Expression of platelet-derived growth factors and their receptors in ovarian clear-cell carcinoma and its putative precursors

ovarian clear-cell adenocarcinoma; PDGFR; autocrine/paracrine; endometriosis; clear-cell adeno- fibroma Among the ovarian cancers, clear-cell adenocarci- noma has been recognized as a distinct clinico- pathological entity because of its characteristic histology, frequent concurrence with endometriotic lesions, and highly chemoresistant nature resulting in an extremely poor prognosis when in high stage.1–3 Surgical debulking of the tumor is the only effective treatment for improving the prognosis of clear-cell adenocarcinoma. Although chemothera- peutic regimens including platinum analogues, taxanes, etoposide, and camptothecin have been developed, mortality of patients with ovarian clear-cell adenocarcinoma has remained largely unchanged. Therefore, more effective treatment options are needed, and these should be based on an understanding of the pathways driving neoplastic transformation and tumor growth. Some of these strategies may involve targeting of growth factors and growth factor receptors, which are important for the growth and development of specific cancer types. Received 19 June 2007; revised and accepted 1 October 2007; published online 14 December 2007 Correspondence: Dr H Tsuda, MD, Department of Basic Pathology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. E-mail: [email protected] Modern Pathology (2008) 21, 115–124 & 2008 USCAP , Inc All rights reserved 0893-3952/08 $30.00 www.modernpathology .org Despite a lack of data on cause–effect relation- ships, histological and epidemiological observations have consistently demonstrated a close association between endometriosis and ovarian clear-cell ade- nocarcinoma.1,4,5 In previous studies, loss of hetero- zygosity on chromosomes 9q, 10q, and 11q was detected in both clear-cell adenocarcinoma and coexisting endometriotic lesions. 6,7 So far, however, there have been few data on the specific molecular or genetic alterations that are consistently involved in clear-cell adenocarcinoma, and it is still unclear whether a specific gene or chromosomal alteration is essential for clear-cell adenocarcinoma develop- ment. On the other hand, clear-cell adenofibroma, a major form of benign or borderline ovarian clear-cell tumor, may be another type of clear-cell adenocarci- noma precursor.8,9 Our previous study demonstrated that clear-cell adenofibroma components coexisted in 21% of surgically resected clear-cell adenocarci- nomas.10 These clear-cell adenofibroma components coexisting with clear-cell adenocarcinoma often contain both apparently benign clear-cell adenofi- broma (clear-cell adenofibroma without atypia) and clear-cell adenofibroma with cellular and structural atypia (clear-cell adenofibroma with atypia or borderline clear-cell adenofibroma). 8–10 Moreover, in comparison with clear-cell adenocarcinoma with- out clear-cell adenofibroma components, clear-cell adenocarcinoma with clear-cell adenofibroma com- ponents shows several distinct clinicopathologic characteristics, that is, a lower frequency of co- existing endometriosis, a higher frequency of histologically low-grade tumor with tubulocystic proliferative architecture, and lower cancer cell proliferative activity. 10 These data suggest that, besides endometriosis, clear-cell adenofibroma components may be another form of clear-cell adenocarcinoma precursor, although there is no evident molecular background to support this spec- ulation. The platelet-derived growth factor receptors (PDGFRs: PDGFR- a and PDGFR- b) are transmem- brane receptor tyrosine kinases that are activated by platelet-derived growth factors (PDGFs). 11 The PDGFs are dimeric proteins composed of two closely related A-chain and B-chain polypeptides encoded by separate genes. The PDGFs selectively bind to receptor subunits via specific epitopes. PDGF-AA binds PDGFR- a, whereas PDGF-AB and PDGF-BB recognize both PDGFR-a and PDGFR-b.11 On binding their ligands, the receptors dimerize and autopho- sphorylate specific tyrosine residues, resulting in activation of a variety of intracellular signaling molecules that control differentiation and cell proliferation.12 Tumor formation may result if these receptors are persistently activated through func- tional mutation or amplification of their genes, or if PDGFRs and PDGFs constitute autocrine/paracrine stimulating loops. Although such mutations and gene amplification have not been described pre- viously in ovarian cancers, recent studies have demonstrated that PDGFR- a, PDGFR- b, and PDGF- AB are commonly expressed in ovarian cancers at frequencies as high as 87, 81, and 67%, respec- tively.13–16 Moreover, Henriksen et al17 have reported that PDGFRs or PDGFs were not detected in any of the benign ovarian tumors or normal ovarian epithelium they examined. Experiments in vivo have shown that PDGFRs activated by PDGFs modulate Akt and MAPK phosphorylation and significantly influence ovarian cancer cell prolifera- tion and tumor expansion. 