Human MutL homolog 1 immunoexpression in oral leukoplakia and oral squamous cell carcinoma: A prospective study in Indian population

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1 Originl Article Humn MutL homolog 1 immunoexpression in orl leukoplki nd orl squmous cell crcinom: A prospective study in Indin popultion Nrendr T Chudhri, Jgdish V Tupkri, Tit Joy, Mnish S Ahire Deprtment of Orl Pthology nd Microiology, Government Dentl College nd Hospitl, Mumi, Mhrshtr, Indi Astrct Bckground: Mmmlin mismtch repir system is responsile for mintining genomic stility during repeted duplictions, nd humn MutL homolog 1 (hmlh1) protein constitutes n importnt prt of it. Vrious isolted studies hve reported the ltered expression of hmlh1 in orl leukoplki (OL) nd orl squmous cell crcinom (OSCC). Reserch is lcking in the quntittive estimtion nd comprison of hmlh1 expression in OL nd OSCC. Aims: To evlute, quntify nd compre hmlh1 immunoexpression in norml orl mucos, OL nd OSCC. Settings nd Design: Thirty ptients of OL nd thirty ptients of OSCC formed the study group nd thirty ptients were included in the control group (norml orl mucos). Formlin fixed prffin wx locks were prepred from the tissue smples. Mterils nd Methods: Immunohistochemistry for hmlh1 ws performed, nd the totl numer of positive cells ws counted in high power fields, nd sed on tht percentge positivity of hmlh1 ws clculted in ll the cses. Sttisticl Anlysis: Kruskl Wllis nd t test were used. P < 0.05 ws considered to e sttisticlly significnt. Results: The men hmlh1 vlue in control group, leukoplki nd OSCC ws 78.26, nd respectively. hmlh1 immunoexpression showed decresing indexes from control group to leukoplki nd then further to OSCC. hmlh1 expression ws significntly lower in OSCC s compred to leukoplki. There ws no significnt correltion of men hmlh1 expression etween different clinicl nd histopthologicl stges of leukoplki nd OSCC. Conclusions: hmlh1 immunoexpression ws inversely relted to the degree of dysplsi. These findings suggest tht there is progressive decrese in hmlh1 expression from control to leukoplki nd further to OSCC. Thus, it cn e concluded tht hmlh1 cn e used s relile iomrker for mlignnt trnsformtion. Key Words: Mismtch repir system, orl leukoplki, orl squmous cell crcinom Address for correspondence: Dr. Nrendr T Chudhri, Nirmn Prk Phse 3, A 6, Room No. 4, Sntoshi Mt Rod, Klyn West, Mumi , Mhrshtr, Indi. E mil: nren.njoi@gmil.com Received: , Accepted: Access this rticle online Quick Response Code: Wesite: DOI: / X This is n open ccess rticle distriuted under the terms of the Cretive Commons Attriution NonCommercil ShreAlike 3.0 License, which llows others to remix, twek, nd uild upon the work non commercilly, s long s the uthor is credited nd the new cretions re licensed under the identicl terms. For reprints contct: reprints@medknow.com How to cite this rticle: Chudhri NT, Tupkri JV, Joy T, Ahire MS. Humn MutL homolog 1 immunoexpression in orl leukoplki nd orl squmous cell crcinom: A prospective study in Indin popultion. J Orl Mxillofc Pthol 2016;20: Journl of Orl nd Mxillofcil Pthology Pulished y Wolters Kluwer - Medknow 453

2 Chudhri, et l.: hmlh1 in orl leukoplki nd OSCC INTRODUCTION Orl squmous cell crcinom (OSCC) is the most common type of hed nd neck cncer, [1] with n nnul incidence of 350,000 cses worldwide. [2] Despite severl tretment modlities in the lst three decdes, it hs 50% survivl rte over 5 yers ecuse of lte dignosis. It is the 11 th most common cncer worldwide nd 8 th most frequent cncer in the world in mles nd 14 th in femles. OSCC ccounts for nerly 3% of ll cncer cses. [1,3] OSCC cn e preceded y orl leukoplki (OL), the min orl potentilly mlignnt disorder. [4,5] The frequency of dysplsi vries from 15.6 to 39.2% in OL. Although the histologicl investigtion is the routinely used method for grding epithelil dysplsi, it is ssocited with interoserver vriility. [6] To overcome this, nd further identify the erly events involved in OL to OSCC cellulr trnsformtion, reserch in iologicl mrkers is necessry. [7] Immunohistochemistry is quick nd chep s compred to genetic