Expression of Fascin and SALL4 in odontogenic cysts and tumors: an immunohistochemical appraisal.

Background Various stemness markers (SOX2, OCT4, and NANOG) have been studied in odontogenic cysts and tumors. However, studies on SALL4 having similar properties of stemness has not been documented. Additionally, insight into fascin as a migratory molecule is less explored. In this study, the expression of SALL4 and fascin were evaluated in ameloblastoma, adenomatoid odontogenic tumor (AOT), odontogenic keratocyst (OKC), dentigerous cyst (DC), radicular cyst (RC), and calcifying odontogenic cyst (COC). Methods Semi-quantitative analysis of fascin and SALL4 immuno-positive cells was done in a total of 40 cases of ameloblastoma (11 plexiform, 12 follicular, 12 unicystic, and 5 desmoplastic) variants, 6 cases of AOT, 15 each of OKC, DC, RC and 5 of COC. Chi-square test was applied to evaluate the association between SALL4 and fascin expression in odontogenic cysts and tumors. Results Fascin immunopositivity was observed in peripheral ameloblast-like cells, and the expression was weak or absent in stellate reticulum-like cells. A moderate to weak immune-reactivity to SALL4 was observed in the cytoplasm of ameloblastoma, epithelial cells of dentigerous and radicular cysts, having a marked inflammatory infiltrate, which was an interesting observation. COC and AOT had negative to weak expressions. No recurrence has been reported. Conclusions Expression of fascin in ameloblastomas elucidate their role in motility and localized invasion. Its expression in less aggressive lesions like DC, COC, AOT will incite to explore the other functional properties of fascin. SALL4 expression in the cytoplasm of odontogenic cysts and tumors may represent inactive or mutant forms which requires further validation.

was done in a total of 40 cases of ameloblastoma (11 plexiform, 12 follicular, 12 unicystic, and 5 desmoplastic) variants, 6 cases of AOT, 15 each of OKC, DC, RC and 5 of COC.Chi-square test was applied to evaluate the association between SALL4 and fascin expression in odontogenic cysts and tumors.

Results
Fascin immunopositivity was observed in peripheral ameloblast-like cells, and the expression was weak or absent in stellate reticulum-like cells.A moderate to weak immune-reactivity to SALL4 was observed in the cytoplasm of ameloblastoma, epithelial cells of dentigerous and radicular cysts, having a marked inflammatory infiltrate, which was an interesting observation.COC and AOT had negative to weak expressions.No recurrence has been reported.

Conclusions
Expression of fascin in ameloblastomas elucidate their role in motility and localized invasion.Its expression in less aggressive lesions like DC, COC, AOT will incite to explore the other functional properties of fascin.SALL4 expression in the cytoplasm of odontogenic cysts and tumors may represent inactive or mutant forms which requires further validation.

Introduction
Odontogenic cysts and tumors are said to originate from odontogenic apparatus or oral epithelium.Ameloblastoma, the most common odontogenic tumor is known for its local but aggressive biological behaviour. 1The 2017 World Health Organisation (WHO) classification on ameloblastomas reclassified them into Conventional, Unicyctic and Peripheral. 2 Literature review states among the odontogenic lesions, ameloblastoma and odontogenic keratocyst are locally aggressive and recurrent lesions, also the commonest and prevalent odontogenic tumor in Indian population is ameloblastoma which ranges from 14.02% to 71.4% when compared to other odontogenic tumors. 3,4The globally, pooled estimate of the incidence rate of ameloblastoma is 0.92 per million population per year. 5The recurrence varies among various populations, 9.8% according to a Chinese study, 6 while in European multicenter study it is reported to be 19.3%, 7 also tumors larger than 6 cm and involving the soft tissues or adjacent anatomical structures are associated with early recurrence irrespective of method of surgery.Also conservatively (marsupialization, enucleation, curettage) treated cases have a high recurrence rate compared to radical treatment. 6However there is no concrete data pertaining recurrence on AOT, they are benign with rare recurrence.The other odontogenic cysts included were developmental viz DC and COC, wherein DC is associated with an impacted tooth while COC is associated with calcifications and ghost epithelial cells.RC an inflammatory odontogenic cyst is commonly associated with carious or non-vital tooth. 2 Research to identify new markers to determine the biological behavior of odontogenic cysts and tumors is ongoing.Literature review reveals many preliminary observations with no concrete evidence of a single marker being specific to these tumors and hence there is a need to determine new markers. 6In this study, we have employed two markers: fascin and SALL4.][9][10] Usually, in normal adult epithelial cells fascin expression is low or absent. 11The gene encoding fascin in humans is located on chromosome 7. SALL4 is a stem cell marker and a master zinc-finger transcriptional factor, and a member of the spalt-like (SALL) gene family. 12SALL4 is mapped to chromosome 20q13.24][15] SALL4 incorporated along with OCT4, SOX2 and KLF4 (OSK) helps in forming stable induction of pluripotent cells (iPS) cells with a higher efficiency. 16][10][11][12][13] Studies have shown the expression of these stem cell markers in Ameloblastoma & OKC except SALL4. 14Most of the studies in SALL4 are related to malignant soft tissue tumors, 15,16 no reports are available of SALL4 expression in odontogenic lesions.SALL4 is activated by various pathways such as Wnt/β-catenin, 15 PI3K/AKT, signalling pathway through targeting PTEN 16 or Notch signalling pathway 17 thus facilitating migration, invasion and proliferation, while Fascin is activated via PI3K/ Akt pathway .
0][21] Hence the present study was done to evaluate and compare the expression of these two biomarkers in various odontogenic tumors (Histopathological variants of Ameloblastoma, AOT) and odontogenic cysts (OKC,DC,RC,COC).

