Rahman et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-024-00500-0(2024) 25:4

BMC Molecular andCell Biology

Open Access

RESEARCH

CTC together with Shh and Nrf2 areprospective diagnostic markers for HNSCC
Md. Mizanur Rahman1,2,3†, Muhammad Mosaraf Hossain2,3*†, Shafiqul Islam2,3,10, Ridwan Ahmed2,3,
Mohit Majumder2,3, Shantu Dey2,3, Md. Kawser2,3, Bishu Sarkar2,3, Md. Ejajur Rahman Himu2,3,
Ali Asgar Chowdhury4, Shakera Ahmed5, Supran Biswas6, Mostafa Mahfuzul Anwar6,
Mohammad Jamal Hussain7, Rajib Kumar Shil2,3, Sunanda Baidya2,3, Ramendu Parial2,3,
Mohammed Moinul Islam2,3, Atul Bharde8, Sreeja Jayant8, Gourishankar Aland8, Jayant Khandare8,
Shaikh Bokhtear Uddin9 and Abu Shadat Mohammod Noman2,3*

Abstract
Background The lack of appropriate prognostic biomarkers remains a significant obstacle in the early detectionof Head and Neck Squamous Cell Carcinoma (HNSCC), a cancer type with a high mortality rate. Despite considerable
advancements in treatment, the success in diagnosing HNSCC at an early stage still needs to be improved. Nuclear
factor erythroid 2-related factor 2 (Nrf2) and Sonic Hedgehog (Shh) are overexpressed in various cancers, including HNSCC, and have recently been proposed as possible therapeutic targets for HNSCC. Circulating Tumor Cell
(CTC) is a novel concept used for the early detection of cancers, and studies have suggested that a higher CTC countis associated with the aggressiveness of HNSCC and poor survival rates. Therefore, we aimed to establish molecular markers for the early diagnosis of HNSCC considering Shh/Nrf2 overexpression in the background. In addition,
the relation between Shh/Nrf2 and CTCs is still unexplored in HNSCC patients.
Methods In the present study, we selected a cohort of 151 HNSCC patients and categorized them as CTC positiveor negative based on the presence or absence of CTCs in their peripheral blood. Data on demographic and clinicopathological features with the survival of the patients were analyzed to select the patient cohort to study Shh/Nrf2
expression. Shh and Nrf2 expression was measured by qRT-PCR.
Results Considering significant demographic [smoking, betel leaf (p-value < 0.0001)] and clinicopathological risk factors [RBC count (p < 0.05), Platelet count (p < 0.05), Neutrophil count (p < 0.005), MCV (p < 0.0001), NLR (p < 0.05), MLR(p < 0.05)], patients who tested positive for CTC also exhibited significant overexpression of Shh/Nrf2 in both bloodand tissue compared to CTC-negative patients. A strong association exists between CTCs and tumor grade. Following chemotherapy (a combination of Cisplatin, 5FU, and Paclitaxel), the frequency of CTCs was significantly decreasedin patients with HNSCC who had tested positive for CTCs. The Kaplan–Meier plot illustrated that a higher numberof CTCs is associated with poorer overall survival (OS) in patients with HNSCC.
†

Md. Mizanur Rahman and Muhammad Mosaraf Hossain contributed equallyto this work.
*Correspondence:
Muhammad Mosaraf Hossain
mosarafbmb@cu.ac.bd
Abu Shadat Mohammod Noman
asmnoman.bmb@cu.ac.bd
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
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Rahman et al. BMC Molecular and Cell Biology(2024) 25:4

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Conclusions Detecting CTCs, and higher expression of Shh and Nrf2 in HNSCC patients’ blood, can be a promising
tool for diagnosing and prognosticating HNSCC.
Keywords CTC​, Shh, Nrf2, HNSCC, Cancer

Introduction
Head and neck squamous cell carcinoma (HNSCC) is acancer that arises from the malignant transformation of
the squamous epithelial cells found in the head, neck,and oral cavity. The tumor originates from various anatomical sites shielded by squamous epithelium, including the aero-digestive tract, oral cavity, paranasal regions,
nasopharynx, hypopharynx, and larynx [1]. HNSCC
ranked as the seventh most prevalent cancer worldwide,with breast cancer being the highest in women and lungcancer in men [2]. In Bangladesh, head and neck cancer is the most common type of cancer in both men andwomen, followed by lung cancer in men and breast cancer in women [3]. HNSCC is significant due to its fiveyear low overall survival rate (50%) and the absence ofsensitive diagnostic and prognostic biomarkers [4]. Several antibody-based biomarkers, such as CA-125, CEA,
PSA, and others, have been developed for detecting various cancers, for example, CA 125 for early detection ofovarian cancer, CEA for colorectal cancer, and PSA for
prostate cancer [5]. However, recent studies demonstrated that overexpression of Shh and Nrf2 contributes
to the development of HNSCC [6].
Sonic hedgehog (Shh) is dysregulated in head and necksquamous cell carcinomas (HNSCC) [6]. Shh overexpression is associated with metastasis and tumor growth [7],maintenance of cancer stem cells (CSCs) [8], and resistance to chemotherapy [9]. During off state, PTCH1 (a12-transmembrane protein) inhibits SMO (a 7-transmembrane protein), while SUFU (Suppressor of fused
homolog) binds to GLI1 (a downstream regulator of
the Shh pathway), preventing transcriptional activation. However, in the active state, when Shh binds to the
PTCH1 receptor, inhibition of SMO is lifted, releasing
GLI1 from SUFU. GLI1 then enters the nucleus, activating target genes such as PTCH1, CCND1 (cyclin D1),
MYC (Master Regulator of Cell Cycle Entry), and others,
resulting in proliferation and suppression of apoptosis[10].
Usually, in the cytoplasm, Nrf2 (Nuclear factor
erythroid 2-related factor 2 (Nrf2) and Keap1 (Kelchlike ECH-associated protein 1) bind each other andcause proteasomal degradation resulting in antioxidant activity, detoxification, and cancer prevention. In
cancer, Nrf2 unbinds from Keap1, enters the nucleus,
and causes activation of transcription factors, resulting
in drug resistance, enhanced cell proliferation, stressadaptation, and activation of metabolic reprogramming[11]. Interestingly, loss of function of KEAP1 or gainof function of Nrf2 has been reported in HNSCC [12].
In HNSCC, Nrf2 is overexpressed in 90% of cases andfound to correlate with poor overall survival [13].
Identification of circulating tumor cells (CTCs) isa liquid biopsy tool [14] approved by the US FDA for
identifying and enriching CTCs from the blood ofcolorectal, prostate, and breast cancer patients [15].
Recent studies have reported the significance of CTCbased diagnosis in various cancers, including HNSCC[16]. In HNSCC patients, the presence of CTCs in the
blood is associated with poor disease-free survival [17].
CTC enumeration has proven helpful in metastatic settings for predicting overall survival, monitoring treatment response, and post-treatment surveillance in
HNSCC [18]. Cancer cells shed from the site of initiation and enter the circulation through a process known
as intravasation. These circulating cells then undergo
extravasation and migration at distant sites, potentiallyleading to distal metastasis [19]. Despite the advancements in chemotherapy and radiotherapy, the overall
5-year survival rate for early and advanced HNSCC
remains approximately 50%, with 20%, respectively. The
number of CTCs correlates with the aggressiveness of
HNSCC metastasis and poorer survival rates [20]. In
HNSCC, the main challenges lie in early metastasis
detection and prompt treatment initiation. Therefore,
overexpression of Shh and Nrf2 and detection of CTCs
could serve as promising prognostic and diagnostic
biomarkers for HNSCC. Moreover, the relationshipbetween Shh/Nrf2 and CTCs has yet to be explored asa potential biomarker for HNSCC patients. The present
study aimed to establish an effective diagnostic andprognostic tool for the early detection of metastasis bylinking biomarkers (Shh/Nrf2) with CTCs in HNSCC
patients. Our findings revealed that Shh/Nrf2 expression was upregulated in the blood and tissues of CTCpositive patients compared to CTC-negative patients.
For HNSCC patients who tested positive for Shh/Nrf2
expression with CTCs, the frequency of CTCs significantly decreased at the post-treatment state. Furthermore, patients with HNSCC who had a higher numberof CTCs exhibited a worse overall prognosis. In conclusion, the combination of Shh/Nrf2 overexpression and
CTC detection can be a potential tool to predict early
diagnosis and prognosis in HNSCC patients.

