Originl Article DOI: 10.17354/ijss/2016/147 Significnce of Silver Binding Nucleolr Orgnizer Regions in Orl Squmous Cell Crcinoms Ritu Shrm 1, Gurv Kumr 2 1 Assistnt Professor, Deprtment of Pthology, Hind Institute of Medicl Sciences, Dr. RML Avdh University, Lucknow, Uttr Prdesh, Indi, 2 Assistnt Professor, Deprtment of ENT, TSM Hospitl & Medicl College, Lucknow, Uttr Prdesh, Indi Astrct Bckground: Cell prolifertion is n importnt fctor in the prognosis of mlignnt neoplsi. The numer of rgyrophilic nucleolr orgnizer regions (AgNORs) per cell hs een considered s n indictor of the cellulr prolifertive ctivity. A study is crried out to exmine whether AgNOR numers relte to the growth rte in squmous cell crcinom (SCC) of the orl cvity. Aim: To verify the reltionship etween totl AgNORs men vlue (magnor) nd/or the percentge of cells exhiiting five or more AgNOR dots/nucleus/100 cells AgNORs (prolifertive index [pagnor]) with histopthologic grding of orl SCC ccording to Broder s grding. Mterils nd Methods: This ws prospective study done over period of 3 yers from My 2012 to June 2015. A totl of 50 cses of orl SCC were grded into three groups ccording to Broder s grding, nmely well-differentited, modertely differentited, nd poorly differentited. For NOR study, 3-5 µm-thick sections were stined with 50% queous silver nitrte solution. The predominnt microscopic pttern of NORs ws determined. Quntittive nd qulittive nlyses of NORs were otined of ll cells present on ech histologicl field. AgNORs were stined y one-step silver method nd exmined in representtive prffin sections from 50 cses of orl SCC. Results: The magnors per nucleus ws 3.3057 ± 0.11 for the well-differentited group, 5.324 ± 0.43 for the modertely differentited, nd 8.167 ± 0.22 for the poorly differentited. The prolifertive index in well-differentited pagnor count is 25%, in modertely differentited count it is 49.7%, nd in poorly differentited count it is 65.4%. Conclusions: AgNOR stining technique seems to e useful dignostic tool since differences in AgNOR numeric vlues cn e identified in the different types of orl SCC. This technique is esy to hndle nd inexpensive, thus justifying its lrge use in histopthology. Key words: Argyrophilic nucleolr orgnizer regions, Nucleolr orgnizer regions, Orl squmous cell crcinom INTRODUCTION In 1941, Broders 1 relted the extent of mlignncy in neoplsms nd emphsized the correltion etween histologicl tumor differentition, their tretment, nd prognosis. The mjority of orl nd phryngel cncers were squmous cell crcinom (SCC). Out of which, 91.6% occurred in the orl cvity. 2 Orl SCC represents the third most common form of mlignncy in the developing www.ijss-sn.com Access this rticle online Month of Sumission : 01-2015 Month of Peer Review : 02-2016 Month of Acceptnce : 02-2016 Month of Pulishing : 03-2016 countries wheres in the developed countries, it is the eighth most common mlignncy. 3,4 The most widespred is the chewing of etel quid with tocco tht hve een demonstrted s n incresed risk for cncers of the orl cvity. Quntifiction of rgyrophilic nucleolr orgnizer regions (AgNORs) is vlule prmeter in tumor pthology. Studies hve shown the higher numer of AgNORs in mlignnt lesions such s SCC, which re ssocited with poor prognosis. NORs re the loop of DNA tht encode riosoml RNA nd re considered importnt in the synthesis of protein. They re locted on the short rm of crocentric chromosomes - 13, 14, 15, 21, nd 22. Mny of them ind silver, property ttriuted to proteins ssocited with these sites, prticulrly the cidic Corresponding Author: Dr. Ritu Shrm, 1404 HIG, Sector-I, LDA Colony, Knpur Rod, Lucknow - 226 012, Uttr Prdesh, Indi. Phone: +91-9455149391. E-mil: drritzshrm@gmil.com 193 Interntionl Journl of Scientific Study Mrch 2016 Vol 3 Issue 12