Patients and tissue samples
Formalin fixed paraffin embedded tissue (FFPE) sections were retrieved from the Department of Oral and Maxillofacial Pathology, Manipal College of Dental Sciences, Manipal, India after obtaining approval from Institutional Ethical Committee, (IEC approval number 360/2019, IEC 156/2014).The samples taken up for the study were from year 2012-REVISED Amendments from Version 4 In this revised version of the manuscript, there are no major changes, and minor comments given by the reviewer have been addressed.
The incubation time for the primary antibodies has been mentioned in the methodology section.
In the discussion, we mentioned the limitations of our study.
In Tables 1 and 2, the p-value has been changed from 1 to >0.999.
Any further responses from the reviewers can be found at the end of the article 2017 which included 40 cases of ameloblastoma with histopathological variants viz plexiform (no:11), follicular (no:12), Unicystic (no:12), desmoplastic (no.5),6 cases of AOT, 15 cases each of OKC, DC, RC and 5 cases of COC.The cases selected did comply to inclusion and exclusion criteria.All the samples taken for the current study were prior to the patient receiving any treatment, cases with recurrence were excluded.The diagnosis of the above said odontogenic cysts and tumors were based on clinical and histological features (using H&E staining) according to WHO guidelines. 2

Immunohistochemistry (IHC)
Immunohistochemical staining of the tissue sections from each of the cases selected was done using the streptavidinbiotin method.In brief, 4 μm sections were mounted on 3-aminopropyltriethoxysilane (APES) coated slides (Novolink Polymer Detection System, Novocastra).Sections were then deparaffinized in xylene, which was done in three grades for 10 minutes and hydrated in different grades of alcohol ranging from absolute alcohol (10 minutes), 95% alcohol (10 minutes), 70% (10 minutes), 50% (10 minutes) each.Sections were then incubated with primary antibodies, rabbit antihuman SALL4 monoclonal antibody at a dilution of 1:100(IgG, clone EP-299, PathnSitu, Livermore, USA), mouse antihuman fascin monoclonal antibody (IgG1,clone 55K-2, SC-21743, Santa Cruz Biotechnology USA, Inc) diluted at 1:200.The incubation time for SALL4 with the primary antibody was for 60 and for fascin, the incubation time was 30 minutes at room temperature.The sections were subsequently washed in tris-buffered saline and incubated with secondary biotinylated antibody and streptavidin-biotin peroxidase complex (Novolink Polymer Detection System, Novocastra) for 30 minutes each.Diaminobenzidine (DAB) was used as the chromogen and the sections were counterstained with Mayer's hematoxylin.Buccal mucosa tissue and oral squamous cell carcinoma were used as positive controls, 22 the basal cells of the epithelium of buccal mucosa were stained positive, and endothelial cells within the lesional tissue were internal controls for fascin antibody (Figure 1), while dysgerminoma was taken as a positive control, for SALL4, for positive nuclear expression (Figure 2).Bud and bell stage of tooth development were also included for the study as controls.The primary antibody was replaced during IHC staining for the negative control as per standard immunohistochemical protocol.The document of the protocol has been uploaded in the repository (Open Science Framework protocol.io). 49munostaining evaluation Presence of brown color at the end of staining was considered as positive reactivity.The slides were evaluated with a light microscope (Olympus BX41) attached with Olympus DP20 microscope camera (Olympus Singapore Pvt Ltd, Singapore) at 20Â & 40Â magnification.The distribution of antibodies was assessed in the cytoplasm and cell membrane of ameloblastic lining of the lesions for fascin while SALL4 staining was evaluated in nuclear areas.In each case, three fields were randomly selected, and two observers independently evaluated the expression of these biomarkers, after selecting the most representative site separately under a light microscope at 200Â and 400Â magnification to eliminate the bias.

Statistical analysis
The data obtained was statistically analyzed with the statistical software program SPSS (version 17.0).The statistical significance of fascin and SALL4 in histopathological types of ameloblastoma was analysed using the chi-square test.P values less than 0.05 were considered to indicate statistical significance.

Results
Immunohistochemically stained sections of various odontogenic cysts and tumors were evaluated for expression of fascin in the cell membrane, between cell boundaries and cytoplasm of peripheral ameloblastic cells, stellate reticulum like cells and stromal cells of 40 cases of ameloblastoma variants while expression of SALL4 was observed in the cytoplasm as well as nuclei of peripheral ameloblastic cells and stellate reticulum like cells.The total IRS score was the main outcome (Table 1, Table 2).Chi-square test was used to compare the frequency of distribution of categorized total IRS score with fascin and SALL4 in various odontogenic tumors (Ameloblastoma and its histopathological variants, AOT) and odontogenic cysts (OKC, DC, COC, RC).The expression of fascin and SALL4 varied from case to case as well as in the same tissue section.Most of the variants of ameloblastoma were strongly positive for fascin but cases of desmoplastic ameloblastoma (5/5) were negative for fascin (Figure 1D).Fascin expression was found to be weak or absent in stellate reticulum like cells (Figure 1).In cases of unicystic ameloblastoma, positivity for fascin was observed in the basal as well as in the suprabasal layers (Figure 1C).However intra-group comparison did not show any significant difference.AOT was immune-positive to fascin in few areas (< 25%) with mild to moderate intensity (Figure 1E).Fascin expression in odontogenic cysts (OKC, RC, DC) (Figure 1G-I) was strongly positive with greater than 75% cells, while intensity ranged from moderate to strong along the cystic lining.COC revealed immune positivity ranging from 25-50% (Figure 1F).The SALL4 positivity was heterogeneous with varied intensity and staining pattern.In most of the histopathological variant of ameloblastoma, the immunopositivity observed, was diffuse in the cytoplasm and less localised to the nucleus (Figure 2A-C).The stromal cells were devoid of its expression except in the endothelial cells.SALL4 expression in odontogenic cysts was strongly positive with greater than 75% cells exhibiting diffuse cytoplasmic staining.Nuclear staining was evident in few cells (Figure 2E-G).COC was immune-negative (Figure 2H).
the higher cell counts were observed in fascin as compared to SALL4 in odontogenic keratocyst.The data regarding the same has been attached as Supplementary files(S1,S2,S3,S4) and has been uploaded in the repository (open science framework).

