Desmoidtumor is locally infiltrative softtissue neoplasm with increased rate of local recurrence following total surgical resection 1.The classic treatmentstrategy of desmoid tumors or as called aggressive fibromatosis is surgerywithaccompanied radiotherapy, but the failure to achieve complete response after surgicalexcision and the possible hazardslinked to radiotherapy in some cases has emerged the need for investigating therole of possible target pharmacological therapy 2, 3. Thehigh percentage ofdesmoid tumors in females (about 80%) and the high number ofreported cases during or following pregnancy and spontaneous regression inpostmenopause have raised the possibility of hormonal factors as major playersin the pathogenesis of desmoid tumors 4. Thedetection of estrogen receptors in desmoid tumors was reported in previousstudies using ligandbinding assays; however, more current studies performingimmunohistochemicalmethodsyielded conflicting results regarding expression of ER? and ER? 5. Thewider distribution of ER? than ER? in several tissues as prostate, thyroid, andmesenchymal tissues and the recently introduced specific antibodies to ER? directed researchersto investigate its role in pathogenesis of desmoid tumors as most studiesreported absence of immunostaining ofER? 5.
.COXenzyme family is involved in prostaglandins production from arachidonic acid.COX-2 is one of the inducible family members that is overexpressed in differenttypes of cancer. It enhances tumorigenesis through inhibiting apoptosis,induction of angiogenesis, invasive potential, and stimulating growth factorssuch as platelet-derived growth factors (PDGF). Accordingly, nonsteroidalanti-inflammatory drugs (NSAIDs) that inhibits COX-2 activity were introducedas target therapy that have beneficial effects in cancer prevention 2, 6. MultipleNSAIDs as indomethacin, have been examined either single or in association withother hormonal treatment such as tamoxifen in treatment of desmoid tumors 4, with reported partial or total response rates ranging from 37%to 57% in multiple studies 7.
Weaimed in the current study to evaluate the immunohistochemical detection of COX-2 andER? in desmoid tumors and assess their correlation with available clinicopathologicvariables. Material and MethodsInthis study, a total of 17 archived paraffin blocks of desmoid tumors wereretrieved retrospectively from Pathology department, faculty of medicine, CairoUniversity in the period from January 2016 till June 2017. All samples includedwere obtained through total surgical excision.
The margins were inadequate in 8cases. Twelve patients were females, while 5 were males. The cases wereclassified according to their site as abdominal, intra- abdominal and extra-abdominal.Of note, the intra- abdominal desmoid tumor included in this study was mesentericmass associated with familial adenomatous polyposis (FAP). Fifteen casesincluded were primary desmoids and two cases were recurrent. The17 retrieved formalin fixed paraffin embedded tissue blocks were cut at 5-?m-thickness,stained by H&E and examined microscopically to confirm the diagnosis ofdesmoid tumor.Immunohistochemical staining of COX-2 and ER?:Twosections were cut at 5-?m-thickness from each of the 17paraffin embedded sections of desmoid cases on immunostaining slides.
Applyingthe standard protocol of Dako; heat mediated retrieval of antigen was performedby applying citrate buffer pH 6 in automated water bath (Dako PT link, PT101).The primary antibodies were COX2 monoclonal antibody (SP21), #MA5-1456,manufactured by Thermofisher Scientific, USA and monoclonal ER beta(PPG5/10),#MA1 81281, manufactured by Thermofisher Scientific, USA. Anautostainer (Dakoautostainer link 48) was used for immunostaining using apolymer-based detection system (DakoEnVision™ FLEX, K8000). Diaminobenzidine(DAB) was applied as chromogen and counterstaining was done using Mayer’s haematoxylin.Afterwards, the cover slips and DPX were used for mounting and preservingtissue sections. The positive control used for COX-2 & ER beta wasurothelial carcinoma positive for COX-2 granulosa cells in normal ovaryrespectively. Anothersection was cut from the paraffin blocks at 5 -?m-thickness,stained with toluidine blue to highlight mast cells. AnOlympus BX51 light microscope equipped with a digital camera was used forexamination and capturing of digital images.
Evaluation of immunostaining of both COX-2 & ER?:Immunostainingof COX-2 was reported as positive if more than 10% of tumor cells showed cytoplasmicpositivity for COX-2 8.TheER? nuclear immunostaining was reported as negative (< 5% positive tumorcells ), low expression (5-25% positive tumor cells) , high expression (>25% positive tumor cells) 9.Theresults of COX-2 and ER? immunostaining were correlated with multipleclinicopathologic factors (age, sex, tumor size, margin status and recurrencestatus).Evaluation of toluidine blue stained sections:TheCOX-2 positive immunostained cells were compared with metachromatically stained mast cells inco-localized touldine blue stained sections to reveal if they coincide or not.Statistical Methods:Statisticalanalysis of all results and available variables were performed using TheStatistical Package for the Social Sciences (SPSS) version 15. The Chi-squaretest was used to assess difference between qualitative variables. P values lessthan 0.
05 levels were considered to be statistically significant.Thestudy took the approval of ethical committee in faculty of medicine, CairoUniversity. ResultsAtotal of 17 cases of desmoid tumors were studied (figure 1).
The patient’s age displayed widerange from 7 years up to 50 years old with median age 33 years. Most patients werefemales (70.6%) while 29.
