This thesis examines the expression of the CD44 cancer stem cell marker in different stages of head and neck squamous cell carcinoma (HNSCC). Samples were collected from 7 HNSCC patients and analyzed using histological staining, immunohistochemistry for CD44 expression, and quantitative RT-PCR of CD44 mRNA levels. Results showed higher CD44 expression in hyperplasia compared to dysplasia and invasive carcinoma samples. CD44 mRNA levels were also higher in tumor samples than normal tissue. This preliminary study suggests CD44 may be a marker for cancer stem cells in HNSCC, though more samples are needed to draw firm conclusions.

1. Immunohistochemical and mRNA expression analysis of CD44
cancer stem cell marker in different stages of Head and Neck
Squamous Cell Carcinoma patients
Defense of Thesis Presented By,
Name: Gourab Mallick
M.Pharm, Pharmacology, 4th Semester
Roll No.– 19320214003
Registration No. – 141932310003 of 2014-2015
Bengal School of Technology
Under Supervision of :
Dr. Chinmay Kumar Panda, Ph.D.
Head and Senior Scientific Officer, Asst. Director Grade
Department of Oncogene Regulation,
Chittaranjan National Cancer Institute (CNCI)
Prof. (Dr.) Himangshu Sekhar Maji, Ph.D
Principal of Bengal School of Technology
Professor (Pharmaceutical Analysis)
Bengal School of Technology

2. Contents:
1. Cancer
2. What is HNSCC? Where it develops?
3. Epidemiology of HNSCC & Etiological Factors in the development
of HNSCC.
4. Histological features of HNSCC
5. Cancer Stem Cell characteristics in HNSCC
6. CD44 CSCs in HNSCC
7. How CD44 act as a marker in CSCs of HNSCC
8. Objective of the Study
9. Methods to achieve the objective
10. Results
11. Discussions and conclusions
12. References

3. Cancer – Cancer is characterized by the uncontrolled cell growth
and spread of abnormal cells.
Six essential alterations inside cell physiology that collectively
termed as “Basic hallmarks of cancer”.
 Self-sufficiency in growth signals.
 Insensitivity to anti-growth signals.
 Evading apoptosis.
 Limitless replicative potential.
 Sustained angiogenesis.
 Tissue invasion and metastasis.

4. What is HNSCC?
Squamous cell carcinoma is a cancer that arises from particular cells
called squamous cells. Squamous cells are found in the outer layer of
skin and in the mucous membranes, which are the moist tissues that
line body cavities.
Head and neck squamous cell carcinoma (HNSCC) develops in the
mucous membranes of the mouth, nose, and throat.
Where it develops?
The oral cavity and oropharynx Squamous cell carcinoma (SCC).
The nasopharynx.
The hypopharynx, larynx, and trachea, and
The nasal cavity and paranasal sinuses.

5.  Epidemiology of HNSCC: Head and neck cancers represent the sixth
most common cancer worldwide with approx. 630,000 new patients
diagnosed annually resulting in more than 350,000 deaths every year. The
international agency for research on cancer has predicted that India's
incidence of cancer will increase from 1 million in 2012 to more than 1.7
million in 2035.
 Etiological Factors:
1. Tobacco Smoking
2. Alcohol
3. Genetic susceptibility
4. Chronic viral infection (HPV-16 and HPV-18)
5. Occupational exposure
6. Malnutrition
7. Poor dental care
8. Mechanical irritation

6. HNSCC arises from a common premalignant progenitor
followed by outgrowth of clonal populations associated with
cumulative genetic alterations and phenotypic progression
to invasive malignancy. The formation of benign and/or
malignant neoplasm can occur during the following stages –
 Hyperplasia
 Dysplasia :
 Mild dysplasia
 Moderate dysplasia
 Severe dysplasia
 Carcinoma in situ
 Micro-invasive carcinoma

8.  A 20 amino acid (aa) signal sequence.
 An extracellular domain (ECD) with a 100 aa hyaluronan-binding disulfide-
stabilized link region.
 A 325-530 aa stem region, a 21 aa trans membrane domain.
 A 72 aa cytoplasmic domain.

