Abstract
Introduction:
Oral squamous cell carcinoma (OSCC) significantly impacts global cancer incidence, with cancer stem cells (CSCs) such as ALDH1 and CD44 promoting tumor growth and self-renewal. This study examines the correlation between these CSC markers ALDH1 and CD44 with clinicopathological factors and survival outcomes in OSCC patients.
Materials and Methods:
The expression of cancer markers in 161 tumor tissue blocks was assessed through Immunohistochemistry (IHC). Fluorescence-activated cell sorting (FACS) was used to identify the immunoexpression of CD44 and ALDH1 in primary oral cancer cell lines derived from patients. The five-year survival outcome was calculated by Kaplan Meier (KM).
Results:
Our five-year study found significant associations between CSC markers and clinicopathological factors in OSCC patients. ALDH1 was linked to lymph node metastasis (P = 0.029), recurrence (P < 0.001), tumor histology (P < 0.001), and higher tumor staging (P = 0.008). Conversely, CD44 correlated with non-tobacoo chewing (P = 0.023), poorly differentiated tumor (P < 0.001), recurrence (P < 0.001) and mortality (P < 0.001). Patients with ALDH1-positive tumors had lower DFS (46.37 vs 62.92), while CD44-positive tumors had lower OS (58.98 vs 63.54). Univariate and multivariate analyses showed high ALDH1 expression associated with lower DFS (HR = 3.35, 95% CI = 1.93-5.82, P < 0.001), and high CD44 levels with lower OS (HR = 2.02, 95% CI = 1.11-3.66, P = 0.021).
Conclusion:
This study aimed to identify ALDH1 and CD44 expressions as cancer stem cell markers and independent predictors of lower five-year disease-free survival (DFS) and overall survival (OS), respectively. These markers effectively predict oral cancer prognosis and treatment response, enabling clinicians to improve patient outcomes.
Keywords: ALDH1, cancer stem cell markers, CD44, clinicopathological factors, oral squamous cell carcinoma, survival analysis
INTRODUCTION
Oral squamous cell carcinoma (OSCC) is the prevalent form of head and neck cancers and ranks as the 13th most frequently diagnosed cancer globally. In 2020, it accounted for 377,713 new cases and 177,757 deaths globally.[1] Notably, India experiences a high burden of this disease, ranking second in incidence with 135,929 new cases and 851,678 deaths annually.[2] Key risk factors include tobacco, alcohol, and areca nut (betel quid) consumption.[3] The prognosis for OSCC remains poor due to its typically late detection, with a five-year survival rate of only 20%.[4]
Despite advancements in therapeutic strategies, oral squamous cell carcinoma (OSCC) continues to exhibit high morbidity and mortality rates. A more comprehensive understanding of its molecular mechanisms is imperative for improving clinical outcomes. The identification and study of cancer stem cells (CSCs) within OSCC populations have become critical, as these cells are thought to drive disease progression, confer treatment resistance, and contribute to unfavorable clinical outcomes. Investigating the role of CSCs in OSCC could provide novel insights into targeted therapy strategies and potentially improve patient prognosis.[5]
Cancer stem cells (CSCs) are a distinct subpopulation of tumor cells characterized by their ability to self-renew and differentiate, thereby sustaining the tumor’s heterogeneous cellular composition.[6] Among the various markers associated with CSCs, ALDH1 (aldehyde dehydrogenase) and CD44 (cluster of differentiation 44) have garnered significant attention in oral squamous cell carcinoma (OSCC).[7] ALDH1 plays a crucial role in initiating tumor growth, progression, and recurrence, and it has been associated with CSC-like properties in a range of cancers, including OSCC.[8] Conversely, CD44, a transmembrane glycoprotein, is involved in cell adhesion, migration, and signaling processes, and is implicated in tumor initiation, progression, and metastasis. These markers are pivotal for understanding the pathology of OSCC and could potentially serve as targets for novel therapeutic strategies.[9]
In light of our findings, targeting ALDH1 and CD44, either individually or in combination therapies, emerges as a promising therapeutic strategy for OSCC. The ability of these CSC markers to predict clinical outcomes suggests their potential role in refining treatment approaches. By systematically evaluating and comparing the expression levels of ALDH1 and CD44 in OSCC, clinicians can gain valuable insights into the prognosis, thereby facilitating more personalized and effective treatment planning for patients.
Research examining both the combined and individual expression of CD44 and ALDH1 in OSCC patient samples, particularly regarding their association with prognosis and clinicopathological factors, is still limited. This study incorporates an extended follow-up period, enhancing our understanding of the survival risks associated with these markers.