nlysis in proving the loss of protein expression. [8] One of the recently evolving iomrkers is the humn MutL homolog 1 (hmlh1) immunohistochemicl stin. [9] The mmmlin mismtch repir (MMR) system is responsile for mintining genomic stility during repeted dupliction. [10] The hmlh1 forms n importnt prt of MMR nd plys mjor role in muttion voidnce. [11] Muttion of hmlh1 gene is seen in hereditry nonpolyposis colorectl crcinom [12] which my occur due to MMR gene muttion or promoter methyltion gene silencing. [13] Microstellite instility (MSI) my result in genomic instility [14,15] nd serve s crucil erly event in crcinogenesis. [16] Altertions such s MSI nd hypermethyltion of the promoter regions of hmlh1 were detected in OL nd OSCC. [17] However, very few studies hve een done in reltion to orl potentilly mlignnt disorders nd orl cncer. Hence, the im of the present study ws to compre the immunoexpression of hmlh1 in different stges of OL nd OSCC with tht of norml helthy mucos, nd to determine whether hmlh1 is relile iomrker for mlignnt trnsformtion. MATERIALS AND METHODS Study group Inclusion criteri Cliniclly suspicious nd dignosed cses of OL nd OSCC Individuls should e otherwise helthy, with no r for ge nd sex Individuls willing to prticipte in the study procedure with written consent. Exclusion criteri Individuls hving ny systemic illness such s dietes nd hypertension. Individuls hving orl ulcertive lesions (trumtic ulcers, herpetic lesions, etc.) which re not cliniclly suggestive of cncer Individuls hving white lesions not ssocited with tocco use Individuls unwilling to prticipte in the study. Control group Inclusion criteri Individuls should e otherwise helthy, with no r for ge nd sex Individuls with no hit history (tocco). Exclusion criteri Individuls hving ny systemic illness such s dietes nd hypertension. Individuls unwilling to prticipte in the study. Tissue smples The study protocol ws pproved y the Ethics Committee of our hospitl. Thirty rndomly selected ptients of OL were cliniclly grouped using LCP clssifiction (L size, C clinicl presenttion nd P pthologicl grding) (Biloor nd Ngesh, 2005). [18] H nd E stined sections were nlyzed y two investigtors nd the degree of epithelil dysplsi ws estlished in ccordnce with the criteri given y WHO. [19] Thirty rndomly selected ptients of OSCC were cliniclly grouped using tumor node metstsis clssifiction. [20] Slides were exmined nd dignosed histopthologiclly s OSCC nd scored s per the criterion given y Anneroth nd Hnsen [21] with modifiction like the the stge of invsion. The depth of tumor cell infiltrtion ws excluded s the mjority of specimens were otined from incisionl iopsy. The scores nd prognosis were determined from the totl mlignncy score [Tle 1]. Thirty ptients were included in control group tht comprised helthy volunteers (norml orl mucos otined from crown lengthening procedure nd third molr surgery). Immunohistochemicl stining Three to four micrometers thick formlin fixed prffin emedded sections were dewxed in xylene nd hydrted with grded ethnol. Antigen retrievl ws done in microwve y plcing slides in th contining 250 ml of Tle 1: Correltion of mlignncy score, grde nd prognosis Mlignncy score Grde Prognosis 5 8 I Good 9 12 II Moderte III Poor 454 Journl of Orl nd Mxillofcil Pthology Sep - Dec 2016 Vol 20 Issue 3

3 Chudhri, et l.: hmlh1 in orl leukoplki nd OSCC working strength ntigen retrievl citr plus solution, turning het to high nd then incuting for min. Slides were trnsferred to room temperture nd wshed with ethylene-dimine-tetr-cetic cid solution with ph of 9.0. Endogenous peroxidse ws locked with 1:1 solution of methnol nd 3% H 2 O 2 for 10 min. The slides were incuted with prediluted primry ntiody nti hmlh1 (monoclonl mouse) (DAKO Corportion, Glostrup, Denmrk, Clone ES05). Detection ws undertken with two step, highly sensitive, redy to use, peroxidse sed system nmed visuliztion system (DAKO Corportion, Denmrk). Rections were reveled with 3,3 diminoenzidine chromogen solution. Hrri s hemtoxylin ws used for counterstining. Negtive controls were otined y the omission of primry ntiody nd