9.  CD44 either alone or in combination has the properties of a cancer
stem cell marker and being a tumor initiator.
 CD44 binds hyaluronic acid (HA) and other ligands within the
extracellular matrix, CD44 is able to bind growth factors and
metalloproteinase, resulting in inhibition of apoptosis, collagen
degradation, invasion and metastasis in HNSCC. In addition, CD44
acts as an anchored reservoir for growth factor receptor ligands.
 By that way, CD44 gene while binds to hyaluronic acid (HA) gives
expression which can be analyzed by immunohistochemistry and
mRNA expression analysis, which is a prognostic marker in CSCs
(Cancer stem Cells) of HNSCC.

10.  Histological analysis of HNSCC biopsy tissue.
 Protein expression analysis by Immunohistochemistry of
CD44 stem cell marker in HNSCC.
 mRNA expression analysis by qRT-PCR of CD44 stem cell
marker in HNSCC.

11.  Samples were collected of seven freshly operated head and neck lesions
prior to any therapy along with adjacent normal tissues of the same
patients.
 Paraffin embedded were done for three (3) samples among the collected
seven samples.
 After that, Micro-dissections were done of three (3) paraffin embedded
samples.
 Then, Haematoxylin and Eosin staining were done of that three samples to
identify the histopathologically progressed region under bright field
microscope in those samples.
 Followed by, Immunohistochemical (IHC) staining were done of those
three samples to score the CD44 expression of the histopathologically
progressed area (hyperplastic zone, dysplastic zone etc.).
 Next, Isolation of Total RNA was done by Trizol reagent from remaining
four (4) pairs HNSCC tissue samples among the seven samples which
were previously collected.

12.  Then, Quantification of freshly obtained RNA from that tissue
samples were done by spectrophotometric method.
 After quantification of RNA, RNA gel electrophoresis were done by
using Formaldehyde-Agarose gel and EtBr as staining dye to
ensure the integrity of the RNA was intact.
 Then, cDNA Preparation from RNA samples were done by RT-PCR
using DNaseI (Promega) enzyme and M-MuLV-Reverse
Transcriptase (Promega) enzyme.
 After that standardization of CD44 and B2M gene were done in a
sample pair (normal and tumour).
 Quantitative gene expression analyses were done by specific
primers using Real-time quantification method using SYBR-Green
dye (Roche) under Light Cycler Platform (Roche). β2-microglobulin
(B2M) was used as the housekeeping gene to determine the
relative level of gene expression of CD44 gene by the comparative
threshold cycle (CT) method.

13.  For performing Real-time PCR based quantification, mRNA specific
primers were designed using NCBI/Primer BLAST tool. Sequence of CD44
(target gene) and B2M (house-keeping gene) forward and reverse primer
and product length is-

14.  Demography of HNSCC patients-

15.  Haematoxylin and Eosin Staining-
.
H&E staining of Normal sample (A, B); Dysplasia (C, D);
Invasive Carcinoma (E, F); (C, D- #650; E, F- #623).
Dysplasia and Invasive carcinoma marked with arrows in the image (C, D, E,
and F).
H and E staining observation-
The nucleus size is larger in the basal
layer, and the size decreases while
moving from the basal to para-basal
and spinous layer. This is because
the basal cells are more proliferative
and have less differentiation and while
moving towards the spinous layer the
proliferation lowers down and the cell
differentiation increases.
Dysplasia (#650) show some residual
structure and Invasive Carcinoma
(#623) show no definitely morphology.

16.  Immunohistochemical (IHC) Staining-
Representative IHC image of CD44 positive cells are
marked with arrow.
(A)– Normal;
(B)– #651 (Hyperplasia) (Moderate intensity);
(C)– #650 (Dysplasia) (Moderate intensity);
(D)– #623 (Dysplasia and invasive carcinoma) (Weak intensity).
IHC staining observation (Scoring)-
The staining intensity (weak, moderate, strong)
and the percentage of positive cells (<1=0, 1-20=1,
20-50=2, 50-80=3 and >80=4) were detected.
The final evaluation of expression was done
(0-2=low/weak,3-5=moderate/intermediate,
6-7=high). [M- Membrane; C- Cytoplasm; N-
Nucleus; Y-presence; X-absence].

