Role of neurotransmitter SP in progression of oral squamous cell carcinoma

1. ROLE OF NEUROTRANSMITTER SUBSTANCE P
IN PROGRESSION OF ORAL SQUAMOUS CELL
CARCINOMA
Amber Hassan
(PhD scholar)
AZNOSTICS The diagnostics
center

2. LIST OF CONTENTS:
oral squamous cell carcinoma (OSCC)
1. Incidence
2. Prevalence
3. Sign and symptoms
4. Risk factors
5. Condition
6. Grading system
7. Stages of OSCC
8. Diagnosis
9. Treatment
10. Tachykinin family
11. tachykinin receptors
12. Tachykinin genes
13. Substance P
14. Role of SP in different system
15. Trigeminal system
16. Trigeminal nerve
17. Aim of study
18. Operational defination
19. Material and methods
20. Results
21. Discussion

3. ORAL SQUAMOUS CELL CARCINOMA (OSCC)
 Oral squamous cell carcinoma (OSCC) that occurs
in any part of the mouth; on the tongue's surface, in
the lips, inside the cheek, in the gums, in the roof
and floor of the mouth, in the tonsils, and also in the
salivary glands.
 6th most common type of cancer.

4. INCIDENCE:
year Number of patients Region
1989 12,212 India
2001-2006 2157 West Yorkshire
2008 11682 United kingdom
2008 270,000 Italy, Spain
2010 10 cases per 100,000 Europe

5. PREVELANCE AND INCIDENCE:
In world wide
12.7 million new cases 2008
7.6 million death 2008
In UK 387,00 patients were diagnosed in 2006.
In Western countries 400,000 new cases.
• 350 million new cases in china
•740 million death in china
•75-80,000 new cases per year in India
•7.6 million death in India
•In Pakistan 38 % cases of oral cancer.

6. SIGNS AND SYMPTOMS:
 Red and white patches in mouth
 Mouth ulcer
 A swelling in the mouth
 A thickening of the skin or lining of the mouth.
 Pain when swallowing
 bleeding.
 Jaw pain.
 Jaw stiffness.
 A sensation that something is stuck in your throat.
 Painful tongue.
 Pain in the neck that does not go away

7. RISK FACTORS OF OSCC:
 Smoking
 Tobacco
 Alcohol
 Human Papilloma virus(HPV)
 Betel quid chewing
 Areca nut chewing
 Naswar
 Paan
 Gutka

8. SMOKING:
 Smoking decreased the gene
expression of anti-oxidant.
 Carcinogenic substances like
nicotine.
Use of cigarette, cigar, pipe smokers.
 Six times more chances to
developed the OSCC.
 USA and Asian countries.
 70% oscc in male and 55% in
female.
 Nicotine an addictive drug
 Damage the DNA
 Abnormal cell division.
smoking
tobacco
smoking
cigar
smoking
smokeless
tobacco
smoking
paan
Gutka
naswar

9. RISK FACTORS OF OSCC:
 Alcohol:
 Causes the carcinogenesis
 Tobacco smoking and use of alcohol have synergetic effect.
 Mouth wash
 Human papilloma virus:
 HPV 16 and 18.
 40 % OSCC
 HPV developed the oral lesions.
 Betal and areca nut chewing:
 Areca nut or betel nut chewing is also an important risk factor
for some south Asian and Chinese populations.
 Sri lanka , India
 Pakistan
 Bangladesh etc.

10. RISK FACTORS OF OSCC:
Naswar, paan and Gutka also cause the development
of OSCC.
Caused the oral lesion.
Mostly used in USA and Asian countries.
Gutka is a mixture of
 Betel nut
 Areca nut
 Lime
 Tobacco
 Caused the oral lesions.

11. CONDITIONS:
 Leukoplakia :
 The condition in which the white patches are formed in the
oral cavity.
 Erythroplakia:
 The condition in which red patches or lesion are formed in the
oral cavity.

12. TNM GRADING SYSTEM:
 Primary tumour staging (T)
 T0 - no evidence of primary tumour
 T1 - tumour 2 cm or less
 T2 - tumour greater than 2 cm and less than 4 cm
 T3 - tumour greater than 4 cm
 T4 - tumour invades adjacent structures.
 Nodal status (N)
 N0 - no regional nodal metastases
 N1 - single node,less than 3cm
 N2 - multiple node, 3-6 cm
 N3 : many nodal metastasis greater than 6 cm
 Metastases (M)
 M0 - no metastases
 M1 - distant metastases present

13. HISTOLOGIC STAGES OF OSCC:
 Stage I - the tumor is under 1 inch in diameter (2 cm)
 Stage II - the tumor is over 1 inch in diameter (2 cm) but less than 2 inches (4
cm).
 Stage III -The tumor is over 2 inches (4 cm) in diameter.
 spread to just one nearby lymph node.
 Stage lV - The cancer has reached tissues around the oral cavity. The cancer
has spread to other parts of the body.

