USE OF ELASTOGRAPHY FOR CERVICAL CANCER DIAGNOSTICS

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1 Experimentl Oncology 37, , 2015 (June) 139 Exp Oncol , 2, USE OF ELASTOGRAPHY FOR CERVICAL CANCER DIAGNOSTICS O.A. Bky*, T.S. Golovko Ntionl Cncer Institute, Kyiv 03022, Ukrine The im of the study ws to investigte the use of elstogrphy for cervicl cncer dignostics. Mterils nd Methods: The ultrsonic study with use of sonoelstogrphy involved 87 ptients with cervicl pthology (cervicitis, n = 11; dysplsi, n = 14; cervicl cncer, n = 62). Results: At non-mlignnt pthology elstic types of elstogrms previled while the tumors were chrcterized y imges of stiff type formtions. Present study hs demonstrted n effectiveness of sonoelstogrphy for vlution of tumor process invsion levels. At detection of tumor invsion into prmetrium, the test sensitivity incresed from 77.1 to 91.4%, specificity from 85.1 to 96.2%, ccurcy from 80.6 to 93.5%; in cse of tumor invsion into vgin these prmeters correspondently chnged: from 75.0 to 83.3%, from 76.9 to 88.4%, from 75.8 to 85.4%, respectively; nd in cse of uterus: from 82.9 to 85.3%, from 85.7 to 95.2%, from 83.8 to 88.7%, respectively. Conclusions: Inclusion of elstogrphy into ultrsound exmintion of cervicl pthologies could significntly improve dignostic qulity of the method. Key Words: sonoelstogrphy, ultrsonic dignostics, cervicl cncer. Significnt increse of cervicl cncer (CC) incidence stipultes necessity of this pthology dignostic methods improvement nd new technologies development. At present, new method of ultrsonic investigtion (USI) sonoelstogrphy estimting tissues elsticity ecomes more nd more populr. In opinion of mny reserchers it is very useful dditionl method for dignostics of differently loclized pthologies [1, 2]. It is known tht elsticity is one of the most importnt tissues chrcteristics, especilly vlule for dignosticin, s it is chnging t different pthologic processes (ltertions, tumors, trums, etc.). Any new formtions with high stiffness re ssocited with incresed risk of mlignncy [3]. Elstogrphy is sed on the sme principle s plption. The pressure cuses deformtion of tissues, more soft tissues would e deformed more profoundly then more stiff ones. Vlution of this deformtion rtes llows otining informtion on tissues elsticity. At elstogrphic investigtion pressure is creted y ultrsonic wve or y light mechnic compression. Ultrsonic detection llows estimting different tissues deformtions due to creted pressure. Otined dt re nlyzed y computer softwre for echo signl processing nd elstogrm producing where different elsticity vlues re mrked with different colors. Sometimes elstogrphy is clled method of visul plption [4]. Thought elstogrphy development strted t the end of 90 s, t present re only few pulictions on this method possiilities t the field of gynecology [5 8]. Elstogrphy use t oncogynecology for cervix pthologies dignostics t present remins insufficiently studied. The im of our present study hs een investigtion of elstogrphy possiilities for cervix tumors dignostics. Sumitted: Jnury 28, *Correspondence: Tel.: E-mil: vgolg@mil.ru Arevitions used: CC cervicl cncer; EG elstogrphy; MRI mgnetic resonnce imging; USI ultrsonic investigtion. MATERIALS AND METHODS The study involved 87 ptients with cervicl pthology in ge from 28 to 61 yers. The medin ge ws 46.5 yers. In 11 cses ws detected cervicitis, in 14 cses dysplsi, in 62 CC. CC stges distriution pttern ws s follows: cncer in situ 4 (6.5%) ptients, Іа stge 5 (8.1%), І stge 9 (14.5%), ІІа 9 (14.5%), ІІ 8 (12.9%), ІІІа 14 (22.6%), ІІІ 11 (17.7%), ІVа 2 (3.2%). CC morphologic structures of ll ptients were verified. Thus in 34 (54.8%) ptients squmous cell crcinom t different stges of differentition ws dignosed, in 12 (19.4%) ptients tumors of ndrogen origin were detected, in 6 (9.7%) ptients other histologicl forms of crcinom (cler cell, smll cell etc.), in 10 (16.1%) ptients undifferentited tumors. Surgiclly treted were 26 women (mong them: cone resection 8 ptients with cncer in situ nd Іа stge, trchelectomy 2 ptients with CC І stge, hysterectomy 16 ptients with CC Іа ІІа stges). 48 ptients with CC ІІ ІV stges otined chemordiotherpy. In 4 ptients, with treted CC, relpse of tumor ws detected. Control group included 30 helthy women (18 of reproductive ge nd 12 in menopus). Complex USI with use of elstogrphy were conducted for ll ptients t Esote MyL Clss C device, Itly, with multi-frequency widend sensors: Convex CA541, trnsvginl EC1123 nd liner LA523. Investigtions were crried out s follows. Firstly stndrd trnsdominl nd trnsvginl USI (B-mode Doppler). After ctivtion elstogrphy regime region of interest ws included into the interrogtion zone. All works hve een crried out ccording to device producers mnul. Region of interest prmeters must e optimized. It should e noted tht the lrger would e interrogtion zone, the lower sptil resolution we otin, nd vice vers. On elstogrm qulity influence depth of oject loction nd its moility (elstogrphy could e the most informtive for dignostics of immoile ojects loclized on the depth from 0.5 to 5.0 cm).

