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INTRODUCTION
GE NERAL PRE VIE W O F ORAL CANCER
Oral cancer is one of t he most common mali gnant neopl asi a
world wi de. Each ye ar about 14 mil lion new oral cancer pati ents are
diagnos ed. The inci dence rat es of oral cancer are hi gher in men as
com pared t o wom en. Squam ous cell carcinoma (SSC ) i s t he m ost
comm on his tologi cal t ype of oral cancer as 90% of oral cancers are
ori ginates from s quamous cel ls ( 1 ). The main s i tes for oral cancer incl ude the li ps, gum s, tongue, buccal mucos a, the floor (bott om) of
the mout h, the hard palat e (bon y top of the mout h ), and gingival
(Fi gure 1.1). Generall y oral cancer is det ect ed b y clini cal examinati on followed b y biops y of tis sues for histopathol ogi cal
conform ation. The development and progressi on of oral squamous
cell carci nom a is a m ulti facet ed compl ex dis eas e invo lvi ng
environm ent al and geneti c factors. Major ri sk associ at ed fact ors for
oral cancer are tobacco, bet el quid and al cohol i ntake.
Earl y pres ent ati ons and precancerous s tage of oral cancer are
oft en observed as red or whit e mucos al les ions defined as oral
er yt hroplakia (red) or oral l eukopl akia (whit e), respectivel y.
Subs equent l y, precancerous lesions advance from h yperpl asia t o
d yspl asi a and then t o carci nom a i n sit u before develop int o i nvasive
oral m ali gnant neoplasi a( 2 ). Oral cancer is generall y categoriz ed into four t ypes accordi ng to t heir t um our, node, and m et ast asis
(TNM ) st ages. Tum our st ages , T0 represent no evi dence of tumour,
T1 si gni f y tumours l ess t han or equal to 2 cm , T2 repres ent t umour
greater than 2 cm , T 3 repres ent greater t han 4 cm and t umours are
considered T4 i f tumours have i nvaded surroundi ng areas.
Moreover, nu m ber of l ym ph nodes (N) and dist ant sit es (M)
occupied b y the tum ours are also included i n the TNM cl as sification
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keratiniz ation of tumour cell s int o four groups, namel y grade - I
(Well di fferenti ated tumours), grade -II (Moderat el y differenti at ed
tumours ), grade - III (poorl y differentiated tum ours ) and grade -IV
(Anaplasti c tumours ) ( 4 , 5 ).
Figu re 1.1:A-showi ng OSCC o f t he Tongue and B-s howing OSCC of the Soft P al at e; and C&D- showing OSCC of the Al veolar Mucos a
Over the past t wo decades, in s pi te of t he m edi cal
advancem ents in local cont rol and the use of different combined
therapi es, the s urvi val rat es for oral cancer have not enhanced
si gni fi cantl y. Dela y in di agnosis i s one of the m ajor factor for low
survival rate in oral cancer patie nts. Therefore, novel diagnosti c
methods of oral cancer need to be devel oped for earl y detecti on and
therap y ( 6 ). A better understandi ng of m echanisms leadi ng to OSCC is needed t o devel op more effective diagnostic and therapeuti c
[image:2.595.111.475.176.493.2]3
Prevalen ce of oral cancer In India
In India, oral cancer is the m ost com mon mali gnanc y amongs t
men (11.28% of a ll cancers), fift h m ost cancer amongst wom en
(4.3% of all cancers ) and t he thi rd most common cancer among m en
and women. The m ean age of oral cancer was 50 years . About 80%
of oral cancers are m ainl y att ribut able to tobacco cons umpti on ( 7 , 8 ).
In Indi a, the preval ence of cancer was m aint aining through by
two cancer regist r y: 1) hospit al -bas ed cancer regist ries, and 2)
popul ati on -bas ed cancer regist ri es (PBCRs ). P BCR s provide
inform ation about the et iology, burden of cancer and the
effectiveness of the treatm ents that have been undertaken to control
cancer. P BCR s are the onl y dat a source which delivers authentic
inform ation on the mort alit y and inci dence of an y cancer in Indi a.
Accordi ng to the cancer regist r y, the overall incidence of oral
cancer is hi ghest i n t he cent ral region of Indi a. In m al es, t he
preval ence of oral cancer is 64.8% and i n females 37.2% at age of
70 years . The next hi ghest preval ence of oral cancer was observed
in northeast regions and west (about 60%) at age of 60 ye ars ( 9 ). Dat a indi cators like age -adjust ed rat es (AAR s) of oral cancer
occurrence of m al e and fem al e in each of the 29 regist ri es of P BCRs
were obt ained from Nati onal C ancer R egis tr y P rogramm e (NCRP)
source and were represent ed regi on -wise. According t o cancer
regist r y in India, age -adjust ed rate (AAR) of m outh cancer is the
hi ghest in the cent ral regi on am ong m al es (64.8%) in the age group
60-69 years fol lowed b y west and northeast regi ons of Indi a with
AAR of 58.4%. Am ong fem al es, oral cancer is the hi ghest in the
nort heas t and cent ral regi ons (60.2% and 37.2%, res pecti vel y), in
the age group of 70 -7 5- years AAR al so indicat es increasi ng
inci dence of oral cancer wit h age. Am ong the different t ype of oral
cancers, AAR of mouth and t ongue cancers i n m al es dis pla y c as es at
a ver y earl y age gr oup (0 –20 years) part icularl y i n t he west ern
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World
Globall y, 369,200 new cas es of oral cancers were report ed
with two-t hirds of the oral cancers identi fi ed i n developing
countries. Worldwide oral cancers are responsibl e for about 145,328
deaths per year. The hi ghest i nci dence of oral cancers i s mostl y
repo rt ed from South and Southeast Asi a. The preval ence of OSCC
vari es si gnifi cantl y, depending on geographical areas whil e
diagnos ed (11 ). Hi gher incidence of oral cancer i s s een in South and
Southeast Asi a i ncl udes Pakist an, Indi a, Tai wan and Sri Lanka and
parts of the W est i ncl udes France. As wel l as, in East ern Europe
incl udes Slovenia, Hungar y, and Slovakia, and Latin America and
the C aribbean includes Urugua y, Brazil and Puerto Ri co and also in
Pacifi c count ri es s uch as P apua New Guinea and Melanesi a.
Accordi ng to t he Human development index of the United Nations
Devel opm ent P rogramme (UNDP ), the incidence al so di ffers. Bas ed
on this i ndex, t he i nci dence is greater in count ri es wit h im proved
growth indicat ors . The GLOBOCAN grouping exposes that the crude
rat e and age -st andardized inci dence rat e (uni vers all y) are great er in
most developed countries, but deat h rat e is s uperior in les s
devel oped count ri es, whi ch exposes soci al inequalit y (2).
Risk factors in oral can cer
Cigarett e sm oki ng and al cohol consumpt ion are the major ris k
fact ors for oral cancer and are report ed t o pl a y a s ynergisti c rol e i n
increasing the risk for oral cancer. Tobacco sm oke contains various
carcinogeni c el em ents whi ch can be basi call y grouped int o 3
groups: nit ros ami nes, aromatic amines, and benz o -p yrenes (BAP)
( 1 2 )
. Thes e el em ent s are t erm ed as pre -carcinogens which whe n
undergoes oxidati ve, enz ym at ic and other non -enz ym ati c
modi ficati ons transforms int o el ect ron defi ci ent s econdar y product s
that can form an adduct i n the DNA b y coval ent bonding ( 1 3 ). Accordi ng to the Int ernati onal Agenc y for R ese arch on
Cancer ( IARC ), 2007 report , snuff cons umption was rel ated to the
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ma y l ead to the dam age of t he oral epithelium due t o the increas ed
production of ox yge n and nit rogen free radi cal s. So m e of the m ost
important carcinogenic arom ati c h ydrocarbons pres ent in t obacco
smoke are the benz o -p yrene and the tobacco -specifi c nit ros amines
such as 4 -(nit rosom eth yl am ino) -1 -(3-pyrid yl ) -1-but anone (NNK)
and N’-nitrosonornicotine (NNN) ( 1 4 )
.
