|Ahead of print publication
Evaluation of myofibroblasts in prognosis of oral squamous cell carcinoma: A systematic review
Priya Shirish Joshi, Swapnita Patil, Madhuri Chougule, Kiran Jadhav
Department of Oral Pathology and Microbiology, Vasantdada Patil Dental College and Hospital, Budhgaon, Maharashtra, India
|Date of Submission||29-Jul-2020|
|Date of Decision||28-Sep-2020|
|Date of Acceptance||25-Dec-2020|
|Date of Web Publication||17-Jul-2021|
Priya Shirish Joshi,
Department of Oral Pathology and Microbiology, Vasantdada Patil Dental College and Hospital, Kavalapur-Sangli, Budhgaon, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Oral squamous cell carcinoma (OSCC), one of the 10 most prevalent cancers worldwide, has a global annual incidence of approximately 300,000 new cases and 145,000 deaths, with considerable geographical and environmental risk factor differences. Myofibroblasts contribute to the tumor stroma through activation and recruitment of resting fibroblasts to the tumor tissue. Studies have shown that myofibroblasts play a significant role in the progression of oral cancers and they also seem to have some prognostic values in OSCC.
Aim: This systematic review aims to recognize the role of myofibroblasts in prognosis/survival of patients with OSCC.
Design: This study design was a systematic review.
Materials and Methods: Studies assessing the prognostic relevance of myofibroblasts (alpha-smooth muscle actin-positive fibroblasts) in patients with OSCC were systematically reviewed using PubMed, PubMed Central, Google Scholar, and Clinical Key databases. The outcomes assessed were prognosis of OSCC patients in terms of overall survival, disease-free survival, and progression-free survival.
Results: Most of the studies assessed myofibroblasts using immunohistochemistry to evaluate its distribution pattern and staining intensity. The presence of high levels of myofibroblasts in the stroma of OSCC patients predicted shortened time to progression of the disease and an overall decrease in survival. Moreover, high presence of myofibroblasts in association with various histopathological prognostic parameters including advanced disease stage (TNM staging), recurrence, tumor grade, depth of invasion, vascular, lymphatic and neural invasion, and extranodal metastatic spread was noted.
Conclusion: The distribution pattern and staining intensity of the myofibroblasts predict its role as a prognostic biomarker.
Keywords: Alpha-smooth muscle actin, epithelial–mesenchymal transition, myofibroblasts, oral squamous cell carcinoma, prognosis, survival
| > Introduction|| |
Oral squamous cell carcinoma (OSCC), one of the 10 most prevalent cancers worldwide, has a global annual incidence of approximately 300,000 new cases and 145,000 deaths, with considerable geographical and environmental risk factor differences. Clinical features including tumor size and cervical lymph node metastasis are the most consistent prognostic factors for OSCC, though prognosis is frequently unpredictable. Among the histological characteristics, depth of invasion, perineural invasion, lymphovascular emboli, tumor necrosis, lymphocyte host response, bone and muscle invasion, as well as worst pattern of invasion have shown prognostic importance for OSCC, leading to incorporation of depth of invasion in the T stage classification in the new edition of the stating manual of the American Joint Cancer Committee. However, OSCC shows significant morbidity and mortality rate that has not changed over recent decades. Therefore, prognostic markers are required to better understand and select more aggressive tumors.
The growth of OSCC cells is influenced by its stroma. Stromal cells such as mast cells (MCs), cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), endothelial cells, pericytes, smooth muscle cells, and lymphocytes promote tumor growth, local infiltration, proliferation, and neovascularization and escape from immune defense. Such cancer-amended stroma is referred to as the tumor microenvironment (TME) and probably leads to cancer cell invasion and metastasis. The concept of TME was first laid down by Paget, when he described his “seed and soil theory” in the 1880s. It basically comprises cancer cells, stromal cells, and the extracellular matrix (ECM). The TME is composed of stromal cells, such as fibroblasts, endothelial cells, and inflammatory cells. Fibroblasts and macrophages are the major cellular components. These cells are influenced by cancer cells via cell–cell adhesions or various soluble factors, which are further responsible for altering their phenotypes. The cells that alter their properties are termed CAFs and TAMs.