Discussion
Researchers have worked on the molecular mechanism to understand the nature of local invasion of ameloblastomas into the surrounding tissues which include molecules degrading the extracellular matrix, those involved in bone remodelling, molecules associated with angiogenesis and molecules related to proliferation. 245][26][27][28] Cell motility is essential for tumor invasion and subsequent dissemination or metastases.This increase in motility occurs via the modulation of actin filaments to form finger-like plasma membrane protrusions termed invadopodia.Numerous actinbinding proteins, including fascin, regulate such dynamic rearrangement of the actin cytoskeleton.Fascin, being one of the actin cross-linking proteins, localizes to filopodia at the leading edge of migratory cells by organising f-actin into wellordered, tightly packed parallel bundles observed in vitro studies. 291][32][33][34] In our study, we observed that a majority of our cases were strongly positive for fascin in the various subtypes of ameloblastoma.
Various in vitro and in vivo studies have observed that fascin has a functional role in cell invasion and motility. 35This could account to the local aggressiveness of ameloblastoma clinically.Few of the ameloblastic follicles did not exhibit fascin, we speculate this could be attributed to loss of antigen during processing or reduced motility in these cells.Fascin expression in various cysts such as DC,OKC,RC and COC could be related to its influence in focal adhesion and cell dynamics. 36 various histopathological grades of ameloblastoma, SALL4 was expressed in the majority of cases.
][39][40][41][42][43][44][45] We observed that the odontogenic epithelial cells were positive for SALL4 in the cytoplasm, stained diffusely, which we speculate could be in an inactive/dormant or mutant form which requires further investigation.Majority of OKC were devoid of SALL4 except in the basal cells.Radicular and dentigerous cysts, having marked infiltration of inflammatory cells had strong immune-positivity for SALL4 in the cytoplasm, an interesting finding of this study.Hence the role of cytokines in stimulating SALL4 needs to be ruled out.Odontogenic tumors, AOT and developmental odontogenic cysts, COC (simple type) were negative for SALL4.Studies have shown that OKCs expressed higher amount of PCNA and Ki-67 when compared to other jaw cysts, indicating its inherently increased proliferative potential of OKC. 46This speculates that various other molecular pathways could play an important role in the disease process.Further studies are required to explore this possibility, since this is a preliminary study.
Normal connective tissue cells such as fibroblasts, vascular endothelial cells, neural and glial cells, brain and splenic tissue expressed fascin, which relates to its function, required to maintain normal homeostasis. 31In embryogenesis, various migratory cells express fascin, except in terminally differentiated squamous cells where its expression is low or absent. 31,47Our previous study on tooth buds showed fascin expression in various stages of tooth development was site and time specific, thus confirming its role in cell remodulation. 3SALL4 expression was not detected in tooth bud stage, however focal positivity was observed in the cytoplasm of the epithelial cells of bell stage, this could attribute to the cells to undergo more differentiated state of the cells. 48The papillary cells in various stages of tooth germ were positive (Figure 2J) and this could relate to stemness due to the pluripotency nature of dental papilla.
"In the current study, one of the limitations was the small sample size.Studies with a larger sample size are needed to understand the molecular (fascin and SALL4) dynamics and functional behavior which is required for further validation and unravel the interaction of the pathways that activate these molecules.The interactivity of these molecules with other stem cell molecules in maintaining the stemness or pluripotency state of the cells needs to be explored." In conclusion, the findings of the present study on the expression of fascin elucidate their role in motility and localized invasion or in maintaining the cellular homeostasis, while the expression of SALL4 remains elusive.

Data availability
Open Science Framework: Expression of fascin and SALL4 in odontogenic cysts and tumors: an immunohistochemical appraisal.https://doi.org/SALL4.While fascin and SALL4 have not been previously reported in odontogenic tumors, some of these pathways have.This relationship should be included in the discussion.Please include how long tissues were incubated with the primary antibody as it has relevance to repeatability.

2.
The authors state "Buccal mucosa tissue was used as a positive control…" Would not this mean these markers are poor biomarkers for odontogenic tumors if they are generally expressed in the oral cavity.

3.
Figures 1 and 2 should be more standardized in appearance and presentation.Some images are darker than others making it difficult to see what is positive.The samples should be presented in the same order and same magnification in each figure for ease of comparison and clarity (i.e., A-D. 4 forms of ameloblastoma, E. AOT, F. COC, G. OKC, H. DC, I. RC, J-K.Positive controls.
Figure 2E does not appear positive as stated and is not clear.7.
The expression of fascin looks quite strong in Figure 1E.Were multiple images used in the pathologic evaluation? 8.
Literature has not shown any clinical significance to histologic variants.A comparison of unicystic ameloblastoma to conventional ameloblastoma would be more relevant.

9.
In the results there is mention of SALL4 nuclear staining.The magnifications shown are too low to see this in the figures presented.Perhaps include an offset at a higher magnification where you wish to demonstrate nuclear staining.