This research explores the expression of ALDH1 and CD44 and their correlation with various clinicopathological factors, aiming to clarify their potential roles in predicting patient outcomes, including disease-free and overall survival.
MATERIALS AND METHODS
Inclusion and exclusion criteria
Between December 2016 and March 2018, we selected patients who had been diagnosed with OSCC at the Surgical Oncology department for a retrospective study. Inclusion criteria included histopathological confirmed OSCC, treatment-naïve status, candidacy for curative-intent surgery, non-metastatic condition, and available histopathology blocks. Exclusion criteria comprised HPV infection, previous chemoradiotherapy or surgery, distant metastasis, recurrent tumors, any other malignancy, and immunodeficiency disorders like HIV/AIDS.
Study setting, design and sample collection
A prospective cohort study was conducted at the Department of Surgical Oncology a tertiary referral center in India. The study included 161 patients with histopathological confirmed OSCC, utilizing paraffin-embedded blocks for immunohistochemistry from surgical resections between December 2016 and March 2018. Among these, six patients were selected based on inclusion-exclusion criteria for immunophenotypic characterization using FACS. The Institutional Ethics Committee approved the study protocol (Ref no1460/Ethics/R.Cell-16), and the research was carried out following the Declaration of Helsinki guidelines. All participants provided written informed consent prior to the collection of their tumor tissue.
Clinical and demographical data collection
Clinical history and patient data were compiled, with follow-up information obtained from the Surgical Oncology Department, K.G.M.U. Patients were followed until March 2023 to evaluate survival. Hospital records provided demographic data, clinical characteristics, oral habits, and follow-up status (OS and DFS), summarized in Table 1. The study’s primary endpoints were OS and DFS—measured from diagnosis to death or recurrence. Secondary endpoints included the correlation of clinicopathological features (tumor differentiation, lymph node metastases, clinical TNM staging, tumor size) with CSC marker expression.
Table 1.
Demographic, Clinico-Pathological Characteristics, and Association between ALDH 1 and CD 44 and clinicopathological factors (n=161)
| Age group | Total no. of patients (n=161) | ALDH 1 |
CD44 |
||||
|---|---|---|---|---|---|---|---|
| Negative (96) | Positive (65) | P | Negative (63) | Positive (98) | P | ||
| Age (Years) | |||||||
| ≤50 | 119 (73.9) | 70 (58.8) | 49 (41.2) | 0.726 | 43 (36.1) | 76 (63.9) | 0.190 |
| ≥50 | 42 (26.1) | 26 (61.9) | 16 (38.1) | 20 (47.6) | 22 (52.4) | ||
| Gender | |||||||
| Male | 135 (83.9) | 78 (57.8) | 57 (42.2) | 0.276 | 54 (40.0) | 81 (60.0) | 0.606 |
| Female | 26 (16.1) | 18 (69.2) | 8(30.8) | 9 (34.6) | 17 (65.4) | ||
| Tobacco Chewing | |||||||
| No | 47 (29.2) | 27 (57.5) | 20 (42.6) | 0.717 | 12 (25.5) | 35 (74.5) | 0.023 |
| Yes | 114 (70.8) | 69 (60.5) | 45 (39.5) | 51 (44.7) | 63 (55.3) | ||
| Alcohol Drinker | |||||||
| No | 146 (90.7) | 85 (58.2) | 61 (41.8) | 0.256 | 57 (39.0) | 89 (61.0) | 0.942 |
| Yes | 15 (9.3) | 11 (73.3) | 4 (26.7) | 6 (40.0) | 9 (60.0) | ||
| Tumor Stage | |||||||
| I-II | 77 (47.8) | 49 (63.6) | 28 (36.4) | 0.339 | 34 (44.2) | 43 (55.8) | 0.258 |
| III-IV | 84 (52.2) | 47 (56.0) | 37 (44.1) | 29 (34.5) | 55 (65.5) | ||
| Lymph node | |||||||
| Negative | 16 (9.9) | 14 (87.5) | 2 (12.5) | 0.029 | 6 (37.5) | 10 (62.5) | 0.999 |
| Positive | 145 (90.1) | 82 (56.6) | 63 (43.5) | 57 (39.3) | 88 (60.7) | ||
| Lymph Node Positive | |||||||
| N1 | 90 (62.1) | 68 (75.6) | 22 (24.4) | <0.001 | 36 (40.0) | 54 (60.0) | 0.987 |
