|Year : 2011 | Volume
| Issue : 3 | Page : 493-494
|Quantitative assessment of expression of cell adhesion molecule (CD44) splice variants: CD44 standard (CD44s) and v5, v6 isoforms in oral leukoplakias: An immunohistochemical study
Pournima Y Godge1, Leena S Poonja2
1 Department of Oral Pathology and Microbiology, Terna Dental College and Hospital, Navi Mumbai, India
2 Department of Oral Pathology and Microbiology, MGM College, Navi Mumbai, India
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|Date of Submission||29-Nov-2010|
|Date of Decision||09-Feb-2011|
|Date of Acceptance||27-Apr-2011|
|Date of Web Publication||3-Nov-2011|
| Abstract|| |
Aim: The aim of this study was to semiquantitatively analyze the immunohistochemical expression pattern of CD44 standard (CD44s) and variant (CD44v) isoforms in leukoplakias using a panel of monoclonal antibodies recognizing epitopes of CD44s and of the variant exons v5 and v6.
Objective: To evaluate the efficacy of CD44s and CD44 v5, v6 immunoexpression as possible molecular markers in detecting high-risk leukoplakias when screening for this oral precancer.
Materials and Methods: Samples of oral leukoplakia (40 cases) and of normal mucosa (10 cases) were evaluated. Oral leukoplakia was graded into: hyperkeratosis without dysplastic change (8 cases), mild dysplasia (13 cases), moderate dysplasia (10 cases), and severe dysplasias (9 cases). Expression of CD44s,v5, v6 was analyzed by immunohistochemistry in a semiquantitative manner. Three areas of epithelium were scored B, S, and C, i.e., stratum basale, stratum corneum, and stratum spinosum, respectively in leukoplakias. Scoring of all specimens followed a two-parameter system, which implemented percentage of positive cells and staining intensities. Statistical analyses for each parameter of all groups and normals, mean, and standard deviation were calculated by using computer software package EPISTAT.
Results: In normal epithelium CD44s, CD44v5, and CD44v6 were expressed as membranous proteins localized on the surface of epithelial cells. Both basal and spinous layer of epithelia expressed strong positive staining of CD44s, v5, v6 which then gradually faded into the negative staining of the superficial keratin layer. Profile of CD44s and v5 revealed that the mean levels of stratum B, S, and C in normal cases were comparable to the study cases and by Student 't' test P>0.05 not significant. There was, however, a statistically significant decrease in the expression of v6 with increasing grades of dysplasias when compared with normal mucosa.
Conclusion: Among CD44s and its variant isoforms,v5, v6, in this study, variant isoform v6 may serve as a marker in detecting high-risk leukoplakias.
Keywords: CD44, dysplasia, immunohistochemistry, leukoplakia
|How to cite this article:|
Godge PY, Poonja LS. Quantitative assessment of expression of cell adhesion molecule (CD44) splice variants: CD44 standard (CD44s) and v5, v6 isoforms in oral leukoplakias: An immunohistochemical study. Indian J Dent Res 2011;22:493-4
Oral cancer is a major health problem in many parts of the world. While its incidence is relatively low in most Western countries, in the Indian subcontinent and in other parts of Asia, however, it remains one of the most common forms of cancer.  Histologically, over 95% of oral cancers are squamous cell carcinomas.  Indeed it has been suggested that the vast majority of squamous cell carcinomas in India arise from preexisting leukoplakias.  The oral leukoplakia (OL) is defined by the World Health Organization (WHO) as "a predominantly white lesion of the oral mucosa that cannot be characterized as any other definable lesion."  The malignant transformation rate of OLs annually is approximately 1%.  It is clear from longitudinal studies describing the natural history of oral cancer that some leukoplakias are neoplastic, but identifying these lesions still remains a big problem. Traditional methods used by the pathologists, such as scoring atypia or dysplasia, are both subjective and lack sensitivity to predict accurately which cancers may progress with time. To date, there have been no reliable markers to identify oral lesions that will progress to malignancy. Various studies on leukoplakias like, Ki-67, argyrophilic nucleolar organizer regions, p53, and the proliferating cell nuclear antigens have been developed as ancillary in this histological assessment.  With the increasing emphasis on the early detection of cancer, the search is on for reliable markers that will be clinically helpful.
