1 gene (CCND1) located on chromosome 11q13 encodes a nuclear protein that is the regulatory subunit of Cdk-4 and Cdk-6. Cyclin D1 plays a major role in cell cycle transition from G1 to S phase by contributing to inactivation of the retinoblastoma (RB) gene product, and overexpression of CCND1 has been reported in 35% to 40% cases of HNSCC2425.

We conducted a study to assess the loss of E-cadherin (tumour suppressor gene) and overexpression of cyclin D1 (proto-oncogene) in primary tumours and metastatic nodes by immunohistochemistry (IHC) to evaluate their role as predictive markers of nodal metastasis and assess clonal expansion in HNSCC patients at our tertiary care centre.

Traditionally, various histopathologic features such as depth of tumour8, pattern of invasion78, tumour differentiation, and TNM stage56 have been used as predictive and prognostic markers in HNSCC. However, the lack of accurate imaging modalities to detect occult or micro-metastasis, combined with the fact that tumours showing the same TNM stage and location behave differently and a lack of consensus on management of draining LNs in TNM stage I and II, has raised serious doubts regarding the adequacy of conventional markers1314. In the past two decades, extensive research has been carried out involving various biomarkers such as p53, beta-catenin, E-cadherin, and cyclin D1 to search for a definitive predictive and prognostic biomarker in HNSCC1819. We studied the importance of E-cadherin and cyclin D1 as combination markers in terms of their relationship to clinicopathologic parameters to examine their role as predictive markers in HNSCC and also examined their expression in metastatic LNs for assessment of clonal proliferation.

The mean age of the study group was 57 years with a standard deviation of 13.1 years, which is similar to the study of Do et al.28 in which the mean age of patients was 61.6 years with no case under 25 years of age and the male:female ratio was 130:16. Loss of E-cadherin expression in primary site tumour (irrespective of comparison of nodal status) was seen in 48.48% cases and cyclin D1 overexpression was observed in 48.45% cases, compared with rates of 77% and 69%, respectively, reported by Do et al.28 and 50.7% and 29.3% reported by Ahmed et al.29. Furthermore, a review of literature revealed great variance in values, ranging from 29.3% to 69%. This variation might be due to different scoring systems used in evaluations, such as the cutoff for cyclin D1 overexpression ranging from 5% to 50% cells. The choice of cutoff values for various markers in HNSCC to distinguish positive from negative results usually lacks a fundamental basis and is rather arbitrary30.

In our study, 29 cases (78.4%) with nodal metastasis showed loss of E-cadherin expression while 26 cases (89.7%) without metastatic nodes showed retained E-cadherin expression (Table 4); this indicates that E-cadherin loss is significantly higher in cases with LN metastasis and is in agreement with various other studies2829303132. Akhtar et al.33 and Kaur et al.34 reported loss of E-cadherin in 5/6 and 7/10 cases (respectively), including cases with LN metastasis. Their results differ from those of Balasundaram et al.35, Wang et al.36, Mattijssen et al.37, and Mehendiratta et al.1, who reported prognostic significance of E-cadherin in HNSCC cases. Regarding tumour differentiation, which is considered to be an independent prognostic marker, we did not find any significant association between tumour grade and E-cadherin expression in our study, which is in concordance with results of several past studies28303839 but differs from some other studies293334. This discrepancy may be due to subjective variation in grading the tumour, a limitation of Broder's system of grading HNSCCs, as a number of stage IV tumours in our study were graded as well differentiated. Also, loss of E-cadherin was significantly higher in advanced TNM stages III and IVA, similar to a report by Ahmed et al.29 involving only laryngeal SCCs. In our study, 94.7% of early-stage cases showed normal expression of E-cadherin whereas only 34% of advanced-stage cases retained E-cadherin expression. However, this finding differs from most previous studies, which have not reported any significant association between E-cadherin expression and TNM stage283035.

Cyclin D1 overexpression was also found to be significantly associated with LN metastasis.(Table 4) These results are comparable to a number of previous studies including those of Do et al.28, Ahmed et al.29, Carlos de Vicente et al.40 and a few Indian studies314142, but in discordance with studies by Michalides et al.43 and Takes et al.30. Regarding TNM stage, few studies in the literature have reported a significant association between TNM stage and cyclin D1 expression282944. In our study we did not find a statistically significant association (P=0.081) although we noted that 68.4% of early-stage tumours did not show cyclin D1 overexpression. No significant association was noted with tumour grade, which is in agreement with previous studies2842, although there are a few reports of a significant correlation between tumour grade and cyclin D1 overexpression2941444546.

Furthermore, among 37 cases with nodal metastasis, 29 showed loss of E-cadherin at the primary tumour site of which 21 cases (72.4%) also had loss of E-cadherin at secondary nodal deposits.(Table 5) This statistically significant association (P=0.007) indicates that the sub-clone of tumour cells that had lost E-cadherin expression proliferated and metastasized to the draining LNs. This is the first report of this finding in HNSCCs. However, 8 cases (27.6%) were negative at the primary site but showed positive E-cadherin expression in the LN deposit. In addition, out of 8 cases with LN metastasis that retained E-cadherin expression at primary site, 7 cases (87.5%) retained E-cadherin expression in the nodal deposit and one case (12.5%) showed loss at the secondary site. These cases point toward heterogeneity of the tumour cells, possibly reflecting additional mutations that play a role in carcinogenesis. Also, a highly significant association (P<0.001) was noted between cyclin D1 overexpression at the primary site tumour and at secondary metastatic deposits in node-positive cases, again pointing to clonal expansion. Similar results suggesting tumour heterogeneity were noted with cyclin D1 expression.(Table 5)

The subgroup of cases with loss of E-cadherin/overexpression of cyclin D1 (negative/positive) showed the highest rate of LN metastasis and higher TNM stages (P<0.001).(Table 6) However, 37.9% of the LN-negative group and 51.4% of the LN-positive group showed both markers as either negative or positive, signifying interplay between additional mutations and these markers.

These results imply that overexpression of cyclin D1 as a cell proliferation marker together with loss of the cell adhesion molecule E-cadherin plays a significant role in metastasis with each marker probably influencing the genesis of mutations in other genes. The combination of these markers might help to predict aggressive disease and prognosis in HNSCC.

E-cadherin and cyclin D1 are potential predictive markers of LN metastasis. The combination of these two markers might help identify early-stage HNSCCs at higher risk of LN metastasis, although tumour heterogeneity may interfere with the association in some cases. With emerging evidence of definite roles of different biomarkers in various malignancies, more randomized trials and meta-analysis studies are required to identify the optimal prognostic and predictive markers for HNSCCs.