16 These observations suggest that the PDGF–PDGFR system may play a functional role in the progression of ovarian cancers through autocrine or paracrine activation within the tumor tissues. In the present study, we histologically reviewed a number of surgically resected cases of ovarian clear- cell adenocarcinoma, and selected endometriotic lesions and clear-cell adenofibroma components synchronous with clear-cell adenocarcinoma. Using immunohistochemistry, we examined the expres- sion of PDGFRs and PDGFs to clarify whether (1) PDGFRs and PDGFs are commonly expressed in clear-cell adenocarcinomas, (2) whether such ex- pression is already evident in the two putative precursor forms of clear-cell adenocarcinoma, and (3) whether the expression status of these molecules is related to morphological changes in the epithe- lium of these putative precursors. It was anticipated that this information would not only lead to better understanding of the development of ovarian clear- cell adenocarcinoma, but also provide insight into potentially promising treatment options for this highly chemoresistant malignancy.

Cases This study was performed with the approval of the Institutional Internal Review Board on ethical issues, and informed consent was obtained from all patients. Sixty-seven cases of primary ovarian clear-cell adenocarcinoma and 21 solitary endome- triotic lesions were identified from the files of the Department of Laboratory Medicine, National Defense Medical College Hospital, Japan. These 67 cases of clear-cell adenocarcinoma had been surgi- cally resected between the years 1987 and 2005, and the patients had not undergone chemotherapy or radiation therapy before surgery. Cases of solitary endometriosis were obtained by salpingo-oophor- ectomy with or without hysterectomy. All speci- mens were formalin-fixed and paraffin-embedded, and 4-mm thick sections were prepared for hematox- ylin and eosin staining. All pathology specimens were reviewed in our institution, and tumors were classified according to the criteria of the World Health Organization. 8 PDGF/PDGFR in ovarian clear-cell adenocarcinomas S Yamamoto et al 116 Modern Pathology (2008) 21, 115–124 Endometriosis Synchronous with Clear-Cell Adenocarcinoma Endometriosis synchronous with clear-cell adeno- carcinoma was defined as (1) endometriosis existing in histological continuity with, or adjacent to, the clear-cell adenocarcinoma, or (2) an endometriotic cyst in which clear-cell adenocarcinoma was observed but histological continuity between the carcinoma and endometriotic epithelium was lacking. With reference to the histological criteria of ‘atypical endometriosis’ described previously, cyto- logical atypia was determined as present if, at most, one of the following features was histologically evident in the endometriotic epithelium: large hyperchromatic or pale nuclei with moderate to marked pleomorphism; an increased nuclear to cytoplasmic ratio; cellular crowding with stratifica- tion or tufting (Figure 1). 5 Consequently, synchronous endometrioses were identified in 21 (31%) of the 67 clear-cell adeno- carcinomas. Of these 21, 15 (71%) had both endometriotic lesions with and without cytologic atypia, 4 (19%) had only endometriotic lesions with atypia, and 2 (10%) had only endometriotic lesions without atypia. Therefore, 17 endometriotic lesions without atypia, 19 lesions with atypia, and 21 clear- cell adenocarcinomas containing synchronous endometriosis were analyzed by immunohisto- chemistry. Clear-Cell Adenofibroma Components Synchronous with Clear-Cell Adenocarcinoma The histological criteria of clear-cell adenofibroma (with or without atypia) have been described previously.10 In short, clear-cell adenofibroma was a surface epithelial–stromal tumor containing tubu- locystic epithelial components embedded in a fibroma-like stroma (Figure 2a). The epithelial