10.
For fascin to be acting as described in the discussion it would need to be staining more organized (with the actin cytoskeleton) rather than diffusely.Please relate your discussion items to your data more clearly.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Odontogenic tumors I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.
Author Response 22 Jun 2024

Dr. Spoorti Kulkarni
We thank Dr. Hope M Amm for reviewing our article and providing valuable feedback.We have carefully considered and addressed the comments.
Reviewer Q1: The authors spend a portion of the introduction telling what pathways regulate fascin and SALL4.While fascin and SALL4 have not been previously reported in odontogenic tumors, some of these pathways have.This relationship should be included in the discussion.
Author Response: The present study evaluated the immuno-expression of two markers Fascin and SALL4.A detailed findings of the current study were discussed.We did emphasize in the discussion the pathways to be explored concerning these markers."In the current study, one of the limitations was the small sample size.Studies with a larger sample size are needed to understand the molecular ( fascin and SALL4) dynamics, functional behavior which is required for further validation and unravel the interaction of the pathways that activate these molecules.The interactivity of these molecules with other stem cell molecules in maintaining the stemness or pluripotency state of the cells needs to be explored." Reviewer Q2: Please include how long tissues were incubated with the primary antibody as it has relevance to repeatability.Response: The incubation time for SALL4 with the primary antibody was for 60 minutes at room temperature and the incubation time for fascin with primary antibody was 30 minutes at room temperature, as per the manufacturer's protocol.The same has been incorporated in the methodology.
Reviewer Q3: The authors state "Buccal mucosa tissue was used as a positive control…" Would not this mean these markers are poor biomarkers for odontogenic tumors if they are generally expressed in the oral cavity.
Author Response: Tooth buds and buccal mucosa were taken as controls also the markers (SALL4& Fascin) employed for the current study are not specific for odontogenic tumors.Moreover, the basal cell of the surface epithelium is one of the proposed pathogenesis contributing to ameloblastomas( Reference: Sivapathasundharam B. Shafer's Textbook of Oral Pathology.9th ed.St. Louis: Elsevier; 2020.Reviewer Expertise: Oral and maxillofacial pathology (oral precancer cancer, odontogenic lesions, dental caries, oral infections, salivary gland lesions, oral developmental disorders), basic 3D medical animation.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Pentti Nieminen
Medical Informatics and Statistics Research Group, University of Oulu, Oulu, Finland The statistical reporting has been improved.A few more points could be improved.1.I could not find any mention of the small number of cases in the discussion section.Please check whether the following sentences have been omitted from the manuscript: "However, in the present study the smaller sample size in odontogenic tumors and odontogenic cysts was one of the limitations.Therefore, for the better understanding of the dynamic and functional behavior of these molecules in the odontogenic lesions, studies on larger sample size and further experimental validation to elucidate their functional significance needs to be done."2. A minor comment: The reporting of results could be improved by replacing p-value 1 with "p>0.999".P-value cannot be equal to 1.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility?Partly

Are the conclusions drawn adequately supported by the results? Partly Introduction
Clarify if fascin and fascin-1 are synonymous terms which has been used.1.

Methods
Resolve the discrepancy between the stated 46 odontogenic tumours samples and the 52 listed in the table.additionally providing descriptions of abbreviations S/NS.

1.
Do mention all-control sample types used.2.

RESULTS -
verify the accuracy of the row entries for IRS, SALL4, and fascin in Tables 1 and 2. 1.
Cite the specific IRS scoring method utilized, considering the multiple such methods are available in the literature .itwill help future researchers.

2.
Include few high magnification microphotographs to show cytoplasmic and nuclear staining.

Discussion-
The authors could enhance the study's value by sharing personal experiences and overcoming difficulties faced, benefiting future researchers. 1.
Overall, this well-executed study could be further strengthened by incorporating the above revisions to improve clarity and account for unique aspects of odontogenic lesions.The authors have produced an impressive body of work with room for refinement.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?I cannot comment.A qualified statistician is required.

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: oral pathology ,oral precancer cancer,odontogenic lesions I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Introduction Q 1: Clarify if fascin and fascin-1 are synonymous terms which has been used.Response: The correction has been made as 'fascin' in the introduction.

Q1:
Resolve the discrepancy between the stated 46 odontogenic tumours samples and the 52 listed in the table.additionally providing descriptions of abbreviations S/NS.Response: The discrepancy has been corrected in Table 1 and the abbreviations have been added for Table 1 and Table 2 in the revised version.
Q2:Do mention all-control sample types used.Response: In the section of immunohistochemistry(IHC), we have mentioned all the positive control as well as internal controls for both the markers in the revised version.

Results
Q1:verify the accuracy of the row entries for IRS, SALL4, and fascin in Tables 1 and 2. Response: Verification has been done and corrected in Table 1 and 2 Discussion-Q1:The authors could enhance the study's value by sharing personal experiences and overcoming difficulties faced, benefiting future researchers.Response: The main focus of the study was to understand one of the cellular processes in terms of motility and migration, which was elucidated by fascin positivity and SALL4 was employed for its stemness behavior.Advanced molecular techniques need to be employed to understand the dynamic behavior of these cells and the various crosstalks with other Tables 1 and 2: You have compared the fascin and SALL 4 expressions using chi-square test.You have not stated this research question in the introduction or methods sections.Why to compare these distributions?

2.
Statistical analysis sub-section: Please clearly state that you have presented the frequency distributions of the fascin and SALL 4 expressions by the sub-types of odontogenic tumors and cysts.Identify the main response variable (total IRS score ) here.