smples of norml orl mucos with known positive rectivity were included s positive controls. Cell counting nd sttisticl nlysis Cells were considered immunopositive, if they presented rown nucler stining, regrdless of intensity. Cell counting ws performed using n eyepiece grid in light microscopy under 400. Counting ws done in suprsl cell lyer in cses of norml orl mucos nd leukoplki, wheres in cse of OSCC, ll tumor cells were counted in prticulr field. According to the nlysis performed y Fernndes et l. to otin hmlh1 index, 16 high power fields ( 400) were nlyzed for ech slide nd positive nd negtive cells were counted. The numer of positive cells ws divided y the totl numer of cells counted in ll the fields, i.e. positive nd negtive ones nd the result ws then multiplied y 100, so the indexes were demonstrted s percentge of positive cells. [9] Sttisticl nlysis ws performed using SPSS softwre version (SPSS Inc., 233 South Wcker Drive, 11 th floor, Chicgo, IL ). Kruskl Wllis test ws pplied to compre men hmlh1 expression in control, leukoplki nd OSCC groups. T test for equlity of mens (2 tiled) ws used to compre the clinicl nd histopthologicl stges of oth leukoplki nd OSCC with tht of control group. The results were considered sttisticlly significnt with P < leukoplki [Figures 2 nd 3] nd 24 of 30 smples of OSCC [Figures 4-6]. Most of the ptients of the study group hd similr hit history, ge group nd site of lesion. In ddition, there were no femle ptients in leukoplki group, hence these prmeters were considered nonsignificnt nd were not compred. The men vlue of hmlh1 expression in norml, leukoplki nd OSCC ptients ws clculted. hmlh1 expression ws significntly higher in control group thn oth leukoplki nd OSCC groups. Between leukoplki nd OSCC groups, hmlh1 expression ws significntly higher in leukoplki s P < 0.05 (Kruskl Wllis test) [Tle 3]. Men hmlh1 expression ws significntly higher in control group s compred to stges 1, 2, 3 nd 4 of leukoplki, s P < 0.05 (t test). There ws no significnt difference of hmlh1 expression etween the different clinicl stges of leukoplki, s P > 0.05 (t test) [Tle 4]. Men hmlh1 expression ws significntly higher in control group s compred to mild s well s moderte Tle 2: The demogrphics nd site distriution of the present study Ptients nd clinicl detils Orl Leukoplki (n=30) OSCC (n=30) Age rnge (yers) Mle/femle 30/0 19/11 Buccl mucos Alveolr ridge 6 Buccl vestiule 3 5 Lil mucos 2 1 Tongue 2 Retromolr re 1 Gingiv 1 1 Plte 1 OSCC: Orl squmous cell crcinom RESULTS The demogrphics nd site distriution of the present study re s presented in Tle 2. Humn MutL homolog 1 immunoexpression hmlh1 positivity ws seen in ll the ten smples of norml orl mucos [Figure 1]; 25 of 30 smples of Figure 1: () Photomicrogrph showing prkertinized strtified squmous epithelium (control) (H&E stin, 100). () Positive humn MutL homolog 1 expression predominntly in sl nd suprsl lyer of norml mucos (control) (IHC stin, 100) Journl of Orl nd Mxillofcil Pthology Sep - Dec 2016 Vol 20 Issue 3 455

4 Chudhri, et l.: hmlh1 in orl leukoplki nd OSCC c d Figure 2: () Orl Leukoplki; Photomicrogrph showing mild dysplsi (H&E stin, 100). () Orl Leukoplki; Photomicrogrph showing mild dysplsi (H&E stin, 400). (c) Orl Leukoplki; positive humn MutL homolog 1 stining in mild dysplsi (IHC stin, 100). (d) Orl Leukoplki; positive humn MutL homolog 1 stining in mild dysplsi (IHC stin, 400) Tle 3: Men vlue of humn MutL homolog 1 expression in norml, orl leukoplki nd orl squmous cell crcinom ptients Group Control Orl Leukoplki Orl Squmous cell crcinom Totl Men hmlh1 percent score n Men rnk χ2 df P hmlh1: Humn MutL homolog 1 Figure 3: () Orl Leukoplki; Photomicrogrph showing moderte dysplsi (H&E stin, 100). () Orl Leukoplki; positive humn MutL homolog 1 stining in moderte dysplsi (IHC stin, 100) P < 0.05 (ttest). There ws no significnt difference of hmlh1 expression etween the different clinicl stges of OSCC, s P > 0.05 (ttest) [Tle 6]. dysplsi in leukoplki, s P < 0.05 (ttest). There ws no significnt difference of hmlh1 expression etween the different histopthologicl grdes of leukoplki, s P > 0.05 (ttest) [Tle 5]. Men hmlh1 expression ws significntly higher in control group s compred to Grdes I, II nd III of OSCC, s P < 0.05 (ttest). There ws no significnt difference of hmlh1 expression etween the different histopthologicl grdes of OSCC, s P > 0.05 (ttest) [Tle 7]. Men hmlh1 expression ws significntly higher in control group s compred to stges I, II, III nd IV of OSCC, s The ssocition of tocco use ws ssessed neither for OL nor for OSCC, s ll the ptients were tocco users 456 Journl of Orl nd Mxillofcil Pthology Sep - Dec 2016 Vol 20 Issue 3