2 140 Experimentl Oncology 37, , 2015 (June) Sonoelstogrphy regime prmeters must e optimized (power, intensifying, color mp). The sensor must e held thwrt to the surfce of the ody, without devitions to the right or left, producing lowmplitude rhythmic compression (comprle with slight tremor), which degree is reflecting t specil disply. Sometimes, externl compression is not necessry, s if ner the re of the survey re lrge vessels, trnsfer ripple is enough. The screen shows the elstogrm where lue hues mpped tighter res, nd red nd green elstic. If desired color scle cn e chnged. It is lso possile to compre the degree of elsticity of the vrious res in the interrogtion zone. For this im on redy elstogrm res of interest must e mrked. Then device clcultes stiffness rtio in selected res. It is thus possile to define stiffness differences in re with chnges in comprison with unchnged tissues. Mgnetic resonnce imging (MRI) of the 58 ptients pelvic orgns ws crried out t tomogrph Philips (Inter). Ultrsound semiotics ws compred with the clinicl courses of the diseses, MRI nd morphologicl studies dt. Tle 1. Types of elstogrms in gynecology Elstogrm description/schemtic denottion Exmples of imges RESULTS Elstogrphy results were nlyzed nd systemtized using the clssifiction proposed V. Gzhonov et l. [9], ccording to which elstic formtions could e chrcterized y colortions types 1 nd 2, while stiff ones y 3 nd 4 (Tle 1). As cn e seen from the Tle 1, type 1 corresponding to liquid ojects, is colored s three-color rtifct. In turn, type 2, corresponding to elstic tissues ojects, hs some vrints: 2а chrcterized very elstic formtions, contining gret mounts of liquid, 2 corresponds to less elstic structures with smll mount of smll rigid inclusions. Mosic type, cont ining oth mny res of high compressiility nd smll rigid inclusions, ws clssified y us s vrint of the 2 type (2c). The type 3 of elstogrm corresponds to stiff formtions, with n equl numer of res of high nd low compressiility. The type 4 of elstogrm is very stiff one usully chrcteristic for mlignnt tumors. During control group ptients investigtions unchnged cervix hs een mpped predominntly green with the ddition of lue nd red foci (type Elstogrm description/schemtic denottion Exmples of imges Type 1 Three-color rtifct (luegreen-red), found in liquid formtions with homogeneous contents Folliculr cyst Type 2c. Mosic structure with inclusions of diffe rent colors Cervix Type 2 corresponds to elstic formtions, with minly green coloring Type 2. With ddition of red nd yellow foci, typicl for very elstic ojects with gret mount of liquids Endometril polypus with multiple cystic inclusions Type 3 corresponds to stiff formtions, with green-lue coloring in lmost equl proportions Hysterofiromyom Type 2. Green coloring with lue foci, typicl for medium elsticity ojects with smll rigid inclusions Uterus ody with smll firous inclusions Type 4 corresponds to very stiff formtions, with previling lue coloring, is more common in mlignnt tumors CC

3 Experimentl Oncology 37, , 2015 (June) 141 2, 2 nd 2c). In cse of mucus presence in the cervix, it ws determined y red colortion long the cnl. Smll endocervicl cysts hve een colored y red, the lrger ones were of the typicl three-color scheme, chrcteristic for liquid ojects. Smll lue foci (corresponding to the connective tissue nd firosis inclusions) were more often defined long the externl os nd the trnsformtion zone. Outer more looser cervicl strom lyer hd green co loring. Around the orgn ws lwys detected red nd corresponding to very elstic prmetril tissue nd to smll mounts of mucus in the vginl vult (Fig. 1). Fig. 1. Norml cervix elstogrm t reproductive period It is known tht the cervix tumors t imnul investigtion re dense enough y touch, which llows the clinicin to differ them (y plption) from unmodified elstic strom [2]. These elsticity differences were lso detected t visul plption elstogrphy. Cervix with ckground nd