{"paper_id":"c995cdcf-213c-45d8-9744-6af211aecb27","body_text":"Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2021 Mar; 165(1):69-79.\n69\nComparative immunohistochemical study of deep infiltrating endometriosis, \nlymph node endometriosis and atypical ovarian endometriosis including \ndescription of a perineural invasion\nJiri Lenza,b,c, Radek Chvatald, Ludek Fialae,f, Petra Konecnac, David Lenzb\nAim. Endometriosis is an inflammatory condition  that shares a number of similarities with malignant diseases, such as \nan abnormal morphology, migration along the nerve bundles and metastatic spread to lymph nodes and distant organs. \nEndometriotic lesions are associated with oestrogen and progesterone imbalance which seems to play a key role in the \npathogenesis of endometriosis. The aim of this  study was to compare the status of both oestrogen and progesterone \nreceptors in tissue of deep infiltrating endometriosis, lymph node endometriosis and atypical ovarian endometriosis \nusing immunohistochemical methods, as well as to investigate the relationship between endometriosis and protein p53. \nMethods.  A total of 40 cases with deep infiltrating endometriosis were included in our study. Based on histopatho -\nlogical analysis of resected specimens, the cases were divided into 2 groups: group 1 - lymph node endometriosis \n(cases with lymph node involvement; n=12) and group 2 – deep infiltrating endometriosis (cases without lymph node \ninvolvement; n=28). As a control group, eutopic endometrium of adenomyosis- and endometriosis-free women were \nused (n=16). Five cases of atypical ovarian endometriosis as well as descriptions of the nerve involvement in endome-\ntriosis were also included. Immunohistochemical staining with a total of 4 markers was performed - oestrogen and \nprogesterone receptors (ER, PR), p53 and Ki-67 (proliferation index). \nResults. The immunophenotype of the cases in groups 1 and 2 and in the control group was virtually identical in the \nproliferative phase - strong nuclear ER and PR expression in more than 90% of endometrial glandular and stromal cells. \nIn the early and mid secretory phase, ER expression only slightly decreased (80%) in endometrial glandular cells in \ngroup 2 and the control group, whereas in the late secretory phase, significant decrease of ER expression only in the \ncontrol group was observed (15-50%; P<0.001). In group 2 and the control group, significant decrease of PR expres -\nsion only in endometrial glandular cells was observed in the mid and late secretory phase (less than 15%;  P<0.001). \nDifferences in receptor content were found only in isolated cases in group 2. In group 1, no secretory changes were \nfound. In all three groups, sporadic and weak nuclear p53 expression in less than 3% in both endometrial glandular and \nstromal cells was detected (regardless of the phase of the menstrual cycle). In atypical ovarian endometriosis, higher \nand strong p53 expression (on average 26%) and decrease in ER (on average 56%) and PR (less than 1%) expression \nwas observed; compared to the control group and groups 1 and 2, the differences for all 3 markers were highly  sig -\nnificant  (P<0.001). In all groups, the proliferation index (Ki-67) reached the highest values in the proliferation phase \nand decreased during the cycle. However, in endometriotic tissue, it was widely variable in the individual phases of \nthe cycle. Perineural spread of endometriosis with significant neural hypertrophy, hyperplasia and involvement of the \nganglia of the autonomic nervous system was detected in 5 cases (12.5%). \nConlusion. From a histological and immunohistochemical point of view, deep infiltrating endometriosis and lymph \nnode endometriosis appear to represent the same entity. For the first time, a simple immunohistochemical panel \nwith antibodies against ER, PR and p53 useful in diagnosing atypical endometriosis has been described. The marked \nendometriosis-associated neural changes (endometriotic neuropathy) could be one of the causes of impaired func -\ntion of the affected organs after debulking surgery with macroscopic negative resection margins as well as pain \nsymptomato logy in macroscopic inapparent endometriotic lesions. \nKey words: deep infiltrating endometriosis, lymph node endometriosis, atypical endometriosis, \nimmunohistochemistry, p53, perineural invasion\nReceived: August 13, 2019; Revised: February 12, 2020; Accepted: February 14, 2020; Available online: March 9, 2020\nhttps://doi.org/10.5507/bp.2020.006\n© 2021 The Authors; https://creativecommons.org/licenses/by/4.0/\naDepartment of Pathology, Znojmo Hospital, Czech Republic\nbCytohisto s.r.o., Breclav, Czech Republic\ncDepartment of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences \nBrno, Czech Republic\ndDepartment of Obstetrics and Gynaecology, Znojmo Hospital, Czech Republic\neObstetrics and Gynaecology Private Clinic, Bulovka Hospital, Czech republic \nfInstitute of Sexology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic\nCorresponding author: Jiri Lenz, e-mail: jiri.lenz@gmail.com\n\nBiomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2021 Mar; 165(1):69-79.