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An Official Publication of the Indian Association of Oral and Maxillofacial Pathologists

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Year : 2022  |  Volume : 26  |  Issue : 3  |  Page : 382-388

Role of Nidogen-2 in diagnosis and prognosis of head and neck squamous cell carcinoma: A systematic review

1 Department of Oral Pathology and Microbiology, Government Dental College and Hospital, Mumbai, Maharashtra, India
2 Department of Oral Pathology and Microbiology, VSPM's Dental College and Research Centre, Nagpur, Maharashtra, India
3 Department of Oral Pathology and Microbiology, Swargiya Dadasaheb Kalmegh Smruti Dental College and Hospital, Nagpur, Maharashtra, India

Date of Submission25-Apr-2022
Date of Decision09-May-2022
Date of Acceptance06-Jul-2022
Date of Web Publication17-Oct-2022

Correspondence Address:
Sanpreet S Sachdev
301, Department of Oral Pathology and Microbiology, Government Dental College and Hospital, P D' Mello Road, Fort, Mumbai – 400 001, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jomfp.jomfp_293_22

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Context: Nidogen-2 (NID-2) hypermethylation has been implicated in many types of cancers, such as lung, bladder, and gastric carcinomas. However, its role has not yet been studied adequately in head and neck squamous cell carcinomas (HNSCC). HNSCCs constituting a major portion of the global cancer load, it is of importance to diagnose and treat them at earliest. This systematic review was performed to assess the role of NID-2 in HNSCCs and assess its utility as a diagnostic and prognostic marker.
Materials and Methods: A systematic search was performed across multiple databases to identify studies pertaining to analysis of expression or methylation of NID-2 in HNSCCs. The sample size, type of cancer/premalignant condition studied, type of tissue/fluid analysed, and the various methodologies used and their results were extracted. PROSPERO registration number: CRD42021245326.
Results: Four studies were identified after a systematic search of literature. The studies analysed NID-2 expression or methylation in conditions such as nasopharyngeal carcinoma, esophageal carcinoma, and oral squamous cell carcinoma (OSCC). NID-2 was found to be a highly specific marker for HNSCCs, and serum NID-2 levels also correlated with poor survival.
Conclusion: Data from the reviewed studies indicate that hypermethylation of NID-2 is highly specific for HNSCC. The high specificity is maintained in salivary and serum samples, facilitating accurate and non-invasive prognostication of HNSCC. The relatively lower sensitivity of NID-2 methylation may be overcome by analysing it along with a panel of multiple biomarkers such as HOX-A2 and YKL20.

Keywords: Epigenetics, hypermethylation, oral squamous cell carcinoma, salivary biomarkers

How to cite this article:
Chettiankandy TJ, Sachdev SS, Khandekar SP, Dive A, Nagpal D, Tupkari JV. Role of Nidogen-2 in diagnosis and prognosis of head and neck squamous cell carcinoma: A systematic review. J Oral Maxillofac Pathol 2022;26:382-8

How to cite this URL:
Chettiankandy TJ, Sachdev SS, Khandekar SP, Dive A, Nagpal D, Tupkari JV. Role of Nidogen-2 in diagnosis and prognosis of head and neck squamous cell carcinoma: A systematic review. J Oral Maxillofac Pathol [serial online] 2022 [cited 2022 Dec 7];26:382-8. Available from: https://www.jomfp.in/text.asp?2022/26/3/382/358731

   Introduction Top

Head and neck squamous cell carcinomas (HNSCCs) constitute about 4–10.3% of all the cancers. About 90% of HNSCCs comprise oral squamous cell carcinoma (OSCCs), making it the most prevalent cancer of the oral cavity.[1],[2] It has been demonstrated that a 6-month delay in diagnosis of OSCC can reduce the chances of survival to half.[3] Therefore, it is of utmost importance to diagnose OSCC in the initial stages of development.

OSCC is generally preceded by various oral potentially malignant disorders (OPMDs), such as oral leukoplakia (OL), erythroplakia, oral submucous fibrosis, and oral lichen planus (OLP) in 80% of cases.[4] OL is the most commonly noted OPMD with a malignant transformation rate of about 2%. It is characterised by dysplastic changes in the epithelium along with hyperkeratinisation. The severity of dysplastic changes has a clinical implication wherein it is indicative of the risk of transition of OL to OSCC. Therefore, identifying the risk of transition would definitely help in determining an apt treatment plan for each case.

