Immunohistochemical analysis of 147 cases of low-grade endometrial stromal sarcoma: refining the immunohistochemical profile of LG-ESS on a large, molecularly confirmed series

Vol.:(0123456789) Vi chows A chi h ps://doi.o g/10.1007/s00428-025-04026-4 ORIGINAL ARTICLE Immunohis ochemical analysis o 147 cases o low-g ade endome ial s omal sa coma: e ining heimmunohis ochemical p o ile o LG-ESS onala ge, molecula ly con i med se ies Mi osla aFlíd o á1· Pa elDund 1· RomanaV ánko á1· K is ýnaNěmejco á1· Da idCibula2· Rena aPonco á2· K ě osla aMichalo á3,4· JiříBouda5,6· JanLaco7· MunachisoNdukwe8· JanuszRyś9· Ma iuszKsiążek10,19· Albe oBe jon11· IgnacioZapa diel12· I anF anin13· An onelaNja o14· Ji kaHausne o á15· Pe aB e o á16· Vladimí Židlík17· Ja osla Klá 18· Zoa dTibo K asznai19· Robe Poka19· Na aliyaVolodko20· I ynaYezho a20· Rado anPilka21,22· RadimMa ek21· Geo ginaKolniko a23· MilanK koška24· MichaelHalaška25· JanaD ozeno á26· Dagma Dolinská27· Vladimí Kalis 28· Ma cinBobiński29· Ma aOs owska‑Leśko30· MagdalenaBizoń31,32· Włodzimie zSawicki32· MaciejS ukan33,34· Ka olinaG abowska33· Ma cinJęd yka35· Tymo euszPop awski36· SimonaS olnicu37· MihaiEmilCăpîlna38· ZuzanaŠpů ko á39· MichalZikán40· F ancescaCicca one41· Gio anniScambia41,42· A chilSha ashenidze43· Mi andaGudadze44· Te ianaPia ny ska45· Iho Va chak45· MichaelaKendallBá ů1 Recei ed: 2 Decembe 2024 / Re ised: 23 Decembe 2024 / Accep ed: 6 Janua y 2025 © The Au ho (s) 2025 Abs ac Low-g ade endome ial s omal sa coma (LG-ESS) can p esen diagnos ic challenges, due o i s o e lapping mo phological ea u es wi h o he u e ine mesenchymal umo s. Misdiagnosis a es emain signi ican , and immunohis ochemical da a o LG-ESS a e limi ed o small se ies and inconsis en an ibody panels. This s udy aimed o e ine he IHC p o ile o LG-ESS by analyzing a la ge, molecula ly con i med se ies o 147 cases using a panel o 24 an ibodies, including newe ma ke s like ansgelin and smoo helin. CD10 and IFITM1, key endome ial s omal ma ke s, we e exp essed in 86% (92% o hose ex ensi ely) and 69% (60% o hose ex ensi ely) o cases, wi h usion-posi i e umo s showing signi ican ly highe exp es- sion. Smoo h muscle ma ke s (α-SMA, desmin, h-caldesmon, calponin, ansgelin) we e a iably exp essed, p edominan ly in ocal o low-in ensi y pa e ns, wi h α-SMA eaching he highes equency o exp ession (44%). Howe e , he in ensi y o smoo h muscle ma ke exp ession was usually e y low. Smoo helin was a ely exp essed. Ho mone ecep o s we e e- quen ly posi i e, wi h PR showing a highe equency (92% s. 83%) and in ensi y han ER. Ma ke s like S-100, HMB45, and CD117 we e la gely nega i e; all umo s we e p53 wild- ype, wi h p ese ed SMARCB1/SMARCA4 exp ession and ALK and ROS1 nega i i y. This wo k ep esen s he la ges molecula ly alida ed IHC s udy on LG-ESS, p o iding a obus diagnos ic p o ile o ou ine pa hology. By add essing key diagnos ic limi a ions and examining newe ma ke s, ou s udy suppo s a mo e s anda dized app oach o diagnosing LG-ESS and unde sco es he alue o immunohis ochemical panels, pa icula ly in usion-nega i e umo s whe e diagnosis elies on mo phological and immunohis ochemical in e p e a ion. These indings con ibu e c i ical da a o imp o ing diagnos ic accu acy. Keywo ds Low-g ade endome ial s omal sa coma· LG-ESS· Immunohis ochemis y· Endome ial s omal ma ke s· Smoo helin In oduc ion Low g ade endome ial s omal sa coma (LG-ESS) is a a e malignan mesenchymal umo which mo phologi- cally esembles p oli e a i e phase endome ial s oma and exhibi s an in il a i e g ow h pa e n [1]. Endome ial s o- mal sa comas cons i u e up o 1% o all u e ine cance s and Ex ended au ho in o ma ion a ailable on he las page o he a icle Vi chows A chi 6–20% o all u e ine sa comas, wi h LG-ESS ep esen ing he second mos common u e ine sa coma a e leiomyo- sa coma [2, 3]. While mos cases a ec he u e ine