Histologically, normal oral mucosa is composed of epithelium and connective tissue. The connective tissue around the epithelium has protective and nutritional roles for the epithelium. The presence of cancer is followed by some changes within the epithelium, which leads the normal stroma to become a reactive one. The formation of reactive stroma is associated with the secretion of cytokines such as TGFβ-1 from cancerous cells. The cancer cells promote initiation of fibroblasts into myofibroblasts, increase in the number of blood vessels, increase in the number of inflammatory cells, cause desmoplasia, decrease the expression of epithelial markers (cadherins), and increase the expression of mesenchymal marker such as vimentin.
A unique group of smooth muscle-like fibroblasts are present within the mesenchymal component which can secrete some cytokines, chemokines, prostaglandins, growth factors, and matrix components. These biologically active, fibroblastic cell types are known as myofibroblasts or reactive fibroblasts. They play a key role in inflammatory, growth, and wound repair processes, which are responsible for further progression of tumorigenesis.
Myofibroblasts, originally identified by Gabbiani et al. in 1971, are derived from fibrotic tissues and they play a major role in healing. They are described to be either mesenchymal stem cell or nonmesenchymal stem cell origin. The latter contributes to the tumor stroma through activation and recruitment of resting fibroblasts to the tumor tissue. Subsequently, a specialized adhesion complex termed the fibronexus junction was described, which consists of aligned myofilament bundles and fibronectin contacting one another through points at the cell surface, where the intracellular actin is linked to the extracellular fibronectin through transmembrane integrins. Myofibroblasts are classified into two types; first type - proto-myofibroblasts, a partially differentiated fibroblast that contains actin stress fibers but no immunohistochemically detectable alpha-smooth muscle actin (α-SMA). The second type expresses α-SMA and is considered a mature myofibroblast. In the cancer stroma, CAFs undergo changes in protein expression that represents an “activated” myofibroblastic phenotype, which typically involves the upregulation of markers such as α-SMA. It has been reported that CAFs are derived from normal cancer stromal fibroblasts under the direct impact of cancer cell-derived cytokines and function to further facilitate local and distant migration, as well as aid in the suppression of the host immune response. The most striking role of CAFs in the TME is the stimulation of EMT which is a necessary step toward invasion, metastasis, and apoptosis resistance. CAFs also promote cancer cell proliferation, invasion, angiogenesis, metastasis, and chemoresistance.
Studies of different cancer types have shown that CAFs are located in the vicinity of tumor cells and are able to enhance tumor growth through the secretion of growth factors (e.g., transforming growth factor-μ, matrix degrading enzymes (e.g., matrix metalloproteinases), and angiogenic factors (e.g., vascular endothelial growth factor). Different CAFs have been described, but the one that is most consistently shown to have an adverse effect on prognosis is myofibroblasts.
Although various biological and molecular markers have been suggested as having utility for determining treatment and predicting prognosis, postoperative management of the patient is determined primarily through detailed pathological examination of the tumor resection specimen and is based on the TNM classification. There is no clear consensus regarding the relative importance of various prognostic histopathological parameters, and attempts have been made to combine various parameters into defined scoring systems. Their use has produced unsatisfactory interobserver agreement. Although patients with advanced disease (large tumors with metastases) show decreased survival, particularly if extranodal spread is present, there is, as yet, no single pathological feature or molecular marker that allows the identification of early-stage aggressive tumors within the heterogeneous OSCC population. Studies have shown that myofibroblasts play a significant role in progression of oral cancers, and they also seem to have some prognostic value in OSCC. This systematic review is planned specifically with the aim to recognize the role of myofibroblasts in prognosis of patients with OSCC with respect to survival outcomes.
| > Materials and Methods|| |
The protocol of this review was based primarily on the PRISMA-P and submitted to PROSPERO under the ID-151170 and is published at PROSPERO 2020 CRD42020151170 (available at: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020151170). PRISMA guidelines were followed to increase the quality and transparency of the search. Questionnaires were separated and organized using the PICOS strategy.