| N2 | 52 (35.8) | 13 (25.0) | 39 (75.0) | 20 (38.5) | 32 (61.5) | ||
| N3 | 3 (2.1) | 1 (33.3) | 2 (66.7) | 1 (33.3) | 2 (66.7) | ||
| Histology Grade | |||||||
| WD | 41 (25.5) | 15 (36.6) | 26 (63.4) | <0.001 | 27 (65.9) | 14 (34.2) | <0.001 |
| MD | 46 (28.6) | 29 (63.0) | 17 (37.0) | 18 (39.1) | 28 (60.9) | ||
| PD | 74 (46.0) | 52 (70.3) | 22 (29.7) | 18 (24.3) | 56 (75.7) | ||
| Composite Stage | |||||||
| 1 | 7 (4.3) | 6 (85.7) | 1 (14.3) | 0.008 | 3 (42.9) | 4 (57.1) | 0.387 |
| 2 | 7 (4.3) | 6 (85.7) | 1 (14.3) | 4 (57.1) | 3 (42.9) | ||
| 3 | 48 (29.8) | 35 (72.9) | 13 (27.1) | 22 (45.8) | 26 (54.2) | ||
| 4 | 99 (61.5) | 49 (49.5) | 50 (50.5) | 34 (34.3) | 65 (65.7) | ||
| Recurrence | |||||||
| No | 102 (63.4) | 77 (75.5) | 25 (24.5) | <0.001 | 46 (45.1) | 56 (54.9) | <0.001 |
| Yes | 59 (36.6) | 19 (32.2) | 40 (67.8) | 17 (28.8) | 42 (71.2) | ||
| Status of Follow up | |||||||
| Alive | 86 (53.4) | 51 (59.3) | 35 (40.7) | 0.996 | 44 (51.2) | 42 (48.8) | <0.001 |
| Death | 70 (43.5) | 42 (60.0) | 28 (40.0) | 14 (20.0) | 56 (80.0) | ||
| Lost to follow up | 5 (3.1) | 3 (60.0) | 2 (40.0) | 5 (100.0) | 0 (0) | ||
Legend: This table presents the clinical and demographic characteristics of patients with oral squamous cell carcinoma (OSCC), categorized based on the expression of ALDH1 and CD44. Statistical significance was assessed using P values. ALDH1 and CD44 expression levels were evaluated to identify potential associations with variables
Immunohistochemical identification of ALDH1 and CD44
Paraffin blocks from 161 oral tumors and corresponding lymph node tissues were prepared for immunostaining. Sections of 3-4 microns were cut and fixed overnight at 56°C. Afterward, they were deparaffinized using xylene and dehydrated through graded ethanol. Antigen retrieval is carried out in a microwave at 98°C for 15 minutes, followed by a 5-minute block of endogenous peroxidase.
The sections underwent incubation with monoclonal rabbit anti-human CD44 (clone EPR1013Y, diluted 1:100; Abcam, Cambridge, UK) and primary antibody anti-ALDH1 (ab131068, diluted 1:200; Abcam) for 1.5 hours, followed by incubation with a secondary poly-horseradish peroxidase antibody for 30 minutes. Following a 10-minute exposure with diaminobenzidine chromogen, the sections were counterstained with hematoxylin, mounted in DPX.
Evaluation of immunostaining
Immunostaining for ALDH1 and CD44 was conducted on whole tissue sections from both control and OSCC samples. A standardized scoring system was applied to assess the staining results. Staining intensity was assessed based on the following scoring system: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). Proportions of tumor cells staining were scored as 0 for ≤5%, 1 for 6%–25%, 2 for 26%–49%, and 3 for ≥50%. Based on this scoring system, expression levels of ALDH1 and CD44 were categorized based on immunoexpression levels into two groups: low (≤2), considered negative, and high (>2).
Primary cell culture
Tissue samples were collected and preserved in Minimum Essential Media (MEM; Sigma, Cat-M0643) supplemented with 0.5% gentamycin and 1% streptomycin, maintained at 2-4°C. The tissues were minced into 1-2 mm fragments and transferred to sterile MEM for single cell suspension preparation. This was followed by enzymatic dissociation using collagenase I, repeated at least three times, with the supernatants collected into a separate tube after each iteration. The digested tissues were then centrifuged at 1500 rpm at 4°C. The resulting cell pellets were resuspended in MEM enhanced with 10% fetal bovine serum (FBS; Fisher Scientific), 0.5% amphotericin B, and cultured in a (5%) CO2 atmosphere at 37°C. The culture medium was refreshed every 3-4 days, and upon reaching 85%-90% confluency, the cells were passaged.