\n70\nINTRODUCTION\nEndometriosis is a common, oestrogen-dependent, in-\nflammatory disease associated with infertility and pelvic \npain. It is defined as the presence of endometrium outside \nthe uterus1.\nThe endometriosis classification of the revised Amer-\nican Society for reproductive Medicine (rASRM) differ -\nentiates between deep infiltratitng endometriosis (DIE), \nperitoneal endometriosis and ovarian endometriosis 2. \nDiagnostic criteria for DIE have been proposed - either \nthey are lesions that affect the intestine, urinary bladder, \nureter, vagina or uterosacral ligaments or lesions that ex-\ntend more than 5 mm below the peritoneal surface3. DIE \nrepresents a subset of endometriosis with an ill-defined \nset of symptoms and lack of specific diagnostic criteria \nwhich frequently cause significant delays in treatment4.\nEndometriosis is traditionally considered a benign dis-\nease. However, some features that are typical of malignant \ndisease have been identified,  including  metastatic spread \nto lymph nodes and distant organs, abnormal morphol -\nogy, migration along the nerve bundles, deregulated cell \ngrowth, cellular invasion, neoangiogenesis, DNA aneu -\nploidy and loss of heterozygosity5.\nLymph node involvement due to  endometriosis is \nuncommon, but it has been documented as a frequent \nevent (25-42%) in bowel, rectosigmoid and rectovaginal \nendometriosis 6-8. Microscopically, two stages of lymph \nnode endometriosis (LNE) are distinguishable – isolated \nendometriotic-like cells (early stage) and metastatic endo-\nmetriotic lesions (late stage) (ref.7). \nA variety of hyperplastic and atypically hyperplastic \nchanges similar to those occurring in the eutopic endo -\nmetrium may appear in endometriotic lesions 9. In view \nof this finding, the term atypical endometriosis was in -\ntroduced.\nThe pathogenesis of endometriosis is not entirely \nclear. However,  oestrogen and progesterone imbalance \nseems to play a key role in the migration, proliferation \nand infiltration of the peritoneum by endometrial cells10. \nHormonal imbalance in endometriosis also represents a \ntarget in the treatment of the disease11.\nThe main goal of this study was to determine and \ncompare the status of both oestrogen and progesterone \nreceptors in a tissue of DIE, LNE and atypical ovarian \nendometriosis using immunohistochemical methods.\nThe possible relationship between endometriosis and \nprotein p53 was also investigated. \nThe p53 molecule is a tumor suppressor that pre -\nvents the outgrowth of aberrant cells by inducing cell \ncycle arrest, DNA repair, or programmed cell death12. Its \ncommonly mutated in several human cancers, but p53 \naccumulation may also occur in non-tumour cells under \nvarious forms of stress (e.g. in a variety of autoimmune \ndiseases) (ref.13). Staining is associated with aneuploidy, \nincreased S phase fraction and genetic instability14.\nMATERIALS AND METHODS\nStudy group and tissue specimens\nA total of 40 patients with DIE were included in our \nstudy, the mean age was 33.1 years (range 22.0 - 47.0); \nten patients were cigarette smokers (25%) (Table 1). All \npatients underwent surgery for DIE at the centre for the \ntreatment of endometriosis at Znojmo Hospital between \n2016-2018. The nature of the surgery varied widely de -\npending on the extent of the disease. A minimally inva -\nsive laparoscopic approach was  always  preferred, but \nin 10 cases, conversion to laparotomy was neccesary. \nFor classification of endometriosis rASRM score and \nENZIAN classification were used.\nAll patients were premenopausal, knowledge of the \ndate of the last menstrual period and the length of the \nmenstrual cycle was derived from the available anam -\nnestic data. Nine of the 40 patients received hormonal \ntherapy at the time of surgery, 8 patients took progestin-\ncontaining medication (treatment for endometriosis, \ndysfunctional uterine bleeding or birth control) and 1 \npatient took gonadotropin releasing hormone analogue \n(GnRH analogue; treatment for endometriosis). The rest  \nhad not  received hormonal therapy for at lest 4 months \nbefore surgery. \nHistological examination confirmed a diagnosis of \nDIE in all selected cases. Based on histopathological \nanalysis of resected specimens, the cases were divided \ninto 2 groups: group 1 - lymph node endometriosis (cases \nwith lymph node involvement; n=12) and group 2 – deep \ninfiltrating endometriosis (cases without lymph node \ninvolvement; n=28). Cases with florid fibrosis, inflam -\nmatory or regressive changes were not included in the \nstudy. Five cases of atypical ovarian endometriosis were \nalso included in our study. As a control group, eutopic \nendometrium of adenomyosis- and endometriosis-free \nwomen was used (n=16). Uterine samples were obtained \nfrom normally cycling women undergoing hysterectomy \nfor cervical intraepithelial neoplasia (n=8), leiomyomata \n(n=4) or chronic pelvic pain (n=4).