cells were polygonal, hobnail or flat in shape, with clear, slightly granular or eosinophilic cytoplasm (Figure 2b). Presence of atypia was determined by histo- logical documentation based on the cellular (ie, nuclear pleomorphism and stratification of the epithelium) and structural (ie, size irregularity and crowding of the tubulocystic architecture) features of each epithelial component (Figure 2c and d). Consequently, of the 67 clear-cell adenocarcino- mas, we identified 10 (15%) cases that had both components of clear-cell adenofibroma with and without atypia. The designation ‘clear-cell adeno- fibroma with atypia’ is synonymous with, or has been referred to historically as clear-cell borderline tumor.8,9 Therefore, 10 lesions of clear-cell adeno- fibroma without atypia, 10 lesions of clear-cell adenofibroma with atypia, and 10 clear-cell adeno- carcinomas containing clear-cell adenofibroma com- ponents were analyzed by immunohistochemistry. Among the 21 solitary endometrioses, reactive changes were identified in 6 (29%) cases on the basis of the histological features described by La Grenade and Silverberg, 18 that is, the epithelia had mildly hyperchromatic or pleomorphic nuclei, but lacked stratification, and were associated with moderate to severe subepithelial inflammation that is rarely seen in atypical endometriosis. Presence or absence of atypia in endometriotic lesions and clear-cell adenofibroma components, and of reactive changes in solitary endometrioses were evaluated by two of the authors (SY and HT), and discussed until a consensus was reached. None of the 21 clear-cell adenocarcinomas with synchro- nous endometriosis and the 10 clear-cell adenocar- cinomas with clear-cell adenofibroma components overlapped. Immunohistochemistry All selected formalin-fixed and paraffin-embedded specimens were cut into 4- mm thick serial sections and analyzed by immunohistochemistry. We used rabbit polyclonal antibodies raised against a peptide mapped at the C terminus of PDGFR- a (RB-16981- R7; prediluted; Neomarkers, CA, USA), PDGFR- b (sc-339; dilution 1/100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and PDGF-A (sc-128; dilution 1/100; Santa Cruz Biotechnology), and against amino acids 136–190 of PDGF-B of human origin (sc-7878; dilution 1/100; Santa Cruz Biotechnology). Sections were deparaffinized and boiled in a microwave oven at 97 1C for 20 min in 0.01 mol/l citrate buffer (pH 6.0), then allowed to cool at room temperature. Endogenous peroxidase was blocked using 5% hydrogen peroxide. The slides were incubated at 4 1C overnight with primary antibodies and then reacted with a dextran polymer reagent combined with secondary antibodies and peroxi- dase (DAKO, Glostrup, Denmark) for 1 h at room temperature. Specific antigen–antibody reactions were visualized with 0.2% diaminobenzidine Figure 1 A case of endometriosis synchronous with clear-cell adenocarcinoma, showing cytologic aytpia. Nuclear enlargement and moderate pleomorphism are evident. H&E staining, original magnification /C2200. PDGF/PDGFR in ovarian clear-cell adenocarcinomas S Yamamoto et al 117 Modern Pathology (2008) 21, 115–124 tetrahydrochloride and hydrogen peroxide, and counterstaining was performed using Mayer’s he- matoxylin. Fallopian tube epithelium or endothe- lium of vessels included in the sections served as a built-in-control for all four antibodies, and as a

for normal expression of the proteins analyzed.19,20 As negative controls, sections without the primary antibody were used. Cytoplasmic immunoreactivity for PDGFs (PDGF- A and PDGF-B) and both membranous and cyto- plasmic immunoreactivity for PDGFRs (PDGFR- a and PDGFR- b) were taken into account for the evaluation. Immunoreactivity in the clear-cell ade- nocarcinomas was scored according to intensity as negative, weak (1 þ ), moderate (2 þ , similar to the control intensity), or strong (3 þ ). If 50% of epithelial components showed an immunoreactive intensity equal to or higher than that of fallopian tube epithelium, that is, 2 þ or 3 þ , the cases (or lesions) were regarded as positive. T wo observers (SY and HT) evaluated the results of the immuno- histochemistry. Statistical Analyses Statistical analyses were performed using StatMate III software (ATMS, Tokyo, Japan). The frequency of immunopositivity for PDGFRs/PDGFs in the various components was compared using Fisher’s exact test or w2-test. Differences at Po0.05 were considered statistically significant.