4% were males. Tumors more than 6 cm in greatestdimension constituted 64.7% of the cases (6/17). The localization of the tumorswas classified as abdominal (11/17), extra-abdominal (5/17) and intra-abdominal(1/17). All desmoid tumors enrolled in this study were treated by surgicalexcision. All clinicopathologic data are summarized in table 1. The majority of the studied cases(16/17) showed positive cytoplasmic immunohistochemical staining for COX-2,while only one case showed negative staining.
TheCOX-2 positive immunostained cells were similar in morphology to tumor cells inhaematoxylin and eosin stained sections. The toluidine blue stained co-localizedsections highlighted only few mast cells with specific metachromatic stainingthat didn’t coincide with COX-2 positive immunostained cells as shown in figure2A and 2B.ER?nuclear staining was expressed in 64.7% (11/17) of cases; 47.1% (8/17)displayed high expression (figure 3) and 19.6% (3/17) displayed low expression(figure 4), while 35.3% (6 /17) of cases showed negative staining. Nosignificant correlation was detected between COX-2 and ER? expression withdifferent clinicopathologic variables including patient’s age, gender, tumorsize, site, margins status and history of recurrence as displayed in table 2and 3 respectively.
COX-2immunohistochemical expression was not significantly correlated with ER?expression as shown in table 4.Desmoidtumors are rare tumors with incidence rate 2 to 4 per million annually 10. Although, the traditional treatment of desmoid tumors is surgery, the infiltrativeborders of these tumors and the absence of tumor capsule makes completesurgical resection with negative margins not always successful 11, 12.Theintroduction of systemic therapy as antihormonal and anti-inflammatory agents have been reportedwith various success rates in recurrent, unresectable and locally advanceddesmoid tumors 13. Severalstudies reported that COX-2 inhibitors induced the shrinkage of desmoid tumorsin clinical trials as COX-2 induces the growth of desmoid tumors throughincreased prostaglandin E2 production14, 15, 16. Moreover,complete regression of desmoid tumors with tamoxifen was reported in otherstudies17 ,18, achievingrecurrence free period up to 9 years in study performed by Maseelall et al 19. Weaimed in this work to examine the expression of COX-2 and ER? and in desmoidtumours using immunohistochemistry as it is practical accessible and cost effective method availablein almost all surgical pathology laboratories. To best of our knowledge,several studies assessed ER? detection in desmoid tumors byimmunohistochemistry but only two studies examined COX2 expression andcontradictory results were reported 14, 16.
Inthe current study, tumor cells expressed COX-2 protein in 92% (16/17 cases) ofdesmoid tumors without statistical significant correlation with allclinicopathologic variables including age, gender, tumor site, size, recurrenceand margin status (p< 0.05). These results coincide withSignoroniet al., results as all of their 14 studied desmoid tumors showed COX-2 positiveimmunohistochemical expression in tumor cells and their results were confirmedby presence of COX-2 mRNA by means of RT-PCR 14. Incontrast, Emori et al. reported that 56% of their 16 studied desmoid casesexpressed COX-2 in mast cells only as clearly co-expressed by tryptase. Thiswas against our findings which were confirmed by toluidine blue staining andproved that COX-2 immnnostained cells don't coincide with few mast cells in colocalized sections 16.
Thiscontroversy in results of COX-2 expression may be owed to different antibodiesused and small sample size enrolled in different studies. Inaddition, COX-2 expression was examined by Poon and his colleagues in cellcultures of 33 cases of aggressive fibromatosis and proved to be positive in 31cases through RT-PCR and in only 3 out of 17 cases of normal tissue dissectedfrom resection margins. Furthermore, the levelsofPGE2 were higher in cell cultures of aggressive fibromatosis than examinednormal margins fibroblasts (p>0.
05) 20.ER?was expressed in 64.7% of our studied desmoid tumor cases (11/17); highexpression was reported in 8 cases (47.1%) and low expression was reported in 3cases (19.
6%). Six cases showed negative ER? expression (35.3%). Thecorrelation between ER? expression and all clinicopathologic variablesincluding age, gender, tumor site, size, recurrence, margin status and COX 2expression as well was non-significant (p> 0.05). Similar figures werereported by Zhang et al.
, 9.as80.5% of their studied desmoid tumors were positive for immunohistochemical expressionof ER?; most (67.
5%) showed high-level expression, and 13% showed low-level expression. Otherinvestigators reported positive ER? expression in almost majority of their casesas Deyrup et al. 5.showedthat 100% of their studied extraabdominal desmoid tumors were positive for ER?.Santos et al., 21,alsostated that 89% of their desmoid tumor cases showed positivity for ER? withoutstatistical significant correlation with all clinical parameters.
Incontrast, Leithner et al. 22, reportedlow expression of ER? in their desmoid tumors (7/80); four out of 46 cases inextra-abdominal, two out of 21 in abdominal, and one out of 13 inintraabdominal cases. Ishizuka et al., 23, alsoreported low percentage of ER? expression in two out of 27 desmoid tumors(7.
4%). Different antibodies used and different sample size may contribute tothis diversity in reported results.ConclusionTosum up, we reported high percentage of COX-2 and ER? immunohistochemicalexpression in tumor cells of the examined desmoid tumors. These observations,recommend antihormonal therapy and COX 2 inhibitors as possible therapeutictargets in management of desmoid tumors. More prospective trials with largersample size and follow up after treatment are needed to validate these results.