|How to cite this URL:|
Godge PY, Poonja LS. Quantitative assessment of expression of cell adhesion molecule (CD44) splice variants: CD44 standard (CD44s) and v5, v6 isoforms in oral leukoplakias: An immunohistochemical study. Indian J Dent Res [serial online] 2011 [cited 2019 Oct 22];22:493-4. Available from: http://www.ijdr.in/text.asp?2011/22/3/493/87080
In the last few years there has been increasing interest in a broadly distributed family of cell surface glycoproteins called CD44. One subfamily of CAMs, the hyaladherins, comprises the CD44 molecule [Hermes antigen, HCAM, pgp-1antigen, ECM-III] and its variant isoforms.  When these molecules were found to be the same, the name CD44 was assigned at the Third International Workshop on leukocyte differentiation antigens.
It is encoded by a single gene containing 20 exons located on chromosome 11p13. Isoform CD44s is the smallest and most abundant member of this polymorphic and monogenic family of proteins. The remaining exons can be differentially inserted into the mature mRNA via alternative splicing and may in theory give rise to hundreds of protein variants. CD44 was originally implicated as a hyaluronic acid and a homing receptor directing the migration of circulating lymphocytes across the high endothelial venular membranes of the lymph nodes and inflamed synovia. It has since been confirmed that CD44 is not only expressed in lymphocytes but also in a wide variety of epithelial tissues. Functionally speaking, CD44 is involved in organ integrity through its ability to contact ECM, is signaling active, and serves as a co-receptor for numerous transmembrane proteins such as matrix metalloproteases, members of the ERB family of receptor tyrosine kinases, and the long known tumor-associated antigens EpCAM (CD326, ESA1)  There have been several immunohistochemical and immunoblotting investigations on CD44 gene expression in a variety of cancers using monoclonal antibodies to epitopes encoded by several different individual exons. These have confirmed that in many types of cancers (with the apparent exception of squamous cell carcinoma of the head and neck and of neuroblastoma), there is overexpression of CD44 protein isoforms in tumor tissues relative to non-neoplastic control tissues.
The nature of this transmembrane adhesion molecule and the role it plays in the tumor development and progression are matters which have interested not only the basic researchers but also cancer clinicians and pathologists. The availability of different exon-specific monoclonal antibodies against CD44 variants has enhanced the ease and accuracy of immunohistochemical analysis. However, there have been comparatively few studies involving CD44 expression and premalignant oral lesions.
Keeping this trend in mind, the current study was designed to investigate the immunohistochemical expression of CD44s and variant isoforms v5, v6 in leukoplakias and to assess their possible significance as markers.
| Materials and Methods|| |
The present prospective study was carried out in the Department of Oral pathology. Formalin fixed, paraffin-embedded specimens from 40 patients with leukoplakias and 10 patients with normal oral mucosa were included in this study. Diagnosis of the lesions was based on the clinical and histological features of hematoxylin-eosin (H and E) stained slides.
In H and E-stained sections, a detailed histological study of epithelium according to the criteria of the WHO (2005) , was carried out. The histological degree of epithelial dysplasia was based on the proportion of the height of the epithelial layer that presented with dysplasic changes. Mild dysplasia was characterized by disturbances in architecture limited to the lower one-third of epithelium, accompanied by minimal cytological atypia. When the lower two-thirds of the epithelium showed architectural and cytological alterations, the lesion was classified as moderate dysplasia. When the entire thickness of the epithelium presented dysplastic changes, OL was graded as severe dysplasia. The architectural changes observed in the classification were as follows: loss of polarity of basal cells, irregular epithelial stratification, dyskeratosis, basal cell hyperplasia, disordered maturation from basal to squamous cells, bulbous drop-shaped rete pegs, and secondary extensions on rete tips. The cellular changes considered were increase of nuclear-cytoplasmic ratio, cellular pleomorphism, cellular and nuclear enlargement, nuclear hyperchromatism and pleomorphism, enlarged and numerous nucleoli, increased mitotic figures, atypical mitotic figures, and increased number and size of nucleoli. All 50 cases were further subjected to immunohistochemical evaluation.
