In this study, we developed and validated CSC gene expression signatures in five independent HNSCC cohorts. We observed that patients in the CSC-HR subgroup had worse prognosis than those in the CSC-LR subgroup, in each cohort. Similar results were observed in the two subgroups of patients with HPV (–) HNSCC. Furthermore, the CSC gene expression signature could accurately predict the outcomes of patients receiving RT. Thus, the CSC gene expression signature could identify patients with HNSCC who do not respond to RT and require intensified or personalized treatment.
Cancer cells within individual tumor masses often represent distinct phenotypic states that differ in their functional attributes . Within this tumor heterogeneity, CSCs are essential for tumor initiation, maintenance, recurrence, and metastasis. To date, the identification of CSCs has mainly been based on CSC surface markers. However, the genes encoding these CSC biomarkers that could predict the prognosis of patients with HNSCC have not been clearly studied. Thus, we focused on the association between CSC biomarker genes and prognosis of patients with HNSCC.
We believed that it would be difficult to predict the prognosis of HNSCC by considering all CSC biomarker genes, since each CSC biomarker gene had different effects on the prognosis, and the proportion of the expression of each CSC biomarker gene is heterogeneous depending on each patient. Thus, we decided to select CSC biomarker genes that satisfied the following criteria: (a) whose corresponding biomarker expression showed clinical significance in more than two studies in the last 10 years and (b) whose high expression of each gene was significantly associated with prognosis in patients with HNSCC. On the above basis, we selected four CSC biomarker genes, CD44, MET, ALDH1A1, and BMI1. We then comprehensively analyzed five independent public cohorts while considering gene signatures associated with these CSC biomarker genes.
CD44 is a transmembrane glycoprotein that is the major receptor for hyaluronan . CD44 is a commonly used CSC marker and is associated with prognosis in various human tumors, including HNSCC . High CD44 expression is associated with poor survival in HNSCC . CD44 is also highly expressed in proliferating cells obtained from N + HNSCC metastasis, thereby highlighting its possible role in tumor progression . In addition, CD44 is a biological factor that is significantly correlated with response to RT, in patients with early stage laryngeal cancer .
The expression of c-MET (a mesenchymal-to-epithelial transition factor) was found to be a CSC marker that is positively correlated with the expression of CD44 in HNSCC clinical databases . Lim et al. found that activation of the c-MET pathway is critical for the proliferation and maintenance of CSC traits in HNSCC . c-MET knockdown significantly decreased the expression of CD44-positive cells . c-MET is expressed in the majority of locally advanced HNSCC, and high expression of c-MET predicts a worse prognosis . High MET expression has also been found to be associated with poor loco-regional tumor control and increased metastasis after post-operative chemoradiotherapy in patients with HPV (–) HNSCC .
Aldehyde dehydrogenase 1 (ALDH1) and B-lymphoma moloney murine leukemia virus insertion region-1 (BMI-1) are two of the most studied CSC markers in HNSCC . ALDH1 is an important stem cell marker in both normal and cancer cells . ALDH1 regulates cellular functions by detoxifying various aldehydes and retinoid signaling. ALDH1 appears to have protective properties against HNSCC . In another study, the positive expression of ALDH1 showed significant correlation with lymph node metastasis and poor prognosis . The positivity of ALDH1 was also found to be correlated with the number of cells undergoing epithelial-mesenchymal transition and metastasis in early stage oral squamous cell carcinoma (OSCC) . However, the association between ALDH1 expression and prognosis is contradictory.
BMI-1 is important for the self-renewal ability of stem cells and is related to epithelial-mesenchymal transition . Rao et al. found a significant positive correlation between ALDH1 and BMI-1 expression in OSCC tissue samples, although the underlying pathways have not yet been elucidated . High expression of BMI-1 was associated with poor prognosis in advanced-stage HNSCC treated with primary chemoradiotherapy . BMI1 is also upregulated after irradiation in OSCC, and is associated with poor prognosis . Based on these results, the CSC biomarker genes selected in this study may play a significant role in the prognosis of HNSCC.
There are genes, other than CSC genes, whose expression is associated with the diagnosis and prognosis of HNSCC. Lohavanichbutr et al. identified and validated a 13-gene expression signature that was strongly predictive of survival in HPV (–) OSCC patients . They first identified 131 genes by comparing the differential gene expression between OSCC and normal control groups . Thirteen of these genes were then further screened using the L1-penalized Cox proportional hazard regression method. Three genes, LAMC2, SERPINE1, KLF7, were found to overlap between the 13 gene expression signatures identified in the study by Lohavanichbutr et al. and the 81 CSC gene expression signatures identified in our analysis. LAMC2, SERPINE1, KLF7 play a role in cell proliferation, migration, and adhesion. High expression of these genes is associated with poor prognosis in HNSCC [41,42,43]. Hypoxia- and ferroptosis-related gene signatures predicting the prognosis of patients with OSCC have also been identified and validated [44, 45]. In this study, we developed and validated signatures associated with CSC biomarker genes, the expression of which was correlated with the prognosis of patients with HNSCC.