precncerous processes (cervicitis, dysplsi) in ll cses remined elstic nd ws colored predominntly y green. At CC levels of elstogrphic symptoms mnifesttions directly depended on tumor sizes nd stge of progress, therefore otined dt nlysis ws crried out in ccordnce with the tumor process stging in oedience to CC clssifiction y FIGO (2009). Thus, with cncer in situ we did not detect dignosticlly significnt differences y elstogrphy investigtion. At CC stge 1а in the mjority of cses sonoelstogrphic imges did not prcticlly differ from norml ones, only t 40% ptients with exophytic tumors in the projections of defets could e seen loclized drk lue res. Tking into ccount smll sizes (up to 5 7 mm) of tumor invsions t this stge, it ws difficult to distinguish them from stiff firous nd connective tissue inclusions s oth of them were colored s drk lue foci t the ckground of unchnged strom. Strting from stge 1, tumors lrger thn 1 cm in ll cses hd the chrcteristic imges, were mpped s zones of lmost solid drk lue color (mostly type 4, sometimes type 3) on ckground of unmodified cervix strom which llowed to distinguish them relily from firous inclusions nd ckground processes. Elstogrphy clerly trced tumor contours. In B-mode it ws not lwys esy to trce this order line, s the difference in tints of grey could e so smll, tht it ws difficult to fix it y the eye. Elstogrphy fcilitted this process s drk lue tumor ws well distinctive from the green unmodified strom nd red prmetril tissue. The etter visuliztion of tumor contours helped to crry out more ccurte mesuring nd locliztion of the tumor. With widespred cncer (II IV stges), the min tsks of the dignostic imging re n estimtion of the process distriution levels nd precise tumor stges definition which could e of gret importnce for oncologists, s it rdiclly influences t choice of tretment tctics nd finl prognosis [10]. CC stge II is chrcterized y the tumor invsion into the uterus nd vgin. Unmodified myometrium hving the sme elsticity s the cervix, is colored in green with smll mount of lue foci (type 2). In cse of tumor invsion into the myometrium, its drk lue imge on elstogrm hs een detected outside of cervix in the ody of the uterus, with well trced order etween invsion re nd elstic myometrium. Invsion of CC into the vginl wll in some cses is difficult to differentite from exophytic tumors protruding into its lumen. At the sence of vgin lesions, its wlls re not chnged, their surfces contin smll mounts of mucus, which re imged t elstogrms y chrcteristic red coloring round the externl os. The min feture for distinguishing ІІ from II stge is the presence of invsion into the prmetril tissue. Its ultrsound visulistion is rther difficult tsk. Undoutedly, devices technicl progress nd wide distriution of Doppler ultrsound dignostics gretly improved the possiilities of ultrsound dignosis in this re [11], ut it is necessry to confess tht t present MRI is the most informtive for estimting prmetrium invsions [12, 13]. Nevertheless, the elstogrphy introduction into complex ultrsound dignostics llows us to otin informing level comprle to MRI. In our study, we oserved correltions etween MRI-tomogrms nd elstogrms. Estimtion of tumor invsion into prmetrium ws crried out hving regrd to the presence of the following sign. In res where the order of the cervix ws preserved, could clerly e seen, green, preserved prt of the strom nd the red strip of unffected prcervicl tissue (Fig. 2). In res of tumor invsion outside the orgn contour ws not trced (typicl imge of cervicl strom nd tissues ws sent nd displced in projection y multiple drk lue foci of the tumor). Fig. 3, 4 compres elstogrms nd MRI-imges of ptients with the invsions of neoplstic processes into prmetrium nd the isthmus of the corpus uteri. In cse of CC invsion into the ldder, the order with this orgn ws not trced, red lyer etween tumor nd ldder ck wll (consisted of very elstic prvesiculr tissue) ws sent on elstogrm.