Immunohistochemical reaction was performed using streptavidin-biotin standard protocol. All kit reagents were equilibrated at room temperature prior to IHC staining procedure. Likewise, humidity chamber was used to prevent evaporation and drying of tissue sections.
Three serial sections approximately 4 μm in thickness were cut from each paraffin-embedded tissue and mounted on Poly-l- lysine (Sigma, Steinheim, Germany) coated glass slides, deparaffinized, and rehydrated. Batches of 20 slides were then microwave treated in 10 mM sodium citrate buffer (pH 6.0) for two 10-min cycles at 700 W for antigen retrieval. This was followed by two washes in TRIS (Hydroxymethyl Amino Methane, Sisco Research Laboratory Pvt. Ltd.) buffer bath. Excess fluid was tapped off, and the slides were treated with 30% H 2 O 2 in the methanol (9 ml/300 ml) for 20 min to block endogenous peroxide activity. The slides were then twice rinsed with distilled water for 5 min.
In all groups, non-specific protein binding sites were blocked by 30min incubation with 1% goat serum (Gibko, BRL) in TBS and then drained.
Optimally diluted monoclonal antibodies anti-human CD44s (clone Hermin-3), CD44v5 (clone VFF8), and CD44v6 (clone VFF18) in ((1:50) working dilution were applied on three slides individually and incubated over night at 4°C. For negative control, goat serum was used instead of primary antibody. A bound primary antibody was detected using LSAB® +system HRP peroxidase kit (Dako Corporation, Carpenteria, CA, K0690).
After washing in 1M TRIS-buffered saline, a biotinylated rabbit anti-mouse IgG antibody in phosphate-buffered saline containing carrier proteins and 15 mM sodium azide was applied followed by incubation for an hour in the humidifying chamber. The slides were then rinsed with three changes of TRIS-buffered saline.
After draining away excess TBS, the slides incubated for an hour with horse radish peroxidase-conjugated streptavidin complex for an hour, and then rinsed. The peroxidase activity was developed by 3.3΄-diaminobenzidine tetrahydrochloride chromaogen (DAB, Sigma Chemical, St. Louis, D5637).
Interpretation of the staining
Presence of a brown colored end-product at the site of the target was indicative of positive reactivity. Staining of the lymphocytes served a positive internal control, whereas omission of primary antibody served as a negative control.
Quantification of the staining
The expression of the antigens investigated was evaluated in a semiquantitative manner. Three areas of the epithelium were scored B, S, and C, i.e., stratum basale, stratum spinosum, and stratum corneum, respectively. The number of positive cells were counted.
The intensity of immunostaining was scored on a scale of 0 (no staining) to 3(heavy staining) and the percentage of cells stained with that intensity. The percentage of positively stained cells was determined by scanning the entire section of the epithelium and recording membrane staining of cells as 10% (1), 11-50% (2), and 51-100% (3). A score was obtained multiplying the intensity of the staining by proportion of cells staining at this intensity (maximum score 9). The scoring of the slides was done by three observers to eliminate interobserver bias, and the average of the observations was taken for the grading.
In this study, statistical analysis, for each parameter of all groups and normals, mean, and standard deviation were calculated by using computer software package Epistat. To find out significant differences between the groups, unpaired "t" was used.
X1 - X2 = Mean of the group
SD1 /SD2 = Standard deviation
| Results|| |
In this study, mean age group of leukoplakia cases was 48.25 years with an age range of 30-70 years. Males were predominantly affected in a ratio 9:1. Majority of the patients, i.e., 90% had a habit of smoking in which 70% of the total cases were bidi smokers. In the above study of 40 patients, 23 patients (i.e., 57.5%) had leukoplakia located on the commisures, 9 patients (i.e., 22.5%) buccal mucosa was involved, followed by the lower anterior vestibule involvement in 4 patients. In two patients, the lesion was observed on the tongue with one patient each on the palate and the floor of the mouth. Out of the total cases that exhibited dysplasia, 32.5% had mild dysplasia, 25% moderate dysplasia, and 22.50% showed severe dysplastic features, whereas in 20% of the total cases dysplasia was absent.