Results of immunohistochemistry are summarized in Tables 1 and 2. PDGFR/PDGF Expression in Clear-Cell Adenocarcinomas PDGFR-a expression was judged as positive in 30 (97%) of the 31 clear-cell adenocarcinomas: 10 (32%) cases scored 2 þ and 20 (65%) scored 3 þ (Figure 3a). PDGFR- b expression was positive in 30 Figure 2 Histological features of clear-cell adenofibroma component without atypia ( a and b) and with atypia ( c and d). (a) A clear-cell adenofibroma component shows simple cystic glands separated by wide fascicles of stromal cells. ( b) Glands are delineated with monolayers of cuboidal, flattened, or in part hobnail glandular cells with clear cytoplasm and uniform nuclei. ( c) Glands are variously sized and shaped, and focally crowded. ( d) Glands are delineated with one to three layers of cells with clear to eosinophilic cytoplasm and mildly pleomorphic, hyperchromatic nuclei. H&E staining, original magnification /C2100 for ( a and c); /C2400 for ( b and d). PDGF/PDGFR in ovarian clear-cell adenocarcinomas S Yamamoto et al 118 Modern Pathology (2008) 21, 115–124 cases (97%): 20 (65%) cases scored 2 þ and 10 (32%) scored 3 þ (Figure 3b). PDGF-A expression was positive in all 31 cases: 21 (68%) cases scored 2 þ and 10 (32%) scored 3 þ (Figure 3c). PDGFR- a, PDGFR-b, and PDGF-A were positive in 95, 95, and 100% of the 21 clear-cell adenocarcinomas asso- ciated with endometriosis, and were always (100%) positive in the 10 clear-cell adenocarcinomas with clear-cell adenofibroma components (Table 1). PDGF-B was determined as positive in only 7 (23%) cases, all of which scored 2 þ (Figure 3d) and showed co-positivity for PDGFR- b. Positivity for PDGF-B in clear-cell adenocarcinomas with clear- cell adenofibroma components (6 of 10, 60%) was significantly higher than that in clear-cell adenocar- cinomas associated with endometriosis (1 of 21, 5%) (P ¼ 0.0017) (Table 2). PDGFR/PDGF Expression in Endometriosis Synchronous with Clear-Cell Adenocarcinomas PDGFR-a and PDGFR- b were positive in 12 (71%) and 8 (47%) of 17 endometriotic epithelia without cytologic atypia, respectively (Figure 4a and b). PDGF-A and PDGF-B were positive in 10 (59%) and 6 (35%) of the 17 endometriotic lesions without atypia, respectively (Figure 4c and d), and all of these showed co-positivity with PDGFR- a and PDGFR-b, respectively (Table 2). Among the 19 lesions showing cytologic atypia in the endometriotic epithelium, PDGFR- a and PDGFR-b were positive in 16 (84%) and 14 (73%) cases, respectively (Figure 4e and f). PDGF-A and PDGF-B were positive in 16 (84%) and 2 (11%) of these 19 lesions (Figure 4g and h). Moreover, 15 (94%) of 16 PDGF-A-positive lesions and 2 PDGF-B- positive lesions showed co-positivity with PDGFR- a and PDGFR- b, respectively. Although PDGFRs (PDGFR-a and PDGFR- b) and PDGF-A tended