3.
Tables 1 and 2: Please indicate in the SALL4 and fascin columns that you report frequencies.Please report also total sample size in the title.The use of statistical significance testing is not motivated in the introduction section and could be removed.The titles still need improvement.

4.
The small number of cases in several subgroups of tumours and cysts is a limitation of your study.You have not addressed this in the discussion section.

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Author Response 09 Dec 2023

Dr. Spoorti Kulkarni
We thank Pentti Nieminen for reviewing our article and giving us valuable comments.We have carefully reviewed the comments and revised the manuscript accordingly.All comments pointed out by the reviewer have been corrected/revised, as below: Q1: Tables 1 and 2: You have compared the fascin and SALL4 expressions using the chisquare test.You have not stated this research question in the introduction or methods sections.Why to compare these distributions?Response: We have put forth the research question in the abstract, the comparison of fascin and SALL4 in the abstract (methodology section).However, we have revised the same in the introduction too.The literature search reveals a crosstalk between the pathway of these biomarkers (fascin and SALL4) which has been explained in the introduction.Hence an attempt was made to evaluate the expression as well as compare the expression of these markers in this study.Q2: Statistical analysis sub-section: Please clearly state that you have presented the frequency distributions of the fascin and SALL4 expressions by the sub-types of odontogenic tumors and cysts.Identify the main response variable (total IRS score) here.Response: Chi-square test was used to compare the frequency of distribution of categorised total IRS score with fascin and SALL4 in various odontogenic tumors (ameloblastoma and its

Qi-Wen Man
Wuhan University, Wuhan, Hubei, China I have carefully reviewed your research on the immunohistochemical expression of Fascin and SALL4 in ameloblastoma, adenomatoid tumor, and odontogenic cysts.The study aims to provide insights into these lesions by examining the expression of specific biomarkers.While the research direction is promising, there are several areas that require further attention to enhance the overall quality of the study: The inclusion of multiple diseases as research subjects is commendable for its comprehensiveness.However, a critical concern lies in the lack of detailed characterization of each disease and their potential interrelationships.Providing a comprehensive description of their specific characteristics and how they relate to each other would enrich the study and offer a more robust foundation for future research. 1.
The research would benefit from a more in-depth discussion of the developmental 2.
prospects of the selected biomarkers, Fascin, and SALL4.Additionally, it is essential to highlight the advantages of these biomarkers compared to other potential candidates.
While you have highlighted Fascin's involvement in the invasiveness of ameloblastoma, further elucidation is necessary regarding the reasons for its overexpression in Dentigerous cysts, Odontogenic Keratocyst, and Radicular cysts, or put forward corresponding conjectures.Similarly, the role of SALL4 in these dental pathologies requires more thorough exploration and explanation.

3.
The absence of normal tissue as a control for IHC staining results is a notable concern.To strengthen the study's validity, it is crucial to include normal tissue controls or appropriate references for comparison.

4.
The statistical tables used to present the IHC results require improvement.Employing more robust statistical methods, such as significance testing, to analyze the differences between lesions would enhance the analysis and provide more reliable conclusions.

5.
Regarding the speculation on the functional roles of the two biological markers, it is suggested to conduct further experimental validation to elucidate their functional significance in the corresponding pathologies.

6.
The background of the IHC staining was not the same which might influence the scores and analysis.

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Immunohistochemistry, RNA sequencing, Odontogenic tumors I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Author Response 05 Sep 2023

Dr. Spoorti Kulkarni
We thank Qi-Wen Man for reviewing our article and giving us valuable comments.I appreciate the time and efforts spent to make our work better.I have carefully reviewed the comments and revised the manuscript accordingly.All comments pointed out by the reviewer have been corrected.
I have responded question wise.1: The inclusion of multiple diseases as research subjects is commendable for its comprehensiveness.However, a critical concern lies in the lack of detailed characterization of each disease and their potential interrelationships.Providing a comprehensive description of their specific characteristics and how they relate to each other would enrich the study and offer a more robust foundation for future research.
Literature review states among the odontogenic lesions Ameloblastoma and Odontogenic keratocyst are locally aggressive and recurrent lesions, also Ameloblastoma are the commonest and prevalent odontogenic tumor in Indian population is ameloblastoma which ranges from 14.02% to 71.4% when compared to other odontogenic tumors 3,4 , while globally, pooled estimate of the incidence rate of ameloblastoma is 0.92 per million population per year 5 .Among the odontogenic cyst we have highlighted only Odontogenic keratocyst, since these lesions are aggressive and recurrent compared to other cysts such as DC, a cyst associated with impacted tooth, RC, a cyst associated with caried or non-vital tooth, COC, a developmental cyst associated with calcification and ghost epithelial cells.We have incorporated the same in introduction.2: The research would benefit from a more in-depth discussion of the developmental prospects of the selected biomarkers, Fascin, and SALL4.Additionally, it is essential to highlight the advantages of these biomarkers compared to other potential candidates.
Response: Literature review confirms fascin contributes for cell motility and migration in many studies (Pubmed 376 articles), SALL4 contributes to stemness along with other stem cell markers 8-13 SOX2, OCT4, and NANOG.Studies have shown the expression of these stem cell markers in Ameloblastoma & OKC except SALL4. 14Most of the studies in SALL4 are related to malignant soft tissue tumors , [15][16] no reports are available of SALL4 expression in odontogenic lesions.Hence the present study aimed to evaluate the expression of fascin and SALL4 in odontogenic cysts & tumors.

Major concerns
The Title refers to odontogenic cysts and tumors, but as it turns out the study includes two tumors and many different cysts.Why did the authors choose to examine those lesions?