Alcohol i s an import ant risk factor for oral cancer which not
onl y acts locall y but also s ystemat ical l y b y increasing the oral
mucosal perm eabili t y, dis solving epit heli al li pid component s
leading t o epit helial atroph y as well as i mpai red DNA s ynthesis and
repair. Alcohol has also been report ed t o have both genot oxic and
mutageni c effects affecti ng t he salivar y flow as well as the abili t y
of t he liver t o detox if y pot ent ial cancer -causing com pounds in the
bod y. R egul ar consumpti on of al cohol has been found to be
associ at ed wit h im paired innat e and acquired immunit y whi ch ma y
cause i ncreased vulnerabilit y to i nfections and neopl asm s ( 1 5 ).
The chewing of bet el l eaf is a common t raditional practi ce i n
man y S out heast Asi an count ries, especi all y i n Indi a where it is
consum ed along wit h other ingredi ent s l ike areca nut, tobacco, and
lime. It is comm onl y term ed as bet el quid. In count ries like
Thailand, vari ous ot her ingredi ent s such as cardamom, anis eed or
cloves are al so added to the bet el quid. In Indi a, apart from bet el
quid tobacco is als o consum ed in vari ous other forms such as
khaini, mi shri, z arda, mawa and guthka. In C ent ral Asi a and the
Middl e East, tobacco is consum ed in the form of nas s and
nas war/ nis war respectivel y. In Saudi Arabi a and S udan, it is
consum ed as shammah and t oombak respectivel y ( 1 6 ). Vari ous reports have previ ousl y descri bed the associ ation of t obacco
consumption wit h t he i ncreased risk for oral cancer and pre
-cancerous s tages s uch as oral submucous fibrosi s, er yt hropl aki a, and
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bet el nut and tobacco product s. In vitro st udi es on oral m ucosal
fibroblasts using DNA dam age, c yt ot oxicit y, and cell proliferati on
assa ys have shown that som e ess enti al bet el quid i ngredients are
genotoxic, c yt ot oxic, and also stimul at e cell proli ferati on. El evat ed
reactive ox ygen s peci es and m eth yl ati ng agents produced due to
bet el quid consum pti on have been obs erved t o induce various DNA
dam age i n t he oral m ucosa cell s ( 1 8 ).
The rol e of di et and nutrition in modif yi ng the ris k for cancer
has been well es tabli shed b y various previousl y conducted
epi demi ological and laborator y studies. According to the IARC
report, it has been affi rm ed that i ndivi duals wi th low frui ts and
veget abl e consum pt ions habits are at an i ncreased ris k for
devel opm ent of cancer ( 1 9 ). Studi es have shown that frequent consumption of green veget abl e as wel l as carrots and tomatoes
hel ps in reducing the ris k for oral and phar yngeal cancer. Food and
food groups other than fruit s and raw veget abl es t hat have a
prot ective effect are fis h, vegetabl e oi l, ol ive oil, et c. However,
foods li ke proces sed meats , eggs , butt er, cakes, et c. have been
report ed t o be as soci at ed with increased cancer ris k ( 2 0 ).
Studi es have report ed i nterpl a y of t he di et ar y habits with the
cult ural risk fact ors in m odi f yi ng the ris k for oral cancer and pre
-cancerous st ages . Cigarett e smokers have been found t o have
decreased β-carotene levels in comparison to the nonsmokers. The serum levels of Vitamin A, C, B12, folate, and β -carotene has been
found t o be lower among bet el q uid chewers i n comparison to the
levels in the non -bet el quid chewers ( 1 2 ).
Virus es have been found t o be strongl y associ at ed with the
onset of mali gnant oral squam ous epithelium t umours. Viral
infections may interfere with the host’s cellular machinery and may
induce m ali gnanc y in t he norm al cell s of t he bod y. These viral
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Viral genes are most l y pres ent as proto -oncogenes whi ch when gets
incorporated into the host’s DNA turns into oncogenes resulting in
the t rans form ation of the norm al cell int o mali gnant cells . The m ost
important vi rus es li nked with oral m ali gnanc y are the Epst ein –Barr
virus, hum an papill omavi rus (HP V), and herpes virus ( 1 2 ).
In oral cancers , the most comm onl y found t ype i s the HPV 16
whi ch encodes E6 and E7 oncoprot eins. These protei ns bi nd to t he
p53 and Rb tum our s uppressor genes and dest ro y them , thus l eadi ng
to the i mpairm ent of the cell c ycl e and DNA repai r pathwa ys ( 2 1 ).
Biomark ers of oral can cers
Cancer bi om arker refers to genet ic, prot eomi c and epi geneti c
charact eri sti cs that indi cate t he pres ence of cancer. A cancer
biom arker m a y be a subst ance s ecret ed b y a tumour or b y a
parti cul ar res ponse of the bod y t o the m ani fes t ati on of
cancer. Recent research studies revealed that increas ed l evels of
interl eukin 8 can be us ed t o recognize OSCC patients from non
-OSCC pati ents , wit h hi gh speci fi cit y ( 2 2 ) . Mali gnant t rans formation of d ys pl asi a predi ct ion is also us ed as a us eful m arker for oral
cancer det ection. Recent st udi es reveal ed that loss -o
f-het eroz ygosit y ( LOH) of chromosomes 3p and 9p i s a predi ctive
marker in OSCC ris k ( 2 3 ).
Several tum our suppres sing genes have been found and
recogniz ed in OSCC progres sion, for example - p53, E-cadher in, and
p120 - cat enin. Whereas, the i ncreased activiti es of tumour
prom ot ers like EGFR, c yclin D1, Src t yros ine kinas es ,
transm embrane recept or podopl anin are frequentl y found.
Especial l y, podopl anin has appeared as a pot enti al biom arker and
functi onall y relevant therapeuti c bi omarker t o treat and prevent oral
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Podoplan in and its rol e in cancer
Podopl anin is a t ype-1 t ransm embrane gl ycoprot ein (38kDa),
and t he nam e podopl ani n was term ed due to its expressi on in kidne y
podoc yt es and in the regul ati on of podoc yt e foot process es .
Podopl anin has an extensive variet y of functi ons including
regul ation of cell m otilit y, organ development, and tum uori genesis
and m et ast asis . Podopl anin has an import ant rol e in inhi bi tion of
cellul ar adhesion, flatt eni ng of podoc yte foot proces ses and the
maint enance of glomerul ar perm eabil it y. It has been found to
associ at ed with oral , lung, ski n, lung, and esophageal cancer ( 2 4 ). Podopl anin gene is 34.2 kilobase (Kb) with 8 exons , which encode
podopl anin prot ei n. The s ub -cell ul ar location of t he podopl ani n is i n
the pl asm a m embrane. Two different speci es of podopl ani n mRNA
were recogniz ed b y northern blotti ng t hat possibl y i niti ates from
alt ernat ive splicing. Podoplanin i s hi ghl y express ed i n l ym phatic
endothelium, but does not express i n the bl ood ves sel endothelium .
Moreover, it is al s o express ed i n a vari et y of neopl asti c hum an
tissues. Podoplanin was detect ed in m an y hum an normal ti ssues,
incl uding l ymphat ic h eart, endot helial cells, mus cl e, lung, pl acenta,
m yoepitheli al cell s of glands, folli cular dendriti c Schwann cel ls ( 2 5 ). It als o i denti fied in the devel opment of t oot h germ and
odont ogenesi s regul ation ( 2 6 ).
Podopl anin prot ein consist s of 162 a.mino acids , wit h a singl e
transm embrane regi on, a four extracel lular plat el et aggregati on
-stimul ating dom ain (P LAG) wit h threoni ne and s erine residues, and
a c yt oplasmi c carbox yl t ail, als o having sites for cAMP and prot ei n
kinase C phos phor yl ation ( 2 7 ) . Podopl ani n does not have enz ym ati c activiti es or functi onal dom ains. Therefore it us es other protei ns,
such as C LEC -2 (C-t ype lecti n -l ike receptor -2), C D44, HSPA9 (heat
shock prot ei n A9), CC L21 (C -C mot if chemoki ne li gand 21),
gal ectin 8, ezri n, m oesi n, CDK5 (c ycl in dependent kinase 5), and
PKA (prot ei n ki nase A). Podopl ani n i nteract wit h t hese bindi ng
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met ast asis . Podopl anin m ainl y i nduces platel et aggregati on b y
bindi ng t o C LEC‐2, whi ch expressed on t he pl atelet surface.