Shrm nd Kumr: Significnce of AgNOR in Orl Squmous Cell Crcinom nonhistone components. NOR stining thus represents ctively trnscriing NORs (thus rdna) nd the frequency of NORs per nucleus my prove to e useful s repliction mrkers. 5,6 AgNORs reflect the stte of ctivtion nd the prolifertion ctivity of the cell nd degree of mlignnt trnsformtion of certin tissues. The mount of AgNOR is proportionl to the prolifertive ctivity of neoplstic cells into the cell cycle, which progressively increse from Go to S phse. 7 A rpidly dividing tumor popultion hd greter proportion of cells in the erly stges of G1. Conversely, tumors with low rte of cell prolifertion disply single NOR. 7 The silver stining technique is not le to recognize rdna nd rrna, ut the cidic proteins ssocited with these sites of rrna trnscription re designted s B23, C23, AgNOR proteins, nd RNA polymerse I. 8 NORs hve got importnce nowdys more ecuse the frequency within the nuclei is much higher in mlignnt cells thn in norml cells, rective, or enign neoplstic cells. 8 nd ), nd poorly differentited (Figure 4 nd ), SCC (Tle 1). The iopsy specimens which were received sujected to proper fixtion in 10% forml sline fter tht followed y Figure 1: () Crcinom of se of tongue, () crcinom of lterl order of tongue Morphologicl Chrcteristics - Qulittive Assessment Morphologicl vritions of AgNORs were ssessed regrding the size nd shpe of the individul AgNOR dots nd their pttern of distriution, s defined y Khn et l., 2006, 9 who identified different ptterns of AgNOR size nd distriution: Figure 2: () Well- differentited squmous cell crcinom (H nd E, 40), () more or less uniform size rgyrophilic nucleolr orgnizer regions (AgNORs) dots/nucleus (AgNOR stin, 100) The grding of size vrition ws performed ccording to Khn et l. 9 nd scores of distriution were given s following: 0 - More or less uniform in size; 1+ - Two different sizes; 2+ - More thn two different sizes (ut not those of 3+); 3+ - Including ll grdes nd sizes. The dots dispersion grding ws performed ccording to Khn et l. 9 nd scores of AgNOR dots were given s following: 0 - Limited to nucleoli; 1+ - Occsionl dispersion outside nucleoli; 2+ - Moderte dispersion outside nucleoli; 3+ - Widely dispersed throughout nucleus. MATERIALS AND METHODS The present study is undertken in the Pthology Deprtment of Mhtm Gndhi Medicl College nd Hospitl, Jipur. A totl of 50 cses were studied from My 2012 to June 2015. The cses re referred from ENT Deprtment hving growth in the orl cvity (Figure 1 nd ) iopsy tken, nd sent for histopthologicl exmintion. These re rodly clssified into three groups ccording to Broder s 1 grding of histopthologicl reports s well differentited (Figure 2 nd ), modertely differentited (Figure 3 Figure 3: () Modertely- differentited squmous cell crcinom (H nd E, 40), () grde 1+ score rgyrophilic nucleolr orgnizer regions (AgNOR) size (AgNOR stin, 100) Figure 4: () Poorly- differentited orl squmous cell crcinom (H nd E, 40), () gret mount nd highly stined rgyrophilic nucleolr orgnizer regions (AgNOR) dots (AgNOR stin, 100) Tle 1: Clssifiction of ptients ccording to Broder s grding Groups Numer of cses Group I (WDSCC) 19 Group II (MDSCC) 22 Group III (PDSCC) 9 WDSCC: Well differentited squmous cell crcinom, MDSCC: Modertelydifferentited squmous cell crcinom, PDSCC: Poorly differentited squmous cell crcinom Interntionl Journl of Scientific Study Mrch 2016 Vol 3 Issue 12 194
Shrm nd Kumr: Significnce of AgNOR in Orl Squmous Cell Crcinom routine prffin sectioning t 3-4 µm thickness. AgNOR stining ws performed s descried y Ploton et l. 6,10 First, the smples were de-wxed in xylene nd then rehydrted through grded ethnols to distilled wter. The silver nitrte solution ws prepred y mixing two prts of 50% silver nitrte solution with 2 g of geltin in 100 ml of 1% formic cid in distilled wter. The sections re incuted in this solution in drk t room temperture for 60 min nd then wshed with de-ionized wter. This is followed y pir dehydrtion in grded lcohol solutions, clered in xylene, nd mounted in Cnd lsm. AgNORs re seen s distinct intrnucler lck dots nd re rndomly counted mnully in 100 nuclei under 1000 mgnifiction with oil immersion in the three groups. Finlly, the men vlue nd stndrd devition of n ech cse re determined. AgNORs re seen s distinct intrnucler lck dots nd were counted mnully in 100 epithelil nuclei under 100 mgnifictions with oil immersion in different groups. Finlly, the men vlue nd stndrd devition of ech group re determined nd tulted. One-wy ANOVA ws used to compre these groups. The men of AgNOR (magnor) is compred in