5 Chudhri, et l.: hmlh1 in orl leukoplki nd OSCC c d Figure 4: () Photomicrogrph of orl squmous cell crcinom Grde I (H&E stin, 100). () Photomicrogrph of orl squmous cell crcinom Grde I (H&E stin, 400). (c) Positive humn MutL homolog 1 stining in orl squmous cell crcinom Grde I (IHC stin, 100). (d) Positive humn MutL homolog 1 stining in orl squmous cell crcinom Grde I (IHC stin, 400) in some or the other forms with vrile durtion nd frequency. DISCUSSION Orl crcinogenesis is multistep process, in which epigenetic chnges form n importnt mechnism in orl cncer development, nd their recognition my help in erly detection s well s development of new therpeutic strtegies. The DNA MMR pthwy corrects replicte mismtches tht escpe DNA polymerse proofreding, nd hence plys n importnt role in the mintennce of genetic stility.[22] DNA repir forms n importnt defense mechnism ginst DNA dmge nd ltertion of MMR proteins such s MutLHomologon 1 (MLH1) tht hve recently een implicted in the development, progression nd metstsis of severl types of hed nd neck neoplsis. MLH1 forms heterodimers with PMS2 nd MLH3 (MutL complex) to discriminte the old from the new DNA strnd nd to signl downstrem repir fctors such s helicses nd exonucleses. Toccoddicted ptients with hed nd neck cncer re more susceptile to gene inctivtion of hmlh1 genes y promoter Tle 4: Men humn MutL homolog 1 expression etween control nd clinicl stges of orl leukoplki Clinicl LCP stge Numer of cses Percentge hmlh1 positivity (men) Stge I Stge II Stge III Stge IV Group Stge I Stge II Stge III Stge IV Ttest for equlity of mens, P vlue (twotiled) Control Stge IV Stge III Stge II Stge I hmlh1: Humn MutL homolog 1, LCP: L size, C: Clinicl presenttion, P: Pthologicl hypermethyltion.[23] Hypermethyltion of hmlh1 ws found in 0 47% of HNSCC[22] nd 14 70% of leukoplki with higher prevlence of MSI in leukoplki showing severe degrees of dysplsi.[9] In this study, no reltionship ws found etween hmlh1 immunoexpression nd gender, ge or smple site etween control, leukoplki nd OSCC groups. This result my well Journl of Orl nd Mxillofcil Pthology Sep - Dec 2016 Vol 20 Issue 3 457

6 Chudhri, et l.: hmlh1 in orl leukoplki nd OSCC c d Figure 5: () Photomicrogrph of orl squmous cell crcinom Grde II (H&E stin, 100). () Fint humn MutL homolog 1 stining in orl squmous cell crcinom Grde II (IHC stin, 100). (c) Photomicrogrph of orl squmous cell crcinom Grde III (H&E stin, 100). (d) Fint humn MutL homolog 1 stining in orl squmous cell crcinom Grde III (IHC stin, 100) Tle 5: Comprison of men humn MutL homolog 1 expression etween control nd histopthologicl grdes of orl leukoplki Histopthologicl grde Numer of cses Percentge hmlh1 positivity (men) Mild dysplsi Moderte dysplsi Group Ttest for equlity of mens, P vlue (twotiled) Control Moderte dysplsi Mild dysplsi Mild dysplsi Moderte dysplsi hmlh1: Humn MutL homolog 1 reflect the sic nd primitive function of the MMR system which is conserved throughout evolution nd unltered y demogrphic vritions.[24] Immunohistochemicl stining procedure ws repeted for the negtive cses to rule out technicl errors owing to the sensitivity of the procedure. However, repeted stining lso showed similr results nd therefore these 11 cses (5 of leukoplki nd 6 of OSCC) were considered negtive for hmlh1. This could e due to promoter hypermethyltion of the hmlh1 gene owing to crcinogens from tocco such s oxygensed free rdicls, peroxides nd peroxinitrite, which cuse severe oxidtive stress. Rective oxygen species cn directly oxidize DNA, resulting 458 in mutgenic chnge nd my dmge some DNA repir proteins.