Rahman et al. BMC Molecular and Cell Biology(2024) 25:4

Method and materials
Place and duration of the study

This cross-sectional prospective study on Head and Neck
Squamous Cell Carcinoma (HNSCC) patients was conducted between February 20, 2020, and September 8,2022, at the Department of Otolaryngology and Head
Neck Surgery, Chattogram Medical College Hospital(CMCH), Bangladesh and the fundamental bench work
was accomplished at the Centre for Research Excellence(CRE), Department of Biochemistry and Molecular Biology, University of Chittagong, Bangladesh. The CTC
detection and analysis was performed at Actorius Innovations and Research Pvt Ltd, Pune, Maharashtra, India.
Sample size determination

Based on GLOBOCAN’s [3] estimate of the prevalence
rate of HNSCC in Bangladesh (~ 7–8%), we included 151
HNSCC patients who met the study’s inclusion criteria,
which included being over the age of 18, being willing toparticipate, and not having any concurrent medical conditions like diabetes, hypertension. A certified pathologist carried out the histopathological examination oftumor tissue sections to confirm the diagnosis of cancer.
We excluded patients who refused to take part in ourstudy. Peripheral blood was drawn from the study participants to measure the expression of Nrf2 and Shh and to
identify Circulating Tumor Cells (CTCs). To compare theShh and Nrf2 expression with the HNSCC patients, 24
healthy controls blood samples were examined. Shh andNrf2 gene expression were also measured using tumor
tissue samples of the study subjects.
Data analysis from TCGA​

To get a glimpse into the expression pattern of Shh andNrf2, cBioPortal and the UALCAN online computational tools were used to analyze Shh and Nrf2 RNA
Sequence data of 523 patients from the TCGA (The Cancer Genome Atlas) dataset. Transcript per million (TPM)
in the TCGA dataset was used to determine the levelof Shh and Nrf2 gene expression profile (TPM < 1). Shhand Nrf2’s mRNA expression patterns were determinedfrom 515 expressed genes, and the data were verified on
all platforms using the ± 1 z-score threshold as a cutoff toidentify high and low levels of Shh and Nrf2.
Blood sample collection for identification of CTCs and gene
expression study

Peripheral blood was collected from the study subjects incooperation with the Department of Otolaryngology andHead Neck Surgery (ENT), Chattogram Medical Collegeand Hospital, Chittagong. A certified phlebotomist was
assigned to draw and collect the HNSCC patient’s blood.
A total of 4.5 ml peripheral blood was drawn from each

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study subject, of which 1.5 ml was used to detect CTCs
using the OncoDiscover platform mediated by antiEpCAM antibody conjugated affinity-based magnetic
nanoparticles approved by the Drug Controller General of India. The rest of the blood was utilized for gene
expression study.
Identification of CTCs in blood samples of HNSCC patients

An aliquot of 1.5 ml blood was used for CTC detection
by the OncoDiscover platform. Briefly, the Oncodiscover
CTC testing platform comprises the following constituents (i) anti-EpCAM (Anti Epithelial Cell Adhesion Molecule Antibody) (Bioss, USA), (ii) crosslinked to ironoxide (Fe3O4) nanoparticles, (iii) PEG, (iv) multiwall carbon nanotube, (v) poly (N-isopropyl acrylamide) (PNIPAM) (vii) glutathione (GSH), and (vii) fourth generation(G4) dendrimers with 64 binding sites (European andIndian Patents, 3259598, 384104 respectively). Magnetic
nanoparticles attached with anti-EpCAM antibody specified to target cancer cells of epithelial origin directed
to capture CTCs. The CTCs were captured by applying
a magnetic field, followed by separation and processing
by immunofluorescence. Finally, CTCs were confirmed
using positive staining of CK 18 (cytokeratin 18) (Abcam,
USA) (Cytokeratin expressed in epithelial cells), DAPI
(Sigma, USA) (for nucleus), and the absence of CD45
(Bioss, USA) signal (for leukocytes).
Assembly of demographics, socioeconomic
and clinicopathological data

As previously indicated, we divided our study cohort into
two groups—CTC positive and CTC negative—basedon the presence of CTCs (circulating tumor cells) in thepatient’s blood. Demographic, socioeconomic, and clinicopathological findings were considered to narrow down
our targeted gene analysis. A clearly defined questionnaire was used to list and store all this data.
Collection of tissue samples for gene expression study

During surgery, HNSCC tumor tissue was obtained frompatients whose cancer had been previously diagnosed byskilled Pathologists. With all aseptic measures and proper
storage conditions, tumor tissue samples were preserved
in RNAlater (ThermoFisher Scientific) for RNA isolation,
followed by measuring the expression of Shh and Nrf2 by
qRT-PCR.
Estimation of hematological parameters

We tested the hematological parameters of our patient
cohort to identify correlations between the biomolecules.
Red blood cell (RBC) was counted in million/cmm using
the Neubauer Chamber method. White blood cell (WBC)count was conducted in thousand/ml using the Neubauer

Rahman et al. BMC Molecular and Cell Biology(2024) 25:4

Chamber method. Lymphocyte, Eosinophil, Monocyte,
Neutrophil, and Platelet counts were determined using
the Leishman staining method. Hematocrit was manuallycalculated using the formula HCT = (RBC x MCV)/10.
Hemoglobin levels were measured using photoelectriccolorimetry. Mean corpuscular hemoglobin (MCH) was
calculated manually by multiplying the percent hematocrit by ten and dividing it by the erythrocyte count.
Serum Creatinine levels were measured using the Jaffe
method. Mean corpuscular hemoglobin concentration(MCHC) was calculated manually by multiplying thehemoglobin result from the complete blood count (CBC)panel by 100 and then dividing it by the hematocrit
result. Serum Glutamic Pyruvic Transaminase (SGPT)
levels were determined using a colorimetric method,and erythrocyte sedimentation rate (ESR) was measured
using the Wintergreen method. Mean corpuscular volume (MCV) levels were measured according to physiological protocol by multiplying the hematocrit by ten anddividing it by the RBC count. Neutrophil-to-lymphocyte
ratio (NLR) and monocyte-to-lymphocyte ratio (MLR)
levels were calculated manually.
RNA isolation from HNSCC patient’s blood and tissue
samples

The chloroform-isopropanol method was used to isolate
RNA from blood. In brief, an aliquot of 500 µL of RBC
lysis buffer was added per 1 ml sample in the tube. Aftera gentle mixing, the mixture was let sit for five minutesat room temperature. The tube was then centrifuged
for 12 min at 4ºC at 2500 rpm (600 g). Once the supernatant was disposed of, 200–300 µL of RBC lysis buffer
was added and carefully blended. The tube was centrifuged for eight minutes at 4ºC and 2500 rpm. This process was repeated until the pellet turned white. Afterwashing the cell pellet in PBS, the tube was chilled to-20ºC. The next day, the tube was vortexed with 800 µL
of TRIzol (Life Technologies, USA) added to it. Following that, 200 µL of chloroform was added and vigorously
vortexed. After that, the tube was kept on ice for twenty
minutes. The tube was then centrifuged for 20 min at
4ºC at 13,000 rpm. The upper aqueous phase was transferred to a new tube. The same volume of isopropanol
was added and vortexed, and then the mixture was
kept at -20ºC for overnight incubation. It was then put
on ice for fifteen minutes. The tube was centrifuged at
13,000 rpm for 20 min at 4ºC the next day. With caution,
the supernatant was aspirated. The 500 µL of ice-cold,
75% ethanol washed over the RNA pellets at 4ºC. The
RNA pellet was allowed to air dry after the supernatant
was carefully drained off. The RNA pellet was then dissolved in the proper volume of RNase-free water. Using ananodrop spectrophotometer (ND, 2000), the RNA wasPage 4 of 17