to be more frequently positive in the endometriotic lesions with cytologic atypia than in those without, the difference was not statistically significant. Table 1 Immunoreactivity for PDGFRs and PDGFs in 31 cases of ovarian clear-cell adenocarcinoma Molecules Number of cases (%) Immunoreaction Negative Positive Score 0 1+ 2+ 3+ PDGFR-a 0 1 (3) 10 (32) 20 (65) PDGFR-b 0 1 (3) 20 (65) 10 (32) PDGF-A 0 0 21 (68) 10 (32) PDGF-B 9 (29) 15 (48) 7 (23) 0 PDGF , platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor. Table 2 Immunoreactivity for PDGFRs and PDGFs in clear-cell adenocarcinoma and in its putative precursor lesions Number of positive cases (%) Immunoreaction positive Total a: PDGFR- a b: PDGFR- b c: PDGF-A d: PDGF-B Both a and c Both b and d I: Immunoreaction in the putative precursors and clear-cell adenocarcinomas Endometriosis synchronous with clear-cell adenocarcinoma Endometriosis without atypia 17 12 (71) 8 (47) 10 (59) 6 (35) 10 (59) 6 (35) Endometriosis with atypia 19 16 (84) 14 (73) * 16 (84) * 2 (11) * 15 (79) * 2 (11) Clear-cell adenocarcinoma 21 20 (95) 20 (95) 21 (100) 1 (5) 20 (95) 1 (5) Clear-cell adenofibroma synchronous with clear-cell adenocarcinoma Clear-cell adenofibroma without atypia 10 0 * 0 * 0 * 0 * 0 * 0 *Clear-cell adenofibroma with atypia 10 10 (100) 9 (90) 9 (90) 3 (30) 9 (90) 3 (30) Clear-cell adenocarcinoma 10 10 (100) 10 (100) 10 (100) 6 (60) 10 (100) 6 (60) Solitary endometriosis 21 3 (14) 6 (29) 4 (19) 13 (62) 1 (5) 5 (24) Endometriosis without reactive change 15 1 (7) 3 (20) 3 (20) 7 (47) * 0 2 (13) Endometriosis with reactive change 6 2 (33) 3 (50) 1 (17) 6 (100) 1 (17) 3 (50) II: Comparison of the PDGFRs and PDGF expression between solitary endometriosis and endometrioses synchronous with clear-cell adenocarcinomas Solitary endometriosis 21 3 (14) * 6 (29) 4 (19) * 13 (62) 1 (5) * 5 (24) Endometriosis synchronous with clear-cell adenocarcinomasa 17 12 (71) 8 (47) 10 (59) 6 (35) 10 (59) 6 (35) PDGF , platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor. *Po0.05. aEndometriosis without cytologic atypia. PDGF/PDGFR in ovarian clear-cell adenocarcinomas S Yamamoto et al 119 Modern Pathology (2008) 21, 115–124 Immunopositivity for PDGFR- a, PDGFR- b, and PDGF-A in endometriotic lesions without atypia was lower than that in clear-cell adenocarcinomas: 71% (12 of 17) vs 95% (20 of 21), 47% (8 of 17) vs 95% (20 of 21), and 59% (10 of 17) vs 100% (21 of 21), respectively. In contrast, PDGF-B was expressed more frequently in synchronous endometriotic lesions without atypia than in clear-cell adenocarci- nomas: 35% (6 of 17) vs 5% (1 of 21), respectively (Table 2I). Consequently, comparison between en- dometriotic lesions without atypia and coexisting clear-cell adenocarcinomas revealed significant dif- ferences with regard to PDGFR- b (P ¼ 0.0011), PDGF-A ( P ¼ 0.0015), and PDGF-B ( P ¼ 0.022). PDGFR/PDGF Expression in the Clear-Cell Adenofibromas Synchronous with Clear-Cell Adenocarcinomas PDGFRs (PDGFR- a and PDGFR- b) and PDGFs (PDGF-A and PDGF-B) were negative in the epithe- lia of clear-cell adenofibroma components without atypia (Figure 4i–l) (Table 2). Of the 10 clear-cell adenofibroma components with aytpia, PDGFR- a and PDGFR-b were positive in 10 (100%) and 9 (90%) lesions, respectively (Figure 4m and n). PDGF-A and PDGF-B were positive in 9 (90%) and 3 (30%) of the 10 clear-cell adenofibroma components with atypia, respectively (Figure 4o and p). Moreover, all 9 (100%) of the PDGF-A-positive lesions and all 3 (100%) of the PDGF-B-positive lesions showed co-positivity with PDGFR- a and PDGFR-b, respectively. PDGFR/PDGF Expression in Solitary Endometriotic Epithelium Among the 21 cases with solitary endometriosis, PDGFR-a, PDGFR- b, PDGF-A, and PDGF-B were positive in 3 (14%), 6 (29%), 4 (19%), and 13 (62%), respectively. One (25%) of the 4 PDGF-A- positive cases and 5 (42%) of the 13 PDGF-B- Figure 3 Expression of PDGFR- a, PDGFR-b, PDGF-A, and PDGF-B in ovarian clear-cell adenocarcinomas. Positive immunoreactions for (a) PDGFR- a (scored 3 þ ), ( b) PDGFR- b (scored 3 þ ), ( c) PDGF-A (scored 3 þ ), and ( d) PDGF-B (scored 2 þ ). Arrowheads indicate the capillary endothelium showing moderate immunoreactivity with each primary antibody. Immunoperoxidase staining, original magnification /C2400. PDGF/PDGFR in ovarian clear-cell adenocarcinomas S Yamamoto et al 120 Modern Pathology (2008) 21, 115–124 positive cases showed co-positivity with PDGFR- a and PDGFR- b, respectively (Table 2). Of the six lesions showing reactive changes, PDGFR-a, PDGFR- b, PDGF-A, and PDGF-B were positive in two (33%), three (50%), one (17%), and six (100%), respectively (Figure 5a and b) (Table 2I). Therefore, PDGFRs and PDGF-B were more fre- quently positive in solitary endometrioses showing reactive changes than in those without, and there was a significant difference with regard to PDGF-B- positivity (47% (7 of 15) vs 100% (6 of 6), P ¼ 0.032) (Table 2I). When the solitary endometrioses and the endo- metrioses synchronous with clear-cell adenocarci- nomas were compared, PDGFRs and PDGF-A were more frequently positive in the latter, and significant differences were evident with regard to PDGFR- a and PDGF-A positivity (PDGFR- a, 14% (3 of 21) in the former vs 71% (12 of 17) in the latter, Po0.001; PDGF-A, 19% (4 of 21) in the former vs 59% (10 of 17) in the latter, P ¼ 0.014) (Table 2II). On the other hand, PDGF-B was more frequently positive in solitary endometriosis than in endometriosis syn- chronous with clear-cell adenocarcinoma, but not to a significant degree (62% (13 of 21) vs 35% (6 of 17), P ¼ 0.096) (Table 2II).