A well-coordinated balance exists between the epithelium and the connective tissue stroma, under normal circumstances. Maintenance of cell adhesive junctions between the epithelial cells and the support provided by extracellular matrix (ECM) to the connective tissue is crucial for maintaining this balance. The basement membrane (BM) also helps in maintaining the integrity of these tissue elements through complex molecular signalling pathways.[5] A breach in the BM and subsequent infiltration of epithelial cells into the underlying stroma is considered as the hallmark of squamous cell carcinoma. Increased proliferation and migratory capacity of cells, coupled with the degradation of BM and ECM, together increase the invasiveness of the cells ultimately leading to tumour progression and metastasis.

Amongst the various genes involved in the BM–ECM signalling, Nidogen-2 (NID-2) has been recently identified as a more suitable biomarker for normal or cancerous state. The protein products of NID-2 gene stabilise the BM by linking collagen IV and laminin to the fibrillar network present in the ECM. They also play a role in maintenance of cell adhesion by connecting to membranous integrins.[6] NID-2 is also considered as a tumour suppressor gene (TSG) as it decreases the colony formation and migratory ability of the epithelial cells.[7] Loss of NID-2 expression due to aberrant hypermethylation of its promoters leads to degradation of BM and ECM, and also increases the invasive ability of the epithelial cells. Consequently, loss of expression of NID-2 has been implicated in various malignant neoplasms, such as gastric, lung, and bladder carcinomas.[7],[8]

In the head and neck region, NID-2 can be detected in tissues, saliva, and serum.[9],[10] Amongst 301 potential TSGs, NID-2 hypermethylation was found to be one of the most specific and sensitive biomarkers for detection of HNSCC.[9] Even so, the number of studies investigating expression of NID-2 in HNSCCs is very limited. In this article, the studies published in scientific literature that have investigated the expression or functional role of NID-2 in HNSCC or OPMDs were systematically reviewed. This systematic review serves to answer the question “does Nidogen-2 play a role in diagnosis and prognosis of HNSCC and OPMDs?” The objective of this review is to discern the feasibility and reliability of NID-2 as a diagnostic and prognostic marker for the most prevalent cancerous and pre-cancerous conditions.

   Materials and Methods Top

The review title and search protocol are registered in the International Prospective Register of Systematic Reviews – PROSPERO under the registration number CRD42021245326.[11] A systematic search for studies pertaining to expression of NID-2 in HNSCC or leukoplakia was performed in the following databases: Medline (Ovid), PubMed, PubMed Central, Web of Science Citation Index Expanded, (SCIEXPANDED), and Google Scholar. The keywords comprised ((Nidogen) OR (NID-2)) AND ((HNSCC) OR Cancer OR OSCC. A second search comprised the keywords ((Nidogen) OR (NID-2)) AND Leukoplakia OR OPMD OR premalignant OR precancer. The cross-references cited in the retrieved literature were also screened for identification of possible studies, in case if any were missed by the search strategy.

The abstracts of search results were screened, and full text articles of the potential studies were scrutinised. Cross-sectional as well as longitudinal studies were considered as eligible for the review that comprised case–control or cohort studies as well as randomised clinical trials. To minimise the risk of bias, the PROBAST tool for assessment of the Risk of Bias and Applicability of Prediction Model Studies was used.[12] To further minimise bias in quality assessment, the authors were divided into two groups (Sanpreet S. Sachdev and Tabita Joy Chettiankandy; Shubhangi P. Khandekar and Alka Dive) that independently evaluated the case reports for their inclusion in this review.

Data extraction

The sample size, type of cancer/premalignant condition studied, type of tissue/fluid analysed, and the various methodologies used to analyse the expression of NID-2 were extracted. The expression of NID-2 noted in normal tissues, OL, and OSCC by various methods used was recorded along with their sensitivity and specificity. The interpretation of results by the respective authors with respect to functional role or clinical significance of NID-2 in OL or OSCC was also elicited. Demographic, clinical, radiological, and histopathological features of all the cases were extracted. The quality of articles included in the review was also assessed using the GRADE approach.[13] The extracted data were entered and tabulated into worksheets (Microsoft Office Excel 2016, Redmond, Washington, USA).

   Results and Discussion Top

The systematic search revealed four studies pertaining to analysis of expression or methylation of NID-2 [Figure 1].[7],[9],[10],[14] The data extracted from these studies has been tabulated in [Table 1]. The most comprehensive demonstration of role of NID-2 in suppressing tumourigenesis was done in the study performed by Chai et al.[7] They demonstrated reduced colony-forming ability to an extent of 43.4% in the cell lines expressing NID-2, by means of 2D and 3D colony formation assays. Even so, NID-2 re-expression was unable to induce significant changes in both the in vitro cell proliferation and in vivo tumourigenicity of the cancer cells. Re-expression of NID-2, however, significantly reduced the migration ability and relative invasiveness of the cells.
Figure 1: PRISMA flow chart indicating selection process of articles for final qualitative synthesis of this systematic review

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Table 1: Summary of studies pertaining to NID-2 expression in HNSCC

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Furthermore, on intrasplenic injection of metastatic cancer cells, metastases were found in the liver of 90% of control mice but not in those expressing NID-2. They also demonstrated weaker expression of NID-2 in Nasopharyngeal Carcinoma (NPC) compared to that noted in non-neoplastic glands of nasopharyngeal mucosa, by means of Immunohistochemistry (IHC). Overall, their study revealed that although NID-2 was unable to induce cell proliferation, it suppressed the colony formation, migration, and invasiveness of the cancerous cells.