then quantified and stored at -40ºC for use in subsequent
analyses, such as cDNA synthesis, quantitative real-time
PCR (qPCR), and agarose gel electrophoresis.
RNA was extracted from the tumor tissue sample using
the Chloroform-Trizol method. A tissue sample weighing
between 50 and 100 mg was taken and put into the Biomasher II tube. Tissue sample was washed using 1 mL of
PBS in the tube. After that, a pestle was placed inside thetube, and 30–400 µL of TRIzol was added. The tissue wasdisrupted by pressing the pestle against the side of the
tube until the sample was fully homogenized. Next, thesample was incubated at room temperature for 2–3 min.
Chloroform (half of Trizol volume) was added andinverted the tube carefully. The sample was subsequently
kept on ice for twenty minutes. The sample was then centrifuged for 30 min at 4ºC at 13000 rpm. A fresh tube wasused to transfer the upper layer. An equal amount of icecold isopropanol (99.99%) was added to the tube. Aftergently inverting the tube, it was set on ice for a duration of 15 to 20 min. Following an overnight incubation
period, the samples were centrifuged for 20 min at 4ºC
at 13000 rpm. Once the RNA pellet was apparent, thesupernatant was cautiously disposed of. The pellet wasthen washed and allowed to air dry with 500 µL of icecold ethanol added. The RNA pellet was then dissolved in
the proper volume of RNase-free water. Finally, the concentration of RNA was measured by a nanodrop spectrophotometer (ND, 2000) and kept at -40ºC for subsequent
tests, such as cDNA synthesis, agarose gel electrophoresis, and quantitative real-time PCR (qPCR).
cDNA synthesis and qRT‑PCR (Quantitative Real‑Time PCR)

Total RNA isolated from blood and tissue samples wasused to synthesize cDNA using the GoScriptTM Reverse
transcription system sourced from Promega (Cat no:
ADM1701 00000654925) per the manufacturer’s protocol. Real-time polymerase chain reaction (PCR) is acommonly used molecular biology technique that amplifies the copy number of a specific gene. The expression
levels of the studied genes (Shh, NRF2) were analyzed
using real-time quantitative PCR with the GoTaq qPCR
master mix (Promega, USA). Working cDNA samples
were prepared by diluting the stock cDNA (1 µg/µl) by
100 times. Forward and reverse primers were then added
to the samples. Finally, SYBR Green (Cat No: A6000, Promega, USA) was added to each sample tube, thoroughly
mixed, and loaded into the RT-PCR machine (BIO-RAD;
CFX96TM). The reaction was carried out using specificconditions optimized for the targeted gene. The dsDNA
was denatured into single-stranded DNA (ssDNA) by
incubating at 95 °C for 3 min. The annealing temperature, which depends on the melting temperature of theprimers, was set at 53 °C to allow for the synthesis of newRahman et al. BMC Molecular and Cell Biology(2024) 25:4

DNA strands from the template. Finally, the extension
step was set at 72 °C to facilitate the extension of the new
DNA strands from the primers. The relative expressionof Shh and Nrf2 was analyzed by the △△CT method,
normalizing with GAPDH. The primers are listed in the
Supplementary Table 1.
Statistical analysis

Graph Pad Prism (Version 8.4.2) was chosen as the statistical tool for all statistical analyses. Experiments related
to qRT-PCR were replicated three times, and results were
presented as mean ± SD. The Mann–Whitney statisticalanalysis was adopted to compare the tested genes’ expression in CTC-positive and CTC-negative patients. An
unpaired t-test was performed to compare hematological parameters between CTC-positive and CTC-negative
patients. One-way ANOVA was done to compare thegrade of CTC-positive patients and the number of CTCs.
Multiple linear regression analysis was done where CTC
was a dependent variable against different hematological parameter coefficients. A log-rank (Mantel-Cox) test
was performed for survival analysis. The values with ap-value < 0.05 were deemed statistically significant.

Results
Expression of Shh and Nrf2 in HNSCC in TCGA data cohort

The TCGA applies high-throughput genome analysis
techniques to identify alterations in the DNA and RNAof various tumor types. We examined the TCGA data
set to investigate the expression pattern of both the Shhand Nrf2 genes in different tumor types. Based on themRNA expression in HNSCC, the results indicated that
Shh and Nrf2 expression levels were altered in 48% and
34% of HNSCC patients, respectively (Fig. 1A and F). Primary tumors exhibited higher levels of Shh expression
than normal tissue, and male HNSCC patients had significantly higher levels than normal individuals (Fig. 1B
and C). Compared to normal controls, Shh expression
was significantly higher in HNSCC patients of 41–60
and 61–80 age groups and in HNSCC patients of stages
2 and 3 (Fig. 1D and E). On the contrary, no compelling disparity was observed in Nrf2 expression between
normal and primary tumor tissue or between males andfemales (Fig. 1G and H). Nrf2 expression was reduced inpatients of different ages compared to normal individuals. However, the reduction was not statistically significant (Fig. 1I). When comparing grade 1 to normal andgrade 2 to grade 1, Nrf2 expression was considerably
downregulated; however, when comparing grade 4 tograde 1 HNSCC patients, the expression was significantly
higher (Fig. 1J). Therefore, Shh and Nrf2 are differentiallyexpressed in HNSCC patients.

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Identification of CTCs from peripheral blood samplesof HNSCC patients

CTC was detected as a single cell or cluster by fluorescence microscopy analysis of peripheral blood
samples from HNSCC patients. Figure 2 represents
CTC images of an HNSCC patient where the first setof images is from a CTC cluster, and the second set isof individual CTCs from the same patient. Strong signals for Cytokeratin 18 (CK-18) and DAPI staining
were observed in the detected CTCs. CK-18 provides
mechanical support to the epithelial cell and is used asa marker for differentiating between various epithelial
cells. The existence of CK-18 and the lack of a CD-45
signal prove that CTC originated from an epithelial lineage. Signals from DAPI staining indicated the CTC’s
well-defined nucleus. The merged image was obtained
from superimposed signals of DAPI, CD-45, andCK-18, where the CK-18 signal showed a homogenous
distribution in CTC.
Demographic and socioeconomic risk factors of selected
CTC‑based HNSCC patients

Demographic and socioeconomic parameters were considered to select the patient cohort for this study. Amongthe selected HNSCC patients, 64 individuals (42%) tested
positive for CTCs, while the remaining 87 (58%) were
CTC negative based on the presence of CTC in theirblood (Fig. 3A). Therefore, we categorized our study subjects into two groups based on the presence of CTC cells(CTC-positive and CTC-negative). The mean age of theCTC-positive group was significantly higher (p < 0.05)
compared to the CTC-negative group (Fig. 3B). Among
the CTC-positive patients, 1 CTC cell was found in
36 HNSCC patients, 2 CTC cells in 18 patients, 3 CTC
cells in 3 patients, 4 CTC cells in 3 patients, 5 CTC cells
in 2 patients, and the highest 6 CTC cells observed in 2
HNSCC patients (Fig. 3C). Regarding the site of tumor
occurrence, buccal mucosa and vocal cord showed ahigher predisposition in CTC-positive patients, whereas
larynx and cervical lymph node involvement were moreprevalent in CTC-negative patients (Fig. 3D, E). CTCpositive patients were most prevalent in the 61–70 age
group, while CTC-negative patients were more prevalent in the 51–60 age group (Fig. 3F, G). Moreover, 64%of the CTC-positive patients had a positive smoking
history, which was statistically significant (p < 0.0001)
compared to CTC-negative patients (Fig. 3H). Amongthe study patients, 45.7% of the HNSCC patients had ahistory of betel nut use, which was significantly higher(p < 0.0001) in the CTC positive group compared to the
CTC negative group (Fig. 3I). Therefore, in Bangladesh,a history of chronic tobacco use (smoking and betel leaf

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Fig. 1 TCGA data analysis for Shh / Nrf2 gene expression in HNSCC patients. A The percentage (%) of HNSCC patients showing altered expressionof Shh. B, C Expression of Shh in HNSCC based on sample type and gender. D, E Expression of Shh in HNSCC patients based on age groupand tumor grade. F The percentage (%) of HNSCC patients showing altered expression of NFE2L2 (Nrf2). G, H Expression of Nrf2 in HNSCC basedon sample type and gender. I, J Expression of Nrf2 in HNSCC patients based on age group and tumor grade. Shh and Nrf2 expressions were
calculated in the number of transcripts per million. The expression pattern of Shh was checked based on the mRNA Z-score. **p-value < 0.005,***p-value < 0.001, ****p-value < 0.0001

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Fig. 2 CTC detection from peripheral blood. Images of CTCs detected in HNSCC patients captured by fluorescence microscope where staining
with DAPI emits a blue signal, and CK-18 emits a green signal. The top panel represents a cluster of CTCs, and the lower panel represents a single
CTC​

consumption) is linked with the presence of CTCs in
HNSCC patients.