4 142 Experimentl Oncology 37, , 2015 (June) is estimted indirectly y mesuring of cervix sizes nd volume, which does not llow evluting stte of tumor process ojectively enough, s the volume of cncer invsion usully differs from the cervix volume itself. c c Fig. 2. CC ІІа stge. Elstogrm of endophytic tumor which significntly ffects the cervix strom nd invdes uterus ody, without leving its mrgins. There is cler order line etween drk lue tumor nd green-colored unchnged strom (). Post-opertive photos of tumor confirm the sence of invsion outside the uterus (, c) It is well known tht t CC stges II IV chemordiotherpy is the min method of tretment [14]. The most importnt tsk for dignostic imging during such tretment is estimtion of its effectiveness t ll its phses. During conservtive tretment monitoring stndrd ultrsound investigtions were complemented y elstogrphy. While estimtion of tumors response to tretment numer of prmeters were tken into ccount: chnges of sizes, volume, echostructure nd chrcter of tumor vsculriztion, the stte of the cervicl cnl, the extent of tumor invsion into the surrounding tissues, djcent orgns, nd the presence of metstses. But the most ojective criterion is the degree of tumor volume regression [15]. Usully, this prmeter Fig. 3. CC ІІ stge. MRI scn: tumor invsion outside the cervix into the uterus front wll nd the ody (); ck contour of the cervix is preserved (), typicl imge of preserved green strom nd red prcervicl tissue is sent t the front side tumor invdes outside the orgn (c) But direct mesuring of tumors sizes nd volumes t rod invsion process is difficult enough, s contours definition during stndrd ultrsound investigtion is often unsimply. Elstogrphy fcilitted this tsk, s improved visuliztion of tumor contours. Furthermore, fter the effective rdiotherpy we registered reduction of drk lue

5 Experimentl Oncology 37, , 2015 (June) 143 foci mount, nd chnges in the tumor strom stiffness comprtively to intct tissue (Fig. 5). ws mpped predominntly green, while scnning of CC, chrcterized y high degree of rigidity, on elstogrms previled lue coloring. Elstogrphy type s distriution t ptients with non-mlignnt nd mlignnt tumors of the cervix is presented t Fig. 7. Fig. 4. CC stge III. Tumors invsion into the isthmus of the corpus uteri: MRI; elstogrm The min tsk of ptients monitoring fter conservtive tretment finishing is possile recurrence (relpse of disese) erly detection [16]. In our investigtions in cse of its sence cervix sonoelstogrphic imge did not differ from the norml one. However, in generl, the numer of lue foci were little greter (predominnt type ws elstogrm 2). Such chnges corresponded to the post-rdition chnges incresed numer of lthough non-mlignnt ut lso non-elstic firous inclusions. Elstogrphic imge of CC recurrence corresponded to the imge of the primry tumor. The ffected re ws mpped in drk lue (elstogrm type 4). In cses of 2 ptients we hd to differentite tumors recurrences from inflmmtory chnges, which could lso cuse n increse in orgn size, the heterogeneity of the structure nd incresed vsculriztion. Inflmmtory infiltrte (unlike the tumor) ws mpped s elstic formtion mostly green colored (Fig. 6). DISCUSSION Thus, our investigtions hs een demonstrted tht the differences in elsticity of cervix nonmlignnt nd mlignnt pthologies re evidently reflected in elstogrphic imges. In non-mlignnt processes cervix in ll cses remined elstic nd Fig. 5. Monitoring of the chemordiotherpy effectiveness fter stge 1 of