A m oti f in the t ransmem brane region drives podoplanin to
form a hom odim er and ci rcul at e t o li pid rafts . Di fferent li gands and
bindi ng agent s like C LEC2, HSP A9, CD44, Galecti n 8, and CC L21
bind to extracellul ar dom ai ns of podopl ani n. C LEC 2 contact sit es
have been localized t o podopl anin residues D48, G45, and D49, with
the hel p of gl ycos yl ation at T52, along with E81, T85, and D82 i n
P LAG4 dom ain. Tet raspanin CD9, t he i ntegral m em brane prot ein
interact s with t he podopl anin transmembrane dom ain. Ez rin and
moes in prot eins int eract wi th the podoplanin c ytopl asmi c tail to
control R OC K/ LIM K pathwa ys acti vities as well as acti n
pol ym erizati on b y Rho GTP ases. The C DK5 and P KA can
phos phor yl at e int racellul ar seri nes t o i nhibit tum our cell m otilit y.
Podopl anin express ed on the surface of t umour cells i nduces plat el et
aggregation b y int eracting wit h C LEC -2 and m a y regul at e t umour
invasion and m etas tasis and therefore, podoplanin mi ght be a
pot enti al target for t herap y of m et ast asis . Podopl ani n expres s ion i s
also i nduced b y tum our promot ers like R AS, TP A, and S rc. S rc i s a
non -receptor prot ei n kinas e that prom ot es cancer cel l growth and
mi grati on requi red for m et ast asis. Therefore, podoplani n pla ys a
vital role i n m an y cancer and in l ym phati c vas cular devel opm ent
met ast asis t hrough i nteracti on with C LE C‐2 ( 2 8 ). Podoplanin is als o express ed in cancer‐associ at ed fibroblasts (CAF) where i t all ows
adenocarci nom a cel l invasi on i n the lung b y acti vat i ng the Rho‐R OCK pathwa y ( 2 9 ).
Podoplan in and squ amou s cel l carcinoma
Podopl anin incl udes an extracell ular regi on, transmem brane
dom ain, and int racellular (IC ) t ail . C LEC -2 int eracts wit h P LAG
dom ains in the extracellul ar region t o induce inflammati on and
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anti bodi es , incl uding NZ -1, MS -1 and podopl anin anti bodi es
(CasM abs ). Anti bodi es can al so t arget podopl anin in order t o stop
trans form ed cell growth and mot ili t y di rectl y, or can be
incorporat ed int o C AR -T cells. Lectins repres ent ed b y M AS L can
also t arget podopl ani n t o block t he tum our progression and
infl amm ation. P rot ei n kinase A (P KA) and c ycl in dependent ki nase
5 (CDK5) can phos phor yl at e s erines on the int racell ular tail to
block cell mi gration, probabl y b y i nhi biting interact ion of ERM
prot ei ns that woul d otherwise l ead to the act ivation of R ho GTPas es
and Rho-as sociated coil ed -coil kinas e (R OCK). Podoplanin
expressi on induces ROC K acti vit y t o promot e squam ous cell
carcinoma survi val and col on y expansi on ( 3 0 ).
Markers Useful for Iden tificati on of Podoplanin
The podopl anin expressi on i s detected m ainl y b y
immunohist ochem ist r y and the most commonl y used ant ibodies is
D2 -40, whi ch i dentifi es t he formalin -resist ant epi tope of
podopl anin. In both normal and neoplas tic t issues , doubl e
immunostaining bas ed on anti D2 -40 or anti -podopl anin and CD34
confi rm ed that the fi nal product of reaction for podopl anin.
Podopl anin -positive sampl es are als o stained with an anti bod y
anti VEGFR -3, and t he final product is mostl y found at the luminal
surface. Lym phati c endotheli al cell s can be considered as
podopl anin -expressi ng cells t hat express CD34 at ver y l ow levels .
Podoplan in as Marker of Lymphati c En doth elial Cells (LE Cs) Since podopl anin is expressed m ainl y b y l ym phat ic
endothelium and not b y norm al blood ves sel endot heli um, therefore,
podopl anin is extensivel y used as a s pecifi c m arker to evaluat e
l ym phati c vess el i nvasion and l ym phati c mi croves sel densi t y.
Lym phat ic vess el i nvasi on (LV I) and l ymphatic mi crovessel densit y
(LMVD) have been found to associ ate with l ymph node me tastasi s,
advanced st age, low survival i n vari ous cancers, includi ng oral
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recept or on pl at elet s and t his binding makes l ym phati c sacs t o
rem ove and separat es it from cardinal veins. This separ ation is ver y
important for t he l ymphatic s yst em to emerge from the embr yonic
cardiovas cul ar s ys t em, whi ch sust ai ns be yond devel opment.
Num erous l ym phat ic endotheli al m arkers , includes prospero -rel ated
hom eobox -1 (P rox -1), podoplanin, vascul ar endot hel ial growth
fact or recept or -3 (VEGFR -3), and l ymphatic vess el endothelial
h yaluroni c acid recept or -1 (LYVE-1) are broadl y utilized in the
diagnos is of l ym phangiogenesis and LV I in s everal t ypes of cancer
incl uding oral cancer. These biom arkers bind wit h each ot her in
several ph ysi ologi cal proces ses.
It was obs erved that the l evel of VEGFR -3 substanti all y hi gh
in l ym phati c endotheli al cells, whereas its expressi on i s decreas ed
in vas cul ar endotheli al cells. S peci fi c m arkers of l ym phatic
endotheli al cells, VEGFR -3, LYVE -1, Prox -1 and podopl ani n, have
provided new di rect ion i nto the cancer diagnos tic and therapeutic.
Man y pri or studi es have report ed the co -expressi on of, VEGFR -3,
Prox -1 and LYVE -1 podopl anin in l ym phatic endot hel ial cells of
norm al adult and t umour tissues ( 3 2 ).
Podoplan in exp ressi on in oral can cer
Combined anal ys is of around 1,000 samples reveal ed that
podopl anin expres si on was ver y low in norm al oral epitheli al cells,
but was found hi gher in oral cancers ( 6 ). Furt hermore, podopl anin expressi on i n oral cancers i s expected to be much hi gher than
actuall y report ed. This is due to podopl ani n expressi on i s observed
mostl y in the i nvasi ve sit es of oral cancers. M an y previous studi es
also reported ver y hi gh podopl anin expressi on in OSCC ( 3 3 , 3 4 ). Podopl anin express i on is induced very earl y in t he mali gnant
trans form ation process , and t herefore ma y be used to det ect
prem ali gnant l esi ons that are bound to devel op i nto oral cancer.
Pooled anal ys is of man y ret rospecti ve studi es on premali gnant oral
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podopl anin expressi on advanced to OSCC. However, very less
percentage (14%) of l esi ons without promi nent podopl ani n
expressi on progress ed t o OSCC. These result s indi cat e that
podopl anin expres sion el evat es the probabili t y of OSCC
devel opm ent b y over 3 -fol d ( 6 ).
Since more suspici ous les ions of OSCC are m ainl y s el ect ed
for biops y, t hese results of podoplanin expression could be
underest imated. P revious stud y also supported thes e
underest imation of Podopl anin expres si on as about 80% of oral
leukopl aki a as that expr ess hi gh l evels of podopl anin devel op i nto
oral cancer ( 3 5 ). M oreover podopl anin expres sion also as soci at es with OSCC m ortali t y, as fi ve year overall survival decli nes from 86
percent for pati ent s with tum ours with undet ect able express ion of
podopl anin level s to 23 percent for cas es with tumours exhi biting
upregul ated podoplanin expressi on ( 3 6 ). These fi ndi ng dem onstrat ed that podopl ani n expressi on prom ot e an around t hreefold decreas e in
five year survi val for oral cancer patient s. P odoplanin is express ed
in man y form s of cancer. Expressi on of podopl ani n expres sion in
OSCC is al so associ ated wit h l ymph node metas tasis and poor
prognosis ( 3 7 ). It al so ass oci at es with sent inel l ym ph node m et ast asis in earl y OSCC ( 3 8 ). Neverthel ess, one of the st udi es dem ons trated that expres sion of podopl anin di dn't infl uence the five - year overall
survival or the fi ve - year dis eas e-free survival ( 3 9 ). A stud y dem onst rated that dual expression of podopl anin in t umour cells and
l ym phati cs with specifi c patt erns connect ed with histopathol ogy and
l ym ph node status in oral cancer, exhibiti ng t he m ol ecular bas is for
testi ng podopl anin as a potenti al t arge t for anti D2 -40 antibod y
bas ed therap y ( 4 0 ).
