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\begin{document}

\title{Predictive value of ALDH1 and CD44 positivity for radiotherapy response
and prognosis in early-stage glottic laryngeal squamous cell carcinoma}



\author[1]{Ozgur Surmelioglu}%
\author[2]{Kivilcim Eren Erdogan}%
\author[1]{Muhammed Dagkiran}%
\author[1]{Suleyman Ozdemir}%
\author[1]{Ozgur Tarkan}%
\author[2]{Aysun Uguz}%
\affil[1]{Cukurova University Faculty of Medicine}%
\affil[2]{Cukurova Universitesi Tip Fakultesi}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Objective: To evaluate of the predictive value of CD44 and ALDH1
expression for prognosis and radiotherapy(RT) response in patients with
early-stage laryngeal cancer receiving RT. Materials and Methods:
Forty-four patients with early-stage laryngeal cancer were included in
the study. Based on retrospective chart review, the patients were
divided into those with local recurrence and those without recurrence
after RT. Correlation between RT response and pre-treatment
immunohistochemical ALDH1 and CD44 staining was evaluated. In addition,
survival times were compared between groups. Results: The mean age was
found 59.8\selectlanguage{ngerman}±9.0 years and 41 were male. There were 20 patients in the
non-recurrent group. Immunohistochemical positivity for ALDH1 was found
to be significant risk factor for RT failure (p=0.0001), whereas CD44
positivity (p=0.114) were not significant. Disease-free survival was
shorter in cases with positive ALDH1 and CD44 staining (p=0.0001).
Conclusion: ALDH1 positivity was identified as a significant predictor
of DFS and RT sensitivity.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Title:} Predictive value of ALDH1 and CD44 positivity for
radiotherapy response and prognosis in early-stage glottic laryngeal
squamous cell carcinoma

\textbf{Running title:} ALDH1 and CD44 in early glottic cancer

\textbf{Authors:}

Ozgur Surmelioglu\textsuperscript{1}, K\selectlanguage{polish}ı\selectlanguage{english}v\selectlanguage{polish}ı\selectlanguage{english}lc\selectlanguage{polish}ı\selectlanguage{english}m Eren
Erdo\selectlanguage{polish}ğ\selectlanguage{english}an\textsuperscript{2}, Muhammed Dagkiran\textsuperscript{1},
Suleyman Ozdemir\textsuperscript{1}, Ozgur Tarkan\textsuperscript{1},
Aysun Uguz\textsuperscript{2},

Cukurova University, Faculty of Medicine, Department of
Otorhinolaryngology, Adana/Turkey

Cukurova University, Faculty of Medicine, Department of Pathology,
Adana/Turkey

\emph{All of the above listed authors approve the final version of the
manuscript and fully agree that the accuracy and integrity of any part
of the work are appropriately investigated and confirmed, and all
questions related to these issues were resolved.}

\textbf{Correspondence:}

Address: Dr. Ozgur Surmelioglu

Cukurova University

Faculty of Medicine

Department of Otorhinolaryngology

Adana/Turkey

Phone: +90 322 3386060

Mobile: +90 532 0557601

Fax: +90 322 3386527

e-mail: surmeli2004@yahoo.com

\textbf{Abstract}

\textbf{Background:} Several prognostic factors can be used for the
evaluation of laryngeal cancer. The aim of this study to evaluate of the
predictive value of CD44 and ALDH1 expression for prognosis and
radiotherapy(RT) response in patients with early-stage laryngeal cancer
receiving RT.

\textbf{Methods:} Correlation between RT response and pre-treatment
immunohistochemical ALDH1 and CD44 staining was evaluated. In addition,
survival times were compared between groups.

\textbf{Results:} The mean age was found 59.8\selectlanguage{ngerman}±9.0 years and 41 were
male. There were 20 patients in the non-recurrent group.
Immunohistochemical positivity for ALDH1 was found to be significant
risk factor for RT failure (p=0.0001), whereas CD44 positivity (p=0.114)
were not significant. Disease-free survival was shorter in cases with
positive ALDH1 and CD44 staining (p=0.0001).

\textbf{Conclusion:} ALDH1 positivity was identified as a significant
predictor of DFS and RT sensitivity.