body, p ima y ex au e ine umo s can also occu , o en associa ed wi h endome iosis, making diagnosis mo e challenging [4, 5]. In mos cases, LG-ESS can be eliably diagnosed based solely on mo phology due o i s dis inc appea ance. How- e e , in some cases, LG-ESS can exhibi signi ican mo - phological he e ogenei y wi h a wide ange o his ological pa e ns, which can also be e lec ed in hei immunohis o- chemical p o ile [6]. Some less common, a ian mo pholo- gies include smoo h muscle-like di e en ia ion, ib oblas ic and/o myxoid ea u es, sex co d-like s uc u es, pseudopap- illa y o ma ions, clea cell change, skele al muscle di e - en ia ion, adipocy ic me aplasia, angioma ous pa e n, and cells wi h habdoid appea ance o biza e nuclei [7–11]. The e o e, he di e en ial diagnosis o LG-ESS p ima ily includes cellula leiomyoma (and po en ially o he smoo h muscle umo s), in lamma o y myo ib oblas ic umo (IMT), and u e ine umo s esembling sex co d-s omal umo s (UTROSCT) [6, 8, 9, 12–15]. Some cases wi h smoo h mus- cle di e en ia ion exhibi a cha ac e is ic “s a bu s ” pa e n, wi h collagen ibe s adia ing h oughou he umo , while o he s may show dispe sed collagen plaques o myxoid changes, which can mimic he eg essi e changes ound in leiomyoma o a myxoid leiomyosa coma [7]. The p esence o epi helioid/ ound cell di e en ia ion may aise suspicion o high g ade endome ial s omal sa coma (HG-ESS) o pe i ascula epi helioid cell umo (PEComa). In some cases, soli a y ib ous umo (SFT), gas oin es inal s omal umo (GIST), o adenosa coma can also s and in he di - e en ial diagnosis. Mo eo e , ecen ly desc ibed sa comas wi h he KAT6B/A::KANSL1 usion usually ha e o e lap- ping ea u es be ween LG-ESS/endome ial s omal nodule (ESN) and smoo h muscle umo s [16–19]. Wi h he inc easing accessibili y o molecula gene ic me hods, especially nex gene a ion sequencing (NGS), and he expanding knowledge o ecu en gene ic al e a ions in u e ine mesenchymal neoplasms, iden i ying cha ac e is ic usions has g ea ly enhanced diagnos ic accu acy. Up o 75% o LG-ESS ha bo ecu en usions, wi h JAZF1::SUZ12 being he mos common, ollowed by JAZF1::PHF1, EPC1::PHF1, and MEAF6::PHF1 [6, 8, 20]. Howe e , ha lea es almos a qua e o LG-ESS which a e no cu en ly associa ed wi h any known ecu en gene ic al e a ion. In a ou ine diagnos ic se ing, access o NGS is s ill a he lim- i ed and o en a ies be ween coun ies, wi h he added ques- ion o inancial a ailabili y. As such, immunohis ochemis y as a me hod which is as ly mo e accessible s ill emains a i al ool in eaching he co ec diagnosis. We conduc ed an ex ensi e immunohis ochemical analy- sis on a la ge, molecula ly examined coho o 147 LG-ESS, all subjec ed o igo ous cen al e iew. Ou s udy es ed a wide panel o immunohis ochemical ma ke s including sex co d-s omal ma ke s, smoo h muscle ma ke s, ho mone ecep o s, and selec ed no el an ibodies, some o which ha e no been assessed in LG-ESS coho s o his size. Ou goal was o de ine he immunohis ochemical p o ile o he la ges coho o LG-ESS o da e, hus p o iding a p ac i- cal diagnos ic guide o pa hologis s, especially in se ings whe e molecula es ing is una ailable. Ma e ials andme hods Samples The samples ep esen a pa o he coho assembled unde he Ra e Gynecological Sa coma (REGYS) s udy, which is an in e na ional p ojec in ol ing 23 pa icipa ing ins i- u ions om 10 coun ies, consis ing mos ly o membe s o he Cen al and Eas e n Gynecology Oncology G oup (CEEGOG). The p ojec included a de ailed assessmen o he mo phological ea u es, immunohis ochemical analysis, and DNA and RNA NGS analysis. A de ailed desc ip ion o he p ojec , he o e all coho assembled, and he molecu- la esul s a e p o ided in a di e en s udy (cu en ly unde e iew) and a e no desc ibed he e. B ie ly, a cen al e iew was pe o med on all hema oxylin and eosin (HE)-s ained slides a ailable o indi idual cases