[24] In ddition, the ntigen levels my e too low for detection y the employed stining method. Loss of ntigenic differentition in some tumors or loss of ntigenicity due to suoptiml or excessive tissue fixtion my result in negtive expression. Immunorectivity is diminished or destroyed when prffin used for emedding process exceeds 60 C.[25] The present study hd few oservtions similr to Fernndes et l.,[8] which re s follows: The study nd control groups hd wide rnge of hmlh1 expression, which my e due to different trnscriptionl nd trnsltionl control of hmlh1 gene[24] In this study, some cses showed hmlh1 immunoexpression in cytoplsm which my e due to ction of the MMR system in mitochondril DNA, similr to tht which occurs in the nucleus,[8] so, only those cses showing distinct nucler immunorectivity were considered positive for hmlh1 The cells of minor slivry glnds, the nucleus of muscle cells nd mononucler leukocytes (when present in strom) showed nucler stining, its significnce is yet unknown[8] The stining pttern oserved in our study ws heterogeneous, i.e. ll the cells in the positive cses did not express hmlh1 Journl of Orl nd Mxillofcil Pthology Sep - Dec 2016 Vol 20 Issue 3

7 Chudhri, et l.: hmlh1 in orl leukoplki nd OSCC c d Figure 6: () Photomicrogrph of orl leukoplki (H&E stin, 100). () Negtive humn MutL homolog 1 stining in orl leukoplki (IHC stin, 100). (c) Photomicrogrph of orl squmous cell crcinom (H&E stin, 100). (d) Negtive humn MutL homolog 1 stining in orl squmous cell crcinom (IHC stin, 100) Tle 6: Comprison of men humn MutL homolog 1 expression etween control nd clinicl stges of orl squmous cell crcinom Clinicl TNM stge Numer of cses Percentge hmlh1 positivity (men) Group Stge 1 Stge 2 Stge 3 Stge 4 Ttest for equlity of mens, P vlue (twotiled) Control Stge 4 Stge 3 Stge 2 Stge hmlh1: Humn MutL homolog 1, TNM: Tumor node metstsis which my e due to different frequency of heterozygosity loss nd MSI in different res of leukoplki nd OSCC ecuse of intrtumor genetic heterogeneity.[8] Men vlue of humn MutL homolog 1 expression in control, orl leukoplki nd orl squmous cell crcinom ptients There ws progressive decrese in hmlh1 expression from control to leukoplki nd further to OSCC [Tle 3], which my e due to the presence of MSI nd epigenetic ltertions in leukoplki s well s occurrence of hmlh1 gene hypermethyltion, leding to reduced immunoexpression of this protein in squmous cell crcinom of the hed nd neck. Cldier et l. (2011) noted tht hmlh1 immunoexpression showed decresing indexes from lesions with lower degrees of dysplsi to lesions with more severe dysplsi. de Oliveir et l. (2014) reported tht higher percentge of epithelil cells expressed hmlh1 in cses of ctinic cheilitis without dysplsi or mild dysplsi s compred to lower lip squmous cell crcinoms. Tocco use would led to epigenetic ltertions of MMR genes in norml orl mucos, which would then ecome more susceptile to mlignnt trnsformtion, possily with MSI phenotype.[26,27] The reduced expression my e ecuse of exhustion of MMR system owing to constnt crcinogenic exposure.[8] Men vlue of humn MutL homolog 1 expression in clinicl stges of orl leukoplki nd control groups Men hmlh 1 expression ws significntly higher in control group s compred to different clinicl stges of leukoplki [Tle 4]. This shows tht MMR system is ffected s the severity of leukoplki increses cliniclly when compred Journl of Orl nd Mxillofcil Pthology Sep - Dec 2016 Vol 20 Issue 3 459

8 Chudhri, et l.: hmlh1 in orl leukoplki nd OSCC Tle 7: Comprison of men humn MutL homolog 1 expression etween control nd histopthologicl grdes of orl squmous cell crcinom Histopthologicl grde Numer of cses Percentge hmlh1 positivity (men) I II III T test for equlity of mens, P vlue (two tiled) Group Control III II I I II III hmlh1: Humn MutL homolog 1 with tht of norml mucos. There ws no significnt difference of hmlh1 expression etween the different clinicl stges of leukoplki. This my e due to the fct tht leukoplki my present different clinicl ehvior nd iologicl evolution, s the predictors of mlignnt trnsformtion