Staining and flow cytometry for marker expression
After establishing primary cell cultures from OSCC patients, we assessed the expression of the surface CSC marker CD44 and the enzymatic activity of ALDH1 in six cell lines. Monolayer cells from these lines were trypsinized, washed, and resuspended in staining buffer before being divided into two aliquots. To measure ALDH1 activity, we utilized the AldeRed ALDH Detection Assay kit (Millipore, Cat. # SCR150, U.S.A.), incubating 1 × 10^6 cells with the ALDH1 substrate in AldeRed Assay Buffer supplemented with verapamil. DEAB (Diethylaminobenzaldehyde), a specific inhibitor of ALDH1, served as the negative control. To assess CD44 expression, cells were incubated with CD44 PE-Cy7 conjugated antibodies at the concentration recommended by the manufacturer for 45-60 minutes in darkness on ice. Post-staining, cells were rinsed with PBS containing 1% BSA. Flow cytometric acquisition and analysis of ALDH1 and CD44 markers were conducted using a FACSAria III cytometer (BD) and analyzed using FlowJo software (version 10, Tree Star, USA).
Statistical analysis
The Chi-square test, or Fisher’s exact test when appropriate, was utilized to evaluate the association between two categorical variables and to determine differences in proportions between groups. Quantitative variables were represented as Mean ± SD. Survival outcomes were assessed using the Kaplan-Meier method, with group comparisons being conducted using the log-rank test. Additionally, the Cox proportional hazards model (multivariate analysis) was employed to identify independent risk factors affecting the survival time of the study patients. A P value of less than 0.05 indicates statistical significance. All statistical analyses were conducted using the SPSS (version 23.0).
RESULTS
Basic characteristics of OSCC patients
The demographic and clinicopathological factors, along with follow-up status for OSCC (n = 161) patients, are detailed in Table 1. Patients ranged from 22-76 years, with a mean age of 42.99 ± 12.52 years and a median of 42 years. A majority were male (83.9%) and under 50 years old (73.9%). Most patients were presented with advanced disease: tumor stage III-IV (52.2%), lymph node involvement N1 (62.1%), and stage IV (61.5%). Poorly differentiated (PD) tumors were noted in 46.0% of cases. Tobacco usage was reported in 114 (70.8%) of patients, while 146 (90.7%) were non-alcoholic. The mean disease-free survival (DFS) and overall survival (OS) were 40.40 ± 17.59 months and 53.13 ± 15.62 months, respectively.
Expression of ALDH1 and CD44
Immunohistochemical analysis revealed the expression of cancer stem cell markers ALDH1 and CD44 in oral squamous cell carcinoma (OSCC) patients, as shown in Figure 1. Among the 161 patients analyzed, ALDH1 was positively expressed in 65 patients, and CD44 was positively expressed in 98 patients, as detailed in Figure 2. In contrast, control samples displayed no expression of either marker, as depicted in Figure 1a and b. The differential expression of ALDH1 and CD44 markers in OSCC patients compared to controls underscores their potential role in the pathogenesis and progression of oral cancers.
Figure 1.
Immunohistochemical Staining of tumor tissue for CD44 and ALDH1 Expression in OSCC samples. Legend: (a) Oral squamous cell carcinoma (primary tumor): Microphotograph showing nests and islands of atypical squamous cells; well differentiated H and E (b) Histological section of tissue stained for CD44 expression: Tumor cells exhibit CD44-negative staining; (c) Histological section showing low CD44 expression, characterized by weak membranous staining in less than 50% of tumor cells; (d) Histological section demonstrating high CD44 expression, with strong membranous staining in more than 50% of tumor cells; (e) Histological section of tissue stained for ALDH1 expression: Tumor cells exhibit ALDH1-negative staining; (f) Histological section showing low ALDH1 expression, characterized by weak cytoplasmic staining in less than 50% of tumor cells; (g) Histological section demonstrating high ALDH1 expression, with strong cytoplasmic staining in more than 50% of tumor cells
Figure 2.
Immunodetection of ALDH1 and CD44 markers in oral squamous cell carcinoma. Legend: The bar chart shows the immunodetection results of ALDH1 and CD44 in oral squamous cell carcinoma. The data illustrate the following intensity levels: intensity level 0 (no staining or < 5% positive cells), intensity level 1+ (mild, 5–15% positive cells), intensity level 2+ (moderate, 16–25% positive cells), and intensity level 3+ (intense, >25% positive cells)
Association of ALDH1 and CD44 with clinico-pathological factors
The analysis of ALDH1 and CD44 staining scores in oral cancer tissues revealed significant correlations of ALDH1 with several clinical parameters. ALDH1 expression was significantly correlated with lymph node metastasis (P = 0.029), well-differentiated (WD) tumors (P < 0.001), recurrence (P < 0.001), and stage 4 composite staging (P = 0.008). However, ALDH1 expression did not show significant associations with age, gender, tobacco use, alcohol use, follow-up status, or general tumor staging.
