This study was designed to confirm the prognostic significance of TSR in OSCC in the largest cohort so far, thus rendering us to proceed to subgroup analysis in a high degree of confidence. Not only that, we show that the prognostic value of TSR was especially sensitive in tumor with grade I and II by means of introducing interaction terms in COX proportional mode. More importantly, we further ascertained that CAFs differ between high- and low-stroma tumors, uncovering the underlying possibilities of poor outcomes in the high-stroma group in a certain extent.
First, TSR was of prognostic value validated in OSCC, specifically its independent prognostic value in different locations. The high-stroma group had worse outcomes than the low-stroma group. Second, gender, N status, and differential grade were significantly correlated with DFS and OCSS through the multivariate Cox regression analysis. Meanwhile, pTNM stage (p = 0.792) illustrated a poor prognostic power for DFS. Additionally, we introduced the interaction terms in the Cox regression analysis, including TSR*gender, TSR*T status, TSR*N status, TSR*pTNM stages, and TSR*differential grade. The results showed that the use of the TSR was most sensitive in high differentiated tumors for DFS and OCSS. No statistically significant differences were observed among the subgroups; however, this does not mean that the prognostic effect of TSR does not differ among the total cohort. Moreover, the concept of interaction term was first used in the clinical research of the TSR in oral cancer. Finally, immunohistochemical analysis reflected that the high-stroma group has higher CFAP+SR and CSMA+SR than the low-stroma group. Additionally, patients with a high CFAP+SR implied a poor outcome, which may be accounted for the adverse outcomes in the high-stroma group.
Moreover, this is the first study to digitally assess the TSR in oral cancer using the Mesker method [10]. Concretely, we chose a 3.2-mm2 circle to perform TSR scoring, which was exactly parallel to the *10 objection in light microscopy. A study has indicated that the diameter of a light microscope does not differ in the final score 11. All slides were scored three times to avoid intra-observer variability. Finally Kappa coefficient reached perfect agreement though in the beginning only 0.762; most initial discordance were settled. Moreover, the remaining slides from four patients were consulted to the third observer to get consensus. Kappa values will further elevate to a perfect level because of the learning curve. Thus, we should digitally examine enough H&E-stained slides to ensure the reliability of the TSR score, which is also mentioned by Vangangelt et al[12]. Through proper training, the TSR as a prognostic tool will be easy, repeatable, and inexpensive.
The tumor stroma, or other the tumor microenvironment (TME), was one of the three key unsolved issues that impede effective clinical therapy for tumors [13], which were heterogeneous and plastic for including the intracellular environment of tumor bulks and the surrounding stromal cells with abundant protumorigenic factors [14]. Though tremendous advance in elucidating the mechanisms underlying tumor-promoting effects of the TME, tackling the complicated mechanisms still has a long way to go. Over the last decade, the prognosis in OSCC has been substantially studied, including biomarkers, such as caveolin-1 [15],MAGE-A11 [16], and clinicopathological parameters, such as histological grade [17, 18] according with our current study. Although accuracy is limited, Tobias Ettl et al. (2016) have found that positive frozen section margins was associated with recurrence [19]. In contrast, the TSR was more practicable as a prognostic tool.
More interestingly, Amol Ramchandra Gadbail et al. (2017) have discovered that the OSCC has a better prognosis in the background of submucous fibrosis [20], which reminded the effects of CAFs implicated in the prognosis in OSCC. The term “cancer-associated fibroblast” were the most abundant stromal component in the TME, originating from at least six cellular categories, including normal fibroblast, quiescent stellate cell, endothelial cell, epithelial cell and pericyte, smooth muscle cell, and adipocyte [4]. Besides, CAFs can secrete a plentiful growth factors, chemokines, and exosomes regulating the course of cancer and immunosuppression, along with interleukin-6 and tumor growth factor-beta [21–23]. Owing to their cellular origins and breadth of functions, CAFs can have either protumorigenic or antitumorigenic effects on different solid tumors [24]. which is the major challenge for CAF-targeting therapies. Preclinical studies have indicated that nonspecific targeting of CAFs does not achieve the desired results in cancer treatment [25]. In view of the abundance and priority of CAFs in the stroma, we set to estimate the CSR in OSCC to attempt to explain the prognostic value of TSR.
Recently, several studies have mentioned the prognostic value of CAF density in OSCC [26]. Moreover, the biological behaviors of CAF were altered by microRNAs to facilitate invasion of OSCC and CD68(+)CAFS predicted poor prognosis illustrated that CAFs is Superior in Prognosis compare to the Epithelial–Mesenchymal Transition(EMT) Score [27–29]. Although molecular studies of CAFs deepen our insight of CAF intratumor heterogeneity and breadth of function, they remain hard to specify for lacking specific markers [4]. Here we chose to use α-SMA and FAP to represent CAF subtypes and found that the high-stroma group has a higher CSR than the low-stroma group. Additionally, the CFAP+SR and CSMA+SR were related to the prognosis, which might be accounted for the negative consequences in the high-stroma group. However, the underlying mechanism remains to be explored. Daniel Öhlund had observed the expansion of stromal fibroblast number, the process was called “stroma-genesis” with concomitant oncogenesis [30, 31]. A study has found fibroblasts encircling early lesions, indicating that the early phase of fibroblasts could be suppressive, and these fibroblasts evolve to tumor-promoting fibroblasts through stroma-genesis [25, 32]. However, the hypothesis was difficult to testify due to the impractical and longitudinal sample of the same lesion and the conversion of the cell states. In this study, the rich-stroma group suggested a worse prognosis than the poor-stroma group, and the rich-stroma group had higher CFAP+SR and CSMA+SR. Moreover, patients with higher CFAP+SR and CSMA+SR had a worse prognosis. Additionally, we found that the low-stroma group has higher positive staining of FAP+ in tumor cells. According to the latest study that overexpression of FAP is related to the epithelial–mesenchymal transition (EMT) in OSCC [33].we inferred that low-stroma tumors might initially be transformed into high-stroma tumors through the events where fibroblasts evolve to protumorigenic fibroblasts and tumor cells via EMT, though the detailed mechanisms remain to be explored.
First, one of the study strengths is manifesting that the prognostic value of the TSR was most prominent in moderately and highly differentiated tumors, and indicating the unique role of the CFAP+SR and CSMA+SR in advancing the prognostic value of TSR. Second, this study has the largest oral cancer sample, which enabled us to render the subgroup analysis in high confidence. Third, the method we used to evaluate the TSR confirmed that we can locate high-stroma areas in the entire slides, and images can be stored to solidify the authenticity of the results compared to those obtained using light microscopy, which are disturbed by uncontrollable factors when scoring the TSR. Limitations of our study are retrospective study design with a short follow-up period and all cases came from a single institution; thus, whether the application of the TSR in a prospective study can obtain reproducible results in OSCC remains to be seen.
In conclusion, the prognostic value of TSR was positively validated in oral squamous cell carcinoma particularly in tumors with moderate or high differentiation. and the prognostic effect of TSR didn’t differ from the prognostic value of the whole cohort in gender, tumor size, lymph node status subgroups. Furthermore, the fact that higher CFAP+SR and CSMA+SR in tumors with stroma-rich simultaneously suggesting the poor outcome, illustrating CFAP+SR and CSMA+SR contributed to the prognostic impact of TSR in OSCC. In a Word, the advantages of TSR were convenient, practicable and lowcost in routine pathological examination and do not need extra staining, which opened a great opportunity for decision-making in individual therapy.