What is the role of myofibroblasts in prognosis of patients with OSCC in terms of survival?
Stromal myofibroblasts play an important role in tumor invasion and metastasis, due to its ability to modify the ECM. The purpose of this systematic review is to evaluate the role of myofibroblasts in prognosis of the patients suffering from OSCC in terms of survival.
The primary outcome considered in this review is to evaluate the role of myofibroblasts in prognosis in terms of survival in patients of OSCC.
The bibliographic searches were conducted in several electronic databases using all publications in PubMed, PubMed Central, Google Scholar, and Clinical Key between January 2009 and September 2019. In addition, a specific electronic search was conducted in Journal of Oral and Maxillofacial Pathology, Anticancer Research, Journal of Oral Pathology and Medicine, Journal of Translational Medicine, International Journal of Oral and Maxillofacial Surgery, Journal of Pathology, International Journal of Oncology, Journal of Oral Diseases, and Asian Pacific Journal of Cancer Prevention. A search for unpublished studies was also conducted (grey literature) [Table 1].
Criteria for considering studies
Condition or domain being studied
TME plays a key role in the development of OSCC. Among the various stromal elements, myofibroblasts play an important role in process of carcinogenesis. The survival rates are poorer in patients with abundant myofibroblasts. Studies were considered eligible for this systematic review when they fulfilled the following criteria: full-text articles in English language from 2009 to 2019 on myofibroblasts and their role in prognosis of OSCC patients.
Outlines according to the PICOS strategy are given below:
- Population (P): Patients diagnosed and treated for conventional primary OSCC and on proper follow-up
- Intervention (I): Role of myofibroblasts in prognosis of participants/population accessed by survival analysis
- Comparison (C): None
- Outcome (O): Role of myofibroblasts in prognosis of OSCC by studying staining intensity and/or pattern of staining of myofibroblasts in correlation to survival during the follow-up period
- Primary outcome: Qualitative studies which aimed to evaluate the role of myofibroblasts in prognosis of OSCC by evaluating changes in staining pattern and intensity of myofibroblasts in correlation to survival during the follow-up period were considered eligible for inclusion. Prognosis is measured in terms of survival and assessed using most widely accepted survival analysis statistical tools such as Kaplan–Meier survival analysis and log rank test.
- Measures of effect: Qualitative studies on staining intensity and staining pattern of myofibroblasts using immunohistochemistry (IHC) technique.
- Study design (S): Prospective, retrospective, observational, and cross-sectional studies.
Survival is defined under following terms:
- Overall survival - The length of time from either the date of diagnosis or the start of treatment for a disease till the time the patient diagnosed with that disease is alive
- Progression-free survival - Survival without progression of disease
- Disease-free survival (DFS) - Length of time after primary treatment for cancer ends that the patient survives without any signs or symptoms of that cancer
- Disease-specific survival - Percentage of people in a study or treatment group who have not died from a specific disease in a defined period of time.
Articles with abstracts only, studies on head and neck carcinomas excluding oral cavity, in vitro studies (cultures), studies with animal experiments, review articles including systematic review, meta-analysis, and duplicate studies were excluded. Studies including patients with variants of OSCC, metastatic or secondary carcinoma, and malignancies other than OSCC and patients who were not on follow-up record were also excluded [Table 2].
The search and screening process was carried out by two independently reviewing authors, following the previously established eligibility criteria; first, analyzing titles and abstracts. In a second phase, complete articles were selected for careful reading and analyzed per eligibility criteria (inclusion/exclusion) for future data extraction. Discrepancies among authors/reviewers were resolved through careful discussion. The search agreement between the two reviewers was evaluated by Cohen's Kappa test. If needed, the authors of the included studies were contacted by e-mail for the clarification of any doubts.