\nThe diagnosis of endometriosis was based on the \npresence of both endometriotic glands and stroma \nwhile diagnosis of atypical endometriosis was based on \nhistopathological criteria reported by LaGrenade and \nSilverberg15 and Czernobilsky and Morris 16. These crite-\nria include eosinophilic cytoplasm, large hyperchromatic \nor pale nuclei with moderate to marked pleomorphism, \nan increased nuclear to cytoplasmic ratio, cellular crowd-\ning and stratification or tufting. Three or more of these \ncriteria were present in all cases (Fig. 1a).\nHematoxylin and eosin stained sections of endome -\ntrial samples were dated by the criteria described by \nNoyes et al.17. As in eutopic endometrium, the histologic \nappearance of the endometrial epithelium and stroma in \nendometriotic lesions was classified as early, mid and late \nproliferative and early, mid and late secretory. Some cases \nwith early proliferative morphology have partly acquired \nan inactive appearance (characterised by small glands \nlined by low columnar epithelium with minimal mitotic \nactivity).\n\nBiomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2021 Mar; 165(1):69-79.\n71\nTable 1. Descriptive statistics of study population including pathologic features of deep infiltrating endometriosis and immuno-\nhistochemical analysis of oestrogen and progesterone receptor expression in deep infiltrating endometriosis (group 2).\nCase Age Medications\nSite  \nof endo-\nmetriosis\nLymph node \ninvolvement\nDay \nof cycle\nHistologic  \nappearance of  \ndeep infiltrating  \nendometriosis\nER  \nexpression*\nPR  \nexpression*\n1 30 GnRH analogue Bladder + 58 EP 100 100\n2† 37 None Vagina - 10 MP 100 100\n3 45 Progestins Sigmoideum - 5 A 90 0\n4 28 None USL - 24 LS 90 15\n5† 35 None Sigmoideum + 11 MP 100 100\n6 40 Progestins USL - 37 I 100 100\n7 29 None Rectum + 10 MP 100 100\n8† 32 None Rectum - 24 MP 100 100\n9 33 None USL - 43 EP 100 100\n10 22 Progestins USL - 11 I 100 100\n11 27 Progestins USL - 30 I 100 100\n12 43 Progestins USL - 31 I 100 100\n13 29 None USL - 14 I 40 70\n14 41 None USL - 22 I 90 80\n15† 34 None USL + 8 EP 100 100\n16 26 None USL + 8 I 100 100\n17 24 None Bladder + 6 EP 90 70\n18 27 None USL - 14 I 100 100\n19 41 None USL - 15 I 90 90\n20† 29 None Vagina - 18 EP 100 100\n21 25 None Vagina - 20 LP 100 100\n22 28 None Vagina - 32 MS 80 1\n23 25 None Bladder - 21 MS 90 5\n24 28 Progestins Bladder - 45 A 80 1\n25 35 None Vagina + 5 MP 100 100\n26 42 None Bowel - Unknown C 90 5\n27 25 None USL + 3 I 100 100\n28 37 None USL - 14 I 100 100\n29 35 None USL - 20 C 90 10\n30 36 None USL - 10 MP 100 100\n31 41 None USL - 7 I 100 100\n32 24 None Rectum + 5 EP 100 100\n33 26 Progestins Rectum + 11 I 100 100\n34 35 None USL - 4 I 100 100\n35 28 None USL - 5 EP 100 100\n36 42 None Sigmoideum + 24 EP 90 100\n37 40 None USL - 17 EP 100 100\n38 31 None Rectum + 15 MP 100 100\n39 47 Progestins Bladder - 48 I 100 100\n40 33 None USL - 8 EP 100 100\n* Percentage (%) of marker positive endometrial glandular cells.\n† Cases with nerve involvement by endometrios.\nA, atrophy with weak secretory changes; C, a combination of proliferative and secretory endometrium; EP, early proliferative; ER, oestrogen \nreceptor; GnRH, gonadotropin releasing hormone; LP, late proliferative; LS, late scretory; MP, mid proliferative; MS, mid secretory; PR, proges-\nterone receptor; USL, uterosacral ligament.\nRoutinely processed formalin fixed and paraffin em -\nbedded tissues were available for immunohistochemical \nanalysis in all cases. The tissue specimens, slides, and \nhistopathology reports were retrieved from the files of the \ndepartment of pathology. All resection specimens were \nfixed in 10% neutral buffered formalin for 24 h. The 4 µm \nthick tissue sections stained with hematoxylin-eosin were \nreviewed by pathologist and representative samples with \nstructures of deep infiltrating endometriosis, lymph node \nendometriosis and atypical ovarian endometriosis were \nselected for immunohistochemical analysis.\nThe study was approved by the ethics comittee of \n\nBiomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2021 Mar; 165(1):69-79.\n72\nZnojmo Hospital. The requirement for patient consent \nwas waived becasuse of the retrospective nature of this \nstudy.\nImmunohistochemistry\nImmunohistochemistry was performed for all cases, \nusing antibodies against oestrogen receptors (clone ER \nSP1, Roche, RTU), progesterone receptors (clone PR \n1E2, Roche, RTU), p53 (clone Bp53-11, Roche, RTU) \nand Ki-67 (clone 30-9, Roche, RTU). All immunohisto -\nchemical staining was performed on the automated immu-\nnostainer BenchMark GX (Ventana Medical System, Inc., \nTucson, AZ, USA); ultraView Universal DAB Detection \nKit was used.\nThe primary antibodies were visualised using hydrogen \nperoxide substrate and 3, 3'- diaminobenzidine tetrahydro-\nchloride (DAB) chromogen. External positive controls \nwere used for all antibodies (invasive breast carcinoma \nfor ER and PR, tonsil for p53 and lymph node for Ki-67). \nNegative controls were prepared by incubating samples \nwithout a primary antibody. Evaluation of immunohisto-\nchemical results was performed using a uniform micro -\nscope and camera setting (Olympus BX43F microscope \nand Promican 3-3CC camera).