In this study, the normal mucosal expressions of CD44s, v5, v6 [Figure 1] were detected as membranous proteins localized on the surface of the epithelial cells. Both basal and spinous layer of epithelial cells expressed strong positive staining of CD44s, v5, v6, which then gradually faded into the negative staining of the superficial keratin layer. On statistical evaluation, it was found that mean level of expression of CD44s of normal patients in stratum basale was 8.40±1.26 (93.33%), in stratum spinosum was 6.90±1.45 (76%) which decreased to 1.40±0.70 in stratum corneum. The CD44s labeling of study cases when compared with normal mucosa was found to be almost same and the difference was not statistically significant [Table 1]. Profile of CD44v5 isoform expression in normal and study cases revealed that the mean expression level of stratum basale in normal mucosa was 8.10±1.45 (90% of cells) which was comparable to the expression in stratum basale of study cases 7.28±1.74 (80.88% of cells). The normal mucosal expression of v5 isoform in stratum spinosum and stratum corneum was also in analogy to the study cases, and the difference was not statistically significant by Student's "t" test P>0.05 not significant; [Table 2].
|Figure 1: Photomicrograph demonstrating a strong membranous staining with CD44v6 in normal mucosa. Only minor component of cytoplasmic and nuclear staining is evident (×400 magnification)|
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In this study group, the CD44v6 isoform expression in all variables, i.e., stratum basale, corneum, and spinosum, mean levels of v6 isoform were significantly lower among study cases when compared with normal cases [Table 3].
Mean expression of stratum basale, corneum, and spinosum was significantly more among those cases where dysplasia was absent when compared with those where dysplasia was mild to moderate to severe. In this study group, mean levels among moderate to severe cases were very low when compared with mild also [Table 4].
|Table 4: Relation between severity of dysplasia and expression of CD44 v6 isoform|
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As the expression of CD44s and v5 was unaltered when compared with normals, their statistical analysis with dysplasia was not performed.
| Discussion|| |
The present phase of fight against cancer is characterized by particular emphasis on early detection. Leukoplakia is the most common premalignant or potentially malignant lesion of the oral mucosa.  The ability to identify precisely which leukoplakia lesions are most likely to progress to malignancy would be of considerable importance in guiding their subsequent clinical management. One of the most important and ubiquitous types of adhesive interactions required for the maintenance of tissues are those that are mediated by the classic CD44 adhesion molecules which has been linked with the development and spread of cancer. It is encoded by a single gene containing 20 exons located on chromosome 11p13 and exists as a number of isoforms generated by alternative splicing.  The data that are available on early premalignant lesions such as dysplastic areas in bladder  and adenomas of the colon,  as well as early stage malignancies from carcinoma in situ to minimally invasive carcinomas indicate that the disorderly expression of this gene begins when the neoplasm is first forming and increases as the tumor becomes larger, invasive, and metastatic. Similarly, the loss CD44 expression in adjacent non-transformed epithelium can be considered as a early phenomenon in tumorogenesis of the tongue.
Specific role of each CD44 variant seems to exist in the gradual process of carcinogenesis and metastatic cascade. 
The potential of CD44 expression in diagnostic intervention indicates a need for research into aspects related to this premalignant lesion.
This study shows the immunoexpression of CD44s and its variant isoforms v5, v6 in OLs.
The extensive expression of CD44s and variant isoforms within many normal epithelia most likely serves an adhesive function relating to the interaction and migration of the resident cells.
The immunolocalization of CD44 molecule in normal oral epithelium shows no regional differences. Epithelium stains strongly for all variants in basal and the suprabasal cells, while cornified and surface layers have negative stain. There is a possibility that the generative cells of epithelia which express the highest levels of CD44v isoforms are the cells that most often undergo malignant transformation. 