rdiotherpy () nd fter 2 stge rdiotherpy (): tumor ws reduced in size, numer of lue foci lowered s soon s the difference etween the stiffness levels in the tumor strom nd intct tissue As cn e seen from the digrm, the strom of the cervix ws mpped y elstogrm types 2 4. Only lrge cysts of the endocervix were colored s elstogrm type 1, chrcteristic for liquid ojects. In cses of non-mlignnt pthologies previled elstogrm types 2, 2 nd 2c. In single cse of cervicl dysplsi we met stiff elstogrm type 3, ssocited with lrge numer of strom firous inclusions fter previuos cone resection. At CC severity of sonoelstogrphic symptoms mnifesttion directly depended on the stge of the process. Thus, t the initil invsion (crcinom in situ, stge I) cervix elstogrms imges were of elstic types, ut just from the I stge tumors were colored minly s type 4 (75.5%) nd type 3 (22.7%). In cses of dvnced cncer presence of green foci t the lue ckground (type 3) could reflect locliztion of tumor res with different levels of stiffness nd more elstic regions of necrosis. In the single cse of stge III CC we determined mosic type 2c, which ws ssocited with significnt necrosis in tumors, nd

6 144 Experimentl Oncology 37, , 2015 (June) plenty of liquid inclusions (zones of disintegrtion with pus). Elstogrm type 4 ws never met with nonmlignnt pthology, ut it ws typicl for CC. Among this it ws estlished, tht chnges in cervix elsticity, cused y chemo-rdiotherpy, were lso detected y elstogrphy. Fig. 7. Elstogrphy type s distriution t ptients with cervix pthologies Fig. 8 demonstrtes elstogrm types distriution during monitoring of the ptients treted with conservtive therpy: fter tretment phse 1, fter its finishing nd with recurrent disese. As cn e seen from the digrm, just fter stge 1 of the chemordiotherpy distriution of elstogrm types shifted (in comprison with strt of tretment) towrds less severe. Elstogrm type 3 ecme more common (50% of cses), type 4 chrcteristic for mlignnt tumors ws determined rrer thn efore tretment (40%). After the tretment finishing elstogrms were similr to tht in non-mlignnt processes with dominted elstic types. But in 25% of cses we met the stiff type 3, reflecting the lrge numer of rigid post-rdition firous inclusions in the cervix. In recurrent CC distriution of elstogrphic types ws similr to tht seen in the primry tumor. 36 (75%) Before tretment 11 (23%) 1 (2%) 24 (50%) After tretment phse 1 19 (40%) 5 (10%) 28 (58%) After tretment finishing Fig. 6. The inflmmtory infiltrte in the vginl stump fter 6 months of comintion therpy. At the Doppler mode (), incresed lood flow is registered, t elstogrphy () formtions hve elstic nture Numer of ptients Cervicitis Type 2 Type 2 Type 2c Type 3 Type 4 Displsi of cervix uteri CC in situ CC I CC I CC II CC II CC III CC III CC IV 3 (75%) 12 (25%) With recurrent disese 1 (25%) 8 (17%) Type 2 Type 2c Type 3 Type 4 Fig. 8. Elstogrphy types distriution during chemiordiotherpy effectiveness monitoring Our studies hve lso demonstrted the enefits of elstogrphy in the evlution of tumor process invsion which improved the ccurcy of CC stges determintion nd ws of gret importnce for tretment plnning. Tle 2 presents dignostic vlue indices of US nd US + EG systems for estimtion cncer invsion into the prmetrium, uterus ody nd vgin. In ptients fter opertions verifiction of ultrsound investigtion ws performed on the sis of surgicl intervention dt nd pthology nlysis, MRI dt ws used s the reference method for the rest of the ptients.