Aberrant promoter hypermethylation of TSGs has been demonstrated to play a critical role in tumourigenesis in various types of cancer.[15] Promoter hypermethylation of TSGs was also demonstrated to be present in premalignant lesions as well as HNSCC, serving as a potential biomarker for early detection.[9] Furthermore, DNA is a highly stable molecule, making it a suitable candidate for studying methylation status of biomarkers. Chai et al.[7] also demonstrated a downregulation in NID-2 expression in 100% NPC and 80% Esophageal squamous cell carcinoma (ESCC) cell lines.

Guerrero-Preston et al.[9] observed hypermethylation of potential TSGs in HNSCC and compared it to leukoplakia and normal tissues. They found that EDNRB, HOXA9, GATA4, NID-2, KIF1A, and DCC genes showed significant differential methylation between OSCC and normal tissues. Of these, the classification performance of HOXA9 and NID-2 was found to be better than the other genes owing to high sensitivity (>70%) and specificity (100%). The several methylated loci harboured by NID-2 in the CpG islands have conferred it to be a top hit promoter hypermethylated gene in NPC as well as ESCC.[9],[16]

Transcriptomic data with respect to NID-2 mRNA as well as proteomic microarray analysis have revealed that hypermethylation is present in cancerous tissues, and its demethylation suppresses the events associated with tumour progression, such as proliferation, migration, and invasion of neoplastic cells.[17] Results from real-time PCR performed by Srisuttee et al.[14] indicated that the NID-2 methylation did not occur over time; supported by the fact that it was absent in the epithelia of smokers as well as patients with OLP. They also found no significant correlation between NID-2 expression and the clinical stage or histological grade of OSCC. Their findings suggested that hypermethylation of NID-2 occurred only when the normal epithelial cells completely transformed into cancerous cells.

On the contrary, Guerrero-Preston et al.[9] demonstrated a gradual increase in hypermethylation from normal to premalignant to cancerous state by means of quantitative methylation specific PCR (qMSP), supporting the association of the level of hypermethylation of NID-2 with carcinogenesis. Further support for the gradual methylation of NID-2 from normal to cancerous state was provided by Chai et al.[7] They found that methylation of NID-2 was present in not only the cancerous tissues but also 34% of the tissues adjacent to NPC or ESCC by means of methylation-sensitive high-resolution melting analysis. The mutagenic changes occurring in the cancerous area also get transmitted to the adjacent apparently normal mucosa, which may later develop the tumour phenotype. The gradual methylation noted in adjacent apparently normal tissues occurs because of field cancerisation.[18]

Histopathological examination of the tissue obtained by means of biopsy is currently the gold standard for diagnosing OSCC. The biopsy procedure is invasive, tends to cause patient discomfort, and may even impair functioning.[19] The histopathological processing of the sampled tissue further requires generous efforts on the part of laboratory personnel and pathologists. Consequently, recent emphasis has shifted on potentially non-invasive modes for diagnosis. These include detection of biomarkers in saliva or serum, which are readily obtained. Analysis of biomarkers is now possible at a genomic or proteomic level without the need of a waiting period needed for histopathological processing. The techniques having developed only recently, the data pertaining to specificity and sensitivity of detecting biomarkers in these fluids through various techniques are quite limited.[10],[14]

Saliva is a rich source of information when considering the cancers occurring in the oral cavity, as it is constantly in contact with the tumour cells. Guerrero-Preston et al. analysed salivary NID-2 methylation in OSCC and Oropharyngeal squamous cell carcinoma (OPSCC) patients by qMSP.[9] A higher sensitivity of the method was found for diagnosing OSCC patients (87%) compared to those with OPSCC (72%). This discrepancy could be attributable to the different aetiological factors of both the lesions. While OSCC commonly arises because of accretion of genetic mutations resulting from tobacco or alcohol, OPSCC is largely related to human papilloma virus.[20] Another contributing factor could be the difference in the extent to which saliva contacts the oral cavity and oropharynx.