17. RNA quality check by formaldehyde-agarose
gel electrophoresis
RNA O.D. Quantification table of oral tumour and adjacent
normal tissue obtained from HNSCC patient’s sample.
Representative image of RNA gel electrophoresis
Lane 1- S/15/7655(N)
Lane 2- S/15/7655(T)
Lane 4- S/15/7185(N)
Lane 5- S/15/7185(T)
Lane 7- S/445/16(N)
Lane 8- S/445/16(T)
RNA Optical Density (O.D) quantification
table

18.  Standardization of CD44 and B2M gene in cDNA obtained from RNA-
To achieve the objective, the mRNA expression quantification of CD44 gene, was first standardized
by PCR based method to check the specificity of the primers and to identify the annealing
temperature for PCR.
Standardization of CD44 Gene for expression study
Lane 1- pUC19 MSPI digested marker
Lane 2- CD44 (141bp)
Standardization of CD44 and B2M gene for expression analysis
Lane 1- pUC19 MSPI digested marker
Lane 2- Expression of B2M Gene in cDNA of normal tissue
Lane 3- Expression of CD44 Gene in cDNA of tumour tissue
Lane 4- Expression of B2M Gene in cDNA of normal tissue
Lane 5- Expression of CD44 Gene in cDNA of tumour tissue

19.  Amplification and Tm Plot (melting curve) of B2M and CD44 Gene by
using qRT-PCR analysis-
Amplification plot for (A) B2M gene and (B) CD44 gene Melting curves (Tm curve) for (C) B2M gene and
(D) CD44 gene
For a quantitative estimation of the CD44 gene , qRT-PCR method was standardized with respect to B2M gene on
RNA samples derived from tumor tissues.

20.  Ct value and relative expression calculation of CD44 gene by using qRT-PCR
method-
CD44 Delta Ct value can be achieved by subtracting the Ct value of B2M gene from Ct value of
CD44 gene.
Delta Ct = [Ct (Target) - Ct (B2M)]
A B
DC
A- Comparison between B2M and CD44 gene in normal tissue sample ; B- Comparison between B2M and CD44 gene in tumour tissue sample; C- Comparison
of CD44 delta Ct value between normal and tumour tissue samples; D- Comparison of CD44 relative expression in normal and tumour samples.

21.  From the experiments conducted and the sample’s details of the patients,
it can be concluded that-
 Among seven patients the number of male patients suffering from Head and Neck
Squamous Cell Carcinoma are higher than that of female patients (57.14% male and
42.85% female). Out of the seven patients invasive carcinoma patients are found to be
71.43%, hyperplasia and dysplasia patients are 14.28% each.
 Form H and E staining of the two sample (# 650 and # 623), it can be concluded that in
tumours, the nuclear size is large compared to the cytoplasm, i.e., the nucleo-cytoplasmic
index is high, indicating that tumor cells are actively dividing.
 Form IHC staining of three samples (#651, #650 and #623) and after scoring of that three
samples, it can be found that the frequency of CD44 positive expressions at the cell
membrane present at the basal layer of patients #651, #650 and #623 are 73.8%, 59.45%
and 46.15% respectively. So, the hyperplasia patient sample (#651) has high CD44
positive cell expression whereas dysplasia patient sample (#650) has moderate CD44
positive cell expression and invasive carcinoma patient sample (#623) has weak CD44
positive cell expression.

22.  From RNA O.D quantification chart, it was found that all the O.D ratio (260/280 ratio)
values of the four pairs of samples are between 1.8-2.0. So, all the samples contain pure
preparations of RNA and integrity remains as intact and there was no DNA contamination
shown in any sample.
 From the standardization of CD44 and B2M gene for expression analysis the PCR bands
were found concordant with the respective amplicon sizes for CD44 gene. The annealing
temperature for PCR of CD44 gene was found to be 56°C. The exponential phase of
amplification plot of both gene starts at PCR cycle no 24 for B2M gene and PCR cycle no
20 for CD44 gene.
 Form melting curve analysis it can be concluded that, the melting curves of all the genes
showed a single pick confirming the specificity of the amplification products is very high in
nature.
 The expression pattern of CD44 gene in normal tissue and in tumour tissue sample
relatively high than B2M gene expression in same sample. By Ct value and relative
expression calculation, it can be concluded that the median value of CD44 delta Ct value
high for normal and low for tumour. But the median value of relative expression of CD44
high is observed for tumour and low in normal sample.
 From the plot of relative expression of CD44 gene it can be concluded that the overall
relative expression is high in tumour sample than normal sample. Hence, lower the Delta
Ct value in the sample causes higher relative expression in the same sample.
 This is a preliminary study of only seven (7) tumor obtained from HNSCC patients.
Though, much study is required in large pool of samples for any concrete conclusion.

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