Furthermore, anot her stud y also report ed a hi gh l evel of
podopl anin expressi on is indi cat ive of hi gh inci dence of l ymph node
met ast asis and imm ature st atus i n the differenti ati on process of
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biom arker in eval uat ing the c yt opl asm -m embrane st aini ng of tumour
cells ( 4 1 ). Anot her previ ous anal ys is also described that podoplanin can be utiliz ed as a biom arker for earl y oral carcinogenesis and for
mali gnant transformation ri sk identi ficati on of pre -cancerous
lesi ons and as a tum our devel opm ent biomarker for advanced grades
of OSC C ( 4 2 ). P revious stud y anal yz ed the podopl anin express ion of oral squam ous cell carcinom a (OSCC ), oral l eukopl akia, oral
submucous fi brosis and norm al buccal mucosa, usi ng monocl onal
anti bod y D2 -40 and found t hat t here was a st atisti call y si gni ficant
upregul ation of podopl ani n expression in OSCC (100%), oral
submucous fibrosi s (90%) and oral l eukoplakia (65%) as com pared
with norm al m ucos a (35%) ( 4 3 ).
Podoplan in as th erapeuti c target
Podopl anin has appeared as an obvious chemotherapeuti c
target for oral cancer. Therefore, com pounds t hat t arget podopl anin
are being enhanced as anti -cancer reagents. Thi s compris es
anti bodi es and cell -bas ed immunotherapi es. Antibodi es agains t
podopl anin h ave exposed pot enti al as ant icancer agents in previ ous
experim ent al st udi es. Thi s experim ent st arted wit h the NZ -1
monoclonal antibody that t argets the P LAG domai ns of hum an
podopl anin. At fi rs t, NZ -1 was est abl ished to obs truct plat el et
aggregation to gli oma cel ls medi ated b y podopl anin -C LEC2
interact ions. A mouse -human chim eri c antibod y against hum an
podopl anin, ch LpM ab -7, was also expos ed to obstruct m et ast asis in
a pulmonar y x enograft mouse model. Ot her ant i -Podoplanin
monoclonal antibodi es s uch as muri ne and hum aniz ed MS -1 al so
numb P odopl anin -C LEC2 int eractions and can effi ci entl y obstruct
lung tumour development b y int ravenous admi nist ration.
Genet icall y modi fi ed chim eri c anti gen receptor (CAR )
-transduced T -cel ls that t arget podopl anin have also bee n revealed to
obst ruct gliobl as tom a progression in m ouse models. For i nstance,
14
vei n for s even da ys aft er gli obl astoma implant ation produced a 3
-fold decreas e i n t umour devel opm ent and 140 pe rcent ex tended
lifetim e than cont rols. Int erest ingl y, t he gl ycos yl ati on patt ern of
podopl anin seems t o change when express ed b y cancerous cel ls,
leading to the growth of cancer specifi c monoclonal ant ibodi es
(CasM abs ). Thes e well s peci fi c reagent s produce improved potenti al
to t arget podopl ani n on cancer cells. For inst ance, the C asM ab
reacts with glioblast oma, mesotheli om a, and lung cancer cell s. Thi s
technology was als o us ed to enhance chLpM ab -23, which can
obst ruct gl iobl ast om a and OSCC devel opment . For instance, hum an
OSCC cell s that were subcut aneousl y admi nist ered int o exposed
mice, followed b y 100 µ g of i.p. adminis tered C hLpMab -23, 6 tim es
above 35 da y period reveal ed a 3.5 -fol d decreas e in t umour wei ght
than controls. This precursor of thi s C asMab a nt ibod y, Lp Mab -23,
also dis pla ys mi ni mum bi nding to nearb y l ym phati c endothelial
cells whi ch express podopl ani n. These cancer -specifi c podoplanin
anti bodi es caus e minimum offsi te t argeti ng to offer a doubl e -edged
tool t hat can be us ed expression in normal ti ssues, and al so as
pot enti al chemotherapeuti c agents wit h reduced side effects .
Podopl anin can also be t arget ed with other bi ological chemicals,
repres ent ed b y M aackiaam urens is s eed l ectin (M AS L). This
com pound is ess ent i all y an unint entional com ponent o f t raditional
medi cines us ed t o treat cancers that have been m ade from the
Maacki aam urensis for decades. The relat i onshi p bet ween M AS L and
NZ-1 s how that both obst ruct OSCC cell mi grati on and vi abi lit y at
similar concentrati ons of nM and µM, respecti vel y. Though, MAS L,
whi ch binds termi nal sialic aci d residues on podopl anin, can be
inject ed vi a mouth to t reat cancer and d ynami call y t arget s OSCC
cells withi n minutes of exposure. Thi s pot enti al for topical and
s yst emi c t reatments could be es peci all y valuabl e for oral cancer and
15
Rational of the s tudy
Oral cancer is a devast ating disorder t hat affects mill ions of
peopl e and thei r families. Over ni net y percent of oral cancers are
OSCC. The l ack of effici ent chem otherapeutics is a s uprem e
problem facing t his pati ent population. Podopl anin has appeared as
a functi onall y perti nent chemot herapeutic target and biomarker.
Tes ting agents are bei ng developed to parti cularl y t arget thi s
extracell ular recept or on cancer cells whil e reducing the
chemot herapeuti c t arget risk factors. The oral health comm unit y i s
now read y t o t ake benefits of thes e t echnologies t o bet ter det ect ,
treat , and prevent oral cancer. Over t he past two decades, i n s pite of
the m edi cal advancement s i n l ocal control and t he use of di fferent
com bined therapies, the s urvival rat es for oral cancer have not
enhanced si gnifi cantl y. R ecent st udi es have al so s hown that
podopl anin m a y be expressed in cert ain t umour cell s, including
squamous cel l carcinom a cel ls, 8 -10 rai sing a possibi lit y that
podopl anin m a y have biol ogi cal rol e in tumour cells. Monocl on al
anti bod y D2 -40 has been used t o i dentify podopl anin expres sion i n
tissue s ampl es wit h prim ar y oral cancer and pot ential correlations
bet ween the expres si on and clini cal and pathologi cal features. Del a y
in di agnosis i s one of the m ajor causes for low sur vival rat e in oral
cancer pati ents . Therefore, novel diagnosis m et hods of oral cancer
need t o be developed for earl y di agnosi s and therap y ( 6 ). A bett er understandi ng of m echanism s leading to OSCC is requi red to
improve effi cienc y of diagnosti c and t herapeutic methods for oral
16
AIM AND OBJECTIVES
1. To evaluate t he expressi on of Podopl ani n Marker in oral
squamous cell carcinoma.
2. To evaluate t he expression of P odoplanin Marker in norm al
tissue (alveol ar mucosa).
3. To eval uat e the ass oci ation of podopl anin expres sion with
different habits -relat ed and clinicopathol ogi cal factors.
4. To ai d in ass essi ng the progressi on of oral squam ous cel l
17
REVIEW OF LITERATURE
Podoplan in overvi ew
Podopl anin is a unique t ransm embrane gl ycoprot ein receptor.