\textbf{Keywords:} Laryngeal squamous cell carcinoma, ALDH1, CD44,
radiotherapy

\textbf{What's already known about this topic?}

\begin{itemize}
\tightlist
\item
  At present, there are various treatment modalities for treating early
  glottic cancer; namely, RT or partial laryngectomy techniques.
\item
  Recent studies have shown similar local control between RT and
  surgery.
\item
  Several prognostic factors can be used for the evaluation of laryngeal
  cancer.
\item
  Some markers such as CD44, CD133, ALDH1, and ABCG2 are used to
  determine of predictivity of prognosis.
\end{itemize}

\textbf{What does this article add?}

\begin{itemize}
\tightlist
\item
  ALDH1 positivity was associated with disease-related survival and RT
  sensitivity.
\item
  CD44 positivity did not differ between patients with and without
  recurrence after RT.
\end{itemize}

\textbf{Introduction}

Although new treatment protocols for head and neck cancer have been
developed in recent years, head and neck cancer is still the sixth most
common cancer worldwide, mainly due to its association with human
papillomavirus (HPV) and tobacco and alcohol usage (1) . The most common
histopathological type is squamous cell carcinoma (SCC) and the oral
cavity, larynx, and pharynx are most frequently involved (2) .

Laryngeal SCC is the most common head and neck cancer, affecting an
estimated 100,000 people per year (3). For patients who present with
advanced disease, the mainstay of treatment is total laryngectomy with
or without adjuvant therapy (4) . However, the survival rate is low.
Among laryngeal cancers, glottic carcinomas are the most common
subgroup, with the glottis being involved approximately 3 times more
often than the supraglottic larynx. Glottic cancers are usually
diagnosed in the early stage due to symptoms of hoarseness.

In early glottic cancer, lymph node metastasis is rarely seen, with an
incidence of clinically positive lymph nodes of nearly zero for stage T1
and \textless{} 2\% for stage T2 disease, and a complete cure can often
be achieved by radiotherapy (RT) or surgery (5-7). Therefore, the goal
is to achieve the best local control leading to a complete cure and
optimal functional results. At present, there are various treatment
modalities for treating early glottic cancer; namely, RT or partial
laryngectomy techniques. Although surgery has been used for decades, its
use has greatly decreased in recent years because of declining
functional results and advances in RT (8). The optimal treatment for
early glottic cancer has remained an issue of debate, primarily due to a
lack of evidence from large prospective randomized trials (9). Recent
studies have shown similar local control between RT and surgery.
Mendenhall et al. reported local control rates ranging from
approximately 80\% to 94\% for T1 tumors and 70\% to 85\% for T2 tumors
with both modalities (10,11).

Several prognostic factors can be used for the evaluation of laryngeal
cancer. Microscopic grade is an independent prognostic factor and
correlates with clinical stage (12). Recurrence is related to aneuploidy
(13). The presence of S100-positive Langerhans cells around the tumor is
called host reaction and has been associated with favorable prognosis
(14).

The most accepted prognostic factors are TNM classification. However,
the TNM system cannot distinguish aggressive tumors from nonaggressive
tumors of the same size. Identifying one or more biomarkers to predict
the biological behavior of head and neck squamous cell carcinomas
(HNSCCs) would be beneficial. Recently, a small population of cancer
cells referred to as cancer stem cells (CSCs) was found to be
responsible for tumor initiation, relapse, and resistance to
chemotherapy or RT; therefore, eradicating CSCs is considered critical
in cancer therapy (15,16). The CSC hypothesis has also been proposed for
HNSCCs; some cell surface markers have been reported as CSC markers in
HNSCC, such as CD44, CD133, ALDH1, and ABCG2 (17-19), and high
expression of these markers is usually regarded as an indicator of poor
prognosis. Among them, CD44 is the most reported CSC marker in HNSCC
(20-22).

In this study, we aimed to evaluate of the predictive value of CD44 and
ALDH1 expression for prognosis and treatment response in patients with
early-stage laryngeal cancer receiving RT.

\textbf{Materials and Methods}

Forty-five patients diagnosed as having early-stage laryngeal cancer and
treated with RT in the otorhinolaryngology department of XXX University
Faculty of Medicine between 2002 and 2016 were included in the study.
Patients were treated with curative radiotherapy with a linear
accelerator with a peak energy of 6 MV. All patients were treated
Patients were treated with 225 cGy /28 days in the same radiotherapy
center. The demographic and clinical data, pathology reports, prognostic
parameters, and survival rates of the patients were retrieved
retrospectively from their medical records. Among patients with
early-stage patients with recurrent and Cure with the same sampling were
studied. Patients with anterior commissure involvement were excluded
from the study. The patients were divided into two groups, those who
showed complete response with RT (non-recurrent group) and those who
developed local recurrence despite RT (recurrent group). The correlation
between treatment response and immunohistochemical staining for ALDH1
and CD44 at time of diagnosis was evaluated. Survival rates were also
compared between the groups.