14. DIAGNOSIS:
 Biopsy:
1. Excisional biopsy
2. Incisional biopsy
3. Needle Aspiration biopsy
CT scan:
 3 dimension image
 MRI:

15. DIAGNOSIS:
 Ultrasonography:
 Doppler Ultrasonography
1. Metastatic condition
 Intraoral Ultrasonography
 Tumor thickness

16. TREATMENT:
 Surgery:
 Preventive surgery
 Diagnostic surgery
 Staging surgery

17. TREATMENT:
 Radiation therapy
 For treatment of cancer
 Ionizing radiation
 Damage the cancerous genetic material
 For treatment of leukoplakia and erythroplakia
 Side effect
 Chemotherapy:
 Drugs used
 Destroy the cancer cells
 Damage the DNA

18. TACHYKININ:
 Is one of the largest family of neuropeptide.
Tachykinin Mammals Non-mammals
Substance P Human -
Neurokinin A Human -
Neurokinin B Human -
Enkephalin Human -
Neuromedin-K Human -
Hemokinin-1 Human -
Substance K Human -
Hylambatin - African frog
Physalaemin - Frog
Eledoisin - Amphibian
Kassinin - Frog

19. TACHYKININ RECEPTORS:
Neurokinin 1
Neurokinin 2
Neurokinin 3
Receptors Gene Ligand
NK1 TAC1 Substance P
NK2 TAC2 Neurokinin A
NK3 TAC3 Neurokinin B

20. G PROTEIN COUPLED RECEPTORS (GPCR):
 All 3 receptors belong to GPCRs.
 GPCRs is a 7 transmembrane protein.
 GPCRs

21. TACHYKININ GENES:
 The preprotachykinin PPT are precursor molecule.
 1300 bases
 Human PPT
 80-85 % beta PPT
 15-20% alpha and gamma PPT
 Expression of PPT gene is
regulated by cyclic AMP.

22. CAMP MEDIATED SIGNALING PATHWAY.

23. SUBSTANCE P:
 Belong to tachykinin family.
 Neuropeptide and neurotransmitter
 It is widely distributed in brain region
 Hypothalamus, cerebral, and cortex region
Amino acid sequence of SP.
Arg
Pro
Lys
Pro Gln
Gln
Pre
Phe Gly
Leu
Met
Tachykinin Substance P
Gene Tac 1
Receptor NK1R
Chromosomal
position
Chromosome # 7
Locus q21-q22

24. ROLE OF SP IN DIFFERENT SYSTEMS:
 Promote the cardiac
hypertrophy
 Prevent myocardial
stunning
 Bone metabolism
 Recover the traumatic brain
injury
 Urinary tract diseases
 Maintain the
gastrointestinal motility
 Regulate the hormones
 Wound healing
 respiratory diseases
 Inflammatory diseases
 etc.

25. TRIGEMINAL SYSTEM:
Trigeminal system are extensively studied in mammals.
Trigeminal ganglions:
In PNS these nerves are distributed in axon terminals.
Gasserian ganglion
Sensory ganglion of the trigeminal nerve
The trigeminal cave ( also known as Meckel's Cave)
It envelop the trigeminal ganglions.
Trigeminal system
1. trigeminal
ganglion
2. trigeminal
Mesencephalon
nucleus

26. TRIGEMINAL NERVE:
 Largest cranial nerve
 Distribute in head and neck region
 Released from trigeminal ganglions

27. TRIGEMINAL NERVE:
 The branches of trigeminal nerve form the short and long descending
tract.
Trigeminal
nerve
Ascending
order
Trigeminal
nucleus
trigeminal
motor
nucleus
Supra
trigeminal
nucleus
Descending
order
Decending
trigeminal
nucleus

28.  But this data is not sufficient and give insufficient
information.
 No study so far has been down in Pakistan.
 The rationale of this study is to see the potential of
SP as a proliferative marker in different grades of
OSCC and progression of OSCC.

29. AIMS OF STUDY:
 1. To evaluate the expression of Substance P in different grades of
oral squamous cell carcinoma.
 2. To check the potential of Substance P as a proliferative marker to
differentiate different grades of oral squamous cell carcinoma
tissues.

30. OPERATIONAL DEFINITION:
 Well differentiated oral squamous cell carcinoma:
 The cells appear similar to healthy cells. This is the initial stage of
oral cancer in which the cells components are layered in an
architectural pattern that looks like a squamous cell carcinoma cell
epithelium.
 Moderately differentiated oral squamous cell carcinoma:
 They look like normal oral squamous epithelium. The tumor cells are
in nests, there are some large, eosinophilic, polygonal cells that are
trying themselves in a squamous way, but the overal resemblance to
normal squamous epithelium is less striking.
 Poorly differentiated oral squamous cell carcinoma:
 They have lost most of its squamous epithelial characteristics and
architecture. This is the disorganized and aggressive stage of oral
cancer.