Podopl anin contains heavi l y gl ycos yl at ed ami no t erminal extra
cellul ar domai n of approxim at el y 130 amino acids , followed b y a
singl e t ransm embrane dom ai n of approximat el y 25 amino aci ds, and
a short int racell ular dom ain of approximat el y 10 amino acids. It
doesn’t include identified functional domains or enzymatic
activiti es . It us es other prot eins, such as C t ype lecti n l ike receptor
-2 (C LEC--2), heat s hock prot ein A9 (HSPA9), CD44, gal ectin 8,
chemokine (C -C motif) l i gand 21 (CC L21), ez rin, m oesin, prot ei n
kinase A (PKA) and c ycl in dependent ki nas e 5 (C DK5), to change
the cell activit y. Thes e prot eins int eract wit h podopl anin to di rect
tumour cell m i grati on, invasion and metastasi s ( 4 7 - 4 9 ).
Martin -Villar et al . (2009) using nort hern bl otting techniques ,
two i soforms of podopl anin have been discovered. The podoplanin
gene com pri ses of 34.4 kb and 8 exons. The y possi bl y resemble a
product of al ternati ve spli ci ng, but the bi ologi cal import ance of thi s
stud y is still to be clarifi ed ( 5 0 ). A t umour promot er i ncludi ng Src, TPA and R AS encourages the podopl anin expressi on. For inst ance,
to induce expressi on of podopl ani n to encourage t umour cell
motilit y, the Src t yrosi ne kinas e us es t he focal adhesion adaptor
prot ei n C as/ BC AR1. Podopl ani n expression is enhanced on th e
trans cription l evel s uch as alternati ve splicing, num erous ini tiati on
sites , and pol yaden ylation and on the post -t ransl ational l evel due to
18
Podoplan in and can cer
Expression of podopl anin i s induced b y s everal t umour
prom ot ers and can be s een in num erous forms of cancer. Hi gh clonal
expansion capacit y i s a characteristi c feature of t umour i nit iating
cells (TIC ) and podopl anin is a T IC m arker for hum an squam ous
cell carcinoma ( 5 3 ). Furt herm ore, the occurrence of l arge col on y formation b y podopl ani n -positi ve cells i s reduced b y t reatm ent with
a Rho-as soci ated coiled -coi l ki nase (ROCK) i nhibitor, while no
difference was found in s ingl e podopl ani n -negative cells ( 3 0 ).
Podopl anin is consi dered as a marker of l ym phati c vess el
endothelium. P odopl ani n -positi ve cel l forms were exposed to be
concurrentl y positive for P rox1 and VEGFR -3. It appears t hat
VEGF-C increas es podopl anin expression through P rox1, which is a
major regul ator gene of l ym phati c ves sel developm ent inducing
LECs differenti ati on from l ym phati c progenitor cell s in embr yonic
vei ns ( 5 4 ). As well as, expression of podopl anin was found to be hi gh in derm al LECs b y IL -3.The si gni fi cant function of podoplanin
in l ymphatic vess el devel opm ent has been done b y t ri als conducted
with podoplanin -defi ci ent mi ce whi ch die at bi rth due t o respi rat or y
fai lure b y i rregul ar alveol ar sac growt h becaus e of the absence of
expressi on of podopl ani n in al veol ar t ype -1 cells ( 5 5 , 5 6 ).
C-t ype l ectin -like receptor -2 (C LEC -2) was dis covered on human
plat el ets as t he fi rst podopl ani n recept or whi c h is vit al for plat el et
aggregating properti es of podopl ani n ( 5 7 ). It was found that t umour cells encourage pl atel et ag gregation which prot ects cancer cel ls
from sheer st ress and host immunologi cal defens e. Thi s m echanis m
ma y result in i ncreas ed tumour development and improved
met ast ati c pot enti al of t he tum ours ( 5 8 , 5 9 ).
Act on et al. (2012) found podopl anin as an i nducer of
dendri tic cell (DC ) mi grati on through i nteracti on with its C LEC -2
19
ret icular cell s (FR Cs) and LEC s wit h C LEC -2 on DCs caused
improved mi grat or y pot ent ial of t he latt er. As well as , this
phenom enon can be si gnifi cant for the mi grat ion of cancer cells
along t he l ym phati c net works whil e the neo -expres sion of C LEC -2
in a hum an A375 m elanoma cell li ne caused protrusion formation in
the cont act of t he C LEC -2 over -expressi ng A375 m el anom a cell l ine
with podopl ani n expressi ng FRC s ( 6 0 , 6 1 ).
Podoplan in exp ressi on and oral can cer
Podopl anin is expressed i n s everal t ypes of cancer. Oral
cancer exemplifi es t he utilit y of podopl anin express ion as a cancer
biom arker. P odoplanin expression increases oral squamous cell
carcinoma cell mi gration, which can l ead to increas ed m etastasis( 3 0 ).
Yuan et al. (2006) invest i gated a st ud y to exami ne t he functi on of podopl anin i n head and neck squam ous cell carcinoma
(HNSCC) in 35 cases including 16 oral t umours and 19 h ypo
-phar yngeal t umours and ot her group of 60 cas es with oral t ongue
cancer b y immunohistochemis tr y and found out ass ociations
bet ween the podoplanin expression stat us and clini cal and
pat hological feat ures of pati ents s uch as s urvival rat e. They f ound
that podoplanin wasn’t expressed in normal oral epithelial cells but
was s een in s ome h yperpl asti c and d ysplasti c lesions. Increas ed
podopl anin expression was found i n 57% of t he 35 t umours and was
more common i n tum ours with l ym ph node met ast as is, es peciall y for
oral t umours. Among 60 oral tongue cancers, increas ed podopl anin
expressi on was found in 60%. P ati ents whose tumours ex press ed
increased l evels of podopl ani n had a statisti call y si gnificantl y
superi or rat e of l ymph node m et ast asi s (P <0.0001). P ati ents with
l ym ph node met as tas is and i ncreased -level podopl ani n di spl a yed t he
shortest disease -specific survi val as compared t o other patient s.
Researchers concl uded that podopl anin pl a y a rol e i n oral
tumouri genesis and m a y offer as a predi ctor for l ym ph node
20
Kawaguchi et al . (2008) conduct ed a stud y t o ass ess the role of podopl anin i n di agnosi ng of oral cancer in 150 oral l eukoplakia
(OP L) pati ent s. The y found that 37% of pati ents s howed podoplanin
expressi on i n t he basal and suprabasal l a yers and were cat egoriz ed
as podopl ani n posit ive. Posi tive result for podopl anin was more
comm on in el derl y cas es , femal es , and d ys pl asti c l esions . OP L
pati ent s that were positi vit y for podopl ani n had si gni ficantl y
increased t he i nci dence of oral cancer as compared to OP L pati ent s
with podopl ani n negative ( 3 5 ).
Fun ayama et al. (2010) conduct ed to evaluat e t he podopl anin expressi on (D2 -40) in pre-cancerous lesi ons, and ass es s ed the
immunohist ochem ical st udi es of podoplanin in oral squamous
epit hel ial lesions. The populat ion of t he stud y was 298 cases with
squamous cell carci noma (SCC ), carcinoma in situ (C IS), epi theli al
d yspl asi a, and h yp erpl asti c and norm al epitheli al les ions b y
immunohist ochem ist r y with D2 -40. The resul t showed that
positi vit y for podoplani n in norm al or h yperpl asti c epit heli a was
none, whereas all of the C IS and SCC patients marked positive.
Approxim atel y t went y-ei ght percent of t he mi ld d ys pl asi a pati ents
and 80 percent of moderat e d yspl asi a pati ents expos ed grade - I
positi ve reactions occurred m erel y in the fi rst l a yer. Grade - II
reactions occurred up to 4t h l a yer were observed in 4% of m oderate d yspl asi a pat ients, 39% of C IS pati ents , and 10% of SCC pat ient s.
Grad e-III reactions occurred up to 5t h l a yer and reacti ons were s een in 47% of C IS pat ient s and 87% of SCC pat ients. Researchers
finalized that the correl ation between the present hist ol ogi cal
categorizati on and podopl anin grade was st atist icall y s i gni ficant.