CD44 expression levels were significantly higher in non-tobacco chewers compared to tobacco chewers (P < 0.023). Similarly, 71.2% of patients who experienced recurrence were CD44 positive, compared to 54.9% of those who did not experience recurrence (P < 0.001). Higher CD44 expression was associated with increased mortality (P < 0.001) and poorly differentiated (PD) tumors (P < 0.001). However, CD44 expression did not show significant associations with age, gender, alcohol consumption, lymph node metastasis, or tumor stage [Table 1].
ALDH1+/CD44+ enrichment in OSCC primary cell lines
The six OSCC primary cell lines were assessed for the ALDH1+ and CD44+ and cell population by flow cytometry analysis. The relationship between ALDH1 and CD44 expression was analyzed using Chi-square test which revealed, no significant association in the expression of ALDH1 and CD44 in oral cancer (P > 0.05). Additionally, the co-localization of ALDH1 with CD44 in oral cancer tissues was examined using FACS showing that some cases of OSCC exhibited co-expression of these markers [Figure 3]. Notably, the CD44 positive cells were found to express ALDH1 in human oral cancer tissues [Table 2].
Figure 3.
Fluorescent-Activated Cell Sorting (FACS) Analysis of Oral Squamous Cell Carcinoma (OSCC) Cells for ALDH and CD44 Marker Expression. Legend: (a) The morphology of the oral cancer cells displays characteristic epithelial features, (b) Unstained cells exhibit negative signals for both CD44 and ALDH1 markers, (c) DEAB-treated cells serving as the negative control for ALDH1 staining, show no positive signal for ALDH1, (d) In contrast, a positive signal for ALDH1 staining indicates that 83.4% of the cells express ALDH1, (e) A positive signal for CD44 staining indicates that 86.1% of the cells express CD44, (f) Additionally, the co-expression analysis demonstrates that both markers, CD44 and ALDH1, are simultaneously expressed in primary oral cancer cells, suggesting concurrent expression in samples from oral cancer patients
Table 2.
Association among ALDH1 and CD44 expression
| Total | CD 44 Expression |
||||
|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | ||
| ALDH expression | |||||
| 0 | 37 | 8 (21.6) | 10 (27.0) | 11 (29.7) | 8 (21.6) |
| 1 | 59 | 7 (11.9) | 16 (27.1) | 25 (42.4) | 11 (18.6) |
| 2 | 33 | 5 (15.2) | 6 (18.2) | 16 (48.5) | 6 (18.2) |
| 3 | 32 | 6 (18.8) | 5 (15.6) | 11 (34.4) | 10 (31.3) |
| Total | 161 | 26 (16.1) | 37 (23) | 63 (39.1) | 35 (21.7) |
0 (negative), no staining or <5% positive cells; 1+ (mild), 5–15% positive cells; 2+ (moderate), 16–25% positive cells and 3+ (intense), >25% positive cells. Chi-square test is used. P=0.637. No Significant association between CD44 and ALDH expression levels
OS and DFS of OSCC patients and its association with clinicopathological factors and CSC marker expression
In a univariate analysis of 161 patients using the Kaplan-Meier method, associations between clinicopathological variables and survival outcomes were evaluated. Sex, CD44 expression, and combined ALDH1-CD44 variables were significantly associated with overall survival (P < 0.05). Furthermore, tumor stage, histology, ALDH1 expression, and combined ALDH1-CD44 variables exhibited a significant correlation with disease-free survival (P < 0.05). Other variables did not show significant associations with either disease-free or overall survival (P > 0.05) [Table 3].
Table 3.