The following data were extracted from the included studies by two independent reviewing authors: publication details, study design, follow-up, number of subjects, role of myofibroblasts in OSCC, prognosis of OSCC patients in terms of survival, author's conclusions, and remarks.
Assessment of the risk of bias and quality
Risk of bias and study quality analyses were performed independently by two reviewing authors. For the analysis of nonrandomized studies (prospective and retrospective cohort studies), the Newcastle–Ottawa scale (NOS) was used. For the categories and results, the studies got a star/point for each item. For the comparison category, two stars/points was assigned. According to NOS, the maximum score assigned to a study was six stars/points. Studies rated five stars and up were considered as high quality.
| > Results|| |
The search of all publications from various electronic databases revealed 18,659 citations [PRISMA Flowchart 1]. Searches of Google Scholar, PubMed Central, and Clinical Key bibliographies of review articles revealed further relevant studies that had not been identified by PubMed search. Similarly, hand searching in the identified journals did not identify any other studies. After adding MeSH terms related to myofibroblasts as cancer associated fibroblasts and tumour associated fibroblasts, oral squamous cell carcinoma and prognosis as disease free survival and progression free survival, a total of 60 articles were retrieved. Further, full-text articles including observational, prospective, and retrospective studies on human species in English language were selected between the publication dates of January 2009 and September 2019. A total number of 47 articles were selected. After reading the abstracts, 20 articles were excluded as they were studies related to cell lines, cell cultures, in vitro studies, and case reports. Further, 27 articles were selected and scanned for the survival outcomes (prognosis of OSCC patients). Studies that did not include prognosis of OSCC patients were excluded, and a total of 12 articles were selected for this systematic review.
The studies included in the present systematic review were mostly retrospective cohort type, and the characteristics of the same are presented in [Table 3]. The number of participants ranged from 26 to 282, with a mean of 102 patients. Ten authors studied myofibroblasts (CAFs) with IHC,,,,,,,,, along with other markers such as p16, p53, p62, EP4, EGFR, αvβ6, integrin, maspin, ki-67, CD68, CD31, CD163, N-cadherin, vimentin, LYVE-I, activin-A, NOTCH3, ROCK2, c-KIT, and PDGFR-β. Four authors concluded that the presence of myofibroblasts along with other associated markers in the cancer stroma was associated with poor prognosis of OSCC patients.,,, Hedbäck et al. studied myofibroblasts (CAFs) by IHC and immunofluorescence as well. Two authors opined that carcinogenesis was associated with distribution pattern of myofibroblasts., Two authors opined that α-SMA expression was an independent marker for OSCC mortality., High p62 correlated with stromal α-SMA expression after chemotherapy. Four authors studied myofibroblasts with molecular methods along with IHC,,, [Table 3].
|Table 3: Characteristics of studies included in investigating prevalence of myofibroblasts in oral squamous cell carcinoma (data extraction sheet)|
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Assessment of quality and risk of bias
NOS was used for the assessment of quality of the articles selected. Regarding the risk of bias across studies, the selected studies used similar methods which reduced the possibility of misinterpretation. Not all studies revealed data related to follow-up of patients which is the cause of bias. Mean follow-up period included in remaining studies was not long enough for outcome to happen. Seven studies presented fair-quality results.,,,,,, Three studies reflected good-quality results,,, and one among them had the highest score reflecting good-quality research. Five studies presented scores which reflect potential risk of bias,,,, and two among them reflected poor-quality research, [Table 4].
|Table 4: Assessment of quality and the risk of bias (Newcastle-Ottawa scale scale)|
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| > Discussion|| |
Summary of evidence
One of the most important steps in tumor progression is epithelial–mesenchymal transition (EMT). EMT is of three types: Type 1 EMT - occurring at the level of embryo and organogenesis; Type 2 EMT - seen during wound healing and tissue regeneration; and Type 3 EMT - associated with cancer progression and metastasis. EMT is a multistep dynamic cellular phenomenon in which epithelial cells lose their cell–cell adhesion and gain migratory and invasive traits that are typical of mesenchymal cells. EMT involves loss of membranous localization of the epithelial marker E-cadherin and an increase in levels of one or more mesenchymal markers, such as vimentin, N-cadherin, α-SMA, or fibronectin, along with a loss of polarized function of epithelial cells.