○
In the Introduction there is some information on three of the lesions investigated (what about DC, RC or COC?) and this is inconsistent, i.e., they present recurrence rate for OKC, but not for ameloblastoma or AOT.The authors should consistently present the same data for each lesion.

○
Fascin and SALL4 seem to be two unrelated.Is there any association between them?Why did they choose to examine them?The authors use some references about the role in malignant neoplasms, however the lesions under consideration are not malignant neoplasms, while most of the references do not include studies on their expression in malignant neoplasms.Please carefully check your references.
The aim of the study should be concise and clear.It should be reconsidered.
○ M&M: "Before the start of the study, statistician was consulted and based on the literature review, availability of the material in the archives of the department and the availability of the budget the sample was decided".Are 6 AOTs or 5 COC's an adequate sample size?

○
The Patients and Tissues samples section needs restructuring.Origin of material, inclusion/exclusion criteria, IRB etc.

○
What was the positive control for Fascin, the basal cells of the oral epithelium (Figure ) or the endothelial cells of the buccal mucosa (text)?Any supporting reference of any of them?

○
In general, please present the findings in a more orderly manner, i.e., what did you see in the positive controls (membranous and cytoplasmic expression in the basal cells of the epithelium, etc.).

○
Follow the same order of the antibodies, i.e., if you choose Fascin/SALL4 then present the results first for fascin and then for SALL4.
○ "40 cases of ameloblastoma variants from the year 2012 to 2017".This information is not proper here.

○
Results should be rewritten in a more orderly manner.

○
Discussion: "Researchers have worked on…".There are no references to support this.

○
Discussion: "the odontogenic epithelial cells were positive for SALL4 in the cytoplasm, stained diffusely, which we speculate could be in an inactive/dormant or mutant form which requires further investigation".Is there any literature supporting that cytoplasmic expression in not a cross reaction with another protein?

○
The last paragraph of the Discussion is interesting but lacks connections with the rest of the Discussion.

Minor considerations:
"Are said to": this is not an "mouth to ear" information, it is well established in the literature.
○ "for its local but aggressive biological behavior": What do they mean by "local" behavior?

○
Please check the classification of ameloblastomas, it is not correct.Furthermore, in the M&M, ameloblastomas are subclassified based on their histopathological features, but there in no mention to them in the Intro.
○ "fascin might contribute for the local migratory behavior of these odontogenic cells".Which cells?
○ "The document of the protocol has been uploaded in the repository (Open Science Framework protocol.io) 26and hydrated".Was the protocol hydrated?Please carefully check the text throughout the manuscript.
○ "To eliminate bias, two observers independently evaluated the expression of these, selecting the most representative site separately under a light microscope..." This information should not be presented here.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound?Partly

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?I cannot comment.A qualified statistician is required.

Are the conclusions drawn adequately supported by the results? No
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Oral pathology I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.
I have responded question wise.
Q: The Title refers to odontogenic cysts and tumors, but as it turns out the study includes two tumors and many different cysts.Why did the authors choose to examine those lesion?
Response: Literature review states among the odontogenic lesions Ameloblastoma and Odontogenic keratocyst are locally aggressive and recurrent lesions, also the commonest and prevalent odontogenic tumor in Indian population is ameloblastoma which ranges from 14.02% to 71.4% when compared to other odontogenic tumors 3,4 ,while globally, pooled estimate of the incidence rate of ameloblastoma is 0.92 per million population per year. 5The Response: Among the odontogenic cyst we have highlighted only Odontogenic keratocyst, since these lesions are aggressive and recurrent compared to other cysts such as DC, RC or COC, we have incorporated the same in introduction.We have also modified the same for Ameloblastoma as well, in 'Introduction': 'The recurrence varies among various populations, 9.8% according to a Chinese study, 6 while in European multicenter study it is reported to be 19.3%, 7 also tumors larger than 6 cm and involving the soft tissues or adjacent anatomical structures are associated with early recurrence irrespective of method of surgery.Also, conservatively (marsupialization, enucleation, curettage) treated cases have a high recurrence rate compared to radical treatment.Q: Fascin and SALL4 seem to be two unrelated.Is there any association between them?Why did they choose to examine them?The authors use some references about the role in malignant neoplasms, however the lesions under consideration are not malignant neoplasms, while most of the references do not include studies on their expression in malignant neoplasms.Please carefully check your references ][10][11][12][13] Studies have shown the expression of these stem cell markers in Ameloblastoma & OKC except SALL4. 14Most of the studies in SALL4 are related to malignant soft tissue tumors, 15,16 no reports are available of SALL4 expression in odontogenic lesions.Hence the present study aimed to evaluate the expression of fascin and SALL4 in odontogenic cysts & tumors.
activated by various pathways such as Wnt/β-catenin, 15 PI3K/AKT, signalling pathway through targeting PTEN 16 or Notch signalling pathway 17 , thus facilitating migration, invasion and proliferation, while Fascin is activated via PI3K/ Akt pathway. 180][21] Hence we have made an attempt to study the expression of these two markers.
inclusion/exclusion criteria, IRB etc.The samples taken up for the study did comply to inclusion and exclusion criteria which included histologically diagnosed cases of odontogenic cysts and tumors from 2012-2017.