CTCs may serve as a prospective prognostic marker forpost-treatment surveillance and early metastasis diagnosis in HNSCC patients.

CTC‑ a biomarker for the diagnosis of early metastasis
and therapeutic management

Clinicopathological features of the study cohort

Through microscopic examination, cancer grading
determines whether cancer cells are regular or aberrant. The more aberrant the cells look, the higher thegrade, and the faster the cancer is likely to spread. We
graded the study patients according to histopathological findings to see the association of CTCs with cancer
grade. The representative pictures of the tissue sections according to grade are displayed in Fig. 4A. Data
reported that grade 3 had the highest number of CTC
compared with grades 1 and 2 (Fig. 4B). Follow-up of
the selected CTC-positive HNSCC patients (n = 10)
for three cycles of chemotherapy, the CTC count was
found to have significantly decreased (p < 0.0005) following treatment (Fig. 4C). Taken together, detecting

Hematological parameters are widely used to diagnose
the disease severity and to compare disease and healthy
conditions. In chronic diseases like cancer, these parameters often deviate from normal ranges. In this study,
the mean red blood cell (RBC) count was found to besignificantly decreased (p < 0.05) in the blood samplesof CTC-positive patients compared to CTC-negative
patients (Fig. 5A). CTC-positive patients had significantly
higher neutrophil counts than CTC-negative patients(p < 0.005) (Fig. 5B). Platelet counts were significantly
lower in CTC-positive groups (p < 0.05) (Fig. 5C). Moreover, CTC-positive patients had higher mean corpuscular volume (MCV) compared to CTC-negative patients(p < 0.0001) (Fig. 5D). The neutrophil–lymphocyte ratio(NLR) and monocyte-lymphocyte ratio (MLR) were also

(See figure on next page.)
Fig. 3 Demographic and socioeconomic risk factors considered to select CTC-based HNSCC patients. A HNSCC patients were categorized
into two groups: CTC-positive (CTCs detected in blood) and CTC-negative patients (CTCs not detected in blood). B The mean age differenceof CTC-positive and CTC-negative HNSCC patients. Data is represented here as mean ± SD. P < 0.05, a two-tailed unpaired t-test was carriedout to compare the ages of CTC-positive and negative patients. C CTC count per 1.5 ml of blood in CTC-positive HNSCC patients. D, E Anatomicalsite distribution of CTC-positive and negative HNSCC patients. The number of HNSCC patients is presented as a percentage. F, G Distributionof CTC-positive and negative HNSCC patients based on age groups. The study subjects were grouped into different age groups with 10-year
intervals. H Comparison of CTC-positive patients to CTC-negative patients based on the duration of smoking. Mann–Whitney unpaired two-tailed
t-test was performed here as mean ± SD. P < 0.05 (I) Duration of Betel leaf use in CTC-positive and CTC-negative groups. Mann -Whitney unpaired
two-tailed t-test was performed here as mean ± SD. P < 0.05. ****p-value < 0.0001

Rahman et al. BMC Molecular and Cell Biology

Fig. 3 (See legend on previous page.)(2024) 25:4

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Fig. 4 Histopathological observation and CTC count in pre/post-chemotherapy in CTC positive HNSCC (A) Histopathological representationof studied subjects based on Grade. Two images in the first column depict the image of two patients (Patients 1 and 2) of Grade I, the middle
column represents the images of two patients (Patients 3 and 4) of Grade II, and the last column represents the images of two patients (Patients
5 and 6) of Grade-III. Histopathological slides were examined using the Hematoxylin and Eosin (H/E) procedure. B Distribution of CTC-positive
patients based on the grades and CTCs count (n = 64). Black circular dots represent the number of CTCs (n = 64). Data is represented here
as mean ± SD and one-way ANOVA was carried out to compare the number of CTCs and the Grade of CTC-positive patients and found highly
significant. C The number of CTCs in blood samples was tested before and after three cycles of chemotherapy (n = 10). Mann–Whitney unpaired
nonparametric unpaired t-test was performed and found highly significant. **p-value < 0.01, ***p-value < 0.005

significantly higher in the CTC-positive group compared to the CTC-negative group (p < 0.05) (Fig. 5E, F ).
No significant correlation was observed while multiple
linear regression analysis was carried out using CTC asa dependent variable against different hematologicalparameters (Table 1). Besides these, no significant correlation was observed in white blood cell (WBC) count,lymphocyte count, eosinophil count, monocyte count,hemoglobin levels, hematocrit levels, mean corpuscular hemoglobin (MCH), serum creatinine levels, mean

Rahman et al. BMC Molecular and Cell Biology(2024) 25:4

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Fig. 5 Enumeration of clinicopathological factors between CTC positive and CTC negative groups. A Red blood cell (RBC) was counted in million/
cmm using the Neubauer Chamber method and compared between CTC-positive than CTC-negative patients. B Leishman staining was used
to count neutrophils, and the results were compared between patients with CTC-positive and CTC-negative patients. C Platelet was counted using
the Leishman staining method and compared between CTC-positive and CTC-negative patients. D The physiological protocol determined mean
corpuscular volume (MCV) levels by dividing the RBC count by the hematocrit and multiplying the result by ten. E, F Neutrophil-to-lymphocyte
ratio (NLR) and monocyte-to-lymphocyte ratio (MLR) levels were calculated manually. *p-value < 0.05, ***p-value < 0.005, ****p-value < 0.0001
two-tailed unpaired t-test

corpuscular hemoglobin concentration (MCHC), serum
glutamic pyruvic transaminase (SGPT) levels, and erythrocyte sedimentation rate (ESR) values between the
CTC-positive and CTC-negative groups (SupplementaryFigs. 1, 2 and 3 and Supplementary Table 2).
Shh and Nrf2 expression in HNSCC patients basedon the presence of CTCs

HNSCC patients who exhibit overexpression of Shhand Nrf2 have shown reduced disease-free survival(DFS) and overall survival (OS) [6]. Additionally, ahigher number of circulating tumor cells (CTCs) has
been associated with more aggressive HNSCC andpoorer survival rates [17]. Therefore, the expressionof Shh and Nrf2 was compared between CTC-positive

and CTC-negative patient groups in blood and tissue samples. In the blood samples, Shh expression was
found to be significantly higher in HNSCC patientscompared to healthy controls (p < 0.01) (Fig. 6A). Shhexpression was found to be significantly higher in CTCpositive patients compared to CTC-negative patients(p < 0.0001) (Fig. 6B). Furthermore, in the tumor tissue samples, Shh expression was significantly higher
in CTC-positive patients compared to CTC-negative
patients (p < 0.0001) (Fig. 6C). Moreover, Shh expression was significantly higher in tissue samples compared to blood samples in both the CTC-positive andCTC-negative groups of HNSCC patients (p < 0.0001)(Fig. 6D, E).