by wo expe ienced gynecopa hologis specialis s (PD, MKB). The equi ed numbe o ep esen a i e slides om he co-ope a ing ins i- u ions o each case was 2 (n = 100). In hose cases which came om he a chi es o ou depa men (as well as cases which we e sen o us as consul a ions, n = 47), he e alu- a ion was pe o med on all slides a ailable om he en i e biopsy/ esec ed specimen. The mo phological e alua ion was combined wi h he esul s o he immunohis ochemical analysis and molecula es ing o each he co ec diagnosis. An immunohis ochemical (IHC) examina ion wi h a b oad panel o 24 selec ed an ibodies was pe o med o each umo . Only cases unequi ocally diagnosed as LG-ESS a e he cen al e iew we e included in he s udy, esul ing in a inal sample se o 147 cases. O hese 147 cases, 133 had RNA NGS esul s a ailable which showed ha 101 cases (75.9%) ha bo ed a ecu en usion. The mos common al e a ion was he JAZF1::SUZ12 usion ound in 67 cases (66.3% o all usions), ollowed mainly by JAZF1::PHF1 (n = 9), MEAF6::PHF1 (n = 8), and EPC1::PHF1 (n = 4). The s udy was pe o med on o malin- ixed, pa a in embedded (FFPE) issue blocks. Hema oxylin and eosin- s ained slides om he FFPE issue blocks we e e iewed, and issue mic oa ays (TMAs) we e cons uc ed om sui - able umo a eas. Two issue co es o a 2.00 mm diame e we e ex ac ed om each FFPE issue block using he TMA Vi chows A chi ins umen TMA Mas e (3DHISTECH L d., Budapes , Hunga y). Immunohis ochemical analysis Immunohis ochemis y was pe o med on 4 µm hick sec ion using he TMAs. In cases whe e he TMA app oach was no possible, pa icula ly due o small umo size o echnical di icul ies du ing sample p ocessing, whole- issue sec ions we e used whe e possible. The IHC was e alua ed indepen- den ly by wo pa hologis s (MKB, MF). The an ibodies used o IHC examina ion we e selec ed based on hei diagnos- ic u ili y, wi h an emphasis on uling ou he o he en i ies mos commonly in ol ed in he di e en ial diagnosis. The whole panel was comp ised o es ogen ecep o (ER), p o- ges e one ecep o (PR), al a-smoo h muscle ac in (α-SMA), desmin, h-caldesmon, calponin, CD10, IFITM1, ansgelin, BCOR, BCORL1, NTRK, S-100, HMB45, CD117, WT1, SMARCA4 (BRG1), SMARCB1 (INI1), SMARCA2, ALK, ROS1, p53, smoo helin, and cyclin D1. The comple e lis o an ibodies used, including hei clones, dilu ion, and manu- ac u e s is p o ided in Table1. The immunohis ochemical esul s we e e alua ed based on he o e all pe cen age o posi i i y (0–100%). Cases we e classi ied as nega i e (comple e absence o s aining o < 1% o posi i e umo cells), 1 + (1–25% o posi i e umo cells), 2 + (26–50% o posi i e umo cells), o 3 + (> 50% o posi- i e umo cells). Fo he an ibodies NTRK and smoo helin, he s aining was e alua ed independen ly as bo h nuclea and cy oplasmic, o WT1 only he nuclea s aining was e alu- a ed. The immunohis ochemical esul s we e also assessed using he H-sco e me hod p e iously desc ibed by o he s [21]. This me hod inco po a es bo h he pe cen age o posi- i e cells and he s aining in ensi y (1 + o weak in ensi y, 2 + o mode a e, and 3 + o s ong). The inal H-sco e is calcula ed by adding he mul iplica ion o he di e en s ain- ing in ensi ies acco ding o he ollowing o mula: [1 x (% o cells 1 +)] + [2x (% o cells 2 +)] + [3x (% o cells 3 +)], esul ing in an H-sco e alue o 0–300. To cha ac e ize exp ession in e ms o posi i e and nega i e cases, a cu - o alue o 1% was used (posi i e: ≥ 1% o cells showing exp ession). Fo compa ing he IHC exp ession wi hin he LG-ESS coho based on he p esence o usion, he cu -o alue was modi ied o 5% o accoun o mino non-speci ic s aining a ia ions. S a is ical analyses All s a is ical analyses we e conduc ed using R so wa e, e sion 4.3.3 (2024–02–29). S anda d desc ip i e s a is ics we e applied o summa ize he da ase : Ca ego ical a iables we e epo ed as equencies and pe cen ages, and con inu- ous a