ech group seprtely using unpired t-test. The dt otined re tulted in the mster chrts of the vrious histologicl grdes/groups in orl SCC. The qulittive ssessment of AgNORs sed on their size, shpe, nd the pttern of distriution ws processed using frequencies, percentges, nd Chi-squre test. The magnors in different grde of orl SCC per nucleus ws 3.3057 ± 0.11 for the well-differentited group, 5.324 ± 0.43 for the modertely differentited, nd 8.167 ± 0.22 for the poorly differentited group (Figure 5). According to one-wy ANOVA, significnt difference ws seen in the numer of AgNOR dots etween the groups (P < 0.001) (Tle 2). The second count ws the prolifertive index (pagnor) which is defined s the percentge of nuclei exhiiting five or more AgNOR grnules/nucleus/100 cells (Figure 6). This count represents the prolifertive ctivity of tumors cells. High prolifertive ctivity in tumors is considered when there is pagnor count of 8% or more (Tle 3). 11 RESULTS Clinicl dt otined from medicl records re compiled ccording to the tumor site such s 23 cses of the lterl order of tongue, 14 of the se of tongue, 6 of retromolr trigone, nd 7 of uccl mucos. The ge of the ptients rnged from 31 to 80 yers t the time of dignosis of neoplsm. Regrding the gender, 36 were mle nd 14 were femle. AgNORs were seen through light microscope Tle 2: Distriution sed on AgNOR counts (men nd rnge) in different grdes/groups Broder s grde of orl SCC Numer of suject magnor count Stndrd devition WDSCC 19 3.3057 0.11 MDSCC 22 5.3241 0.43 PDSCC 9 8.1677 0.22 SCC: Squmous cell crcinom, WDSCC: Well differentited squmous cell crcinom, MDSCC: Modertely differentited squmous cell crcinom, PDSCC: Poorly differentited squmous cell crcinom, AgNORs: Argyrophilic nucleolr orgnizer regions 10 5 0 G I (WDSCC) 3.3057 Men AgNOR Count G II (MDSCC) 5.324 8.1677 G III (PDSCC) Figure 5: Distriution sed on men rgyrophilic nucleolr orgnizer regions count in different grdes of orl squmous cell crcinom Figure 6: Prolifertive index of rgyrophilic nucleolr orgnizer regions count in different grdes/groups of orl squmous cell crcinom inside the cell nuclei s lck to rownish dots s the yellow stining llowed esy visuliztion of individul NORs. The numer nd dimeter of the NORs, usully round, were vrile nd either diffusely distriuted ll over the nucler re or grouped in wide, round, nd less intensely stined structure. Qulittive ssessment ws done ccording to Khn et l. 9 nd scores of dot distriution were given 195 Interntionl Journl of Scientific Study Mrch 2016 Vol 3 Issue 12
Shrm nd Kumr: Significnce of AgNOR in Orl Squmous Cell Crcinom Tle 3: Comprison of magnor count, pagnor, size, nd distriution per cell etween different grdes/groups of orl squmous cell crcinom Grdes Numer of cses magnor count per cell pagnor (percentge of nucler with >5 AgNOR dots/nucler) AgNOR vrition in size per cell AgNOR dispersion per cell WDSCC 19 3.306 25 0.263 0.263 MDSCC 22 5.324 49.7 0.454 0.41 PDSCC 9 8.167 65.4 1.22 0.88 SCC: Squmous cell crcinom, WDSCC: Well differentited squmous cell crcinom, MDSCC: Modertely differentited squmous cell crcinom, PDSCC: Poorlydifferentited squmous cell crcinom, pagnors: Prolifertive index rgyrophilic nucleolr orgnizer regions, magnors: Men rgyrophilic nucleolr orgnizer regions s: 0 - more or less uniform in size; 1+ - two different sizes; 2+ - more thn two different sizes (ut not those of 3+); 3+ - including ll grdes nd sizes. The grding dots dispersion ws performed ccording to Khn et l. 9 nd scores of dispersion of AgNOR dots were given s following: 0 - limited to nucleoli; 1+ - occsionl outside nucleoli; 2+ - moderte outside nucleoli; nd 3+ - widely throughout the nucleus. DISCUSSION Although conventionl histologicl stining with hemtoxylin nd eosin my e useful in the determintion of dysplstic chnges in precncerous lesions nd grding of SCC, sometimes it is difficult to differentite these lesions with this stining technique. In such cses, AgNORs stining seems to e useful. The AgNOR counts increse with incresed cell ploidy nd with incresed trnscriptionl ctivity in the stges of ctive cell prolifertion. 