7 Experimentl Oncology 37, , 2015 (June) 145 Tle 2. Dignostic vlue indices of US nd US + EG systems for estimtion cncer invsion t CC Detection of tumor Sensitivity, % Specificity, % Accurcy, % invsion US US + EG US US + EG US US + EG Into the prmetrium Into vgin Into uterus ody Note: US ultrsound dignostics; EG elstogrphy. As cn e seen from the tle 2, the use of elstogrphy in the dignostics hs een given us n dvntge in estimtion CC invsion processes in comprison with stndrd ultrsound exmintion, which llowed to increse this methods informtiveness to MRI indices levels. The use of elstogrphy investigtion profited most (ccording to ll indices) for the ssessment of tumor invsion: sensitivity, specificity, nd ccurcy of the detection incresed 14.3; 11.1 nd 12.9%, respectively. Elstogrphy, like ny other method hs its strengths nd weknesses s soon s in specific ppliction res. Elstogrphy limiting fctor is its low informtiveness t detection of CC erly stges. It is difficult to differentite tumor invsions of very smll sizes from firous inclusions, lso hving high rigidity. It should e emphsized tht elstogrphy differentites elstic formtions from stiff ones ut not non-mlignnt from mlignnt. Also, limiting is the depth of the formtions locliztion nd their moility. Elstogrphy profits most for the dignosis of inctive formtions, loclised t depth of 0.5 to 5 cm. CONCLUSIONS Thus, our investigtions demonstrted tht the sonoelstogrphy inclusion into the complex ultrsound studies extended the dignostic possiilities of these methods, llowed to increse the informtiveness t estimtion of cncer process stges (t the detection of tumor invsion into the prmetrium sensitivity incresed from 77.1 to 91.4%, specificity of 85.1 to 96.2%, ccurcy from 80.6 to 93.5%; into the vgin: from 75.0 to 83.3%, from 76.9 to 88.4%, from 75.8 to 85.4%, respectively; into the uterus ody: from 82.9 to 85.3%, from 85.7 to 95,2%, from 83.8 to 88.7%, respectively). Its use in complex with other methods of investigtion my improve dignostic qulity t thretening pthologies such s CC. This elstogrphy method is inexpensive, simple in use, does not occupy plenty of time in the routine study, non-invsive, sfe. Currently elstogrphy technique is in the process of development nd requires further study. REFERENCES 1. Botr JC, Vsilescu D, Dmin L. Musculoskeletl sonoelstogrphy. Pictoril essy. Med Ultrson 2012; 14: Admietz BR, Meier-Meitinger М, Fsching P, et l. New dignostic criteri in rel-time elstogrphy for the ssessment of rest lesions. Ultrschll Med 2011; 32: Bot S, Spore I, Sirli R, et l. Intr- nd interopertor reproduciility of coustic rdition force impulse (ARFI) elstogrphy preliminry results. Ultrsound Med Biol 2012; 38: Brr RG, Destounis S, Lckey LB, et l. Evlution of rest lesions using sonogrphic elsticity imging: multicenter tril. J Ultrsound Med 2012; 31: Ard K, Ciledg N, Akts E. Quntittive ssessment of norml soft-tissue elsticity using sher-wve ultrsound elstogrphy. Am J Roentgen 2011; 197: Switkowsk-Freund M, Preis K. New methods of ultrsound exmintion of uterine cervix efore lor induction. Gin Pol Med Project 2010; 3: Switkowsk-Freund M, Preis K. Elstogrphy of the uterine cervix: implictions for success of induction of lor. Ultrsound Ostet Gynecol 2011; 38: Molin F, Gomez L, Florido J. Quntifiction of cervicl elstogrphy. A reproduciility study. Ultrsound Ostet Gynecol 2012; 39: Gzhonov V, Churkin S, Lukynov E, et l. Clinicl ppliction of new method sonoelstogrphy in gynecology. Кremlin medicine. Clinicl Herld 2008; 2: (in Russin). 10. Vn den Bosch T, Coosemns A, Morin M. Screening for uterine tumours. Best Prct Res Clin Ostet Gynecol 2012; 26: Blleyguier C, Sl E, D Cunh T, et l. Stging of uterine cervicl cncer with MRI: guidelines of the Europen Society of Urogenitl Rdiology. Eur Rdiol 2011; 21: Mitchell DG, Snyder B, Cokley F, et l. Erly invsive cervicl cncer: MRI nd CT preditors of lymphtic metstses in the ACRIN 6651/GOG 183 intergroup study. Gynecol Oncol 2009; 112: Kesic V. Mngement of cervicl cncer. Eur J Surg Oncol 2006; 32: Mneo A, Colomo A, Lndoni F, et l. Tretment of stge IIIB cervicl crcinom. A comprison etween rdiotherpy, concurrent chemo-rdiotherpy nd neodjuvnt chemotherpy. Minerv Ginecol 2005; 57: Houveneghel G, Lelievre L, Gonzgue-Csinc L, et l. Long-term survivl fter concomitnt chemordiotherpy prior to surgery in dvnced cervicl crcinom. Gynecol Oncol 2006; 100: Akyir O, Corcioglu A, Numnoglu C. Preopertive ssessment of myometril nd cervicl invsion in endometril crcinom y trnsvginl ultrsound. Gynecol Oncol 2011; 122: Copyright Experimentl Oncology, 2015