iables we e desc ibed using means wi h s anda d de ia ion (SD) o medians wi h in e qua ile ange. Co ela- ions be ween he exp ession o IHC ma ke s (ca ego ized as posi i e s. nega i e) and usion s a us (p esence s. absence o usion) we e assessed using Pea son’s chi-squa ed es o Fishe ´s Exac es based on expec ed alues. All es s we e wo-sided, and a p alue < 0.05 was conside ed s a is ically signi ican . Resul s A de ailed o e iew o he IHC esul s is p o ided in Table2. The IHC es ing o all an ibodies was no possible in all o he cases due o limi ed ma e ial. Rep esen a i e images o selec ed IHC ma ke s a e p o ided in Figs.1, 2, 3, and 4. The coho consis ed o 122 cases (83%) o LG-ESS wi h he usual mo phological pa e n, 5 cases (3%) wi h pu e ib oblas ic pa e n, and a single case each (0.6%) displaying p edominan smoo h muscle-like mo phology o a glandu- la pa e n. Fou een umo s showed a mixed pa e n—mos commonly usual + ib oblas ic (n = 7.5%) and usual + smoo h muscle-like (n = 2.1%), while 6 cases (4%) showed a sex co d-s omal componen o a iable ex en . Two cases (1%) we e made up o a mix u e o ib oblas ic and myxoid pa - e ns, and one case displayed a combina ion o usual, ib o- blas ic, and myxoid mo phology. As expec ed, he umo s showed high le els o exp es- sion o he endome ial s omal ma ke s CD10 and IFITM1. CD10 exp ession was seen in 86% o cases, wi h a majo i y o he posi i e cases (92%) exhibi ing di use, ex ensi e s aining o a high in ensi y (median H-sco e 196). The o he ma ke o endome ial s omal di e en- ia ion IFITM1 was posi i e in a lowe numbe o cases (69%) wi h a he e ogenous ex en o exp ession, which was mos ly di use bu o a ying in ensi y. The exp ession o he s anda d smoo h muscle ma ke s (α-SMA, desmin, h-caldesmon, calponin, and ansgelin) was p esen o a a iable deg ee in all o he examined umo s, wi h α-SMA eaching he highes equency o posi i e cases (44%). The ex en o exp ession o hese ma ke s was ypically on he opposi e ends o he spec um—ei he only a e and ocal (wi h posi i ely s aining a eas comp ising less han 25% o he umo issue) o ex ensi e and di use (wi h mo e han 50% o he umo showing exp ession). How- e e , he in ensi y o he smoo h muscle ma ke exp ession was usually e y low, wi h he highes a e age H-sco e o 46 (obse ed o α-SMA) and he median H-sco e 0 o all examined ma ke s. Smoo helin showed exp ession in only wo cases o LG-ESS, which was only ocal and o a weak in ensi y. Ho mone ecep o s we e exp essed in a high p opo ion o he umo s, wi h PR eaching a sligh ly highe equency (92% s. 83%) and also in ensi y Vi chows A chi Table 1 Lis o an ibodies used o immunohis ochemical analysis An ibody Clone Dilu ion P oduce Pla o m De ec ion E alua ed exp ession ALK D5F3 1:100 Cell Signaling Tech- nology, Dan e s, Massachuse s, USA Ven ana BenchMa k ULTRA (Roche, Basel, Swi ze land) Op iView Cy oplasmic BCOR C-10 1:50 San a C uz Bio ech- nology, Dallas, Texas, USA Ven ana BenchMa k ULTRA (Roche, Basel, Swi ze land) Op iView Nuclea BCORL 1 Polyclonal abbi 1:200 A las an ibodies, B omma, Sweden Dako Omnis, (Agi- len , San a Cla a, CA, USA) EnVision FLEX (Dako) + Linke Nuclea SMARCA4 (BRG1) EPNCIR 111A 1:200 Abcam, Camb idge, UK Dako Omnis, (Agi- len , San a Cla a, CA, USA) EnVision FLEX (Dako) Nuclea H-caldesmon h-CALD 1:800 San a C uz Bio ech- nology, Dallas, Texas, USA Dako Omnis, (Agi- len , San a Cla a, CA, USA) EnVision FLEX (Dako) Cy oplasmic Calponin CALP 1:400 Dako, Glos up, Denma k Dako Omnis, (Agi- len , San a Cla a, CA, USA) EnVision FLEX (Dako) Cy oplasmic CD10 56C6 1:50 No ocas a, Leica Biosys ems, We - zla , Ge many Ven ana BenchMa k ULTRA (Roche, Basel, Swi ze land) Op iView Cy oplasmic CD117 c-ki 1:200 Dako, Glos up, Denma k Ven ana BenchMa k ULTRA (Roche, Basel, Swi ze land) Ul aView Cy oplasmic Cyclin D1 EP 12 RTU Dako, Glos up, Denma k Dako Omnis, (Agi- len , San