Rahman et al. BMC Molecular and Cell Biology(2024) 25:4

Page 11 of 17

Table 1 Multiple linear regression analysis using CTC as a dependent variable against different hematological parameters C­ oefficientsa
Parameters

Unstandardized coefficientStandardized
coefficient

B

Beta

Standard Error

Intercept

1.782

4.433

WBC

-3.155e-005

0.0001044

0.1477tSignificance (P 95% CI for B
value)
Lower Bound

Upper Bound

Normalitytest*(α = 0.05)

0.40200.6896-7.146

10.71

Passed0.30220.7639-0.0002418

0.0001787

PassedLymphocyte

0.01038

0.028130.28190.36880.7140-0.04629

0.06704

PassedEosinophil

0.27610.14230.21211.940

0.0587-0.01057

0.5627

PassedMonocyte

0.06533

0.12230.27320.53440.5957-0.18090.3116

PassedHematocrit-0.01717

0.040310.35450.42590.6722-0.09836

0.06402

PassedHemoglobin

0.043860.17160.13670.2556

0.7995-0.3018

0.3895PassedMCH

0.023410.032230.31700.72620.4715-0.04151

0.08833

PassedS. Creatinine-0.30471.114

0.17410.27350.7857-2.548

1.939

Passed

MCHC-0.06824

0.07537

0.31640.90530.3701-0.2201

0.08357

Passed

SGPT-0.02754

0.020090.23921.371

0.1772-0.06800

0.01292

PassedESR

0.021540.011750.24171.833

0.0735-0.002134

0.04521

PassedMore details on Supplementary Table 2
CI confidence interval*

Normality test was used using Kolmogorov–Smirnov (distance) testaDependable variable is the CTC value

On the other hand, the expression of Nrf2 wasfound to be significantly higher in the blood samples of HNSCC patients compared to healthy controls(p < 0.0001) (Fig. 6F). The expression of Nrf2 was seen
to be significantly higher in the blood samples of CTCpositive patients compared to CTC-negative patients(Fig. 6G). Furthermore, Nrf2 expression was significantly higher in the tumor tissue samples of CTCpositive patients compared to CTC-negative patients(p < 0.0001) (Fig. 6H). Additionally, Nrf2 expression
was significantly higher in tissue compared to blood
samples of the CTC-positive and CTC-negative groupsof HNSCC patients (p < 0.0001) (Fig. 6I, J). Therefore,
there is a correlation between Shh and Nrf2 expression
and CTCs in HNSCC patients.

Expression of CTC with Shh and Nrf2 expression in HNSCC
during pre/post‑treatment surveillance

Radiation therapy and/or chemotherapy with a regimen
consisting of Cisplatin, 5FU, and Paclitaxel is the standard course of treatment for HNSCC in Bangladesh [19].
We further examined the blood samples of our patient
cohort (n = 10) and grouped them into two groups, before
treatment and after treatment, to determine whether the
expression of Shh and Nrf2 changes following treatment.
Our results showed that following chemotherapy and/or
radiation therapy, there was a significant decrease in theexpression of both Shh and Nrf2 (Fig. 7A, B).
Association of CTCs in overall survival of HNSCC patients

Survival analysis is a widely used statistical techniquein health research to analyze the time until an event ofinterest occurs. The Kaplan–Meier curve, a graphicalrepresentation of the probability of survival over time,(See figure on next page.)
Fig. 6 Expression of Shh and Nrf2 in blood and tissue samples based on CTCs. A Shh expression was quantified by qRT-PCR normalizedagainst GAPDH in blood samples of HNSCC patients and compared with healthy control. B, C qRT-PCR was used to measure Shh expression
in study subjects’ blood and tissue samples, normalized against GAPDH. The results were compared between patients who tested positive for CTC
and those who did not. D, E qRT-PCR was used to quantify Shh expression in both CTC-positive and CTC-negative HNSCC patients. The results
were normalized against GAPDH and compared between tissue and blood samples. F The expression of Nrf2 was measured in blood samples
from study participants using qRT-PCR, normalized against GAPDH, and compared to healthy control subjects. G, H The expression of Nrf2
was also measured in study subjects’ blood and tissue samples using qRT-PCR, normalized against GAPDH, and compared between patients who
tested positive for CTC and those who tested negative. I, J Quantitative real time PCR was carried out to determine Nrf2 expression in both bloodand tissue samples from patients with HNSCC who were CTC-positive and negative, normalized against GAPDH. Data represented as mean ± SD
and the Mann–Whitney test was carried out to compare Shh and Nrf2 expression among the study subjects. **p-value < 0.01, ****p-value < 0.0001,
two-tailed unpaired t-test (Mann–Whitney test)Rahman et al. BMC Molecular and Cell Biology

Fig. 6 (See legend on previous page.)(2024) 25:4

Page 12 of 17

Rahman et al. BMC Molecular and Cell Biology(2024) 25:4

Page 13 of 17

Fig. 7 Expression of Shh and Nrf2 before and after chemo and/or radiotherapy (A) Quantitative real-time (qRT-PCR) was used to measure Shh
expression in study participants’ blood samples before and after treatment, normalized against GAPDH. B Nrf2 expression was also quantified
by qRT-PCR normalized against GAPDH in blood samples of study subjects at pre- and post-treatment phage. Data is represented here
as mean ± SD, and the Mann–Whitney test was carried out to compare Shh and Nrf2 expression before and after treatment. * p-value < 0.05, ***p-value < 0.005, two-tailed unpaired t-test

also revealed that, out of the HNSCC patients that were
examined, 18 patients from Group A (CTC ≤ 2) and15 patients from the CTC-negative group were at risk
(Supplementary Table 3). Survival analysis of HNSCC
patients with different CTC scores revealed that patientswith a higher number of CTCs have poor overall survival
in HNSCC (Fig. 8).

Fig. 8 Survival analysis using Kaplan-Meyer (KM) plotter. Kaplan
Meier analysis for overall survival in the patient cohort according
to the number of CTCs to analyze the survival differencewith statistical significance. We categorized CTC negative as control
and CTC positive divided into two groups: namely group A (CTC ≤ 2)
and group B (CTC > 2). * p < 0.05

is frequently used in cancer survival analysis. We categorized the CTC-positive patients as the test group andthe CTC-negative patients as the control group. Next,
we plotted the survival curves after stratifying the CTCpositive patients into two groups: Group A (CTC ≤ 2)
and Group B (CTC number > 2). We performed a Logrank (Mantel-Cox) test and found a statistically significant Chi-square value of 11.85 (df = 2) with a p-valueof 0.0027. The median survival time differs between
groups A (40) and B (22). The study’s survival analysis

Discussion
Head and neck squamous cell carcinoma (HNSCC) is adevastating and heterogeneous cancer [21]. Worldwide,
HNSCC is the sixth deadliest cancer, with almost a million new cases detected every year and 50% mortality in
low-income countries. The five-year overall survival of
HNSCC is less than 50% [22], which warrants emphasison early diagnosis along with monitoring of treatment
outcomes. To address this concern, we aimed to develop
novel diagnostic, prognostic, and therapeutic markers
for head and neck squamous cell carcinoma. By using
the TCGA dataset, we revealed that Shh (48%) and Nrf2(34%) had been altered in HNSCC (Fig. 1). The alteration
is higher in male compared to female patients (Fig. 1C,G). Alterations of Shh and Nrf2 in HNSCC patients wereobserved in all age groups, suggesting that Shh and Nrf2
expression is independent of age in HNSCC (Fig. 1D, H).
Our data, too, has suggested a similar trend where both
Shh and Nrf2 are overexpressed in HNSCC. We observedthat males are more susceptible to HNSCC than females
in both CTC-positive and CTC-negative cases (Fig. 3B).
Canning M et al. reported that males are at greater risk
for the development of HNSCC and the metastatic progression of HNSCC [23]. The mean age of CTC-positiveRahman et al. BMC Molecular and Cell Biology(2024) 25:4