Association of clinicopathological variables, CD44, and ALDH markers with patients’ survival (n=161)
| Variables |
P
|
|
|---|---|---|
| Overall Survival | Disease-Free Survival | |
| Age Groups (<50, ≥50) | 0.926 | 0.082 |
| Sex (Male/Female) | 0.004 | 0.981 |
| Tobacco Chewing (Yes/No) | 0.333 | 0.737 |
| Alcohol Consumption (Yes/No) | 0.451 | 0.14 |
| Lymph node (Negative vs. Positive) | 0.429 | 0.708 |
| Tumor Stage (I-II vs. III-IV) | 0.467 | 0.006 |
| Composite Stage (1 vs 2 vs 3 vs 4) | 0.935 | 0.17 |
| Histology (WD vs. MD vs. PD) | 0.366 | 0.002 |
| ALDH Expression Group (Negative vs. positive) | 0.902 | <0.001 |
| CD44 Expression Group (Negative vs. Positive) | 0.010 | 0.603 |
| ALDH_CD44 (Both Positive, ALDH Positive, CD44 Positive, Both Negative) | 0.021 | <0.001 |
Kaplan Meier Method was used. P<0.05 statistically significant
Independent predictors of DFS and OS
Variables significantly associated with overall and disease-free survival, as detailed in Table 3, were used to estimate the median survival times and their 95% confidence intervals. These variables were subsequently included in multivariate Cox proportional hazards models to identify independent predictors of disease recurrence and mortality risk, quantified by hazard ratios.
Table 4 indicates that patients with Stages 3 and 4 tumors, well-differentiated (WD) histopathology, positive ALDH1 expression, and simultaneous CD44 and ALDH1 expression experienced significantly shorter disease-free survival times. Multivariate analysis further identified Stages 3 and 4 tumors and positive ALDH1 expression as significant predictors of reduced disease-free survival [Figure 4].
Table 4.
Disease Free Survival time and associated Independent Predictors of the Study Cancer Patients (n=161)
| Variable’s | Value | 95% CI |
P | |
|---|---|---|---|---|
| Lower | Upper | |||
|
Survival time (months) | ||||
| Univariate analysis | ||||
| Tumor stage | ||||
| 1 and 2 | 61.39 | 56.90 | 65.89 | 0.006 |
| 3 and 4 | 50.37 | 45.63 | 55.12 | |
| Histology | ||||
| WD | 45.68 | 38.53 | 52.83 | 0.002 |
| MD | 59.94 | 53.54 | 66.34 | |
| PD | 57.31 | 52.67 | 61.96 | |
| ALDH expression | ||||
| Negative | 62.92 | 59.04 | 66.79 | <0.001 |
| Positive | 46.37 | 41.25 | 51.49 | |
| ALDH_CD44 Expression | ||||
| Both Positive | 48.32 | 42.14 | 54.50 | <0.001 |
| ALDH Positive | 41.36 | 33.18 | 49.55 | |
| CD 44 Positive | 61.17 | 56.08 | 66.26 | |
| Both Negative | 64.74 | 59.92 | 69.57 | |
|
Hazard Ratio | ||||
| Multivariate analysis | ||||
| Tumor stage | ||||
| 3 and 4 | 1.77 | 1.01 | 3.10 | 0.047 |
| 1 and 2 | Reference | |||
| ALDH expression | ||||
| Positive | 3.35 | 1.93 | 5.82 | <0.001 |
| Negative | Reference | |||
Kaplan Meier Method was used to estimate the mean (95% CI) of the disease-free survival time (Univariate analysis). The Cox Proportional Hazard model was used to estimate hazard ratio (95% CI) for the independent predictors (Multivariate analysis). Key: WD, well differentiated; MD, moderately differentiated; PD, poorly differentiated; HR, hazards ratio; CI, confidence interval; The hazards ratio indicated above was assessed relative to the reference category. P<0.05 is statistically significant
Figure 4.
CD44 and ALDH1 as Independent Predictors for Disease-Free Survival (DFS) and Overall Survival (OS). Legend: CD44 has been identified as an independent predictor for disease-free survival (DFS). (a) Kaplan-Meier survival curves are plotted to show cumulative DFS over months, stratified by CD44 expression (negative versus positive). (b) Similarly presents Kaplan-Meier survival curves for cumulative DFS over months, differentiated by clinical stage (Stages 1 and 2 versus Stages 3 and 4); ALDH1, on the other hand, is an independent predictor for overall survival (OS), (c) illustrates Kaplan-Meier survival curves for cumulative OS over months, according to ALDH1 expression (negative versus positive). Additionally, (d) displays Kaplan-Meier survival curves for cumulative OS over months, stratified by gender (male versus female)
Table 5 reveals significantly reduced overall survival times in patients who are female, and those with positive CD44 and combined CD44_ALDH1 expressions. Multivariate analysis further highlights female sex and positive CD44 expression as significant risk factors impacting overall survival [Figure 4].
Table 5.