\nEvaluation of immunostaining\nIn the endometrial tissue, nuclear immunostaining \nfor ER, PR, p53 and Ki-67 was regarded as positive. The \npercentages of marker positive cells were evaluated using \na light microscope at ×200 magnification. At least five \nfoci of eutopic/ectopic endometrium were analyzed and \nthe percentage of marker positive cells of the covered area \nwas determined. The intensity of immunoreaction with \nantibodies against ER, PR and p53 was also investigated; \nweak, moderately and strong staining was distinguished.\nStatistical analysis\nStandard descriptive statistics were used to summarise \nthe data. Continuous variables  were expressed as means \nand standard deviation, and statistically significant dif -\nferences (at the level of α=0.05) between  means were \nevaluated using the non-parametric Mann-Whitney rank \nsum test with continuity correction where appropriate. \nFig. 1. Atypical ovarian endometriosis and immunohistochemical expression of ER, PR and p53 in atypical ovarian endometriosis.\nA: Atypical ovarian endometriosis consisting of cells with eosinophilic cytoplasm, large hyperchromatic or pale nuclei with mod-\nerate to marked pleomorphism, an increased nuclear to cytoplasmic ratio, cellular crowding, stratification and tufting. Note the \nabsence of the acute inflammatory infiltrate (hematoxylin-eosin staining, original magnification ×200).\nB: Nuclear expression of ER in approximately 40% of endometrial glandular cells with only moderately staining (immunohisto -\nchemistry, original magnification ×200).\nC: Nuclear expression of PR in less than 1% of endometrial glandular cells (immunohistochemistry, original magnification ×200).\nD: Strong nuclear expression of p53 in approximately 28% of endometrial glandular cells (immunohistochemistry, original mag -\nnification ×200).\n\n\nBiomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2021 Mar; 165(1):69-79.\n73\nRESULTS\nDIE was diagnosed in all 40 patients with incidentally \nremoved lymph nodes. In our series, which was a total of \n118 coincidentally resected lymph nodes; the endometri-\notic lesions were noted in 15 of them (12.7%). \nPathologic features of lymph node endometriosis  \n(group 1) (Table 2) \nAll cases of LNE consisted of both endometrial glan-\ndular and stromal elements located in the lymph node \ncapsule or cortex. The glands were mostly separated, fo -\ncally cystic dilated, occasionally formed papillary struc -\ntures and in one case surrounded by a lesser amount of \nhypocelular dense fibrous stroma. No microcalcification \nor  psammoma bodies were  found. The endometriotic \nglands were lined with weak proliferative endometrial \nepithelium with mild cellular atypia (early proliferative \nappearance), focal ciliated metaplasia and low mitotic \nactivity; secretory changes were not found. Only in the \ncase of stromal fibrosis did the epithelium seem to be \nflattened (atrophic). Haemosiderin-laden macrophages \nand pseudoxanthoma cells were a common finding in the \nstroma (Fig. 2a-c).\nPathologic features of deep infiltrating endometriosis \n(group 2) (Table 1)\nThe predominant pattern of the endometriotic tissue \nwas early proliferative (10 cases, 25%) or  inactive (15 \ncases, 37.5%). The others were classified as mid prolifera-\ntive (7 cases, 17.5%), late proliferative (1 case, 2.5%), mid \nsecretory (2 cases, 5%) and late secretory (1 case, 2.5%). \nIn two cases, a combination of proliferative and secretory \nendometrium was found (5%). In 9 patients receiving hor-\nmonal therapy, the inactive pattern was found in 7 cases, \nwhile the atrophic pattern with weak secretory changes \nwas found in 2 cases (5%).\nWhen more than one endometriotic focus was exam-\nined in the same patient, the appearance of the specimens \ndid not differ significantly.\nThe appearance of endometriotic tissue was synchro-\nnous with the dating of the menstrual cycle in 11 cases \n(27.5%) – early proliferative (5 cases), mid prolifera -\ntive (4 cases), mid secretory (1 case) and late secretory \n(1 case).\nThe most common changes in DIE were ciliated (tub-\nal) metaplasia, cystic changes and focal endometrial hy -\nperplasia without atypia. Cystic changes were found in 19 \ncases (47.5%), endometrial hyperplasia without atypia in \n2 cases (5%) and a combination of both in 6 cases (15%). \nIn a total of 13 cases (32.5%), no structural changes were \nfound. Endometrial hyperplasia was characterised by out-\npouching and dilatation of glandular epithelium (simple \nhyperplasia), but with an increasing degree of architec -\ntural abnormality, glands become complex, branched, \ncrowded or back-to-back (complex hyperplasia). The lin-\ning cells were columnar, mostly stratified, with oval or \nelongated bland nuclei with smooth contours, fine even \nchromatine and variable mitotic activity (no more than \nseven mitotic figures per 10 high-power fields). Only in 1 \ncase (2.5%), was mucinous metaplasia  found.\nEutopic endometrium was available in 6 cases of DIE. \nIn 4 (67%), the histologic appearance of endometriotic \ntissue was synchronous with the corresponding eutopic \nendometrium.