Histopathological analysis

For each patient, slides stained with hematoxylin-eosin were reviewed by
the same pathologist, tumor tissues were selected, and ALDH1 and CD44
immunohistochemistry was performed on these tissues. For
immunohistochemical staining, 4-micron thick sections were obtained from
paraffin-embedded blocks in positively charged glasses and then
deparaffinized. Rehydration, blockade with hydrogen peroxide, and 20
minutes of antigen retrieval was performed with sodium citrate buffer in
a microwave. Samples were incubated overnight at 2--8°C with anti-ALDH1
rabbit monoclonal antibody (ab9883, Abcam) (1:500) (5 \selectlanguage{greek}µ\selectlanguage{english}g/ml) and CD 44
antibody (1:50) (Santa Cruz). After incubation with antibody, samples
were stained with DAB chromogen. Finally, all samples were stained with
Mayer hematoxylin and washed with distilled water and PBS.

For immunohistochemical analysis, a semiquantitative evaluation was
performed. The intensity and extent of staining were scored for ALDH1.
Density was scored as 0: no staining; 1: weak staining; 2: moderate
staining; 3: strong staining. Extent of staining was evaluated based on
the percentage of positive cells. A total score was obtained by adding
the density and extent scores. The evaluation of CD44 staining was based
on membranous staining and was performed using the same scoring method.

\emph{Statistical analyses}

Chi-square test or Fisher's exact test were used to compare the groups.
The Kaplan--Meier method and Cox proportional regression model were used
to estimate the mean/median survival rates, failure rates, and hazard
ratios (HRs). Log-rank test was used to compare the survival
distributions between groups. The prognostic ability of ALDH1 and CD44
were evaluated for DFS in both univariate and multivariable analyses.
DFS was defined as the time from diagnosis to any documented clinical
progression, relapse, or death from any cause. The results were reported
as mean\selectlanguage{ngerman}±SD, median, number (\emph{n} ) and percent (\%). A \emph{p}
value \textless{} 0.05 was considered significant. The analyses were
performed using the IBM SPSS Statistics version 22.0 (IBM Corp., Armonk,
NY).

\textbf{Results}

Forty-four patients with early-stage laryngeal cancer and treated with
RT were retrospectively evaluated. Their mean age was 59.8±9.0 (43--81)
and 41 were men. Before treatment, all patients were confirmed as having
early-stage disease according to the findings of indirect laryngoscopic
and radiologic evaluation and were reported as SCC according to
pre-treatment biopsy. Thirty-four patients had T1 glottic tumor and 11
had T2 glottic tumors. There were not anterior commissure involvement
seen at all patients. All of the patients were diagnosed as early-stage
glottic carcinoma according to the classification system of AJCC 2017
(23).

There were 20 patients (all men) in the non-recurrent group and 24
patients (22 men and 3 women) in the recurrent group. Median follow-up
time was 48 months (22--88 months). Treatment failure was observed in 24
cases. Table 1 shows the demographic and clinical features of the
patients.

Table 2 shows demographic and clinical features of the patients
according to treatment response. While positive ALDH1 staining
(p=0.0001) was found to be significant risk factor for treatment
failure, positive CD44 staining (p=0.114) and age group (p=0.287) were
not significant factors.

Means and medians for disease-free survival (DFS) time are shown in
Table 3. The mean OS was 60.7 months (median: 62.0 months). Cumulative
proportion of surviving patients was 81\% at 12 months (1 year), 56\% at
24 months (2 years), and 42\% at 36 months (3 years). Figure 1 shows
total DFS times. DFS was shorter in cases with positive ALDH1 staining
and CD44 staining (p=0.0001); Figure 2A shows DFS times according to
ALDH1 positivity and Figure 2B shows DFS times according to CD44
positivity.

Table 4 shows results of the Cox regression analyses. Two Cox regression
models were created using significant parameters detected in univariate
survival analysis: age, ALDH1 positivity, CD44 positivity. According to
Cox regression model, only the ALDH1 positivity was found to be a
significant independent factor for DFS, with positivity associated
significantly increasing the poor prognosis HR:16.4 (95\% CI: 3.6-73.5,
p=0.0001).