a Cla a, CA, USA) EnVision FLEX (Dako) Nuclea , cy oplasmic Desmin D33 1:200 Dako, Glos up, Denma k Ven ana BenchMa k ULTRA (Roche, Basel, Swi ze land) Op iView Cy oplasmic ER SP1 1:200 Zy omed Sys ems GmbH, Be lin, Ge many Ven ana BenchMa k ULTRA (Roche, Basel, Swi ze land) Op iView Nuclea HMB45 HMB 45 1:50 Dako, Glos up, Denma k Dako Omnis, (Agi- len , San a Cla a, CA, USA) EnVision FLEX (Dako) Cy oplasmic IFITM1 Polyclonal abbi 1:400 Abcam, Camb idge, UK Dako Omnis, (Agi- len , San a Cla a, CA, USA) EnVision FLEX (Agilen ) Cy oplasmic SMARCB1 (INI1) MRQ-27 RTU Cell Ma que, Rocklin, CA, USA Ven ana BenchMa k ULTRA (Roche, Basel, Swi ze land) Op iView Nuclea p53 DO-7 1:400 Dako, Glos up, Denma k Ven ana BenchMa k ULTRA (Roche, Basel, Swi ze land) Op iView Nuclea , cy oplasmic PR clone 16 1:100 No ocas a, Leica Biosys ems, We - zla , Ge many Ven ana BenchMa k ULTRA (Roche, Basel, Swi ze land) Op iView Nuclea ROS1 D4D6 1:100 Cell Signaling Tech- nology, Dan e s, Massachuse s, USA Ven ana BenchMa k ULTRA (Roche, Basel, Swi ze land) Op iView Cy oplasmic S-100 4C4.9 1:400 DCS, Hambu g, Ge many Dako Omnis, (Agi- len , San a Cla a, CA, USA) EnVision FLEX (Agilen ) Cy oplasmic α- SMA 1A4 1:1600 Dako, Glos up, Denma k Dako Omnis, (Agi- len , San a Cla a, CA, USA) EnVision FLEX (Agilen ) Cy oplasmic Vi chows A chi RTU eady o use Table 1 (con inued) An ibody Clone Dilu ion P oduce Pla o m De ec ion E alua ed exp ession SMARCA2 1:800 A las an ibodies, B omma, Sweden Dako Omnis, (Agi- len , San a Cla a, CA, USA) EnVision FLEX (Dako) + Linke Nuclea Smoo helin R4A 1:50 Ze a Co po a ion, Mon o ia, CA, USA Ven ana BenchMa k ULTRA (Roche, Basel, Swi ze land) Op iView Cy oplasmic, nuclea NTRK EPR17341 1:100 Abcam, Camb idge, UK Ven ana BenchMa k ULTRA (Roche, Basel, Swi ze land) Op iView Nuclea , cy oplasmic T ansgelin 2A10C2 1:300 Cell Ma que, Rocklin, CA, USA Ven ana BenchMa k ULTRA (Roche, Basel, Swi ze land) Op iView Cy oplasmic WT1 6F-H2 1:400 BioSB, San a Ba - ba a, CA, USA Dako Omnis, (Agi- len , San a Cla a, CA, USA EnVision FLEX (Agilen ) Nuclea , cy oplasmic Table 2 De ailed o e iew o IHC exp ession o he an ibodies mos commonly used in he di e en ial diagnosis o LG-ESS SD s anda d de ia ion, cy opl. cy oplasmic. The numbe o analyzed cases o each s ain di e s due o he amoun o a ailable issue. Pe cen - ages a e ounded up/down. Pe cen ages o 1 + /2 + /3 + posi i i y a e coun ed only om he posi i e cases IHC ma ke Nega i e (0–1%) n (%) Posi i e (≥ 1%) n (%) Posi i i y 1 + (1–25%) n (%) Posi i i y 2 + (26– 50%) n (%) Posi i i y 3 + (> 50%) n (%) H-sco e mean (SD) H-sco e median ( ange) ER (n = 143) 25 (17%) 118 (83%) 6 (5%) 8 (7%) 104 (88%) 118 (87.3) 100 (0–300) PR (n = 144) 11 (8%) 133 (92%) 8 (6%) 5 (4%) 120 (90%) 201 (106.5) 223 (0–300) α-SMA (n = 142) 80 (56%) 62 (44%) 20 (32%) 7 (11%) 35 (56%) 46 (75.4) 0 (0–300) Desmin (n = 141) 106 (75%) 35 (25%) 15 (43%) 6 (17%) 14 (40%) 24 (66.8) 0 (0–300) H-caldesmon (n = 141) 121 (86%) 20 (14%) 12 (60%) 3 (15%) 5 (25%) 7 (28.0) 0 (0–200) Calponin (n = 143) 94 (66%) 49 (34%) 22 (45%) 4 (8%) 23 (47%) 31 (72.7) 0 (0–300) CD10 (n = 141) 20 (14%) 121 (86%) 6 (5%) 4 (3%) 111 (92%) 171 (110.9) 196 (0–300) IFITM1 (n = 143) 45 (31%) 98 (69%) 28 (29%) 11 (11%) 59 (60%) 65 (77.2) 20 (0–300) T ansgelin (n = 141) 110 (78%) 31 (22%) 13 (42%) 6 (19%) 12 (39%) 16 (49.7) 0 (0–300) BCOR (n = 142) 135 (95%) 7 (5%) 2 (29%) 1 (14%) 4 (57%) 3 (19.3) 0 (0–170) BCORL1 (n = 141) 107 (76%) 34 (24%) 31 (91%) 2 (6%) 1 (3%) 2 (7.9) 0 (0–70) NTRK nuclea (n = 145) 137 (94%) 8 (6%) 5 (63%) 3 (38%) 0 (0%) 2 (11.9) 0 (0–100) NTRK cy opl. (n = 145) 141 (97%) 4 (3%) 2 (50%) 1 (25%) 1 (25%) 1 (8.8) 0 (0–100) S-100 (n = 143) 143 (100%) 0 (0%) 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) HMB45 (n = 143) 140 (98%) 3 (2%) 3 (100%) 0 (0%) 0 (0%) 0 (1.8) 0 (0–20) CD117 (n = 146) 145 (99%) 1 (1%) 