31. MATERIAL AND METHODS:
 study design:
 Cross sectional study
 Study duration
 One year
 Sample size
 40 cases of OSCC
 Inclusion criteria
 All Patients of OSCC were included.
 Patients of all age group and both gender were included.
 Exclusion criteria
 Tissues which were not processed properly
 Sample collection
 The tissue samples was collected from Mayo hospital, Ittefaq
Hospital and Fatima Memorial Hospital Lahore.

32. MATERIAL AND METHODS:
 Processing of OSCC tissue:
 Sectioning:
 microtome
 4-5 µm
Fixation
10%
phosphate
buffered
formalin
Different
grades of
alcohol
Embedding
in paraffin's
Cool down
in fridge

33. HEMATOXYLENE STAINING:
Cutting the tissue
Hot air chamber for 15
min
xylene I,II,III for 3 min
Alcohol I,II,III for 3 min
Distilled water for 3 min
Tap water for 1 min
Haematoxylene for 3
min
Tap water 1 dip only
Acid alcohol 1 dip only
Running water for 5
min
Ammonia water for 1
min
70% Alcohol for 1min
Eosine for 1min
4 jars of alcohol for 1
dip only
Mounting
Labelling

34. IMMUNOHISTOCHEMISTRY:
Cutting the tissue
Hot air chamber for 45-50 min
Xylene I,II,III for 3 min in each
Alcohol (different grades) for 3 min in each
Distilled water for 2 min
Target retrievel solutioin
Hot water chamber 45-50 min
Washing with wash buffer for 10 min
Blocking reagent 10-15 min
Primary antibody 45-50 min
Washing with wash buffer for 10-15 min
Secondary antibody 45-50 min
washing with wash buffer for 10- 15 min
DAB chromogen 1-5 min
washing with distilled water for 2 min
Haematoxylene for 30 sec- 1min
wash with tap water for 1 min
3 jars of Alcohol for 5 min
Xylene I,II,III for 5 min
Mounting
Labelling

35. RESULTS:
 40 biopsies were included. Cases were divided into 3 groups
 Well differentiated OSCC
 Moderately differentiated OSCC
 Poor differentiated OSCC
 Total # of OSCC
 Age wise and Gender wise distribution of OSCC.
well, 14
cases, 35%
mod, 14
cases, 35%
poor, 12
cases, 30%
Pie chart showing total No.
of OSCC cases
well
mod
poor

36. RESULTS:
Distribution of Sp cases
according to Sp expression
Age wise distribution of
OSCC according to SP
expression
SP+ve, 25
cases,
62%
SP-ve, 15
cases,
38%
20-40 41-60 61-80 total
SP +ve 4 11 10 25
SP -ve 3 6 6 15
total 7 17 16 40
0
5
10
15
20
25
30
35
40
45
No.ofOSCCcases
Agewise distribution of
OSCC cases according to
SP expression

37. RESULTS:
Gender wise distribution of
OSCC according to SP
expression
Gender wise distribution of
oscc according to grading
well mod poor total
SP +Ve 1 13 11 25
SP -ve 13 1 1 15
total 14 14 12 40
0
5
10
15
20
25
30
35
40
45
No.ofOSCCcases
gradewise distribution of
OSCC cases according to
SP expression
well mod poor total
female 6 3 2 11
male 8 11 10 29
total 14 14 12 40
0
5
10
15
20
25
30
35
40
45
No.ofcases
Genderwise distribution of
OSCC cases according to
grading

38. RESULTS:
SP +VE SP-VE total
female 5 6 11
male 20 9 29
total 25 15 40
0
5
10
15
20
25
30
35
40
45
No.ofOSCCcases
Genderwise distribution
of OSCC cases according
to Sp expression
well mod poor total
total 14 14 12 40
61-80 5 5 4 14
41-60 6 6 7 19
20-40 3 3 1 7
0
10
20
30
40
50
60
70
80
90
No.ofOSCCcases
Agewise distribution of
OSCC cases according to
grading

39. RESULTS:
0 1+ 2+ 3+
poor 0 2 4 6
mod 1 1 12 0
well 13 1 0 0
0
2
4
6
8
10
12
14
16
18
No.ofOSCCcases
gradewise distribution of
OSCC cases according to
intensity of SP
expression
well mod poor
SP+ve 1 13 11
SP-ve 13 1 1
0
2
4
6
8
10
12
14
No.ofOSCCcases
Gradewise distribution of
OSCC cases according to
SP expression