In the present study, most (97%, 30 of 31) of the ovarian clear-cell adenocarcinomas analyzed were positive for either PDGFR- a or PDGFR- b, and all of the 30 PDGFR- a-positive carcinomas showed co-positivity for the PDGF-A chain, which is a component of PDGF-AA or PDGF-AB. Although several reports have documented the frequent Figure 4 Expression of PDGFR- a, PDGFR-b, PDGF-A, and PDGF-B in putative precursor lesions for ovarian clear-cell adenocarcinomas. Immunohistochemistry for PDGFR-a (a, e, i, and m), PDGFR-b (b, f, j, and n), PDGF-A ( c, g, k, and o), and PDGF-B ( d, h, l, and p) in clear- cell adenocarcinoma-associated endometriosis without cytologic atypia ( a–d) and with cytologic atypia ( e–h), clear-cell adenocarcinoma- associated clear-cell adenofibroma components without atypia ( i–l), and with atypia ( m–p). PDGFR-a, PDGFR-b, and PDGF-A are positive in the epithelial components of endometriosis without atypia ( a–c) and with atypia ( e, f–g), and in clear-cell adenofibroma with atypia (m–o). PDGF-B is positive in epithelial components of endometriosis without atypia ( d) and clear-cell adenofibroma with atypia ( p), but negative in endometriosis with atypia ( h), and clear-cell adenofibroma without atypia ( l). Immunoperoxidase staining, original magnification /C2200. PDGF/PDGFR in ovarian clear-cell adenocarcinomas S Yamamoto et al 121 Modern Pathology (2008) 21, 115–124 expression of PDGFRs in ovarian carcinomas, the number of clear-cell adenocarcinoma cases included in those studies was generally small. 13–15,17 The largest series by Matei et al 16 demonstrated that 17 (90%) and 18 (95%) of 19 clear-cell adenocarcino- mas were immunohistochemically positive for PDGFR-a and PDGF-AB, respectively, the former being almost concordant with our data. In contrast, the frequency of positivity for the PDGF-B chain, which is a component of PDGF-AB or -BB, was 23% of clear-cell adenocarcinomas in the present study, being much lower than the frequency of PDGF-AB expression (95%) reported by Matei et al.16 This discrepancy may have resulted from the differences in the antibody used, protocols and evaluation of the immunohistochemistry. PDGFR-b, which is a receptor for PDGF-AB or PDGF-BB, was mostly (97%) positive in the present series of clear-cell adenocarcinomas. However, PDGFs including PDGF-BB are natural ligands present in stromal cells such as fibroblasts, and the smooth muscle and endothelium of blood vessels, which constitute the major proportion of the stroma necessary for tumor growth and invasion, and these were constantly positive for PDGF-A and PDGF-B in the present study. 19,20 These observations appear to support the hypothesis that not only an autocrine loop but also a paracrine loop of PDGFRs/PDGFs contributes to tumor progression of clear-cell ade- nocarcinoma. Although not studied in the present series, PDGF-D, which has been recently cloned, binds PDGFR- b and shows mitogenic properties. 21 The expression of PDGF-D in clear-cell adenocarci- noma would therefore be of interest. In the endometriotic lesions, synchronous with clear-cell adenocarcinomas, the frequencies of PDGFR (PDGFR- a and PDGFR- b) and PDGF-A expression increased in accordance with the acqui- sition of cytologic atypia by the epithelium, and a similar trend was also evident in solitary endome- triotic lesions. Moreover, except for PDGF-B, ex- pression of PDGFs and PDGFRs was more frequent in endometriotic lesions synchronous with clear- cell adenocarcinomas than in solitary endome- trioses. These findings suggest that the establish- ment of autocrine/paracrine loops of PDGFRs/ PDGFs, especially those mediated by PDGFR- a, might be an early event in the development of endometriosis-associated clear-cell adenocarcino- mas, and that activation of PDGFR has already occurred by the stage of solitary endometriosis without cellular atypia. In contrast to the endometriotic lesions, PDGFs and PDGFRs were not detected in the epithelium of apparently benign clear-cell adenofibroma compo- nents (clear-cell adenofibroma without atypia). However, similarly to endometriotic epithelium, expression of PDGFs and PDGFRs was frequently detected in clear-cell adenofibroma components when cytologic atypia was present. These findings suggest that establishment of autocrine/paracrine loops for PDGFR activation is not essential for the formation of clear-cell adenofibroma without atypia, but is highly associated with the acquisi- tion of cytologic atypia by the epithelium of clear-cell adenofibroma and the development of clear-cell adenofibroma-associated clear-cell adenocarcinomas. Our previous study demonstrated that clear-cell adenocarcinomas with clear-cell adenofibroma com- ponents were more frequently low-grade histologi- cally, showed