1 (100%) 0 (0%) 0 (0%) 0 (1.7) 0 (0–20) WT1 (n = 140) 84 (60%) 56 (40%) 18 (32%) 4 (7%) 34 (61%) 25 (43.3) 0 (0–200) Smoo helin nuclea (n = 142) 141 (99%) 1 (1%) 1 (100%) 0 (0%) 0 (0%) 0 (0.1) 0 (0–1) Smoo helin cy opl. (n = 142) 141 (99%) 1 (1%) 1 (100%) 0 (0%) 0 (0%) 0 (0.1) 0 (0–1) Cyclin D1 (n = 141) 62 (44%) 79 (56%) 39 (49%) 26 (33%) 14 (18%) 25 (48.6) 2 (0–300) Vi chows A chi Vi chows A chi o s aining, compa ed wi h ER. Rega ding he an ibodies use ul in di e en ial diagnosis such as S-100, HMB45, and CD117, hese showed almos comple e nega i i y. O hose ma ke s no included in Table2, all cases we e p53 wild ype, and ALK and ROS1 nega i e. The exp ession o SMARCB1 (INI1) and SMARCA4 (BRG1) was p ese ed, while se en cases showed an isola ed loss o SMARCA2 (BRM). As he molecula p o ile o hese umo s was known, none o hem ha bo ed BRM al e a ions. The di e ences in exp ession o all he s udied ma ke s be ween he umo s wi h a con i med usion (n = 101) and hose wi hou a ecu en usion (n = 32) we e also ana- lyzed (Supplemen a y Table1). The esul s showed ha only he endome ial s omal ma ke s CD10 and IFITM1 di e ed signi ican ly be ween he usion posi i e and nega- i e g oups, wi h usion posi i e umo s showing a mo e equen posi i e exp ession o bo h CD10 (p < 0.001) and IFITM1 (p = 0.007). Due o he imbalanced numbe o cases showing a ian mo phologies compa ed wi h he usual pa e n, i was no possible o s a is ically analyze he di e ences in exp ession be ween hese g oups. Tumo s wi h a leas a componen o ib oblas ic mo phology (n = 13) showed he exp ession o α-SMA, desmin, h-caldesmon, calponin, and ansgelin in 5/13 (38%), 4/13 (31%), 3/13 (23%), 5/10 (50%), and 3/13 (23%) cases espec i ely. CD10 was posi i e in 11/13 (85%) cases and IFITM1 in 7/13 (54%) cases. Tumo s wi h a sex co d-s omal componen (n = 7) showed he exp ession o α-SMA, desmin, h-caldesmon, calponin, and ansgelin in 5/7 (71%), 3/7 (43%), 2/7 (29%), 3/7 (43%), and 2/7 (29%) cases, espec i ely, while CD10 was posi i e in 6/7 (86%) cases and IFITM1 in 5/7 (71%) cases. The h ee umo s wi h smoo h muscle mo phology we e all nega i e in desmin and h-caldesmon, and he exp ession o α-SMA, calponin, and ansgelin was seen in 2/3 cases. All cases we e CD10 posi i e, wi h IFITM1 exp ession in 2/3 cases. Discussion Low g ade endome ial s omal sa coma p esen s a diag- nos ic challenge due o i s signi ican mo phological o e - lap wi h o he spindle cell mesenchymal umo s o he u e us. Recen s udies e-e alua ing he pa hological diag- noses o u e ine mesenchymal umo s ha e epo ed ha LG-ESS can be misdiagnosed in up o 20% o cases [22]. Cu en knowledge abou he immunohis ochemical p o ile o LG-ESS is de i ed om s udies wi h a ela i ely low numbe o examined cases, wi h he la ges immunohis o- chemically analyzed se ies including ewe han 50 cases [23]. Much o he published da a comes om single-case s udies and small an ibody panels, limi ing he a ailabili y o obus da a. Addi ionally, LG-ESS is bu dened wi h a ela i ely high in e obse e a iabili y, as some cases can only be eliably diagnosed using molecula es ing. This in oduces bias in o olde da a, pa icula ly due o e ol ing e minology and de ini ions, as some olde s udies only use he e m “endome ial s omal sa coma,” which makes he compa ison o hese esul s p oblema ic. Gi en hese limi a ions, ou aim was o p o ide eliable da a om a ca e ully selec ed, molecula ly examined se ies o LG- ESS, he la ges se ies desc ibed in he li e a u e o IHC p o iling o da e. Reliable da a on he immunop o ile o LG-ESS a e especially c i ical in ou ine p ac ice, pa icu- la ly o cases wi h equi ocal ea u es o cases whe e only limi ed umo issue is a ailable (e.g., small diagnos ic biopsies o u-cu biopsies). The main IHC ma ke s o diagnosing endome ial s o- mal umo s a e CD10 and he mo e ecen ly in oduced ma ke IFITM1 [24, 25]. The exp