In this study, the normal mucosal expressions of CD44s, v5 and v6, were detected as membranous proteins localized on the surface of the epithelial cells. [Figure 1] Both basal and spinous layer of epithelial cells expressed strong positive staining of CD44s, v5 and v6, which then gradually faded into the negative staining of the superficial keratin layer.
Particular role of each CD44 variant seems to exist in the gradual process of carcinogenesis. 
Like over expression of CD44 v5 has been reported in colorectal, gastric, and breast carcinomas, whereas a downregulation was found in endometrial carcinomas. 
Piffko et al.  designed a study to immunolocalize CD44v6 and v5 isoforms in normal, dysplastic, and malignant oral mucosa as well as in primary metastatic oral squamous cell carcinomas. Their results strongly suggested that CD44 v5 and v6 isoform expression is not altered and they seem irrelevant factors in prediction of prognosis in this type of cancer.
Results from another studies suggest that CD44v6 and CD44v5 isoform expression is not altered during development and progression of oral carcinomas.
Spafford et al.  reported that decreased expression CD44s is correlated with decreased survival and an increase in metastasis in laryngeal squamous cell carcinoma.
Herold Mende  analyzed the immunoexpression of CD44s and variant isoforms in normal and dysplastic squamous epithelia, as well as in primary and metastatic squamous cell carcinoma. He found same pattern of expression of CD44v5 and CD44v6 in normal and in dysplastic epithelia.
In this study, expression of CD44 v5 and standard expression was comparable to those of the normal mucosa. In all types of dysplasia, i.e., mild [Figure 2] and [Figure 3], moderate, [Figure 4] and [Figure 5] and severe [Figure 6], [Figure 7] and [Figure 8] dysplastic cells expressed CD44 s and CD44v5 isoforms uniformly strongly on all cell surfaces. No significant downregulation of expressions of CD44s and v5 was noted in any of the cases. Thus, they seemed to be irrelevant factors in predicting the prognosis of this oral precancerous lesion.
|Figure 2: Photomicrograph showing immunohistochemical staining of CD44s in a section of leukoplakia with mild dysplasia. Cells in basal and spinous layer show strong staining pattern with CD44s (×100 magnification)|
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|Figure 3: Photomicrograph showing immunohistochemical staining of CD44v5 in a section of leukoplakia with mild dysplasia. Cells in basal and spinous layer show strong staining pattern with CD44v5 (×120 magnification)|
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|Figure 4: Photomicrograph of a leukoplakia with moderate epithelial dysplasia. Immunohistochemical staining demonstrating strong expression of CD44s (×100 magnification)|
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|Figure 5: Photomicrograph of leukoplakia with moderate dysplasia. Immunohistochemical staining demonstrating unaltered expression of CD44v5 (×120 magnification)|
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|Figure 6: Photomicrograph of leukoplakia with moderate to severe dysplasia. Immunohistochemical staining demonstrating unaltered expression of CD44s (×120 magnification)|
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|Figure 7: Photomicrograph demonstrating unaltered expression of CD44s in moderate to severe dysplasia (×120 magnification)|
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|Figure 8: Photomicrograph of moderate to severe dysplasia demonstrating unaltered immunoexpression of CD44v5 (×120 magnification)|
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Since CD44s and CD44v5 expression were unaltered in dysplasias, no statistical analysis were performed.
The CD44v6 adhesion molecule has been linked to progression of various carcinomas, but its role in relation to oral cancer development is not clear.
Changes in CD44v6 expression have correlated with poor prognosis in several human malignancies. Elevated CD44 v6 expression has been observed in benign and malignant colorectal epithelium, primary brain tumors,  cutaneous lymphomas,  and certain types of gastric adenocarcinomas.  Other investigators have reported, however, that epithelium expressing CD44v6, under normal conditions, showed a tendency toward down regulation during tumor progression and metastatic spread.
This tendency has been reported in human tumors that originated from squamous epithelium of the skin,  endometrium,  urothelium,  laryngeal mucosa,  and oral mucosa. 