patients is greater than that of CTC-negative patients(Fig. 3C). Only two individuals in our study had the highest number of CTCs (6 CTCs). In contrast, most patients
had one or two CTCs (Fig. 3D). According to Yu et al.,metastatic breast cancer patients had more than 5 CTCsper 7.5 mL of blood [24]. Also, higher CTC numbers correspond with aggressive disease, less time to relapse, andincreased metastasis [25].
Circulating tumor cell (CTC) identification is a novel
concept in diagnosing many cancers, including HNSCC
at the early metastatic stage and surveillance of cancer
treatment. It is a minimally invasive liquid biopsy technique to detect early metastasis and monitor treatment
outcomes [26]. Our study found that the age range of
61–70 years was typical for CTC positivity, whereas theage range of 51–60 years was typical for CTC negativity(Fig. 3F, G). However, Wu et al. reported no significant
correlation between CTCs, age, and gender in HNSCC
patients [27].
Smoking is directly associated with the developmentof HNSCC, including a more extended period of smoking [28], and the use of betel leaf is a self-reliant risk factor for the occurrence of HNSCC [29]-the use of tobacco
accounts for 1.6 million deaths each year in Southeast
Asia. Approximately 10–20% of the world’s population
consumes betel quid and tobacco; approximately 81% of
this population is from Southeast Asian Regions, including Bangladesh. Our study tested whether using these
two tobacco products is associated with detecting CTCs.
Our data demonstrated that HNSCC patients having ahistory of smoking and betel nut consumption correlatedwith the presence of CTC (Fig. 3H, I). A similar result
was observed in a study where they reported that smoking alone is an independent important risk factor for thegenesis of HNSCC [30]. Another study also reported that
betel nut chewing history is a significant risk factor for
locally advanced HNSCC [31]. Therefore, smoking, andbetel nut use are independent risk factors for the detection of CTCs in HNSCC.
The number of CTCs was correlated with the advancing clinical stage of the disease in many epithelial-originated cancers, including HNSCC. Buglione et al. showedthat stage IV patients had higher CTCs than stage I-III[32]. Our data demonstrated that Grade-3 HNSCC
patients exhibit the highest number of CTCs comparedto grade 1 and grade 2 (Fig. 4B). A study showed that thepresence of CTCs is only correlated to the T and M stages[33], while another study concluded that CTC counting isrelated to the N stage rather than the T or M stage [34].
Hence, it is evident that the number of CTCs is increasing in higher-grade HNSCC patients compared to low
grade. Identification of CTCs may aid in evaluating the
response to cancer treatments. Our result has shown that

Page 14 of 17

the number of CTCs decreased significantly after threecycles of chemotherapy (Fig. 4C). McMullen et al. also
reported a similar finding in HNSCC patients, mentioning CTC as a potential maker for treatment response in
HNSCC [35].
Next, in our study, we also analyzed the clinicopathological features of our subjects based on their CTC count.
In our recent clinical trial in 152 oral cancer patients,
we observed that the preoperative CTC levels showed astronger correlation with adverse clinicopathological factors. The outcome suggested the role of CTCs as a sensitive prognostic marker to predict survival outcomes anddisease progress [20]We tested multiple hematological parameters of CTCpositive and negative HNSCC patients. Anemia is aprevalent physiological feature in all cancers, including
HNSCC [36]. Our study showed a significant reductionin RBC count in CTC-positive patients compared to
CTC-negative patients (Fig. 5A). The neutrophil countin patients positive for CTC was significantly higher thanin negative patients (Fig. 5B). There was a significant
decrease in platelet count in CTC-positive patients compared to CTC-negative patients (Fig. 5C). Szczerba et al.
reported a significant association between CTCs andneutrophils in breast cancer patients and animal models[37]. This observation supports our findings that there
is an association between CTCs and neutrophil levels in
HNSCC [38]. In a recent study, it was observed that there
is a positive correlation between platelet count and CTC
count in prostate cancer. The same study also reported
that the total lymphocyte count was positively correlatedwith the CTC count in prostate cancer [39]. MCV itself
is correlated with the survival of HNSCC patients [40].
There was a significant difference in MCV levels between
CTC-positive and CTC-negative groups (Fig. 5D). The
neutrophil–lymphocyte ratio (NLR) is one of the significant predictors in many cancers, including HNSCC [41].
Our study showed a significant relation of NLR in both
CTC positive and CTC negative patients, which may be
due to both groups being from HNSCC (Fig. 5E). Monocyte-lymphocyte ratio (MLR) in the peripheral blood of
HNSCC also serves as a worse prognostic marker [42].
Our study observed significant MLR in both CTC-positive and CTC-negative patients (Fig. 5F). For the firsttime, we have shown the difference in multiple hematological parameters based on the presence or absence of
CTCs in blood in HNSCC patients.
One of our main objectives was to investigate and compare Shh and Nrf2 expression in HNSCC patients’ tumor
tissue based on the presence or absence of CTCs. Shhand Nrf2 were reported to be overexpressed in HNSCC
patients [43, 44]. CTC is a novel concept in the early diagnosis of HNSCC to extend the survival rate of HNSCC

Rahman et al. BMC Molecular and Cell Biology(2024) 25:4

patients. We reported the novel correlation between Shh
and Nrf2 expression with CTCs in HNSCC. Diagnostically and prognostically essential markers such as Shhand Nrf2 are not investigated in the context of CTCs
in HNSCC. Previously, our group showed that Shh andNrf2 are potential therapeutic and prognostic biomarkers
for HNSCC [6]. Therefore, we planned to investigate theassociation between the expression of Shh/Nrf2 and thepresence of CTCs to establish these biomarkers as prognostic markers for early diagnosis and therapeutic management in HNSCC.
Sonic hedgehog is overexpressed in many cancers,
including HNSCC [45]. In our study, we found that Shh
was overexpressed in the blood of HNSCC (both CTC
positive and negative patients) compared to controls(healthy volunteers) (Fig. 6A). Shh overexpressed in
the blood of CTC-positive patients compared to that of
CTC-negative patients (Fig. 6B). Shh was also shown tobe significantly overexpressed in the tissue of patients
who had CTCs as opposed to those who did not (Fig. 6C).
CTC-positive and CTC-negative HNSCC patient groups
showed considerably increased Shh expression in tissue
samples compared to blood (Fig. 6D, E). In our study,
Nrf2 was also observed to be overexpressed in HNSCC(both CTC-positive and CTC-negative) patient bloodcompared to healthy subjects (Fig. 6F). Overexpressionof Nrf2 was observed in CTC-positive patient’s bloodcompared with CTC-negative patients’ blood (Fig. 6G).
Nrf2 was also shown to be significantly overexpressed in
the tissue of patients who had CTCs as opposed to those
who did not (Fig. 6H). CTC-positive and CTC-negative
HNSCC patient groups showed considerably increased
Nrf2 expression in tissue samples compared to blood(Fig. 6I, J).
We further analyzed our patient cohort and categorized it into two groups before and after treatment. We
observed that Shh and Nrf2 expression significantly
decreased after chemotherapy and or radiotherapy(Fig. 7A, B). The Kaplan-Meyer survival analysis curve
showed a poor survival outcome for patients with higher
CTC (Fig. 8). According to Kim H et al., overall survival(OS) was inversely correlated with the total number of
CTCs following chemotherapy, and a lower number of
CTCs after chemotherapy was substantially linked to alonger OS in pancreatic ductal adenocarcinoma [46]. The
sample sizes for men and women in this study were notcomparable. The sample sizes for the high and low grades
were also unrelated. It would be rational if only one histopathologist rated the HNSCC patients rather than several
pathologists.