Overall Survival time and associated Independent Predictors of the Study Cancer Patients (n=161)
| Variable’s | Value | 95% CI |
P | |
|---|---|---|---|---|
| Lower | Upper | |||
|
Survival time (months) | ||||
| Univariate analysis | ||||
| Sex | ||||
| Female | 53.09 | 45.98 | 60.20 | 0.004 |
| Male | 62.75 | 59.93 | 65.58 | |
| CD44 expression | ||||
| Negative | 63.54 | 59.55 | 67.52 | 0.010 |
| Positive | 58.98 | 55.64 | 62.32 | |
| ALDH_CD44 Expression | ||||
| Both Positive | 60.53 | 55.30 | 65.76 | 0.021 |
| ALDH Positive | 58.37 | 50.50 | 66.24 | |
| CD 44 Positive | 57.71 | 53.37 | 62.06 | |
| Both Negative | 66.19 | 62.21 | 70.17 | |
|
Hazard Ratio | ||||
| Multivariate analysis | ||||
| Sex | ||||
| Female | 1.95 | 1.15 | 3.31 | 0.014 |
| Male | Reference | |||
| CD44 expression | ||||
| Positive | 2.02 | 1.11 | 3.66 | 0.021 |
| Negative | Reference | |||
Kaplan Meier Method was used to estimate the mean (95% CI) of the disease-free survival time (Univariate analysis). The Cox Proportional Hazard model was used to estimate hazard ratio (95% CI) for the independent predictors (Multivariate analysis). Key: WD, well differentiated; MD, moderately differentiated; PD, poorly differentiated; HR, hazards ratio; CI, confidence interval; The hazards ratio indicated above was assessed relative to the reference category. P<0.05 is statistically significant
DISCUSSION
In a study of 161 oral cancer patients, we assessed the correlation between ALDH1 and CD44 expression, clinicopathological factors, and survival outcomes (DFS and OS). Our findings indicate that OSCC cells exhibit cancer stem cell properties, with ALDH1 expression linked to lymph node metastases, well-differentiated tumors, recurrence, higher tumor stages, and decreased DFS. CD44 expression was associated poorly differentiated tumors, recurrence, increased mortality, and reduced OS. These results underscore the critical roles of ALDH1 and CD44 positive expressions in OSCC progression.
In our study, we found higher CD44 expression in non-tobacco chewers, contrary to other studies reporting increased expression in tobacco chewers.[10] Although numerous studies have reported an increased CD44 expression among tobacco chewers, our findings show a lower expression in this group. This discrepancy may stem from not considering other oral habits like betel nut use. Chou et al.[11] highlighted that CD44 expression can be influenced by carcinogenic habits, such as betel quid chewing, even without tobacco use, suggesting that CD44 may play a role in oncogenesis independent of tobacco use.
In this study, 40.4% of OSCC cases exhibited positive ALDH1 expression, which is consistent with findings from previous research.[12] However, disparity exists, as another study reported ALDH1 positivity in 70% of OSCC patients,[13] while CD44 expression was observed in 60.9% of our cases, aligning with prior studies.[14] It’s important to note that ALDH1 and CD44 expressions can vary even within the same sample, with ALDH1 located in the cytoplasm and CD44 on the cell membrane.[7] Such variations in expression levels may result from differences in review methodologies, cutoff points, sampling techniques, study populations, and the sites from which samples are taken.
Our research findings depict the strong association between high ALDH1 expression and lymph node metastasis in OSCC patients, aligning with findings from previous studies on epithelial tumors.[15,16] A significant proportion of our cases with positive lymph node status also demonstrated ALDH1 overexpression. Supporting this, Michifuri et al.,[17] Tamatani et al.,[12] and Chen et al.[8] reported increased ALDH1 expression in OSCC cases with lymph node metastasis. ALDH1 activity is known to facilitate tumor progression and invasiveness by disrupting the basement membrane and invading the stroma through the lymphatic and blood-vascular systems.[18] This link is further supported by studies showing significant associations between ALDH1 immunoexpression and advanced clinical stages.[19,20] Our findings also reveal a correlation between ALDH1 expression and overall stage of tumors, suggesting that ALDH1-expressing cells may have a significant impact on the invasiveness and progression of advanced OSCC.
The results indicate a correlation between well-differentiated tumor grades and ALDH1 expression, suggesting that ALDH1-positive cells may sustain a population of less differentiated, stem-like cells within the tumor. This is in contrast to the expression of CD44, which is predominantly associated with poorly differentiated tumors. However, previous studies present mixed findings regarding the correlation between ALDH1 expression and tumor histology, with references[12,17,18,21] showing varying results. These discrepancies may stem from factors such as small sample sizes and differences in sampling sites among the studies, underscoring the need for further exploratory research to elucidate role of ALDH1 in tumor development and heterogeneity.