a tubulocystic architectural pattern, and had lower cancer cell proliferation activity than clear-cell adenocarcinomas without clear-cell ade- nofibroma, which were mostly (68%) endometrio- sis-associated.10 In the present study, the incidence of PDGF-B positivity was higher in clear-cell adenofibroma-associated clear-cell adenocarcino- mas (60%) than in endometriosis-associated clear-cell adenocarcinomas (5%). All non-atypical clear-cell adenofibroma components studied were negative for PDGF-B. In contrast, PDGF-B positivity was relatively high in both solitary endometrioses (62%) and non-atypical endometrioses synchronous with clear-cell adenocarcinoma (35%). Therefore, it Figure 5 A case of solitary endometriosis showing positive immunoreactivity for ( a) PDGFR- b and ( b) PDGF-B. Immuno- peroxidase staining, original magnification /C2200. PDGF/PDGFR in ovarian clear-cell adenocarcinomas S Yamamoto et al 122 Modern Pathology (2008) 21, 115–124 can be speculated that the role of PDGFR-b and PDGF- BB (or PDGF-AB) expression may be associated with cell differentiation rather than cell proliferation or neoplastic transformation, although how the differ- ence in PDGF-B expression status between these clear-cell adenocarcinomas contributes to the bio- logical nature of the tumors is largely unknown. Ovarian carcinoma is one of the human epithelial malignancies showing histological heterogeneity, and clear-cell adenocarcinoma is one of its repre- sentative variations. 1 Therefore, the possibility that areas of apparently benign endometriosis-like or clear-cell adenofibroma-like lesions represent well differentiated or ‘mature’ components of the carci- noma cannot be ruled out, although previous reports have indicated that stromal features of clear-cell adenofibroma (fibroma-like) could be an aid for differentiating clear-cell adenofibroma from clear- cell adenocarcinoma with a prominent tubulocystic pattern.9,10 In the present study, expression of PDGF- A and PDGFRs was detected less frequently in the endometriotic epithelium than in clear-cell adeno- carcinomas, and was not detected in apparently benign clear-cell adenofibroma components. These observations suggest that the apparently benign areas coexisting with clear-cell adenocarcinomas, that is, non-atypical endometriosis and clear-cell adenofibroma without atypia, may be true precur- sors of ovarian clear-cell adenocarcinomas. More- over, the striking differences in the frequency of expression of PDGFRs/PDGFs between endometrio- tic lesions and non-atypical clear-cell adenofibro- mas support the idea that clear-cell adenofibroma and endometriosis are two distinct forms of pre- cursor for ovarian clear-cell adenocarcinoma. The incidence of frequent high-intensity expres- sion of PDGFRs in clear-cell adenocarcinomas demonstrated in this series appears to throw light on the molecular background involved in clear-cell adenocarcinoma formation, and also provides new insights into possible molecular-based therapies for this highly chemoresistant malignancy. PDGFRs, as well as KIT, belong to the type III receptor tyrosine kinases, and can be specifically treated by tyrosine kinase inhibitors such as imatinib mesylate (STI571).22 A recent study has demonstrated that imatinib mesylate inhibits ovarian cancer cell pro- liferation and PDGF-induced S-phase entry through PDGFR-a and Akt inactivation. 23 Although a phase II trial of imatinib mesylate as a single agent failed to produce satisfactory efficacy against recurrent and platinum-resistant ovarian cancer, 24 cases with clear-cell adenocarcinoma were not enrolled and, therefore, the effect of imatinib or other tyrosine kinase inhibitors on ovarian clear-cell adenocarci- noma remains undetermined. In the present study, we have demonstrated that PDGFRs and the PDGF-A chain are frequently expressed in ovarian clear-cell adenocarcinomas. Epithelial–epithelial and epithelial–stromal inter- actions via autocrine and/or paracrine activation of PDGFRs might drive the development of clear-cell adenocarcinoma during the process of multistage carcinogenesis. The present results also suggest biological differences between clear-cell adenocar- cinomas that arise in association with clear-cell adenofibroma vs endometriosis.

This work was supported in part by a grant-in-aid for special research from National Defense Medical College, and by a grant-in-aid for cancer research from the Ministry of Health, Labor, and Welfare, Japan. We are thankful to Dr David Douglas (Douglas’ Scientific Editorial Services, Tokorozawa, Japan) for his professional review of the final version of the manuscript. Disclosure/conflict of interest We indicate no potential conflicts of interest.

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