40. RESULTS:
8%
93% 92%
well mod poor
Histogram showing Sp
positive OSCC cases
SP +ve
4%
48%
48%
Pie chart showing SP
positive OSCC cases
well
mod
poor

41. RESULTS:
 Well differentiated OSCC, Substance P IHC. A)
showing intercellular bridges, SP negative B)
intercellular bridges and no atypia, SP negative C)
strand of cytoplasmic epithelium showing a typical
cell, SP negative D) sheet of cells showing
intercellular keratinization, SP negative E)
intercellular bridges and showing typical cell, weak
SP positivity seen, intensity +1, F) intercellular
bridges and moderate amount of cytoplasm.
 Well differentiated oral squamous cell
carcinoma, H &E. A) Moderate amount of
cytoplasm . B) Few cells showing individual cell
keratinization C) intercellular bridges D) strands
of cytoplasmic and diffused epithelium showing
a typical cell E) few cells showing intercellular
keratinization and keratin pearls. F) intracellular
bridges.

42. RESULTS:

 Moderately differentiated OSCC H& E A) Epithelium
dysplastic strands of neoplastic cells in underline
dermis. B) Neoplastic cells in underline dermis C) sheets
of cluster cells in neoplastic cells and keratin pearls D)
sheet of cluster cells and moderate amount of Atypia. E)
sheet of cluster in malignant cells F) sheet of cluster
cells, small intercellular bridges .
 Moderately differentiated oscc, SP IHC. intensity is
+2 A) strands in epithelium and underline epithelium
cells B) neoplastic cells and 70% tumor stands in
the cytoplasmic region C) stands of tumor present
and Malignant cells D) 50% tumor are present in
cytoplasm. E) Abundant cytoplasm and malignant
cells. F) stands of neoplastic cells in epithelium cells.

43. RESULTS:
 Poorly differentiated oral squamous cell
carcinoma, H & E. A) cluster are medium in
size and penetrating in dermis cells B) nest in
neoplastic cells C) Diffused sheets of
neoplastic cells infiltrating in dermis cells D)
sheet of neoplastic cells in epithelium cells.
 Poorly differentiated oscc SP, IHC. All cases SP
positive with intensity of expression +3, strong
expression A) showing 80-90% tumor in the cells.
B) maximum atypia. C) sheet of neoplastic cells
and atypia D) Bundle of malignant cells. E) no
keratin pearls and intercellular bridges but high
atypia. F) Bundles of malignant cells.

44. COMPARISON OF WELL, MODERATELY AND
POOR DIFFERENTIATED OSCC:
 A) Well differentiated OSCC, SP weak
positive, +1 intensity, Intercellular
bridges and moderate amount of
cytoplasm at 10X, B) at 40X, C)
Moderately differentiated OSCC, SP
intermediate positive, +2 intensity,
strands in epithelium and underline
epithelium cells D) at 40X, E)
Moderately differentiated OSCC, SP
positive, +2 intensity of SP expression,
F) at 40X, G) Poorly differentiated
OSCC, SP strongly positive, intensity
+3, showing 80-90% tumor in the cells
and maximum atypia at 10X, L) Poorly
differentiated OSCC, SP strongly
positive, +3 intensity, sheet of neoplastic
cells and atypia, Bundle of malignant
cells.
 W-Well differentiated, M-Moderately
differentiated, P-Poorly differentiated

45. DISCUSSION:
 Previous studies reported the expression of SP in different cancers and in OSCC. But
there is insufficient information available. No study clearly demonstrates the expression
of SP in different grades of OSCC.
 Increased the level of SP Apoptosis cell proliferation and cell
 division increased cancer
 SP phosphorylation protein kinase Akt Antiapoptosis.
 In tumor cell more SP expression more NK1R
 SP migration of tumor cells and Angiogenesis proliferation of
 endothelial cells increased
 In my study SP level increased
 Well (less) moderate (increased) poor ( double increased)

46. CONCLUSION:
 Prevalence of oral cancer in males suggests substance abuse
e.g pan, cigarette, beetle nuts, chewing gum and other such
things as the etiology of oral cancer in males in our region.
 We suggest Substance P as a potential biomarker for the
proliferation of oral squamous cell carcinoma cases.
 We can also differentiate different grades of oral cancer on the
basis of its expression.
 It has a strong role in progression and development of tumor.

47. FUTURE ASPECTS:
 Substance P should be studied as a proliferative
marker for the detection of other cancers also
 The expression of Substance P should be studied
at serum levels
 Tachykinin 1 gene should also be studied in future
for any kind of mutations
 Expression of NK-1R should also be studied in oral
as well as other cancers and the use of its
antagonists can block the pathways leading to
cancer

48. PAPER SUBMITTED:
 Journal of cellular physiology
 Impact factor: 4.218