Conversely, increased levels of CD44 have been linked to high-grade tumors, aligning with our findings.[22] This contrasts with the results of Sato et al.[23] and Krump et al.,[24] who observed that low CD44 expression correlated with high-grade tumors. Notably, in head and neck squamous cell carcinoma, no significant association was found between tumor grade and CD44 expression,[25] and similar findings were reported by Hema et al.,[26] who found no correlation between CD44 expression and tumor stage. These inconsistencies highlight the complex relationship between CD44 expression and tumor pathology, further emphasizing the need for comprehensive studies to clarify these associations.
Our study identified a correlation between poorer OS and DFS with a subpopulation of CD44+ cells that are also ALDH1+ in OSCC. Similar findings have been observed in other OSCC and HNSCC studies, where a tumorigenic cancer stem cell (CSC) subset within the CD44+ population is distinguished by ALDH1 expression.[27,28] Additional research is required to gain a comprehensive understanding of the tumorigenic features of this specific subgroup.
Our research indicates a correlation between ALDH1 expression and the advanced tumor staging both are associated with a lower 5-year disease-free survival, while female gender and CD44+ expression correlate with worse overall survival (OS). Similar findings were reported in tongue carcinoma and head and neck squamous cell carcinoma (HNSCC), where ALDH1 positivity adversely affected survival outcomes.[29,30] The primary reason for poor DFS and higher recurrence rates in the ALDH1+ population is their resistance to therapy. This resistance is often attributed to ALDH1 roles in retinoic acid biosynthesis, detoxification of harmful aldehydes, and scavenging of reactive oxygen species (ROS), which are increased by chemotherapy and radiotherapy.[31,32,33]
Our study discovered that high CD44 expression is significantly correlated with a high recurrence rate, compared to negative CD44 expression. Similar research has shown that CD44 overexpression in oral cancer correlates with recurrence and decreased overall survival highlighting its potential prognostic value in predicting patient outcomes and its role in recurrence and prognosis in OSCC patients.[34,35] In other study, high CD44 expression in oral cavity squamous cell carcinoma patients is associated with increased recurrence and decreased overall survival, especially when co-expressed with xCT.[36]
Thus, CD44 presents a viable therapeutic target to lower mortality in OSCC. Our findings also highlighted a significantly higher risk of death and recurrence in patients with high expressions of CD44 or ALDH1, with CD44 serving as an independent predictor of overall survival and ALDH1 as a predictor of disease-free survival in oral cancer.
A notable strength of this study is the utilization of primary cell lines isolated from oral cancer patients, allowing for the identification of biomarkers in a setting that closely mimics the in vivo disease environment. This approach enhances the reliability of our findings. Additionally, the extensive follow-up period, exceeding five years, is another significant strength. This prolonged observation allowed researchers to identify subtle correlations that may not be apparent in shorter studies. Conducting such a long-term follow-up is both time-consuming and challenging, yet the insights gained provide a solid foundation for future research. However, there are limitations to consider. The study may be subject to selection bias due to stringent inclusion and exclusion criteria. Furthermore, it does not establish causal relationships or include functional experiments to elucidate the mechanistic roles of the identified biomarkers in cancer stem cell behavior or prognosis. This limitation could affect the external validity of our findings.
CONCLUSION
Our research establishes the prognostic significance of ALDH1 and CD44 as cancer stem cell markers in OSCC. The findings demonstrate that the expression of these markers significantly affects survival rates, lymph node metastasis, tumor differentiation, and disease recurrence in oral cancer patients. Based on these results, developing risk stratification guidelines could help clinicians identify high-risk patients early, allowing for more targeted follow-up and intervention strategies. This proactive approach has the potential to significantly improve patient outcomes.
Approval
Ethical Clearance was obtained from King George's Medical University, Lucknow Institutional Ethical Committee with Ref no 1460/Ethics/R.Cell-16 dated 14.01.2017.
Abbreviations
OSCC: Oral Squamous Cell Carcinoma
HNSCC: Head and Neck Squamous Cell Carcinoma
CSC: Cancer Stem Cell
OS: Overall Survival
DFS: Disease Free Survival
HR: Hazard Ratio
IHC: Immunohistochemistry
FACS: Fluorescence-activated single-cell sorting
KM: Kaplan-Meier
SPSS: Statistical Package for the Social Sciences.
Conflicts of interest
There are no conflicts of interest.
Acknowledgments
I want to express our special thanks to the Centre for Advance Research and the Department of Pathology at King George’s Medical University for their invaluable assistance in research work.
Funding Statement
Nil.
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