Overall, a salivary sample obtained from the oral cavity would contain more representative cells with NID-2 hypermethylation compared to the oropharynx, accounting for the difference in sensitivity. The specificity of detecting NID-2 hypermethylation in salivary samples was found to be much lower (21%) and with low inter-classification agreement (k = 0.23), compared to tissue specimens (specificity = 100%, k = 0.60). Unlike other sites, such as oesophagus or nasopharynx, the oral cavity is readily accessible, which facilitates obtaining swabs from the tumour area.

Srisuttee et al.[14] detected NID-2 methylation in OSCC patients and observed a 100% specificity by both the methods. They also found that the oral swab method was more sensitive (90.91%) compared to oral rinse (79.07%). However, oral swab could only be obtained from patients with an obvious lesional area, thereby limiting its application as a screening or diagnostic procedure. Care must also be taken to ensure that the clinical technique for obtaining swab is proper and covers the cells beyond the necrotic tissue over the lesion, to avoid a false-negative outcome. Furthermore, obtaining oral swab is more invasive compared to oral rinse and may cause physical discomfort for the patients.

The source of NID-2 in serum is largely unknown although a few theories have been postulated.[10] Considering the fact that the BM is disrupted in carcinomas, the NID-2 linked with integrins could be carried into the bloodstream along with the other disintegrated components. It is also possible that the tumour microenvironment may not be a major source of serum NID-2, and only contributes a small amount of the protein. The downregulation of NID-2 noted in the ECM may be sensed by stromal or endothelial cells. A feedback mechanism signalling the cells to replenish the NID-2 in ECM may be triggered, which could account for the elevated levels of the protein in serum.

Chai et al.[10] observed significantly elevated serum NID-2 levels in patients with ESCC compared to healthy individuals by means of Enzyme-linked immunosorbent assay (ELISA). They also classified the patients into three groups – “high” (32.60 μg/L), “medium” (20.0–32.60 μg/L), and “low” (<20.0 μg/L) based of their serum NID-2 levels. It was found that “high” serum NID-2 levels were associated with poorer survival rate with a hazard ratio of 1.984. There was no correlation noted between the serum NID-2 level and clinicodemographic factors, such as age, gender, Tumor-Node-Metastasis stage, or histopathological grade of the tumour. Their results pointed towards the effective yet limited utility of serum NID-2 as a diagnostic as well as prognostic marker.

The sensitivity and specificity of serum NID-2 as a diagnostic biomarker for tumours did not significantly outperform other existing biomarkers, such as YKL20, CEA, CYFRA21-1, and SCCA.[21] In their study, Guerrero-Preston et al.[9] also reported an improved sensitivity (94%) and specificity (97%) when analysing hypermethylation of HOXA2 and NID-2 together as a panel, compared to the individual genes. Therefore, future studies can analyse and correlate expression of NID-2 and other specific biomarkers such as YKL20 by means of methods such as multiplex IHC. Such studies would yield a highly sensitive and specific panel, ultimately improving the diagnosis and prognostication of HNSCCs.

Another clinical applicability of analysing NID-2 expression would be to evaluate the effectivity of cancer therapy. Care must be taken, however, to avoid false-positive or false-negative outcomes when assessing post-chemotherapy margins of the tumour. It is possible that the drugs used may alter the biochemistry of tissues yielding unreliable results. In general, epigenetic alterations are related, to some extent, to systemic conditions. Disorders causing damage to mucosa BM may be able to induce false-positive results. Other systemic conditions or viral infections may also cause epigenetic alterations leading to uncertain predictability of the methylation status.[22] Because NID-2 is only hypermethylated when the tissue cells completely transit into cancer cells, a negative detection in dysplastic or non-invasive aggressive lesions must not be trifled with. At present, because of limited number of studies, NID-2 expression in OPMDs and other systemic conditions such as AIDS is largely unknown. An extensive study of NID-2 in various diseased states and validation across laboratories would minimise this bias or error in assessment in future.

   Conclusion Top

NID-2 is a tumour suppressor gene playing a vital role in limiting the invasive ability of cells. The studies analysing NID-2 expression or methylation in HNSCC patients are very limited. Data from the reviewed studies indicate that hypermethylation of NID-2 is a highly specific and sensitive marker for HNSCC. The high specificity is maintained in salivary and serum samples, facilitating accurate and non-invasive prognostication of HNSCC. The relatively lower sensitivity of NID-2 methylation may be overcome by analysing it along with a panel of multiple biomarkers, such as HOX-A2 and YKL20. Further studies with respect to NID-2 methylation in OPMDs and systemic conditions would further improve the diagnostic and prognostic utility of NID-2.

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Conflicts of interest

There are no conflicts of interest.

   References Top

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