Expression podopl anin i s recogniz ed t o be ass ociat ed wi th the
21
Kreppel et al (2010) conduct ed to examine t he effect of expressi on of podoplanin on prognosis and m et ast ati c l ymphatic
spread in oral squamous cell carcinoma (OSCC ) patient s with
immunohist ochem ist r y. The y st udi ed rel ations hips bet ween the level
of podopl ani n expres sion and di fferent cli nicopathologi c
paramet ers. In 84% of pati ent s, podopl ani n was express ed on t he
tumour cell s. 24% of pati ents exposed i ncreased levels of
expressi on. The five year overall survi val rat e 31% for patient s wi th
increased l evels of podopl anin expres si on was si gnifi cantl y less er
as com pared t o 93% and 65% for patient s wi th decreas ed and
moderat e podopl anin expression res pectivel y. There was a
correl ation bet ween the express ion of podopl ani n and t he frequenc y
of cervi cal l ymph node m etast ases. C ervi cal l ym ph node m etastas es
were obs erved i n 79 percent patients wi th increas ed express ion of
podopl anin, whil e onl y 22% of pat ients with decreas ed expression
of podopl ani n had m et ast as es (p< 0.001) ( 6 3 ).
Margari tes cu et al. (2010) report ed that expression of podopl anin was very l ow in basal cell s, parti cularl y i n resection
margins of OSCC patients grown in the lower li p areas .
Neverthel es s, a hi ghl y vari abl e D2 -40 expres sion in t um our -free
resecti on margins rel ated with h yperpl asti c or d ys plas tic lesi ons
was recogniz ed. Additionall y, express ion of podopl anin al so
devel oped to the basal l a yer of the l ower lip s kin appendages, t he
m yoepitheli al cells of aci ni and duct s of minor s alivar y glands, and
other part s from the oral cavit y ( 6 4 ).
Shi et al. (2010) invest i gated to est abli sh prot ein expressi on of podopl anin and ATP -bi ndi ng cas set te, G2 s ubfamil y (ABC G2) i n
oral li chen pl anus (OLP ) pati ents and as sess thei r us e as biomarkers
for O LP mali gnant transform ation complication. In 120 pat ient s,
110 with unt ransformed OLP and 9 with mali gnant transform ed OLP
(mean fol low -up of 5.1 years). They found that podopl anin
22
88.9% of pati ents of t ransformed OLP. ABCG2 expres si on was
obs erved in 20.9% of pati ent s of untrans formed OLP and in 66.7%
of pat ients of transform ed OLP . The ris k of OLP mal ignant
trans form ation was si gnifi ca ntl y great er wi th co -expressi on of
podopl anin and ABCG2 as compared t o without co -expres sion of
podopl anin and ABC G2. Thi s study concluded that since
expressi ons of podoplanin and ABC G2 in O LP were cons iderabl y
associ at ed wi th m ali gnant t rans form ation ris k , therefore, podoplanin
and ABC G2 m a y be utilized as biom arkers for ri sk ass es sm ent of
oral m ali gnant trans form ation i n O LP pati ents ( 6 5 ).
Huber et al . (2011) t argeted to eval uat e the rel ati ons hi p bet ween expres sion of podoplanin i n the prim ar y tum our b y
immunohist ochem ist r y using tis sue mi croarra ys and occurrence of
met ast as es in t he s entinel l ym ph node (S LN) of pat ients wit h earl y
head and neck squamous cell carci nom a (HNSCC ) of the oral cavit y
and orophar ynx. Expressi on of podopl anin was comput ed b y the
intensit y reacti vit y score and cl assified int o expres sion and non
-expressi on. S LN as s essm ent di spl a yed occult m etast asis in 37.5% of
cases. 24.2% of cas es of prim ar y HNS CC expos ed express ion of
podopl anin. Expres si on of podopl anin as soci at ed si gnifi cantl y with
S LN m etastasis and rem ained a considerable predi ctor for l ym ph
node st at us even aft er cont roll ing for tumour st age. As a predicti ve
marker for S LN m et astasi s, t hough, expression of podopl anin gai ned
a s ensit ivit y of an onl y 36% and a speci ficit y of 83%. Podoplanin
expressi on is rel at ed wit h metastasis to l ymph nodes in vivo.
podopl anin immunohistochemistr y in earl y HNSCC of t he oral
cavit y and orophar ynx ma y aid to choose pati ent s for the S LN
assess ment and to fi nd pati ents with hi gh ris k for pres ence of occult
23
Shi mamura et al. (2011) eval uated the podopl anin and fas cin expressi on in oral d ysplasi a and carcinom a i n situ (C IS ). Expressi on
of podopl ani n was scored into 5 previousl y recogniz ed
cl assi fi cations and expression of fasci n was s cored into 4 ori gi nal
cl assi fi cations. The immunost aining s cores for podopl anin and
fasci n were si gni fi cantl y great er i n C IS and d ys pl asi a as compared
to beni gn diseas e (p=0.00036 and 0.0011). In all invas ive SCC
pati ent s, podopl ani n was expres sed i n the c yt opl asm and membrane
of the tum our cells . This st ud y concl uded t hat it would be us efu l for
devel oping t he di agnosti c accurac y of oral d ys plasi a and C IS b y
evaluati ng t he expressi on of podoplani n and expres sion of fas cin
immunohist ochem icall y ( 6 7 ).
Bartuli et al. (2012) demonstrat ed that podopl anin expression rai ses in the earl y stages of tum ouri genesi s and i t appears to be
rel at ed with a great er ris k of head and neck cancer. While i n SCC,
expressi on of podopl anin decreas es at the tim e of t umour
devel opm ent . These data help a function for expressi on of
podopl anin in t he begi nning but not in the developm ent of cancer.
Authors concluded t hat podopl anin i s concerned in oral oncogenesi s
and can be a forecas ter for l ym ph node met ast asis in as ym pt omati c
pati ent s. Hist ology and podoplanin anal ys is can be ve r y hel pful to
find the ri sk of growth, invasion and met ast ati c progressi on of a
tumour i n pati ents with oral cancer ( 6 8 ).
de S ousa et al (2012) demonst rated that majorit y cas es with nodal i nvol vement offered a hi gh peritumoural LVD. Moreover, a
strong relat ions hip of LVD wit h siz e and location of prim ar y
tumours could also be recognized. MVD was st ati sti call y rel at ed
with m et ast asis , and a si gni ficant rel ati ons hip bet ween the
l ym phangiogenic factors and the vess els densit y i n th e intra
-tumoural areas was also found. The wel l -mult ipli ed tum ours didn't
express podoplanin. LVD and MVD were great er in m etast ati c
24
devel oped vas cul ar s yst em in m et ast ati c l ym ph nodes em phasiz es
the earli er report s of l ym phangiogenesi s occurrence in l ym ph nodes.
Addit ionall y, t he podopl ani n expressi on b y m an y undifferentiat ed
tumour cel ls recom mends that this protein could be a m arker of
tumour aggress iveness ( 6 9 ).
Fen g et al (2012) found the expressi on of podopl anin and ABCG2 was 44.1% and 61.8% res pectivel y, and m ul tivari at e
anal ys is showed that expression patt ern of podopl anin and ABC G2
was relat ed with 6.31 -fol d and 14.39 -fold i ncreas ed the risk of
trans form ation, res pectivel y. Thi s st ud y concluded that the
podopl anin and ABCG2 expression were relat ed with oral cancer
progres sion, recom mending that podoplani n and ABC G2 ma y be
useful forecast ers for ass ess ing oral cancer ri sk ( 7 0 ).
Kreppel et al (2012) evaluated whether expressi on patt ern of podopl anin i n pre -t reatment bi opsi es could provide as a biomarker
to fi nd t he ris k of m ali gnant t ransformation oral leukoplakia
pati ent s b y imm unohi stochemistr y. The y found i ncreas ed
podopl anin expressi on pat tern in pre -t reatm ent bi opsi es was rel at ed
with mal i gnant trans form ation (p= 0.003) and risi ng S IN
-cl assi fi cation (p=0.009). The five - year OCFS rat e reduced from 100
percent for pati ents wit h no expressi on patt ern of podoplanin to
41.7% for pati ent s with t he m aximum l evel of express ion patt ern of
podopl anin ( 7 1 ).