Patients with HPV (–) HNSCC have a worse prognosis in terms of OS and RFS rates than those with HPV ( +) HNSCC . However, each patient with HPV (–) HNSCC has a different prognosis owing to various risk factors. We confirmed that the CSC gene expression signature was an independent prognostic factor of non-oropharyngeal HNSCC. Since many non-oropharyngeal HNSCC patients did not include HPV status, we indirectly analyzed the role of CSC gene expression signature in HPV (–) HNSCC using information about non-oropharyngeal HNSCC patients. In addition, the CSC-HR subgroup showed a significantly worse prognosis than the CSC-LR subgroup, among patients with HPV (–) HNSCC. Next, we investigated whether the CSC gene expression signatures influence the prognosis of patients with HPV ( +) HNSCC. In these patients, the 5-year OS rates tended to be lower in the CSC-HR subgroup than in the CSC-LR subgroup; however, the differences were not significant. This may be due to the relatively small size of the HPV ( +) HNSCC cohort (n = 128). There is a need for further studies in larger HPV ( +) HNSCC cohorts, to confirm the association between CSC gene expression signatures and prognosis of patients with HPV ( +) HNSCC.
CSCs can regulate their proliferative and self-renewal capacity, and are thus, involved in metastasis, cancer development, and resistance to RT . However, the association between various CSC biomarker genes and the response to RT in HNSCC has not been studied. Only the mRNA expression of CD44 has been shown to be a significant predictor of local recurrence after RT in early stage laryngeal cancer . Thus, we hypothesized that the overexpression of a specific mRNA of CSC biomarker genes in HNSCC might be correlated with response to RT. However, each patient heterogeneously expresses various CSC biomarker genes, and thus, might respond heterogeneously to RT. Our results showed that compared to the CSC-HR subgroup, the CSC-LR subgroup benefited significantly from RT. These results indicated that the CSC gene expression signature might help to program a RT schedule, if further research is conducted on the response to various doses of irradiation in CSC-HR and CSC-LR HNSCC cell lines.
A limitation of our study is that we analyzed CSC gene expression signatures using five different public HNSCC cohorts. Thus, there was a difference in the essential information that was available for each cohort. In particular, the HPV status was missing in about 40% in TCGA cohort and all patients in MDACC and Greece cohorts. Thus, it was not possible to accurately evaluate the effect of the CSC gene expression signature in prognosis of HNSCC patients with HPV (–) status. Instead, we hypothesized that analysis of non-oropharyngeal HNSCC regardless of the HPV status might help find independent prognostic factors of HPV (–) HNSCC patients. In addition, detailed treatment modality methods or doses, such as post-operative RT, concurrent chemoradiotherapy, and induction chemoradiotherapy with surgery, were not included in each cohort. To compensate for the missing information, we conducted an additional analysis on the CSC gene expression signature and found that the CSC gene expression signature was associated with the prognosis of patients with HPV (–) HNSCC and the response to RT in HNSCC. Finally, the mRNA expression of selected CSC biomarker genes showed very low values for AUC as well as sensitivity and specificity that were below the thresholds required for decision-making in clinical settings (AUCs were less than 0.6 for CD44, MET, ALDH1A1, and BMI1). A possible reason for the same seems to be that the prognosis of HNSCC is not entirely changed by the mRNA expression of only a single gene, because the cancer is caused by the accumulation of multiple mutations in various pathways. However, these four genes have shown clinically significant association with the expression of corresponding CSC biomarker proteins in HNSCC over the past 10 years [5, 8, 22,23,24,25,26,27,28]. Thus, we analyzed and confirmed the actual association between mRNA expression of these genes and prognosis in TCGA HNSCC cohort, by referring to these ROC curves.
To the best of our knowledge, this is the first study to assess the prognosis of patients with HNSCC using various CSC biomarker genes. Each CSC biomarker gene influences the prognosis of patients with HNSCC, but the proportions of these genes are highly heterogeneous in each patient. Thus, we first clarified that the gene expression signatures of the four reference CSC biomarker genes, CD44, MET, ALDH1A1, and BMI1, were significantly related to the prognosis of patients with HNSCC. In addition, the Cox proportional hazards model showed that the CSC gene expression signature was an independent prognostic factor that influenced the OS of non-oropharyngeal HNSCC patients.