12 Vritions in the size nd numer of the AgNOR dots my depend on the stge of the cell cycle, the numer of NOR-ering chromosomes in the kryotype, or the trnscriptionl nd metolic ctivity of the cell. In rpidly proliferting cell, AgNOR distriution nd the chromosoml remin disorgnized with the resultnt formtion of smll, multiple, nd dispersed nucleoli. Actively proliferting cells hve impired nucleolr ssocition, nd therefore, they exhiit higher AgNOR count, regrdless of the ploidy stte of the cell. In the present study, results of 50 cses were compred ccording to ge, sex, sites of lesions, evluted quntittive, nd qulittive AgNOR counts in the different grdes of orl SCC with the study done y other workers, nd it ws suggested tht AgNOR stining cn e used to differentite different grdes of orl SCC. CONCLUSION The present study shows the importnce of AgNORs in the vrious grdes of orl SCC. Although conventionl histologicl stining with hemtoxylin nd eosin my e useful in the grding of SCC nd determintion of dysplstic chnges in precncerous lesions, sometimes it is difficult to differentite these lesions with this conventionl stining technique. In such cses, AgNORs stining seems to e eneficil nd useful. It hs een estlished tht quntifiction of interphse AgNORs cn ctully represent vlule tool for cell kinetics evlution. The AgNOR counts increse with incresed cell ploidy nd with incresed trnscriptionl ctivity in the stges of ctive cell prolifertion. Of the vrious newer techniques which were used for ssessing the tumor tissue sed on nucler studies, the stining of AgNORs y silver compound hs ecome populr for its: Simplicity thn other vrious techniques Ese of use nd to crry out Low cost of stining Good correltion with other prolifertive mrkers of tumors. Finlly, AgNORs (magnor nd pagnor) counting hve direct reltionship with Broder s histopthologic grding, leding us to suggest tht AgNOR technique cn e used s iologicl mrker of orl SCC tumor progression. REFERENCES 1. Broders AC. The microscopic grding of cncer. Surg Clin North Am 1941;21:947-61. 2. Chmni G, Zrei MR, Rd M, Hshemipoor M, Hghdoost AA. Epidemiologicl spects of orl nd phryngel cncer in Kermn Province, South Estern Irn. Irn J Pul Helth 2009;38:90-7. 3. Johnson NW. How gret is the risk? In: John NW, editor. Risk Mrkers for Orl Diseses. Orl Cncer. Detection for Ptients nd Lesions t Risk. Vol. 2. Cmridge: University Press; 1991. p. 3-27. 4. Bhosle RB, Murti PR, Gupt PC. Tocco hits in Indi. In: Control of Tocco Relted Cncers nd Other Diseses. Proceedings of n Interntionl Symposium, 5-19 Jnury. Bomy: Oxford University Press; 1990. p. 25-46. 5. Chttopdhyy A. AgNORs in tumorl pthology. Review of literture nd oservtions on the technic nd rection in norml orl epithelium. Indin J Dent Res 1993;4:47-53. 6. Crocker J, Boldy DA, Egn MJ. How should we count AgNORS? Proposls for stndrdized pproch. J Pthol 1989;158:185-8. 7. Crini RL, Schwint AE, Mendez A, Femopse F, Lnfrnchi H, Itoiz ME. Morphometric study of nucleolr orgnizer regions in humn orl norml mucos, ppillom nd squmous cell crcinom. J Orl Pthol Med 1992;21:275-9. 8. Underwood JC, Giri DD. Nucleolr orgnizer regions s dignostic Interntionl Journl of Scientific Study Mrch 2016 Vol 3 Issue 12 196
Shrm nd Kumr: Significnce of AgNOR in Orl Squmous Cell Crcinom discriminnts for mlignncy. J Pthol 1988;155:95-6. 9. Khn SA, Chudhry NA, Khlid AW, Akhtr GN, Ine-Rs SN. Ptterns of rgyrophilic nucleolr orgniser regions in pleurl nd peritonel effusions. J Coll Physicins Surg Pk 2006;16:412-5. 10. Orrell JM, Evns AT, Grnt A. A criticl evlution of AgNOR counting in enign nevi nd mlignnt melnom. J Pthol 1991;163:239-44. 11. Mourd WA, Vllieres E, Chuen J, Alroish A. Cell kinetics nlysis of surgiclly resected non-smll cell crcinom of the lung using the AgNOR silver stin. Ann Sudi Med 1997;17:161-6. 12. As NF, As EA, Al WE. Imge cytometric nlysis of men nucler re nd nucleolr orgnizer regions (AgNORs) in orl squmous cell crcinom. Egypt Med J NRC 2002;1:141-57. How to cite this rticle: Shrm R, Kumr G. Significnce of Silver Binding Nucleolr Orgnizer Regions in Orl Squmous Cell Crcinoms. Int J Sci Stud 2016;3(12):193-197. Source of Support: Nil, Conflict of Interest: None declred. 197 Interntionl Journl of Scientific Study Mrch 2016 Vol 3 Issue 12