Response
All the samples taken for the current study were prior to the patient receiving any treatment, cases with recurrence were excluded.The diagnosis of the above said odontogenic cysts and tumors were done based on clinical and histological features (using H&E staining) according to WHO guidelines.
Q:What was the positive control for Fascin, the basal cells of the oral epithelium (Figure ) or the endothelial cells of the buccal mucosa (text)?Any supporting reference of any of them Response: Buccal mucosa tissue 22 (Figure 1), was considered as external positive control while endothelial cells with in the samples to be tested was considered for internal positive control for Fascin expression.The reference article is as below.Response: For fascin, the positive control used was buccal mucosa, the cytoplasm of the basal cells of the epithelium were stained positive.In case of SALL4 dysgerminoma was taken as positive control where in nuclear expression in the cells is seen.
Q:Follow the same order of the antibodies, i.e., if you choose Fascin/SALL4 then present the results first for fascin and then for SALL4.

Response: Correction is done as follows:
Immunohistochemically stained sections of various odontogenic cysts and tumor tissue were evaluated for expression of fascin in the cell membrane, between cell boundaries and cytoplasm of peripheral ameloblastic cells, stellate reticulum like cells and stromal cells of 40 cases of ameloblastoma variants while expression of SALL4 was observed in the cytoplasm as well as nuclei of peripheral ameloblastic cells and stellate reticulum like cells.The total IRS score was the main outcome (Table 1, Table 2).The expression of fascin and SALL4 varied from case to case as well as in the same tissue section.Most of the variants of ameloblastoma were strongly positive for fascin but cases of desmoplastic ameloblastoma (5/5) were negative for fascin (Figure 1D).Fascin expression was found to be weak or absent in stellate reticulum like cells (Figure 1).In cases of unicystic ameloblastoma, positivity for fascin was observed in the basal as well as in the suprabasal layers (Figure 1C).However intra-group comparison did not show any significant difference.AOT was immune-positive to fascin in few areas (< 25%) with mild to moderate intensity (Figure 1E).Fascin expression in odontogenic cysts (OKC, RC, DC) (Figure 1G-I) was strongly positive with greater than 75% cells, while intensity ranged from moderate to strong along the cystic lining.COC revealed immune positivity ranging from 25-50% (Figure 1F).The SALL4 positivity was heterogeneous with varied intensity and staining pattern.In most of the histopathological variant of ameloblastoma, the immunopositivity observed, was diffuse in the cytoplasm and less localised to the nucleus (Figure 2A-C).The stromal cells were devoid of its expression except in the endothelial cells.SALL4 expression in odontogenic cysts was strongly positive with greater than 75% cells exhibiting diffuse cytoplasmic staining.Nuclear staining was evident in few cells (Figure 2E-G).COC was immune-negative (Figure 2H).
Regarding the evaluation of the statistical significance test, in all the odontogenic tumors, the staining intensity of fascin is similar compared to SALL4.With regard to the stained cell count, higher counts are observed with fascin as compared to SALL4.In relation to odontogenic cysts, odontogenic keratocyst and dentigerous cyst,the intensity of fascin is more than SALL4.Also the higher cell counts were observed in fascin as compared to SALL4 in odontogenic keratocyst.Q:Discussion: "the odontogenic epithelial cells were positive for SALL4 in the cytoplasm, stained diffusely, which we speculate could be in an inactive/dormant or mutant form which requires further investigation".Is there any literature supporting that cytoplasmic expression in not a cross reaction with another protein?
10 minutes and hydrated in different grades of alcohol (ranging from absolute alcohol (10 minutes), 95 % alcohol (10 minutes), 70% (10 minutes), 50% (10 minutes) each).Sections were then incubated with primary antibodies,rabbit antihuman SALL4 monoclonal antibody at a dilution of 1:100(IgG, clone EP-299, PathnSitu, Livermore, USA ),mouse antihuman fascin monoclonal antibody ( IgG1,clone55K-2, SC-21743, Santa Cruz Biotechnology USA, Inc) diluted at 1:200.The sections were subsequently washed in tris-buffered saline and incubated with secondary biotinylated antibody and streptavidin-biotin peroxidase complex (Novolink Polymer Detection System, Novocastra) for 30 minutes each.Diaminobenzidine (DAB) was used as the chromogen and the sections were counterstained with Mayer's hematoxylin.Buccal mucosa tissue was used as positive control and endothelial cells were internal controls for fascin antibody (Figure 1), while dysgerminoma was taken as a positive control, bud and bell stage of tooth development were also included for the expression of SALL4 (Figure 2).The primary antibody was replaced during IHC staining for the negative control as per standard immunohistochemical protocol.The document of the protocol has been uploaded in the repository (Open Science Framework protocol.io)48Q:"incubated with primary antibodies (rabbit monoclonal IgG for SALL4 and mouse monoclonal IgG1 for fascin) 26 against fascin (clone SC-21743, Santa Cruz Biotechnology USA, Inc) diluted 1:200".Please correct.

Response:
We corrected the same as follows: Sections were then incubated with primary antibodies,rabbit antihuman SALL4 monoclonal antibody at a dilution of 1:100(IgG, clone EP-299, PathnSitu, Livermore, USA ),mouse antihuman fascin monoclonal antibody ( IgG1,clone55K-2, SC-21743, Santa Cruz Biotechnology USA, Inc) diluted to 1:200 Q:"To eliminate bias, two observers independently evaluated the expression of these, selecting the most representative site separately under a light microscope..." This information should not be presented here

Response:
We have removed the sentence from results and incorporated in 'Immuno staining evaluation'.Immunostaining evaluation Presence of brown color at the end of staining was considered as positive reactivity.The slides were evaluated with a light microscope (Olympus BX41) attached with Olympus DP20 microscope camera (Olympus Singapore Pvt Ltd, Singapore) at 20× & 40× magnification.The distribution of antibodies was assessed in the cytoplasm and cell membrane of ameloblastic lining of the lesions for fascin while SALL4 staining was evaluated in nuclear and cytoplasmic areas.In each case, three fields were randomly selected, and two observers independently evaluated the expression of these, selecting the most representative site separately under a light microscope at 200× and 400× magnification.
Response: Yes, OKC, dentigerous are developmental while radicular cyst is inflammatory in origin In this study, the expression of SALL4 and fascin were evaluated in odontogenic tumours and cysts.The findings of the study on fascin expression clarify their role in motility and localized invasion or maintenance of cellular homeostasis.However, the expression of SALL4 remains unclear.
The focus of this review is mainly on data reporting.My comments to the authors are as follows: One positive aspect of your manuscript was that it was a short report, which kept the focus on the main objective. 1.
I enjoyed reading the introduction section of this manuscript.The research question related to was well described.