Page 15 of 17

Conclusions
Shh and Nrf2 were highly expressed in HNSCC patients’blood and tumor tissue samples compared to CTC-negative HNSCC patients. Shh and Nrf2 were overexpressedin the tumor tissue of both CTC-positive and CTC-negative patients compared to blood. Smoking and betel leaf
users were at risk of having CTC in HNSCC patients,
and non-smokers and non-chewers had no CTC or fewer
CTCs. In CTC-positive patients compared to CTC-negative patients, RBC and Platelet counts were dramaticallydecreased, although Neutrophil count, MCV, NLR, andMLR were significantly increased. In our study subjects,
buccal mucosa and vocal cord are common sites of cancer
occurrence in CTC-positive patients. Ages 61 to 70 are
more susceptible to CTC positivity. In HNSCC patients
who tested positive for CTCs following treatment, thefrequency of CTCs considerably decreased. It was also
observed that Shh and Nrf2 expression was decreased in
post-treatment (combination regimen of Cisplatin, 5FU,
and Paclitaxel). In conclusion, CTC detection, Shh, andNrf2 overexpression could be ideal biomarkers for prognostic, therapeutic, and diagnostic purposes in HNSCC.
Abbreviations
Anti-EpCAM	Anti Epithelial Cell Adhesion Molecule AntibodyCBC	Complete blood countcDNA	Complementary DNA
CTC​	Circulating Tumor Cell
DAPI	4′,6-Diamidino-2-phenylindole
DFS	Disease-free survival
ESR	Erythrocyte sedimentation rate
ENT	Otolaryngology and Head Neck Surgery
5-FU	5-Fluro uracil
GAPDH	Glyceraldehyde 3-phosphate dehydrogenase
GLI1	GLI Family Zinc Finger 1
HNSCC	Head and Neck Squamous Cell Carcinoma
Keap1	Kelch-like ECH-associated protein 1
MCH	Mean corpuscular hemoglobin
MCHC	Mean corpuscular hemoglobin concentration
MCV	Mean corpuscular volume
MLR	Monocyte-to-lymphocyte ratio
MYC	Master Regulator of Cell Cycle Entry
Nrf2	Nuclear factor erythroid 2-related factor 2
NLR	Neutrophil-to-lymphocyte ratioOS	Overall Survival
PTCH1	Protein patched homolog 1
PCR	Polymerase chain reaction
qRT-PCR	Quantitative real time PCR
RBC	Red blood cell
SGPT	Serum Glutamic Pyruvic Transaminase
Shh	Sonic Hedgehog (Shh)SMO	Smoothened
ssDNA	Single-stranded DNA
SUFU	Suppressor of fused homolog
TCGA​	The Cancer Genome Atlas
TPM	Transcript per millioncaWBC	White blood cell

Rahman et al. BMC Molecular and Cell Biology(2024) 25:4

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12860-​024-​00500-0.
Additional file 1.
Additional file 2.
Additional file 3.
Additional file 4: S1 Table. List of primers used for qRT-PCR.
Additional file 5: S2 Table. Summary of hematological investigations.
Additional file 6: S3 Table. Patients at risk.
Acknowledgements
The Department of Otolaryngology and Head Neck Surgery, Department ofRadiotherapy, Chattogram Medical College Hospital (CMCH), and the Centre
for Research Excellence, Department of Biochemistry & Molecular Biology, University of Chittagong, Bangladesh, have been acknowledged by the authors assources of patient support and excellent research infrastructure, respectively.
The authors would also like to thank Actorius Innovations and Research
Pvt Ltd, Pune, Maharashtra, India, for their support in CTC detection. We aresincerely appreciating Dr. Md. Saiful Islam, Associate Professor of Pathology at
Rangamati Medical College, for his help in grading the histopathological slides
and providing images.
Authors’ contributions
MMR: Conceptualization and design of the study; experiment; data curation;formal analysis; original draft writing. MMH: Conceptualization and design ofthe study; experiment; data curation; validation; manuscript review & editing;project administration; supervision. SI: Conceptualization and design of thestudy; Collection of samples; experiment. RA, MM, SD, MK, BS, MERH: Collection of samples; experiment. AAC, SA, SB, MMA, MJH: Resources (hospital);
investigations. RKS, SB, RP: Data curation; formal analysis; supervision. MMI:
Data curation; formal analysis; supervision; fund acquisition. AB, SJ, GA, JK:
Experiment (CTC detection); supervision. SBU: Supervision; ASMN: Conceptualization and design of the study; manuscript review & editing; Supervision.
Funding
This work was partly supported by Research and Publication Cell (Ref:301/2021–22/First Call/25/2022) University of Chittagong (CU), Bangladesh.
Actorius Innovations and Research (AIR), India supported the CTC detection
expenses.
Availability of data and materials
No datasets were generated during the current study. Data will be available
on request.

Declarations
Ethics approval and consent to participate
This study was conducted in accordance with the Declaration of Helsinki andapproved by the National Research Ethics Committee (NREC), Bangladesh
Medical Research Council (BMRC) [Approval ID- BMRC/ERB/2018-2028/99(1100)]. All the participants were verbally informed about the research to bedone before being enrolled in this study, and their signed informed consent
was obtained and properly documented before enrollment. The study featured no minor research subjects.
Consent for publication
Participants in the study and the funding body confirmed approval for publication of the data.
Competing interests
Jayant Khandare has a CTC platform patent and is a part of Actorius Innovations and Research (AIR) and others do not have any conflicts of interest to
declare about this work.

Page 16 of 17

Author details
1
Rangamati Medical College, Rangamati, Bangladesh. 2 Department of Biochemistry & Molecular Biology, University of Chittagong, Chattogram 4331,
Bangladesh. 3 EuGEF Research Foundation, Chattogram, Bangladesh. 4 Department of Radiotherapy, Chittagong Medical College, Chattogram, Bangladesh.
5
Department of Surgery, Chittagong Medical College, Chattogram, Bangladesh. 6 Department of Otolaryngology and Head Neck Surgery, Chittagong
Medical College, Chattogram, Bangladesh. 7 Department of Otolaryngology
and Head Neck Surgery, Rangamati Medical College, Rangamati, Bangladesh.
8
Actorious Innovations and Research Pvt. Ltd., India and Simi Valley, Pune, CA,
USA. 9 Department of Botany, University of Chittagong, Chattogram, Bangladesh. 10 Present Address: Stem Cell Genetics, Institute of Life and Medical
Sciences, Kyoto University, Kyoto, Japan.
Received: 4 December 2023 Accepted: 29 January 2024

References
1. Chow LQ. Head and neck cancer. N Engl J Med. 2020;382(1):60–72.
2. Barsouk A, Aluru JS, Rawla P, Saginala K, Barsouk A. Epidemiology, risk
factors, and prevention of head and neck squamous cell carcinoma.
Med Sci (Basel). 2023;11(2):42.
3. Global Cancer Observatory. 2020. https://​gco.​iarc.​fr.
4. Huang G, Pan ST. ROS-mediated therapeutic strategy in chemo-/
radiotherapy of head and neck cancer. Oxid Med Cell Longev.
2020;2020:5047987.
5. Bottoni P, Scatena R. The role of CA 125 as tumor marker: biochemical
and clinical aspects. Adv Exp Med Biol. 2015;867:229–44.
6. Noman AS, Parag RR, Rashid MI, Rahman MZ, Chowdhury AA, Sultana
A, et al. Widespread expression of Sonic hedgehog (Shh) and Nrf2 inpatients treated with cisplatin predicts outcomes in resected tumors
and are potential therapeutic targets for HPV-negative head and neckcancer. Ther Adv Med Oncol. 2020;12:1758835920911229.
7. Kelly RJ, Ansari AM, Miyashita T, Zahurak M, Lay F, Ahmed AK, et al. Targeting the Hedgehog pathway using itraconazole to prevent progression of Barrett’s esophagus to invasive esophageal adenocarcinoma.
Ann Surg. 2021;273(6):e206–13.
8. Iriana S, Asha K, Repak M, Sharma-Walia N. Hedgehog signaling: implications in cancers and viral infections. Int J Mol Sci. 2021;22(3):1042.
9. Kumar V, Vashishta M, Kong L, Wu X, Lu JJ, Guha C, Dwarakanath BS. The
role of notch, hedgehog, and Wnt signaling pathways in the resistance oftumors to anticancer therapies. Front Cell Dev Biol. 2021;9:650772.
10. Flemban A, Qualtrough D. The potential role of hedgehog signaling in
the luminal/basal phenotype of breast epithelia and breast cancer invasion and metastasis. Cancers. 2015;7(3):1863–84.
11. Telkoparan-Akillilar P, Panieri E, Cevik D, Suzen S, Saso L. Therapeutic targeting of the NRF2 signaling pathway in cancer. Molecules.
2021;26(5):1417.
12. Namani A, Matiur Rahaman M, Chen M, Tang X. Gene-expression
signature regulated by the KEAP1-NRF2-CUL3 axis is associated with apoor prognosis in head and neck squamous cell cancer. BMC Cancer.
2018;18(1):46.
13. Zhou J, Ding J, Ma X, Zhang M, Huo Z, Yao Y, et al. The Nrf2/KEAP1 pathway modulates nasopharyngeal carcinoma cell radiosensitivity via ROS
elimination. Onco Targets Ther. 2020;13:9113–22.
14. Lianidou ES, Mavroudis D, Georgoulias V. Clinical challenges in themolecular characterization of circulating tumour cells in breast cancer. Br
J Cancer. 2013;108(12):2426–32.
15. Lianidou ES, Strati A, Markou A. Circulating tumor cells as promising novel
biomarkers in solid cancers. Crit Rev Clin Lab Sci. 2014;51(3):160–71.
16. Ahmed S, Bharde A, Hossain MM, Parial R, Nayeema NJ, Das M, et al. Circulating tumor cells (CTCs) detection and isolation in different subtypesof early-stage breast cancer patients from Bangladesh. J Clin Oncol.
2023;41(16_suppl):e12529.
17. Spector ME, Farlow JL, Haring CT, Brenner JC, Birkeland AC. The
potential for liquid biopsies in head and neck cancer. Discov Med.
2018;25(139):251–7.