\nPerineural and intraneural invasion (PNI) was detect-\nTable 2. Pathologic features of lymph node endometriosis and immunohistochemical analysis of oestrogen and progesterone \nreceptor and p53 expression in lymph node endometriosis (group 1) and atypical ovarian endometriosis.\nLymph node  \nendometriosis\nAtypical ovarian  \nendometriosis\nCase\nHistologic \nappearance \nof lymh node \nendo metriosis\nER  \nexpression*\nPR  \nexpression*\np53  \nexpression* Case ER  \nexpression*\nPR  \nexpression*\np53  \nexpression*\n1 EP 100 95 1 1 40 1 20\n2 EP 100 100 1 2 55 0 26\n3† A 90 90 0 3 70 0 27\n4 A 95 95 1 4 55 1 28\n5 EP 90 90 0 5 60 0 27\n6 EP 100 95 0 - - - -\n7 EP 90 95 1 - - - -\n8 EP 95 90 0 - - - -\n9 A 90 90 1 - - - -\n10† EP 100 90 1 - - - -\n11 EP 95 95 0 - - - -\n12 EP 100 90 0 - - - -\n* Percentage (%) of marker positive endometrial glandular cells.\n† Involvement of 2 lymph nodes by endometrios.\nA, atrophic; EP, early proliferative; ER, oestrogen receptor; PR, progesterone receptor. \n\nBiomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2021 Mar; 165(1):69-79.\n74\nFig. 2. Immunohistochemical expression of ER, PR and p53 in lymph node endometriosis.\nA: Strong nuclear expression of ER in more than 90% of endometrial glandular cells. Periglandular fibrosis, haemosiderin-laden \nmacrophages and pseudoxanthoma cells are noted (immunohistochemistry, original magnification ×200).\nB: Strong nuclear expression of PR in more than 90% of endometrial glandular cells (immunohistochemistry, original magnifica-\ntion ×200).\nC: Weak nuclear expression of p53 in less than 1% of endometrial glandular cells (immunohistochemistry, original magnification \n×200).\nFig. 3. Endometriotic neuropathy.\nA: Intraneural spread of endometriotic gland (visible in the lower right corner) with marked neural hypertrophy and hyperplasia \n(hematoxylin-eosin staining, original magnification ×100).\nB: Involvement of the parasympathetic ganglion of the autonomic nervous system by endometriosis. Numerous bundles of nerve \nfibers, cell body of the postsynaptic neuron (visible in the right half of the field) and deposits of  haemosiderin are noted (hema-\ntoxylin-eosin staining, original magnification ×100).\n\nBiomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2021 Mar; 165(1):69-79.\n75\ned in a total of 5 cases (12.5%) – rectal endometriosis \n(1 case), rectosigmoid endometriosis (1 case), vaginal \nendometriosis (2 cases) and endometriosis of the utero -\nsacral ligament (1 case). The nerve involvement due to \nendometriosis occurred in the organ (bowel or vaginal) \nwall and  also in the adjacent tissue. PNI was a multifo -\ncal finding with concomitant marked neural hypertrophy \nand hyperplasia (Fig. 3a). An interesting finding was the \ninvolvement of the parasympathetic (terminal) ganglia \nlocated in, or close to, the wall of the affected organs \n(Fig. 3b).\nAnalysis of ER, PR, Ki-67 and p53 expression  \nin the control group (Table 3)\nExpression of ER in glands of normal endometrium \nwas maximal in the proliferation phase (more than 90%), \nslightly decreased in the early and mid secretory phase \n(about 80%) and lowest being in the late secretory phase \n(15-50%). Expression of PR was identical in the prolif -\nerative and early secretory phase, a significant decrease \noccurred in the mid secretory (less than 10%) and the late \nsecretory phase (less than 1%; P<0.001). For both types \nof hormonal receptors, expression in endometrial stromal \ncells was high, regardless of the phase of the menstrual \ncycle (90%). The intensity of staining in both endometrial \nglands and stroma for ER and PR was moderate or strong.\nThe proliferation index (Ki-67) in glands also showed \na decrease during the cycle, the highest values being \nreached in the mid proliferative phase (50-90%, on aver -\nage 70%), lower in the late proliferation phase (1-40%, on \naverage 22%), while a significant decrease with minimal \nexpression was observed in mid and late secretory phase \n(about 1%; P<0.001).\nWeak nuclear expression of p53 in less than 3% in \nboth type of cells was detected.\nAnalysis of ER, PR, Ki-67 and p53 expression  \nin the lymph node endometriosis (group 1) (Table 2)  \nand deep infiltratring endometriosis (group 2) (Table 1)\nThe results of immunohistochemical staining with an-\ntibodies against ER, PR, Ki-67 and p53 were somewhat \nsimilar to those in the control group. In both groups, \nstrong nuclear expression of ER and PR in more than \n90% of cells in both endometrial glands and stroma in \nthe proliferative phase was observed (i.e. there were no \ndifferences between LNE and DIE in the proliferative \nphase) (Fig. 2a,b and Fig. 4a,c). Only in one case in group \n2, decrease in the PR expression only in glandular cells \n(70%) was found. In group 2, in the mid and late secretory \nphase, ER expression only slightly decreased (to more \nthan 80%) (Fig. 4b), whereas a significant decrease of \nPR in endometrial glands was detected (on average 7%; \nP<0.001) (Fig. 4c). \nIn group 1, all cases with atrophic morphology showed \nexpression of ER and PR in more than 90% of glandular \nand stromal cells.