ession o CD10 has long been es ablished as one o he key diagnos ic ma ke s, as i is sensi i e (75–100%) and has been epo ed in 258/294 (88%) o all cases o LG-ESS wi h he a ailable IHC esul s o da e. Howe e , CD10 is no highly speci ic o LG-ESS, as a small po ion o LG-ESS can be nega i e, especially in cases wi h poo ixa ion [26]. CD10 exp es- sion is also epo ed in up o 30% o smoo h muscle umo s (including cellula leiomyoma), as well as in PEComa and he sa coma ous componen o adenosa coma [10, 27–30]. Cu en ly, he e is limi ed da a on he exp ession o IFITM1 in LG-ESS. Two s udies epo IFITM1 exp es- sion in 83% (10/12) and 100% (16/16) o LG-ESS cases, compa ed wi h 30% (6/20) and 40% (12/30) o smoo h muscle umo s [25, 31]. Bo h s udies concluded ha IFITM1 speci ici y (70% and 86,7%) su passes CD10, al hough each s udy examined a ela i ely small se ies o cases. IFITM1 exp ession, while no en i ely es ic ed o LG-ESS, ends o be weak and ocal in smoo h muscle umo s. Zhao e al. also emphasized IFITM1’s supe io sensi i i y and speci ici y o e CD10, al hough also based Fig. 1 Examples o CD10 and IFITM1 exp ession in di e en mo - phological a ian s o LG-ESS. All mic opho og aphs aken a 100 × magni ica ion. A Di use, s ong exp ession o CD10 in a usual LG-ESS (no usion de ec ed). B Focal exp ession o CD10 o a a i- able in ensi y in a case wi h p edominan smoo h-muscle mo phology (JAZF1::SUZ12 usion). C Mode a e o s ong CD10 exp ession in an LG-ESS wi h a sex co d s omal-like mo phology (JAZF1::SUZ12 usion). D Di use, s ong exp ession o CD10 in a myxoid LG-ESS (JAZF1::SUZ12 usion). E Di use and s ong exp ession o IFITM1 in LG-ESS wi h a usual mo phology (SVIL::EPC1 usion). F Com- ple e IFITM1 nega i i y in a case wi h p edominan ly smoo h muscle mo phology (JAZF1::SUZ12 usion). G Dispe se IFITM1 exp es- sion o a a iable in ensi y in an LG-ESS wi h sex co d s omal-like mo phology. Same case as depic ed in 1C (JAZF1::SUZ12 usion). H Focal, occasional g anula exp ession o IFITM1 in a case wi h myx- oid ea u es. Same case as depic ed in 1D (JAZF1::SUZ12 usion) ◂ Vi chows A chi on a small se ies [32]. Ou s udy ound IFITM1 exp ession in 98/143 cases (69%), mos ly ex ensi e o di use wi h a iable in ensi y. IFITM1 seems o ou pe o m CD10 in dis inguishing LG-ESS om smoo h muscle umo s; how- e e , la ge s udies a e s ill needed o con i m i s u ili y. A use ul app oach would he e o e include using bo h CD10 and IFITM1 as a pa o he IHC panel. In e es ingly, ou s udy ound ha bo h CD10 and IFITM1 we e signi ican ly mo e commonly exp essed in LG-ESS cases wi h a ecu - en usion, which unde sco es he diagnos ic challenges o Fig. 2 Examples o he exp ession o selec ed smoo h muscle ma k- e s in di e en mo phological a ian s o LG-ESS. A α-SMA in a usual LG-ESS, 100 × magni ica ion (JAZF1::SUZ12 usion). B Focal i egula exp ession o α-SMA in LG-ESS wi h p edominan ly smoo h-muscle mo phology, 200 × magni ica ion (JAZF1::SUZ12 usion). C Occasional a e exp ession o α-SMA in indi idual cells in a myxoid a ian o LG-ESS (wi h posi i ely s aining essels in he ield), 100 × magni ica ion (JAZF1::SUZ12 usion). D Almos comple e nega i i y o ansgelin in a usual LG-ESS (wi h posi- i ely s aining admixed myome ium), 100 × magni ica ion (no usion de ec ed). E Focal exp ession o ansgelin smoo h muscle a ian o LG-ESS, 200 × magni ica ion (JAZF1::SUZ12 usion, same case as 2B). F T ansgelin in an LG-ESS wi h sex co d s omal-like mo phol- ogy showing i egula bu ex ensi e weak o mode a e exp ession, 100 × magni ica ion (JAZF1::SUZ12 usion) Vi chows A chi usion-nega i e umo s gi en he lack o ecu en gene ic al e a ions and po en ially mo e equi ocal IHC esul s. Ho mone ecep o s, speci ically ER (ERα) and PR, a e ano he c ucial pa o he diagnos ic panel. The a ailable li e a u e indica es ha ER is exp essed in 84,5% o LG- ESS, wi h PR exp ession eaching 