Al meida et al. (2013) perform ed a st ud y to ass ess the cli ni cal si gni fi cance of expression pattern of podopl anin and vas cul ar
endotheli al growth factor -C (VEGF-C ) in well -differentiated OSCC.
The result showed t hat st rong expres sion of podopl ani n was rel at ed
with m al e (p=0.037) and with earl y cli nical st age (1 -2) (p=0.027).
Strong expressi on of podopl anin was more oft en est ablished in
25
the st rong expressi on patt ern of podopl anin and VEGF -C b y
mali gnant cells is rel at ed with perineural invasion in OSCC s ( 7 2 ).
de Vicen te et al . (2013) observed that 38% of cases were categorized as podoplanin -positive, and the rem aining 62% l esions
were as podoplani n -negati ve. The podopl anin express ion was
associ at ed wit h the grade of d yspl asi a (p<0.0005), and wit h t he ris k
of developm ent of oral cancer (p<0.0005). This s tud y concluded that
podopl anin coul d be a us eful biomarker for ris k ass es sm ent of
mali gnant t rans formation in OP L along with his tologi cal
evaluati on( 7 3 ).
Chandra et al. (2014) i nvesti gat ed the function of podoplanin in m ali gnant transform ation of potenti all y m ali gnant oral lesions
(PMO Ls ) and elucidat ed the prognosti c and clini cal si gnifi cance of
both LVD and M VD in PM OLs in devel opm ent of oral cancer. In
this stud y, LVD is referred as of podopl ani n detect ed positive
l ym phati c vess els and MVD as t he densit y of C D 31 positi ve micro
-ves sel s. The y found that LVD is a more val uable tool as com pared
to MVD. Aut hors concl uded t hat podopl anin is contri buted i n
mali gnant trans form ation t o oral cancer in PM OLs pati ent s and can
be a predict or for l ymph node m et ast asi s in oral cancer cas es ( 7 4 ).
Cirligeriu et al. (2014) found out expres sion of podoplanin in tumour cells of li p, oral cavit y, tongue and phar ynx SCCs, toget her
with l ym phati c vess els di stributi on, m orphology, densit y and their
effect on tumour progression. Esti mat ion of podopl anin b y D2 -40
immunohist ochem ist r y assessm ent on 56 pati ents of oral cancers
expos ed two different expressi on patt erns i n t umour cell s bas ed on
thei r sit e. P eri -tumour and int ra -tumour l ym phati c vess els densit y,
morphol ogy and di st ribution were associ at ed with l ym ph node st atus
but not with tumour stage. The m aximum number of l ym phatic
vessel s was found in grade - III SCCs. Dual expression of podopl anin
26
with hist opathology and l ym ph node st at us in oral cancer, ex hibiting
the mol ecul ar basis for assessi ng podopl ani n as a pot enti al target
for anti D2 -40 anti bod y based treatm ent ( 4 0 ).
Garcia et al. (2014) aim ed to asses s the rel ati ons hip between expressi on of podopl ani n and ezrin b y i nvasi ve tumour front cells in
the lip cancer. 48 SCCs arisi ng i n the inferior lip were
immunehis tochemi call y eval uated for the m embranous and
c yt opl asmi c expressi ons of podopl ani n and ezrin in peripheral and
cent ral areas of t he tumour front . The relationship between t he
membranous and c yt opl asmi c expression of podoplanin and ez rin b y
mali gnant cel ls was ass essed b y chi -square t es t and Spearman's
correl ation coeffici ent with a s i gni fi cance level of 5% for both
assess ments. The resul ts found that a strong m embranous and
c yt opl asmi c podopl ani n expression b y peripheral cells of the
invasive tum our front, with no expression of thi s prot ein b y cent ral
cells. A st atist icall y si gni fi ca nt vari ati on was est ablis hed between
the podopl ani n expressi on i n peripheral and central t umour cells
(p<0,001). This st ud y concl uded t hat the tum our cells in t he
invasive front tumour express ed s trongl y bot h podopl anin
(membranous and c yt opl asmi c) as cyt o pl asmi c ezrin and it
recommends an involvement of thes e prot ei ns i n the process of
invasion in lip cancer ( 7 5 ).
Inoue et al. (2014) as sessed paraffin -embedded tis sue specim ens of 69 pri mar y and 29 corresponding met ast ati c l esions b y
using antibodies against podoplanin and α -SMA. Podoplanin
positi ve strom al fi brobl as ts were detected i n 73.9 % of t he 69
primary OSCCs and 82.8 % of the 29 lymph nodes metastases. α
-SMA immunoreactivit y was found in 56.5 % of t he prim ari es and
82.8 % of the m et ast ases. Further eval uat ion exposed t hat 74.5 % of
the pri mar y l esions and 95.8 % of the met ast a ses with podopl anin
27
stronger. P odopl anin -positi ve C AFs are consi dered to be
m yofibrobl ast s t hat m a y involve in the developm ent of oral
cancer( 7 6 ).
Logeswari et al . (2014) i nvesti gat ed to eval uate t he podopl anin expressi on i n OSCC, l eukopl akia and normal oral t issues
and t o find it s us e as a bi om arker. The y found that t he absence of
expressi on of podoplani n in the epit heli um of all t he oral s ampl es
(Group 1). Positive podopl anin expression observed in 59.4%
pati ent s of l eukopl akia as Group 2 and 82% pati ent s of OS CCs as
Group 3. The podoplani n expressi on am ongst di fferent groups was
hi ghl y s i gnifi cant . This stud y concluded that t he podopl anin ma y be
considered as a forecast er in evaluati ng mali gnant t rans form ation of
pre-m ali gnanci es and prognos is of oral mali gnanc y. Cert ainl y, it is
thought that podopl ani n mi ght pl a y a role i n tumour devel opm ent
whil e correct mechanis m is not complet el y expl ained. Advance
studi es are needed to know it s precise pat hoph ys iology ( 7 7 ).
Swain et al . (2014) invest i gat ed a revi ew stud y t hat summ ariz es the rel ationship of over -expressi on of podopl anin in
oral pot ent ial l y mali gnant disease and oral cancer wit h speci al
prom inence on its put ative rol e i n carci nogenesis and al so its
pot enti al us e i n t argeted t reat m ent. Podopl anin, an im port ant
molecul e in t he si gnali ng pat hwa y contributed in progress ion of
epit hel ial d ys pl asi a -carcinom a s equence, can act as a good
forecast er in eval uat ing risk of cancer growth in pot ent ial m ali gnant
diseas es of oral cavi t y. With a famili ar role i n encouragi ng cancer
cell mi gration and m et ast asis al ong wit h its specifici t y i n ass essi ng
l ym phovas cul ar i nvasion, podoplanin can be proj ected as a new
28
Ochoa -Alvarez et al. (2015) target ed to s tud y t hat how a monoclonal antibody (NZ -1) and l ectin (MAS L) that target
podopl anin affect human OSCC cell m otilit y and vi abil it y. Bot h
testi ng agents bl ocked t he mi gration of podopl anin expressi ng
OSCC cells at nanomolar concent rati ons prior t o blocki ng t he cell
viabilit y at mi cromolar concentrati ons. M oreover, both testing
agents encou rage mit ochondri al mem brane perm eabilit y t ransi tion to
dest ro y OSCC cells t hat express podopl anin b y caspas e independent
nonapoptoti c necros is. Additi onall y, M AS L showed an am azingl y
vi gorous skil l to t arget podoplanin on OS CC cells wi thin mi nutes of
exposure, and si gni ficantl y bl ocked hum an OSCC diss emi nat ion i n
zebrafish embr yos. This st ud y concl uded t hat hum an OSCC cell s
produced tum ours t hat express ed podopl ani n in mi ce, and i nduced
podopl anin expres sion i n infilt rati ng host m uri ne CAFs.
Collecti vel y, t hese s tudi es suggest t hat anti bodi es and l ect i ns ma y
be ut ilized to fi ght OSCC and ot her t ype of cancers that expres s
podopl anin ( 7 9 ).