\nIn both groups, Ki-67 expression in endometrial \nglands was quite variable. In the early proliferative phase \nit ranged between 1 and 50% (on average 34.2% for both \ngroups), in the mid proliferative phase it ranged between \n10 and 70% (on average 44% only for group 2) and in \nthe late proliferative phase it was 10% (only group 2); no \nTable 3. Immunohistochemical analysis of oestrogen and progesterone receptor, p53 and Ki-67 expression  \nin eutopic endometrium (control group).\nCase Day of cycle Stage of cycle ER  \nexpression*\nPR  \nexpression*\np53  \nexpression*\nKi-67  \nexpression*\n1 6 EP 100 100 0 40\n2 8 EP 100 100 2 60\n3 10 MP 100 100 1 70\n4 9 MP 100 95 3 50\n5 9 MP 100 100 0 70\n6 11 MP 100 100 3 90\n7 13 LP 95 90 1 1\n8 13 LP 90 90 1 40\n9 12 LP 100 100 1 10\n10 16 ES 90 90 1 30\n11 18 ES 100 100 1 25\n12 22 MS 80 10 0 1\n13 20 MS 80 2 0 1\n14 19 MS 75 1 0 1\n15 23 LS 15 0 0 1\n16 23 LS 50 1 1 1\n* Percentage (%) of marker positive endometrial glandular cells.\nEP, early proliferative; ER, oestrogen receptor; ES, early secretory; LP, late proliferative; LS, late secretory; MP, mid proliferative; MS, mid secre-\ntory; PR, progesterone receptor.\n\nBiomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2021 Mar; 165(1):69-79.\n76\nFig. 4. Immunohistochemical expression of ER, PR and p53 in deep infiltrating endometriosis in the mid proliferative and mid \nsecretory phase of the cycle.\nA: Diffuse strong nuclear expression of ER in both endometrial glands and stroma in the mid proliferative phase of the cycle (im-\nmunohistochemistry, original magnification ×200).\nB: Diffuse strong nuclear expression of ER in both endometrial glands and stroma in the mid secretory phase of the cycle (im -\nmunohistochemistry, original magnification ×200).\nC: Diffuse strong nuclear expression of PR in both endometrial glands and stroma in the mid proliferative phase of the cycle (im-\nmunohistochemistry, original magnification ×200).\nD: Loss of PR expression in endometrial glands but not in endometrial stroma in the mid secretory phase of the cycle (immuno-\nhistochemistry, original magnification ×200).\nE: Absence of p53 expression in both endometrial glands and stroma in the mid proliferative phase of the cycle (immunohisto -\nchemistry, original magnification ×200).\nF: Absence of p53 expression in both endometrial glands and stroma in the mid secretory phase of the cycle (immunohistochem-\nistry, original magnification ×200).\ndifferences between LNE and DIE in the proliferative \nphase were found (in group 1, no secretory changes were  \nfound). As in the control group, a significant decrease in \nthe secretory phase in group 2 was observed (less than \n15%; P<0.001). \nIn both groups, weak and sporadic nuclear p53 expres-\nsion in less than 1% in endometrial glands and stroma was \ndetected (Fig. 2c and Fig. 4e,f).\nIn group 2, the immunophenotype of inactive appear-\ning glands was as follows:  strong nuclear expression of \n\nBiomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2021 Mar; 165(1):69-79.\n77\nboth ER and PR in more than 90% of cells, minimal Ki-67 \nexpression (less than 1%) and absence of p53 expression \n(0%). In one case, significant decrease in ER expression \n(40%), and in two cases a decrease in PR expression \n(70 and 80%) was observed.\nIn group 2, in the two cases of atrophic pattern with \nsecretory changes, ER expression was observed in more \nthan 80% of glandular cells, while PR expression was al -\nmost lost (less than 1%). In endometrial stromal cells, \nhigh expression of both hormonal receptors was detected \n(more than 80%).\nIn the remaining two cases in group 2 (a combination \nof proliferative and secretory endometrium) ,,secretory \npattern‘‘ of hormonal receptor expression in endometri-\notic glands was observed (high ER expression and very \nlow PR expression).\nAnalysis of ER, PR, p53 and Ki-67 in atypical ovarian \nendometriosis (Table 2)\nA different immunophenotype in all 5 cases of atypi-\ncal ovarian endometriosis was found. Compared to the \ncontrol group and groups 1 and 2, differences were related \nto ER, PR and p53. Expression of ER was on average 56% \n(range 40-70%) (Fig. 1b), whereas expression of PR was \nless than 1% (Fig. 1c). Differences for both hormonal \nreceptors were statistically significant (P<0.001); the stain-\ning was moderately or strong.\nOnly in the lesions of atypical ovarian endometrio -\nsis was a strong nuclear p53 expression detected. The \nnumber of positive glandular cells ranged between 20 and \n28% (on average 26%) (Fig. 1d). Compared to the control \ngroup and groups 1 and 2, differences reached statistical \nsignificance (P<0.001).\nThe Ki-67 values ranged between 5-50% (on average \n32%). \nAnalysis of ER, PR, p53 and Ki-67 in simultaneously \nsampled eutopic endometrium \nEutopic endometrium was available in 6 cases of DIE. \nWhen the immunophenotypes of simultaneously sampled \neutopic endometrium and endometriotic foci were com -\npared, there were 3 cases where differences only in the \nexpression of Ki-67 were found; a significant decrease in \nendometriotic foci was found (17% vs. 67%).