\textbf{Discusion}

Recently, a small population of cancer cells referred to as CSCs has
been implicated in tumor initiation, relapse, and resistance to
chemotherapy or RT; as a result, eradication of CSCs is considered
essential in cancer therapy (15,16). The CSC hypothesis has also been
coined for HNSCC in the head and neck; some cell surface markers have
been reported as CSC markers in HNSCC cancers, such as CD44, CD133,
ALDH1 and ABCG2 (17-19), and high expression of these markers is usually
considered an indicator of poor prognosis. Among them, CD44 is the most
reported CSC marker in HNSCC (20-22). Chen et al. reported that
ALDH1-positive cells are phenotypic and functional precursors of CSCs.
These cells develop from ALDH1 cell lines and have more proliferative
activity (24). In the same study, it was shown that ALDH1-positive cells
had similar genetic structure to mesenchymal stem cells. Guided by these
studies, ALDH1 expression is thought to be a good biomarker for CSCs in
head and neck cancers (24,25).

A study by Chen et al. showed that colony formation was stimulated with
0--10 Gy radiation in isolated ALDH1-positive cells. The study also
compared CD44-positive, CD24-negative cells that were positive and
negative for ALDH1. A total of 226 HNSCC patients who were positive for
ALDH1 were evaluated, and ALDH1 levels were found to be associated with
advanced-stage disease and undifferentiated tumors. In addition, it was
determined that patients who were positive for ALDH1 had poorer survival
with oncological treatment (26). In an experimental study, authors
reported that a small portion of the population of CD 44 possitive
cancer cells, which typically comprise \textless{} 10 \% of the cells in
a head and neck squamous cell carcinoma tumor, but not the CD44 negative
cancer cells, gave rise to new tumor in vivo. Immunohistochemistry
showed that the CD44 possitive cancer cells have a primitive cellular
structure and costain with the basal cell marker Cytokeratin 5/14,
whereas the CD44 negative cancer cells resemble differentiated squamous
epithelium and express the differentiation marker Involucrin. The tumors
that arose from purified CD44 possitive cells reproduced the original
tumor heterogeneity and could be serially passaged, thus demonstrating
the two defining properties of stem cells: ability to self-renew and to
differentiate (27).

In our study, the prevalence of ALDH1 positivity was significantly
higher among patients with tumor recurrence due to RT failure. Al-Assar
et al. reported that several biomarkers such as CD44, CD24, CD133, and
epithelial-specific antigen may be related to radiosensitivity. In this
study, it was found that CSCs, except those that were CD24-negative, did
not have a radioresistant phenotype (28). In a meta-analysis, ALDH1
expression was reported to be associated with low overall and
disease-related survival (29). In contrast, Lopez-Gonzales reported that
patients with stage 1-2 lung cancer and ALDH1 nuclear expression showed
good survival (30). This seems completely opposite of our study, in
which ALDH1 positivity was significantly more common among patients with
failed RT while CD44 positivity was not significantly different between
the two groups. ALDH1 positivity was found to be an independent risk
factor for DFS and significantly associated with poor prognosis.

This study has limitations such as the small sample size, and not
specifying the smoking status and comorbidities of the patients. Because
patients with anterior commissure involvement excluded from the study,
sample size is small.

\textbf{Conclusion}

In conclusion, the results of our study demonstrate that ALDH1
positivity was associated with disease-related survival and RT
sensitivity. CD44 positivity did not differ between patients with and
without recurrence after RT. In our study, sample size is small and
needs to be done larger cohort studies for the powered the predictive
effects of these markers. For ALDH1 positivity to be used as a biomarker
when making treatment decisions, it is necessary to conduct studies with
a larger patient series and determine its correlation with other
biomarkers.

\textbf{Acknowledgements}

We would like to thank Prof. Dr. Gulsah Seydaoglu for statistical
analysis.

\textbf{Conflicts of Interest}

The authors report no conflicts of interest.

\textbf{Financial support and sponsorship}

This research did not receive any specific grant from funding agencies
in the public, commercial, or not-for-profit sectors.

\textbf{Ethics}

Ethics committee approval of the study was obtained from the ethics
committee of the University of Çukurova (\textbf{approval number-March
3,2017;62/27).} The study was carried out in accordance with the
principles of the Helsinki Declaration.

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\textbf{Figure legends}

Figure 1: Total DFS curve.

Figure 2: DFS curves according to ALDH1 positivity (A) and CD44
positivity (B).\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-2a/Figure-2a}

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\end{figure}\selectlanguage{english}
\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/Figure-2b/Figure-2b}

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