87% [15, 23, 33–38]. Mos s udies epo mo e ex ensi e, s onge PR exp ession han ER, which is consis en wi h ou indings. The exp es- sion o and ogen ecep o (AR) has also been desc ibed in a high pe cen age o LG-ESS, hough he numbe o s udies is limi ed [35]. While ER and PR a e ypically less use ul o dis inguishing smoo h muscle neoplasms, hey can aid in uling ou o he non-Mülle ian o igin umo s, pa icula ly o ex au e ine LG-ESS. Ho mone ma ke exp ession (especially ER and PR) may also ha e p edic i e signi icance, wi h hei high exp ession a es in LG-ESS suppo ing ho mone he apy such as high- dose p oges ins, a oma ase inhibi o s, o GnRH analogues as a iable ea men op ion [39–41]. A ecen me a-analysis ound ha adju an ho mone he apy can educe ecu ence isk in pa ien s wi h FIGO s ages I–II disease, bu wi h no bene i conce ning o e all su i al [42]. Ano he s udy sug- ges ed ha ho mone he apy can also educe ecu ence isk e en in s ages II–IV LG-ESS, ecommending 12 mon hs o high-dose p oges in ea men pos -su ge y [43]. While he e ec i eness o ho mone he apy is deba ed, epo ing umo ho mone s a us emains essen ial as i can help guide ea - men decisions. Con lic ing e idence also exis s ega ding ER/PR exp ession as a p ognos ic ac o , equi ing alida- ion h ough a molecula ly con i med se ies [35, 43, 44]. In ou ine p ac ice, one o he common diagnos ic pi - alls lies in di e en ia ing cellula leiomyoma (CL) om LG-ESS. The co ec diagnosis o CL e sus LG-ESS is o ex eme clinical impo ance, gi en he di e en biologi- cal na u es and beha io o hese umo s. In which case, a combina ion o smoo h muscle ma ke s such as α-SMA, desmin, h-caldesmon, calponin, ansgelin, and smoo he- lin, and endome ial s omal ma ke s, such as CD10 and IFITM1, is essen ial. Smoo h muscle ma ke s a e equen ly posi i e in LG-ESS, especially in cases wi h smoo h muscle Fig. 3 Examples o he exp ession o selec ed smoo h muscle ma k- e s in di e en mo phological a ian s o LG-ESS, con inued. A Nega i i y o desmin in a usual a ian o LG-ESS, 100 × magni ica- ion (no usion de ec ed, same case as 2D). B Dispe se s ong exp es- sion o desmin in a myxoid a ian o LG-ESS, 100 × magni ica ion (JAZF1::SUZ12 usion, same case as 2C). C) Focal weak o mode a e exp ession o h-caldesmon in an LG-ESS wi h smoo h muscle mo - phology, 200 × magni ica ion (JAZF1::SUZ12 usion). D Smoo helin in a usual LG-ESS, 200 × magni ica ion (no usion de ec ed, same case as 2D and 2G) Vi chows A chi 32 Chai andDepa men o Obs e ics, Gynaecology andGynaecological Oncology, Medical Uni e i y o Wa saw, Wa saw, Poland 33 Depa men o Gynecological Oncology, Pome anian Hospi als, Gdynia, Poland 34 Su gical Oncology Clinic, Medical Uni e si y inGdansk, Gdansk, Poland 35 Depa men o Gynecological Oncology, W oclaw Medical Uni e si y, W oclaw, Poland 36 Depa men o Oncological Gynecology, Lowe Silesian Oncology, Pulmonology andHema ology Cen e , W oclaw, Poland 37 Depa men o Pa hology, Uni e si y o Medicine, Pha macy, Sciences andTechnology GE Palade, Ta guMu es, Romania 38 Depa men o Gynecology, Uni e si y o Medicine, Pha macy, Sciences andTechnology GE Palade, Ta guMu es, Romania 39 Depa men o Pa hology, Uni e si y Hospi al Bulo ka, P ague, CzechRepublic 40 Depa men o Gynecology andObs e ics, Cha les Uni e si y - Fi s Facul y o Medicine andUni e si y Hospi al Bulo ka, P ague, CzechRepublic 41 Gynecologic Oncology Uni , Depa men o Woman andChild Heal h andPublic Heal h, Fondazione Policlinico Uni e si a io ’A. Gemelli’ IRCCS, Rome, I aly 42 Sec ion o Obs e ics andGynecology, Uni e si y Depa men o Li e Sciences andPublic Heal h, Uni e si à Ca olica del Sac o Cuo e, Rome, I aly 43 Caucasus Medical Cen e, Onco-gynecological Depa men , Tbilisi, Geo gia 44 Caucasus Medical Cen e, Megalab,Tbilisi, Geo gia 45 Depa men o Gynecologic Oncology, Khmelny skyi egional an i umo cen e , Khmelny skyi, Uk aine