\nDISCUSSION\nIn our study, we retrospectively reviewed a series of \n120 cases of ovarian endometriomas; the diagnosis of \natypical endometriosis was determined in 5 (4.2%). In \nall these cases, the criteria described by LaGrenade and \nSilverberg15 and Czernobilsky and Morris 16 were strictly \nfollowed for diagnosis. However, despite these histological \ncriteria, severe epithelial atypia may be of reactive origin \nand the distinction is therefore problematic and limited. \nSome studies concluded that atypical endometriosis is \nnot a result of reactive inflammation but rather possesses \na precancerous potential because epithelial atypia occur \nirrespective of severe stromal inflammation, and are con-\nsidered the precursor lesion for endometriosis associated \ncarcinomas (most commonly clear cell or endometrioid). \nNone of our cases of atypical endometriosis exhibited \nmore than a scant inflammatory infiltrate (mostly rep -\nresented by rare intraepithelial neutrophils). The endo -\nmetrioid epithelium was focally denuded and, in some \ncases, it was problematic to distinguish between moderate \n(reactive) and severe celular atypia. In these cases, we \nused a simple immunohistochemical panel with antibod-\nies against ER, PR, p53 and Ki-67. The immunophenotype \nof endometriotic lesions with severe celular atypia was as \nfollows: significantly higher strong p53 expression and \nlower ER and PR expression with only moderate staining. \nThe immunophenotype of cases with reactive (moderate) \natypia as well as the Ki-67 values were comparable to non-\natypical endometriotic lesions. Based on these findings, \nall ovarian endometriotic lesions with such an immuno -\nphenotype and severe celular atypia were classified as \natypical endometriosis. A significant decrease in receptor \ncontent indicates a greater degree of autonomy of atypi -\ncal endometriotic lesions. These findings, together with \na strong and significantly higher p53 expression support \nthe preneoplastic potential of the atypical endometriotic \ntissue. Although the diagnosis of atypical endometriosis \nshould be based primarily on morphology, finding of such \nan immunophenotype (in the appropriate morphological \ncontext) is a very useful ancillary method.\nDiagnosis of atypical endometriosis should be fol -\nlowed by careful long-term observation of the patient; \nthe reason is the possible subsequent development of \nneoplasia. When searching the literature, the incidence \nof atypical endometriosis in ovarian cancer ranges from \n4.4 to 22.8% and without neoplasm involvement from 1.7 \nto 32.3% (ref.18-21). At our hospital, the frequency of atypi-\ncal endometriosis was 4.2% between 2016-2018; so far no \nneoplasm has developed in any of these cases.\nThe possibility of malignant transformation of endo -\nmetriosis was originally mentioned by Samson in 1925 \n(ref.22) and later by Scott in 1953 (ref. 23). However, the \nassociation between atypical endometriosis and ovarian \nneoplasm was originally described by LaGrenade and \nSilverberg in 1988 - clear cell carcinoma and endometri-\noid carcinoma have been found in continuity with atypical \novarian endometriosis15.\nA number of studies immunohistochemically analyzed \nthe ER and PR expression in endometrial tissue. In the \nstudy of Bur et al., it was conluded that ER expression \nin most endometriotic lesions corresponds to their mor -\nphological appearance 24. These results are fully consis -\ntent with the results of the presented study. In another \nstudy, Lessey et al. found that receptor content in endo -\nmetriotic tissue was more heterogeneous, unlike eutopic \nendometrium, and did not undergo predictable changes \nin response to endogenous hormones. The authors also \ndescribed a significant decrease in ER and PR in both \nthe glands and stroma in the endometriotic tissue of pa -\ntients treated with hormonal therapy relative to untreated \npatients25. In our series, 8 women took progestins. In 2 \nof them progestin-mediated morphologic changes in en -\ndometriotic tissue were observed (atrophic pattern with \n\nBiomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2021 Mar; 165(1):69-79.\n78\nweak secretory changes). In these two cases, loss of PR \nexpression (only in glands) and a slight decrease in ER ex-\npression (in both stroma and glands) were noted. The his-\ntologic  appearance of the remaining 6 cases was inactive. \nIn general, the morphological appearance of the eutopic \nendometrium and endometriotic tissue after progestin \ntreatment is influenced by several factors, in particular \nthe dose and duration of hormonal treatment, as well as \nthe status of the endometrium26. \nSome studies describe an overall reduction in PR \nand ER expression in endometriotic lesions relative to \neutopic endometrium27,28. We  observed this finding only \nin isolated cases. PR expression decreases significantly in \nthe mid and late secretory phase in both endometriotic \ntissue and eutopic endometrium. Lower concentrations \nof PR receptors were observed in only 2 cases of DIE \nwith early proliferative and inactive appearance, but not \nin LNE. In terms of oestrogen receptors, significant dif -\nferences were found in only one case of DIE with inactive \n appearance, compared to LNE, where all cases showed \nstrong ER expression