The biological properties and functions of myofibroblasts in tumor progression and metastasis have been extensively reported in several studies. Owing to the substantial weight of evidence indicating a protumorigenic role, myofibroblasts have been suggested as a promising therapeutic target in various cancers. However, data on the prognostic value of myofibroblasts particularly in OSCC are limited. In the current systematic review, we included articles discussing or evaluating the role of myofibroblasts in prognosis (survival outcome) of OSCC patients. A comprehensive search was carried out including electronic search, manual search, and grey literature. Most of the studies have concluded that the presence of CAF in the stroma of the tumors was consistently associated with several related clinicopathological features such as aggressiveness, staging, grading, and recurrence of tumors; depth of invasion; vascular, lymphatic, and neural invasion; and extranodal metastatic spread.
Myofibroblasts have been studied by various modalities including IHC, immunofluorescence, and molecular methods. Luksic et al. studied α-SMA staining reaction for myofibroblasts by IHC method. They are of the opinion that an increase in the number of myofibroblasts and a change in their distribution pattern (network arrangement) occur during carcinogenesis which can be an expression of their role in high tumor invasiveness and weaker prognosis. Myofibroblasts (CAFs) have been studied with IHC along with other markers such as p16, p53, p62, EP4, EGFR, αvβ6, integrin, maspin, ki-67, CD68, CD31, CD163, N-cadherin, vimentin, LYVE-I, activin-A, NOTCH3, ROCK2, c-KIT, and PDGFR-β.,,,,,,,,, Ding et al. have noted that α-SMA + myofibroblasts generally surrounded the tumor islets and were closely associated with lymphatic metastasis and poor survival in patients with oral tongue SCC (OTSCC). Furthermore, their study was first of its kind showing that the expression of α-SMA was associated with expression of N-cadherin, vimentin, and LYVE-1. They postulate that these myofibroblasts might play a dual role in promoting EMT process and lymphogenesis of premetastasis microenvironment in OTSCC. Kelner et al. studied whether the presence of CAFs and the expression of activin A as well as clinicopathological features are predictive of occult lymph node metastasis in early-stage tongue SCC. They have noted that high density of CAFs is a strong predictor of poor prognosis and further showed that CAFs in the stroma of oral carcinomas may influence proliferation and invasion, resulting in a more aggressive tumor. In vitro studies demonstrated that TGFβ-1 released by oral carcinoma cells induces myofibroblast transition, suggesting that the emergence of CAFs within tumor stroma is coordinated by tumor cell invasion. In support of this hypothesis, majority of samples classified as CAF-negative were those with the shortest depth of invasion, whereas high CAF density was found in tumors with more than 5 mm depth of invasion. Attramadal et al. have correlated mast cell density to other stromal cells at the invasive front. They did not find any correlation of mast cells with CAFs at the invasive front, but a high CAF density was apparently associated with increased recurrence in the patients receiving postoperative radiotherapy. Matsuoka et al., Bello et al., and Fujii et al. studied myofibroblasts in correlation to prognosis in OSCC patients. They opined that high myofibroblasts (CAFs) expression in the tumor stroma contribute to poor prognosis in patients with OSCC and resistance to chemoradiotherapy. Dourado et al. have studied the association of ROCK2 and CAF, and they have suggested that the organization of a more invasion-permissive microenvironment facilitates tumor progression and metastasis. They revealed ROCK2 positivity in both tumor and stromal cells and a significant association between ROCK2 expression by tumor cells and the density of CAF. Liang et al. have investigated CAFs and p62 expression in OSCC after chemotherapy. They concluded that high p62 expression in OSCC cells statistically correlated with stromal α-SMA expression after chemotherapy, and postchemotherapy, p62 expression was associated with poor prognosis in OSCC patients.