depend on severl fctors such s the durtion of the lesion, ptient s ge nd gender, the ffected site, clinicl ppernce, smoking hit nd presence of epithelil dysplsi. [9] Men vlue of humn MutL homolog 1 expression in histopthologic grdes of orl leukoplki nd control groups Men hmlh1 expression ws significntly higher in control group s compred to mild s well s moderte dysplsi in leukoplki group [Tle 5]. These findings suggest tht there is n ltertion in DNA repir pthwy, prticulrly in hmlh1 gene, with n increse in the severity of dysplsi of leukoplki when compred to norml mucos. [26] There ws difference of men hmlh1 expression etween the different histopthologicl grdes of leukoplki, ut it ws not sttisticlly significnt. This could e due to sujective nd lck of inter nd intr oserver reproduciility in the grding of dysplsi. [28] Leukoplki with similr histologicl phenotypes my show different iologicl ehvior. [26] Men vlue of humn MutL homolog 1 expression in the clinicl stges of orl squmous cell crcinom nd control groups Men hmlh1 expression ws significntly higher in control group s compred to different stges of OSCC [Tle 6]. These results suggest tht hmlh1 ctivity reduces s the severity of OSCC increses cliniclly when compred with norml mucos. There ws no significnt difference of hmlh1 expression mong the different clinicl stges of OSCC which my e due to the lck of ccurte nd relile strtifiction of hed nd neck cncers ecuse of the numerous ntomic sites nd susites from which tumors cn rise nd the diversity of histologic types of tumors in these loctions. [29] Furthermore, ll the prmeters were not ssessed for the clinicl stging due to prcticl difficulties such s lck of mgnetic resonnce imging scn. Men vlue of humn MutL homolog 1 expression in histopthologic grdes of orl squmous cell crcinom nd control groups Men hmlh1 expression ws significntly higher in control group s compred to vrious grdes of OSCC [Tle 7]. Fernndes et l. reported n overexpression of hmlh1 in well differentited OSCCs s compred to poorly differentited OSCCs which showed reduced hmlh1 expression. The reduced expression of hmlh1 in poorly differentited OSCCs suggests sturtion of the MMR system. On the other hnd, the protein overexpression my reflect n ttempt on the prt of the MMR system to correct the multiplicity of mismtches. [8] There ws no significnt difference in hmlh1 expression mong the different histopthologicl grdes of OSCC. This could e explined y the fct tht the vlidity of histopthologic grding s mrker of prognosis remins controversil due to tumor heterogeneity, interoserver disgreement nd vritions in the size of the high power field in different microscopes. [30] In spite of our efforts, uniform smple size could not e chieved in different stges which ffected sttisticl nlysis. CONCLUSIONS Identifiction of erly moleculr mrkers tht precede phenotypic ltertions will help in the prediction of cncer development. Hence, despite its possile role in the development nd progress of dysplstic phenotype, hmlh1 lone is not sufficient to grde epithelil dysplsi s n mple rnge of hmlh1 vlues were seen in the present study within the sme group for OL nd OSCC. In the present study, significntly, lower hmlh1 expression is seen in leukoplki, which further decreses in OSCC s compred to norml orl mucos. Moreover, significntly reduced hmlh1 expression is seen in different clinicl nd histopthologicl stges of oth leukoplki nd OSCC with respect to norml orl mucos. Therefore, ltered expression of hmlh1 in leukoplki seems to e n erly event in crcinogenesis. In ddition, reduced hmlh1 expression in OSCC my reflect the sturtion of DNA repir pthwy nd highlight the role of hmlh1 in the progression of crcinogenesis nd cn e considered useful mrker of poor prognosis. Moleculr reserch studies on MMR system might e helpful in understnding the precise mechnism of hmlh1 in potentilly mlignnt disorders nd cncer. Finncil support nd sponsorship Nil. Conflicts of interest There re no conflicts of interest. 460 Journl of Orl nd Mxillofcil Pthology Sep - Dec 2016 Vol 20 Issue 3

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