Parh ar et al. (2015) found that normal epi thelium exposed negli gible podopl ani n expressi on, whereas the expression ex panded
si gni fi cantl y at t he bas al l a yer and the s uprabas al l a yer or above at
one or multi ple regi ons in pot ent iall y m ali gnant l es ions . P odopl anin
expressi on i n OSCC displ a yed two various patt erns i ncl ude diffus e
and focal. A statisti call y si gnifi cant ris e in average LVD was found
from norm al epit helium to OP M Ls (P<0.001) and to OSCC
(P<0.022) whereas a non -si gni ficant ris e was obs erved (P<0.594)
bet ween OPM Ls and OSCC. On t he whole, no si gnifi cant
rel ati ons hip was seen bet ween D2 -40 epi theli al positi vit y and LVD
29
Patil et al. (2015) found t hat st ati sti cal l y hi ghl y s i gnifi cant ris e of the express ion of podopl ani n from mild to severe d ysplasi a
and from well to poorl y different iat e OSCC b y one -wa y ANOVA
test (P < 0.01). Hi ghl y si gni fi cant difference was obs erved between
grades of mil d to m oderate d ys plasia, moderate t o s evere d ys plas ia,
wel l to poorl y di fferentiat ed OSCC , and moderat el y t o poorl y
differenti at ed OSC C b y Turke y HSD test (P < 0.01). Authors
concl uded t hat podopl anin can be uti lized as a bi omarker for
prem ature oral carci nogenesis and for m ali gnant t rans form ati on risk
assess ment of pre -m ali gnant l esi ons and as a t umour devel opment
biom arker for hi gher grades of OSCC ( 8 1 ).
Prasad et al. (2015) found that 27 pati ents of SCC dis pl a ye d expressi on of podopl ani n and t here is no expressi on i n rem ai ning 3
pati ent s. In OSCC , 7 pati ent s s howed weak expressi on offered
immunoreacti ve s core ( IRS ) 0 to 3, fi ft een pati ents s howed
moderat e expres sion offered IRS S core 4 to 7 and five pati ents
showed hi gh expres sion offered IRS S core great er than 8. The
evaluati on of expressi on of podoplanin in the c yt opl as m, the
membrane and both (c yt opl asm and m embrane) of tumour cel ls
expos ed overall hi gh positi vit y i n the c yt opl asmi c followed by both
and the m em brane ( 8 2 ).
Sgaramell a et al. (2015) inves ti gat ed t o evaluat e the expressi on of podoplani n using immunohistochemis tr y. The mRNA
levels were ass es sed in twent y -seven pat ient s of tongue SCC with
adj acent cli nicall y t umour -free tongue t issue and fourt een t ongue
samples were collect ed from health y pati ents . Increas ed podoplanin
levels were found in tumours compared t o bot h normal tongue and
clini call y norm al t ongue i n the t um our vi cinit y. This stud y
concl uded t hat podopl anin level s in prim ar y tongue SCC s aren't
rel at ed with l ym ph node m et ast ases. Though, t ongue SCCs emerging
in young pati ent s are m o re probabl e t o express hi gh podopl anin
30
pati ent s. The st ud y recomm ends that podopl anin has a characteristi c
functi on i n young pati ent s, who are recognized to have a poor
diagnos is, t hes e pat ient s m a y benefit from podopl ani n inhibitor y
treatm ents (8 3).
Habiba et al. (2016) revealed that HuR and podopl ani n expressi on was associ ated wi th a 2.93‑fold and 2.06‑fold i ncreas e
in risk of m ali gnant trans form ation, res pectivel y. The risk of OP L
mali gnant t rans form ation was si gnifi cantl y increas ed with t he co
-expressi on of HuR and podopl anin com pared wit h t he histologi cal
grading. R es earchers concluded that the expres si on of HuR and
podopl anin correl ated with m ali gnant t rans form ation and
recommends t hat t he prot eins ma y be used as bi omarkers to i dentif y
OP L pat ients with a hi gher risk of developing cancer ( 8 4 ).
Ugorski et al. (2016) des cri bed podopl ani n is a tin y membrane gl ycoprot ein with a huge number of O -gl ycosi de chains
and consequentl y, it bel ongs t o m uci n -t ype prot eins . It can be found
on t he s urface of numerous t ypes of norm al cell s em erging from
various germ l a yers . It is found m ainl y on the endotheli um of
l ym phati c vess els , t ype I pneum oc yt es and glom erul ar podoc yt es .
Hi gh l evel s of podopl anin or its neo -expressi on have been
est abli shed i n s everal t ypes of hum an carci nom as, but it is
parti cul arl y wides pread i n SCCs, incl udes cervi cal, lar ynx , oral
cavit y, and lung and ski n cancer. This ti n y sialomuci n i s als o found
on the surface of CAFs i n lung adenocarcinom as, and al so in breast
and pancreat ic t umours . Littl e is recognized about the biologi cal
functi on of podo pl anin. However, res earch done on mi ce with a
knock -out gene of t his gl ycoprot ein dis pla ys that the presence of
podopl anin det ermines norm al devel opm ent of l ungs , the l ymphatic
s yst em and heart . P odopl anin on carci nogenesis and C AFs appears
31
cancers. However, i ts rol e in these process es is both uncl ear and
controvers ial ( 8 5 ).
Deepa et al . (2017) eval uated t he podoplanin expression i n oral leukopl aki a, oral sub -m ucous fibrosis and OSCC wit h that in
norm al buccal mucosa b y i mmunohis tochemi str y wit h D2 -40. The
expressi on of podoplani n was graded from grade 0 -4. Thi s stud y
found that st ati sti cal l y t here was a si gnifi cant increas e of the grades
of expressi on of podopl ani n in OSCC as 100%, oral s ub -mucous
fibrosi s as 90% and ora l l eukoplakia as 65% when compared t o that
in norm al m ucos a as 35%. Expression of podopl ani n increas ed wit h
decreas e in grades of di fferenti ati on in OSCC. Expres sion of
podopl anin i n the s amples of oral sub -m ucous fibrosi s was s uperior
to that in oral l euk oplaki a. This stud y concluded t hat the ass es sment
of expres sion of podopl anin in t he epit helial cells of oral d ys plas tic
lesi ons m a y offer useful i nform ati on to fi nd thei r m ali gnant
trans form ation risk ( 8 6 ).
Liu et al. (2017) performed a stud y t o evaluat e t he expressi on patt ern of ABCG2 and podopl anin in OSCC tis sues and
corres ponding cervical l ym ph node, and ass ess t he diagnosti c value
of ABCG2 and podoplanin in OSCC for carcinogenesis , hist ologi cal
stage and cli ni cal di agnosis. ABCG2 displa yed a mix ed m em branous
and c yt opl asmi c pattern of stai ning i n cert ain areas of s tratum
corneum and the cent er of well -di fferenti at ed t umour, whereas
merel y ex pres sed i n c yt opl asm of poorl y different iat ed tumour.
Podopl anin expos ed strong st ai ning in LV endot helia, the peripher y
of well -differenti at ed cancer and t umour strom al fi brobl asts
encom passing t umour nests . The ABC G2 expression, part icul arl y
c yt opl asmi c ABCG2, and podopl anin protei n expos ed si gni ficantl y
superi or t endenc y t o l ym ph node m etast asis (P<0.05). At the sam e
time, podopl anin positive rat e raised wi th the reducing degree of
histol ogi cal di fferentiat ion (P <0.05 ). ABC G2 and podopl ani n
32
Authors s uggest ed t hat c yt oplasmi c ABCG2 wit h podopl ani n have
clini cal prospective i n st abl e m ol ecule di agnosi s for OSC C ( 8 7 ).
Gis si et al. (2018) conduct ed a st ud y t o eval uate a possi bl e rel ati on bet ween alt ered podopl ani n expressi on and d ys pl asia b y
immunohist ochem ist r y and biom arkers s uch as p53 and Ki67 in 40
OPL patients. 32 cases didn’t expose dysplasia, while 8 lesions
pres ent ed wit h d ys pl asi a. The y found t hat al l three bi om arkers were
positi ve i n 7 of 8 d yspl asti c oral l es ions. Among the pati ents
without d ys pl asi a, Ki67 and p53 expos ed pos itivit y i n 21 and 10
samples res pecti vel y, whereas pos itivit y for podoplanin was found