We could retrieve four articles conducting molecular studies along with IHC evaluation for myofibroblasts. Important among these is the study by Marsh et al. They have opined that TNM staging has limited prognostic value, and they suggest that an SMA-positive, myofibroblastic stroma is the strongest predictor of OSCC mortality. Whether used independently or as part of a prognostic model, SMA identifies a significant group of patients with aggressive tumors, regardless of disease stage. Bello et al. are of the opinion that there is a strong association between increased CAF density and higher mortality in mobile tongue SCC. They recommend routine assessment of CAF density for disease course prognosis and that it should be included as an integral part of treatment protocols.
Hedbäck et al. studied the presence of miR-21 in myofibroblasts of tumor stroma by molecular methods using combined fluorescent in situ hybridization for miR-21 and immunofluorescence for α-SMA. They observed that high levels of myofibroblasts in the stroma of OSCCs are strongly associated with increased mortality from oral carcinomas, regardless of disease stage, and miR-21 expression in myofibroblasts is indicative of a parallel pathway of activation. Thus, high miR-21 expression is related to a decreased DFS and may be used as an independent prognostic biomarker. Kayamori et al. have studied NOTCH3 expression in CAFs. They demonstrated that OSCCs with NOTCH3-positive CAFs were associated with significantly larger tumor size and lymph node metastasis compared to OSCCs without NOTCH3-positive CAFs, and they have suggested from their study that OSCCs promote angiogenesis by inducing NOTCH3 expression in CAFs. They have concluded that NOTCH3 expression in CAFs can be used as a prognostic marker of OSCCs.
Thus, it can be suggested that the presence of myofibroblasts in OSCC has an inductive phenomenon. Different growth factors released by malignant epithelial cells and epithelial–stromal interactions (EMT) induce the formation of myofibroblasts (CAFs). Studying the staining intensity and distribution pattern of myofibroblasts can be correlated with prognosis of OSCC in terms of survival, and the same should be considered along with various other recorded histopathological findings to help in treatment planning of OSCC patients. Further research is needed to understand the molecular mechanisms by which myofibroblasts affect the biologic behavior of OSCC. As the current evidence is based on retrospective studies, researchers are encouraged to conduct prospective, observational, and cross-sectional studies with uniform long-term follow-up in evaluating the role of myofibroblasts in prognosis of OSCC.
This systematic review presents several strengths such as a previous record of protocol, homogeneity in study design of included articles, data extraction, and quality assessment performed in duplicate. We could identify articles presenting with high risk of bias in this systematic review, and they should be interpreted with caution., We can state certain limitations of this systematic review like no uniformity in the follow-up period among the included articles, which may give an inaccurate survival rate in OSCC patients. It was also noted that authors have not commented on patients who were lost to follow-up. Heterogeneity in the data collected by included studies such as number of subjects included and methods of studying α-SMA noted. Studies did not report important data regarding the selection process of the patients (e.g., age, gender, occupation, socioeconomic status, habits, medical history, any medications, local etiological factors, and potentially malignant disorder present, if any).
| > Conclusion|| |
Based on the available data, it can be concluded that the distribution pattern and staining intensity of myofibroblasts predict its role as a prognostic biomarker in survival studies of OSCC patients. Consideration of IHC and immunofluorescent analysis of myofibroblasts should be considered as an adjuvant to standard diagnostic procedures, which will further be useful in treatment planning of OSCC patients.
Recommendations to further research
We recommend research studies to be conducted with homogeneity in number of subjects included, years of follow-up of the selected subjects, and methods of studying α-SMA. We also propagate systematic reviews based on molecular studies related to myofibroblasts. Meta-analysis should be performed on systematic reviews analyzing various prognostic parameters.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]