Rahman et al. BMC Molecular and Cell Biology(2024) 25:4

18. Habli Z, AlChamaa W, Saab R, Kadara H, Khraiche ML. Circulating tumor
cell detection technologies and clinical utility: challenges and opportunities. Cancers. 2022;12(7):1930.
19. Jethwa AR, Khariwala SS. Tobacco-related carcinogenesis in head andneck cancer. Cancer Metastasis Rev. 2017;36(3):411–23.
20. Qayyumi B, Bharde A, Aland G, D’Souza A, Jayant S, Singh N, et al. Circulating tumor cells as a predictor for poor prognostic factors and overall
survival in treatment naïve oral squamous cell carcinoma patients. Oral
Surg Oral Med Oral Pathol Oral Radiol. 2022;134(1):73–83.
21. Gasparoni LM, Alves FA, Holzhausen M, Pannuti CM, Serpa MS. Periodontitis as a risk factor for head and neck cancer. Med Oral Patol Oral Cir Bucal.
2021;26(4):e430–6.
22. Zolkind P, Lee JJ, Jackson RS, Pipkorn P, Massa ST. Untreated headand neck cancer: Natural history and associated factors. Head Neck.
2021;43(1):89–97.
23. Canning M, Guo G, Yu M, Myint C, Groves MW, Byrd JK, et al. Heterogeneity of the head and neck squamous cell carcinoma immune landscapeand its impact on immunotherapy. Front Cell Dev Biol. 2019;7:52.
24. Yu M, Stott S, Toner M, Maheswaran S, Haber DA. Circulating tumor
cells: approaches to isolation and characterization. J Cell Biol.
2011;192(3):373–82.
25. Plaks V, Koopman CD, Werb Z. Cancer. Circulating tumor cells. Science.
2013;341(6151):1186–8.
26. Chalfin HJ, Pramparo T, Mortazavi A, Niglio SA, Schonhoft JD, Jendrisak A,
et al. Circulating tumor cell subtypes and T-cell populations as prognostic
biomarkers to combination immunotherapy in patients with metastatic
genitourinary cancer. Clin Cancer Res. 2021;27(5):1391–8.
27. Wu XL, Tu Q, Faure G, Gallet P, Kohler C, Bittencourt MD. Diagnostic andprognostic value of circulating tumor cells in head and neck squamous cell carcinoma: a systematic review and meta-analysis. Sci Rep.
2017;6(1):1–9.
28. Hernandez BY, Zhu X, Goodman MT, Gatewood R, Mendiola P, Quinata K,
et al. Betel nut chewing, oral premalignant lesions, and the oral microbiome. PLoS One. 2017;12(2):e0172196.
29. Alotaibi M, Valova V, Hänsel T, Stromberger C, Kofla G, Olze H, et al. Impactof smoking on the survival of patients with high-risk HPV-positive HNSCC:
A Meta-analysis. In Vivo. 2021;35(2):1017–26.
30. Su YY, Chien CY, Luo SD, Huang TL, Lin WC, Fang FM, et al. Betel nut
chewing history is an independent prognosticator for smoking patientswith locally advanced stage IV head and neck squamous cell carcinoma
receiving induction chemotherapy with docetaxel, cisplatin, and fluorouracil. World J Surg Oncol. 2016;14(1):1–9.
31. Johnson DE, Burtness B, Leemans CR, Lui VW, Bauman JE, Grandis JR.
Head and neck squamous cell carcinoma. Nat Rev Dis Primers. 2020;6:92.
32. Buglione M, Cavagnini R, Di Rosario F, Sottocornola L, Maddalo M, Vassalli
L, et al. Oral toxicity management in head and neck cancer patientstreated with chemotherapy and radiation: dental pathologies and osteoradionecrosis (Part 1) literature review and consensus statement. Crit Rev
Oncol Hematol. 2016;97:131–42.
33. Gröbe A, Blessmann M, Hanken H, Friedrich RE, Schön G, Wikner J, Effenberger KE, Kluwe L, Heiland M, Pantel K, Riethdorf S. Prognostic relevanceof circulating tumor cells in blood and disseminated tumor cells in bonemarrow of patients with squamous cell carcinoma of the oral cavity. Clin
Cancer Res. 2014;20(2):425–33.
34. Hsieh JC, Lin HC, Huang CY, Hsu HL, Wu TM, Lee CL, et al. Prognostic valueof circulating tumor cells with podoplanin expression in patients withlocally advanced or metastatic head and neck squamous cell carcinoma.
Head Neck. 2015;37(10):1448–55.
35. McMullen KP, Chalmers JJ, Lang JC, Kumar P, Jatana KR. Circulating tumor
cells in head and neck cancer: a review. World J Otorhinolaryngology
Head Neck Surg. 2016;2(02):109–16.
36. Borsetto D, Polesel J, Tirelli G, Menegaldo A, Baggio V, Gava A, et al.
Pretreatment high MCV as an adverse prognostic marker in nonanemic
patients with head and neck cancer. Laryngoscope. 2021;131(3):E836–45.
37. Szczerba BM, Castro-Giner F, Vetter M, Krol I, Gkountela S, Landin J, et al.
Neutrophils escort circulating tumour cells to enable cell cycle progression. Nature. 2019;566:553–7.
38. Zhang X, Wang Y, Gari A, Qu C, Chen J. Pan-cancer analysis of PARP1
alterations as biomarkers in the prediction of immunotherapeutic effects
and the association of its expression levels and immunotherapy signatures. Front Immunol. 2021;12:721030.

Page 17 of 17

39. Hayes B, Brady L, Sheill G, Baird AM, Guinan E, Stanfill B, et al. Circulating tumour cell numbers correlate with platelet count and circulating
lymphocyte subsets in men with advanced prostate cancer: data fromthe ExPeCT clinical trial (CTRIAL-IE 15–21). Cancers. 2021;13(18):4690.
40. Huang SJ, Zhan PF, Chen SB. Mean corpuscular volume as a prognostic
factor for patients with habitual alcohol or tobacco use after esophagectomy. Front Oncol. 2021;11:752229.
41. Wong BW, Stafford ND, Green VL, Greenman J. Prognostic value of theneutrophil-to-lymphocyte ratio in patients with laryngeal squamous cell
carcinoma. Head Neck. 2016;38(S1):E1903–8.
42. Song Q, Wu JZ, Wang S. Perioperative change in lymphocyte countand prognosis in esophageal squamous cell carcinoma. J Thorac Dis.
2019;11(6):2332.
43. Dimitrova V, Arcaro A. Targeting the PI3K/AKT/mTOR signaling pathway inmedulloblastoma. Curr Mol Med. 2015;15(1):82–93.
44. Stacy DR, Ely K, Massion PP, Yarbrough WG, Hallahan DE, Sekhar KR, et al.
Increased expression of nuclear factor E2 p45-related factor 2 (NRF2) inhead and neck squamous cell carcinomas. Head Neck. 2006;28(9):813–8.
45. Doheny D, Manore SG, Wong GL, Lo HW. Hedgehog signaling and truncated GLI1 in cancer. Cells. 2020;9(9):2114.
46. Kim H, Heo CM, Oh J, Chung HH, Lee EM, Park J, Lee SH, Lee KH, Lee KT,
Lee JK, Cho YK, Park JK. Clinical significance of circulating tumor cells after
chemotherapy in unresectable pancreatic ductal adenocarcinoma. Transl
Oncol. 2022;16:101321.

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