2.
Introduction, last sentence: Please consider specifying the aims of your study.It would help your readers if you stated that the aim was to estimate the prevalence of two biomarkers, SALL4 and fascin, in histopathological variants of ameloblastoma, AOT and various odontogenic cysts.In addition, your aim was to compare the prevalence of biomarkers between different tumours and cysts.

3.
As a short report, the statistical intensity of the manuscript was lower than the average of articles published in visible general medical journals.However, the quality of statistical reporting and data presentation was weak: I gave it a score of 3 on a scale of 0 (poor) to 10 (very high).In particular, the quality of data presentation in tables should be improved.

4.
Consider including a table describing the distributions of the basic characteristics of the cases, their clinical history, and the main outcome variables.If many characteristics of human cases are described, the details of the cases and the number of cases contributing to the analysis are best included in a tabular presentation.

5.
Tables 1A and 1B are not well prepared.Tables with proper titles, clear labelling and optimally presented data will enable readers to scrutinise the data.The tables did not have clear titles.The current formatting of these tables resembled a spreadsheet, and the lines of 6.
In relation to odontogenic cysts viz odontogenic keratocyst and dentigerous cyst, the intensity of fascin staining is more than SALL4.Also the higher cell counts were observed in fascin as compared to SALL4 in odontogenic keratocyst.
7. Statistical analysis section should help a knowledgeable reader to judge the appropriateness of the methods for the study and to verify the methods reported.You should improve the description of statistical methods.State clearly that total staining scores of SALL4 and fascin are your main outcome measures.Then describe that the prevalence of these response variables was estimated in different tumour and cyst groups.Clarify the variables and methods for each significance test performed in the study.

Response:
We have uploaded the modified tables and these tables are self-explanatory, as per the advice of the reviewer.Fischers exact test was used to compare the distribution of staining intensity and cell count across SALL4 and Fascin.
8. The small number of cases in several subgroups of tumours and cysts is a limitation of your study.Please address this in the discussion section.

Response:
The small number of cases in the present study with regard to odontogenic tumors and its subgroup as well as in odontogenic cysts were limitation of this study.
Further studies with large sample size is required for the better understanding of these biomarkers in these lesions.
Competing Interests: No competing interests were disclosed.
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Reviewer Q4 :
Figures 1 and 2 should be more standardized in appearance and presentation.Some images are darker than others making it difficult to see what is positive.The samples should be presented in the same order and same magnification in each figure Sandhya Tamgadge D.Y. Patil University School of Dentistry, Navi Mumbai, Maharashtra, India I have no further comments to make.The authors have diligently addressed the concerns which were raised and incorporated the necessary revisions, resulting in a significantly improved and enriched manuscript.The revisions have enhanced the clarity and depth, making it a valuable contribution to the field of oral pathology.While this study has provided valuable insights and advancements, there are still avenues for further exploration.Future research could focus on more molecular markers as stated by authors to gain a more comprehensive understanding.The authors are encouraged to continue this research and build upon the foundation laid by this study.Is the work clearly and accurately presented and does it cite the current literature?Partly Is the study design appropriate and is the work technically sound?Partly Are sufficient details of methods and analysis provided to allow replication by others?Partly If applicable, is the statistical analysis and its interpretation appropriate?Partly Are all the source data underlying the results available to ensure full reproducibility?Partly Are the conclusions drawn adequately supported by the results?Partly Competing Interests: No competing interests were disclosed.
. Q1.Cite the specific IRS scoring method utilized, considering the multiple such methods are available in the literature .itwill help future researchers.Response: Citation for IRS scoring method has been given as a modified version.(Ref: Klein M, Picard E, Vignaud JM, Marie B, Bresler L, Toussaint B, Weryha G, Duprez A, Leclere J. Vascular endothelial growth factor gene and protein: strong expression in thyroiditis and thyroid carcinoma.Journal of Endocrinology.1999 Apr 1;161(1):41-50).
(S1,S2,S3,S4,Table1,Table2) Q:Results should be rewritten in a more orderly manner.Response: Correction has been made Q:40 cases of ameloblastoma variants from the year 2012 to 2017".This information is not proper here.Response: The above sentence has been removed as instructed.Q:Discussion: "Researchers have worked on…".There are no references to support this Response: The reference article for support, Ref no 23: Fuchigami T, Ono Y, Kishida S, Nakamura N.Jpn Dent Sci Rev. 2021 Nov;57:27-32.: Molecular biological findings of ameloblastoma.

Table 1 .
Expression of Fascin and SALL4 in odontogenic tumors.

Table 2 .
Expression of Fascin and SALL4 in odontogenic cysts.
archives of the department did have other odontogenic lesions which were taken up for study

:
Samples were retrieved from the Department of Oral and Maxillofacial Pathology, Manipal College of Dental Sciences, Manipal, India after the approval from the