in more than 90% of both glandular \nand stromal cells. \nIn our study, considerable attention was paid to the \nanalysis of PNI. We found a total of 5 cases of the nerve \ninvolvement due to endometriosis. Regardless of the \nanatomical location of PNI, significant changes in the af-\nfected nerve tissue were detected. We  introduced the term \n,,endometriotic neuropathy“, which is defined by distinct \nneuronal hypertrophy and hyperplasia with concomitant \ninvolvement of the ganglia of the autonomic nervous sys-\ntem (ANS) located in the wall of the affected organs as \nwell as in the adjacent fibroadipose tissue. These ganglia, \nconsisting of a population of both Schwann and ganglion \ncells, were easily detectable on the tissue sections stained \nwith hematoxylin-eosin. Immunohistochemically, neural-\nappearing bundles and ganglia were strongly positive for \nS100 protein and neuron-specific enolase (NSE).  \nRegarding the organization of the ANS in the ab -\ndominopelvic organs, sympathetic presynaptic fibers lead \nfrom the lower thoracic and upper lumbar segments of \nthe spinal cord via abdominopelvic splanchnic nerves to \nthe vertebral and prevertebral ganglia, where postsynaptic \nneurons have their cell bodies. Parasymphatetic presyn -\naptic neurons send axons from the sacral segments of \nthe spinal cord to visceral ganglia that contain bodies of \nthe postsynaptic neurons and are generally located in the \nwalls of the organs, such as the ganglia of the submuco -\nsal (Meissner’s) plexus, the deep submucosal (Henle’s) \nplexus and the myenteric (Auerbach’s) plexus in the ali-\nmentary canal29. It follows from the previous comments \nthat, for all five of our cases, the ganglia affected by en -\ndometriosis are part of the parasympathetic branch of \nthe ANS.\nIn general, the autonomic  nervous system, which is \nsubdivided into a sympathetic, parasymphatetic and enter-\nic divisions, conducts impulses to smooth muscle, cardiac \nmuscle, and glandular epithelium. The enteric division of \nthe ANS controls motility, exocrine and endocrine secre-\ntions, and blood flow through the gastrointestinal tract. It \nalso regulates immunologic and inflammatory processes. \nIn addition to muscle fibers and the neural network, a \npopulation of the interstitial cells of Cajal (ICC) are criti-\ncal for bowel motility and their function is modulated by \nboth sympathetic and parasympathetic neural inputs 30. \nWhen nerve fibers and ganglia of the ANS are affected \nby endometriosis, all of these functions can be impaired. \nAs mentioned above, in our study PNI was a multifocal \nfinding detected in the areas of macroscopically apparent \nlesions but also outside the confines of these areas. PNI \nallows the spread of endometriosis over a large distance \nand thus, PNI can make R0 resection of endometriotic \nlesions more difficult. These facts could be one of the \ncauses of impaired functions of the affected organs after \ndebulking surgery with macroscopic negative resection \nmargins as well as pain symptomatology in macroscopic \ninapparent endometriotic lesions. Coversely, the absence \nof PNI could be the cause of the well-known situation of \nextensive organ involvement due to  endometriosis and \nno pain at all. \nCONCLUSION\nFrom a histological and immunohistochemical point \nof view, deep infiltrating endometriosis and lymph node \nendometriosis appear to represent the same entity.\nFor the first time, a simple immunohistochemical \npanel with antibodies against ER, PR and p53 useful in \ndiagnosing atypical endometriosis has been described.\nThe marked endometriosis-associated neural changes \n(endometriotic neuropathy) could be one of the causes of \nimpaired functions of the affected organs after debulking \nsurgery with macroscopic negative resection margins as \nwell as pain symptomatology in macroscopic inapparent \nendometriotic lesions. \nABBREVATIONS\nANS, Autonomic nervous system; A, Atrophy; C, \na combination of proliferative and secretory endometrium; \nDAB, Diaminobenzidine tetrahydrochloride; DIE, Deep \ninfiltrating endometriosis; DNA, Deoxyribonucleic acid; \nEP, Early proliferative; ER, Oestrogen receptors; GnRH, \nGonadotropin releasing hormone; ICC, Interstitial cells of \nCajal; LNE, Lymph node endometriosis; LP, Late prolifer-\native; LS, Late secretory; MP, Mid proliferative; MS, Mid \nsecretory; NSE, Neuron-specific enolase; PNI, Perineural \ninvasion; PR, Progesterone receptors; p53, Protein 53; \nrASRM, Revised American Society for reproductive medi-\ncine; RTU, Ready to use; USL, Uterosacral ligament. \nAuthor contributions: JL: project development, manu -\nscript writing; RCH: manuscript writing, critically revis-\ning the article; LF: statistical analysis, manuscript editing; \nPK, DL: manuscript writing, description of the histopa -\nthology findings, literature (PubMed) search.\nConflict of interest statement: The authors state that there \n\nBiomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2021 Mar; 165(1):69-79.\n79\nare no conflicts of interest regarding the publication of \nthis article.\nREFERENCES\n 1. 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