In our study, mLSCC were most often occurred in men, mainly in elders. The vocal cord was the most common site with right predominancy. Grossly, mLSCC were mainly flat with a few cases showing exophytic or verrucous papillary configurations. Histomorphologically, the growth pattern at the invasive front of mLSCC is mainly infiltrative, but the expansive growth pattern are not uncommon. Three types of tumor associated stromal changes were observed in our study, typical desmoplastic stromal changes, densely chronic inflammatory infiltrates, and no distinct stromal changes assembling adjacent normal lamina propria. Among these, desmoplastic reaction is the most common feature observed in mLSCC.
As the major component of tumor microenvironment, CAFs are found to be present in a variety of tumors such as esophageal cancer, lung cancer, hepatocellular carcinoma, and kidney cancer. Recent studies showed that the detection of CAFs can aid in the diagnosis of microinvasive ductal carcinoma, endoscopically removed invasive colorectal adenocarcinoma, and assist in differentiating between pancreatic ductal adenocarcinoma and chronic pancreatitis [11-13]. Study from Kumar et al and New et al using a mouse xenograft model of head and neck squamous cell carcinoma demonstrated CAFs play a significant role in invasion and the progression of squamous cell carcinoma [14, 15]. In our study, majority of mLSCC (29/41, 70.7%) showed positive reactivity of α-SMA on CAFs. In contrast, there was no or few immunoreactivity identified in laryngeal SIL and PEH (0/20, 0% ; 2/20, 10%). SIL and PEH are lesions that often need to be differentiated from mLSCC. Although it is said that SIL showed an intact basement membrane, but this is difficult to identify histologically. SIL with inverted growth pattern or tangentialing section can also be difficult to distinguish from invasive carcinoma. PEH, a not uncommon benign lesion occurring in the laryngeal mucosa, can be seen in chronic irritation, non-specific inflammation, tumors such as granular cell tumor, laryngeal tuberculosis and fungal infections, or keratosis [16-18]. As the name PEH implies, the histomorphologic appearance assemble to invasive carcinoma and characterized with mucosal epithelial hyperplasia showing sub-epithelial extension in lamina propria. The irregular epithelial projections in PEH is difficult to distinguish from the invasive nests of well-differentiated squamous cell carcinoma. In our study, the α-SMA expression pattern in stromal fibroblasts of mLSCC were significantly different both from those of SIL and PEH. This support that, the detection of CAFs by immunohistochemistry against α-SMA plays a valuable role in confirmatory diagnosis of mLSCC, and would be used as a reliable marker for the diagnosis of mLSCC and differentiated from SIL and PEH.
The origin of CAFs has been under debate for a long time. Several hypotheses have been proposed for the origin of CAFs such as resident tissue fibroblasts, bone marrow-derived mesenchymal stem cells, or epithelial cells . A commonly accepted theory for CAFs origin points to resident tissue fibroblasts, which are activated by TGF-β1 and converted into CAFs . TGF-β1, mainly localized in exosomes released by cancer cells, can promote the proliferation and expression of CAF markers . Then CAFs secrete various cytokines, chemokines and inflammatory mediators such as stromal cell-derived factor 1 (SDF-1/CXCL12) influence the growth and invasion of tumor . This can explain why CAFs can be widely detected by α-SMA in mLSCC but rarely in laryngeal SIL and PEH. As for the two cases of PEH with SMA positivity in the interstitium, we believe that this is due to interstitial myofibroblasts transformation caused by fibrosis. Fibrosis in vascular vocal cord polyps seems not uncommon. As for the positive case of keratosis, the patient had undergone surgical resection months ago, and the fibrosis should be the repair for injury caused by the previous surgery. Different from the SMA positive fibroblasts in mLSCC appearing around the invasive epithelial tumor nests, positive cells in PEH can appear in lamina propria covered with flat epithelium or in the interstitium away from epithelium. And fibroblasts surrounding the downward growing epithelial nests in PEH may be SMA negative. The different distribution characteristics of SMA -positive fibroblasts can be used to distinguish CAFs from myofibroblasts in fibrosis. The driving force of myofibroblast differentiation in fibrosis are mainly mechanical tension and TGF-β1 which is released from a variety of inflammatory cells and platelets in the microenvironment of damaged or fibrotic tissue .
Comparing the histomorphologic features with the results of immunohistochemical staining of mLSCC, it was found that α-SMA positive cases, especially diffusely positive cases, were concentrated in cases showed flat/non-exophytic gross appearance, infiltrative growth pattern, or with desmoplastic stromal reaction in the stroma. While cases showed exophytic gross appearance, expansive growth pattern or dense lymphoplasmacytic infiltration in the stroma tend to be focal, scattered positive or negative. Especially the interstitial dense lymphoplasmacytic infiltration, our study support that there is a negative correlation between this characteristics and the presence of CAFs in LSCC. Similar findings were reported by Zidar N et al . In our study, six of 7 cases of mLSCC with dense lymphoplasmacytic infiltration were stromal negative for α-SMA, and the remaining 1 case was focally positive. Furthermore, 5 of these 6 cases with α-SMA negative labelling showed expansive growth pattern at the invasive front. It has been reported that pushing borders and good cohesion of the deep invasive front, and high levels of tumor-infiltrating lymphocytes are indicators of good prognosis for laryngeal squamous cell carcinoma [25, 26]. This may be related to less or no CAFs formation in stroma under these conditions, for it is reported that overexpression of CAFs correlates with poor prognosis in some tumors [27, 28]. However, due to small sample and not enough long term follow-up data available in current study, a larger scale study is needed to provide a further confirmatory.
In our cohort, a small percentage (12/41, 29.3%) of mLSCC showed no immunoreactivity identified with α-SMA in the stroma, 6 of which showed dense lymphoplasmacytic infiltration as we said above, and the remaining 6 showed no distinct stromal changes. While, study from Kojc N et al reported that all cases of LSCC contain α-SMA positive stromal cells . This difference may arise from the variabilities of sampling tissue and cases selection in some degree. There was no information about depth of invasion of the LSCC mentioned in their study. In our cohort, we collected mLSCC with depth of invasion not greater than 3 mm. We speculate that it may be due to the poor stromal response when tumor invades superficially. In our study, we compared positivity rate of α-SMA between cases with DOI less than 1 mm and DOI greater than 1mm, we found that the higher positive frequency of α-SMA was observed in mLSCC with DOI greater than 1 mm (P<0.05) . This indicates CAF formation is gradually enhanced along with the depth of tumor invasion. In addition, of the 6 α-SMA negative mLSCC cases showed no distinct histological changes in the stroma, 5 cases belonged to Group 1-1 (DOI≤1 mm) and 1 case belonged to Group 1-2 (1 mm＜DOI ≤2 mm ). This most likely due to the fact that the depth of tumor invasion is too superficial, resulting in a too weak stromal response to be detected and the tumor morphological characteristics have not yet been shown, which further support our hypothesis.
In summary, mLSCC is predominantly flat/non-exophytic grossly, and often histologically shows an infiltrative growth pattern. Varying degrees of desmoplastic stromal reaction is observed in most cases, but in a few cases it is replaced by dense lymphoplasmacytic infiltration in the stroma. Our study support that there is a negative correlation between dense lymphoplasmacytic infiltration in the stroma and the presence of CAFs in LSCC. Our study further supports α-SMA, as a surrogate marker for CAFs, plays a reliable role in confirmatory invasion for mLSCC.