Salmi et al.  found that there was reduced expression of the v6 domain SCC, the loss being greatest in the least differentiated regions.
Bahar et al.  studied the expression of CD44 variant 6 (CD44v6) protein in benign, premalignant, and malignant (SCC) oral epithelial lesions as oral carcinomas compared with the pattern in normal mucosa. He found that CD44v6 was clearly downregulated in cases of severe premalignant lesions as well as in most of SCCs.
Different pathological patterns are included in the term dysplasia, so further distinguishing and grading of dysplastic lesions are useful in the study as long as the specimen is large enough to preserve the levels of statistical significance.
Using more discrete criteria in epithelial dysplasia with early features of invasion not yet developed into squamous cell carcinoma, Sneath  revealed downregulated expression of CD44v6, comparing with benign epithelial lesions and normal tissue.
On the other hand, Rautava  found no statistically significant correlation between CD44v6 expression and dysplasia but his results mirrored disorderly epithelial maturation in dysplasias and carcinomas.
Gao et al.  investigated the expression of CD44v3 and CD44v6 in oral premalignant epithelia. They concluded that low level expression of CD44v3 and v6 exists in moderate and severe dysplasias.
In our study, the analysis of CD44v6 expression in leukoplakia showed a significantly altered expression from its normal positive pattern to a downregulated state [Figure 9]. On correlating the expression pattern with the degree of dysplasia, it was found that in moderate to severe dysplasias, wide areas through all sections showed overall downregulation [Figure 10] and [Figure 11], however, focal areas with downregulated expression were also observed in some cases [Figure 12]. Highly pleomorphic cells showed downregulation to almost negative expression, whereas less pleomorphic or normal cells expressed CD44v6 positively in the same section. This tendency toward downregulation of expression on correlating with the degree of dysplasias was found to be statistically significant (P<0.05).However in mild cases of dysplasia a very slight decrease in the degree of staining intensity was noted [Figure 13].
|Figure 9: Photomicrograph showing significant downregulation of membranous staining with CD44v6 in severe dysplasia (×400 magnification)|
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|Figure 10: Photomicrograph leukoplakia with moderate to severe dysplasia. Immunohistochemical staining showing significant downregulation of expression with v6 isoform (×120 magnification)|
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|Figure 11: Photomicrograph showing significant downregulation of membranous staining with CD44v6 in moderate to severe dysplasia (×120 magnification)|
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|Figure 12: Photomicrograph of leukoplakia with moderate dysplasia. Immunohistochemical staining demonstrating focal downregulation of expression with CD44v6 (×120 magnification)|
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|Figure 13: Photomicrograph of leukoplakia with mild dysplastic features demonstrating a slight decrease in the immunohistochemical staining intensity of cells in basal and spinous layer with CD44v6 (×120 magnification)|
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Our results are in consensus with the previous studies reported by Bahar et al.  and Gao et al.  that downregulation of CD44v6 occurs in premalignant lesions with epithelial dysplasias.
This is also in sharp contrast to some previous reports, and these may rely on different technologies used to visualize CD44 molecule and also on use of more discrete criteria for grading of dysplasia.
The correlation between the degrees of dysplasia and CD44v6 downregulation might reflect the fact that early cellular change from normal cell-cell and cell-matrix interactions toward a bizarre, pathophysological heterotypic cell surface adhesion property might predispose the cells to achieve invasiveness when they become fully malignant.
Thus, our results suggest that loss of CD44v6 may facilitate loss of cell-cell cohesion, detachment from basement membrane, and subsequent invasion into the underlying tissue.
Hence in conclusion, it can be inferred that among CD44s, v5, v6, isoform v6 may serve as molecular marker for detecting high-risk leukoplakias. Since the study sample was small, we have tried to avoid overinterpretation of the results. Further studies involving a larger sample size are essential to corroborate our results.
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Pournima Y Godge
Department of Oral Pathology and Microbiology, Terna Dental College and Hospital, Navi Mumbai
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13]
[Table 1], [Table 2], [Table 3], [Table 4]
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