IGFBP-1 is expressed speciﬁcally in ovarian clear cell ...diagnostic-pathology-tsukuba.jp/assets/templates/... · and gynaecological organs.5,6 In the gynaecological ﬁeld, they

IGFBP-1 is expressed specifically in ovarian clear celladenocarcinoma

Shintaro Sugita,1,2 Yukio Morishita,1,2 Junko Kano,1 Shuichiro Furuya,2 Aya Shiba-Ishii1 &

Masayuki Noguchi1,2

1Department of Pathology, Institute of Basic Medical Science, Graduate School of Comprehensive Human Science,

University of Tsukuba, and 2Department of Pathology, Tsukuba University Hospital, Amakubo, Tsukuba, Ibaraki, Japan

Date of submission 21 April 2010Accepted for publication 12 August 2010

Sugita S, Morishita Y, Kano J, Furuya S, Shiba-Ishii A & Noguchi M

(2011) Histopathology 58, 729–738

IGFBP-1 is expressed specifically in ovarian clear cell adenocarcinoma

Aims: To investigate the specific expression of insulin-like growth factor binding protein-1 (IGFBP-1) inovarian clear cell adenocarcinoma (CCA).Methods and results: Immunohistochemistry and in situhybridization for IGFBP-1 were performed in normalendometrium, placenta, and 100 surgically resectedcases of ovarian cancer including 31 CCAs and 69 non-CCAs. Immunohistochemistry for hepatocyte nuclearfactor-1 (HNF-1b) was also examined in all cases.Specific expression of IGFBP-1 was confirmed in secre-tory endometrium, decidua of placenta and Arias-Stellaglands of miscarriage material. Among ovarian can-cers, almost all cases of CCA showed expression of bothIGFBP-1 protein and mRNA, but non-CCA hardly

expressed IGFBP-1. There was a significant differencebetween CCA and non-CCA in the expression of IGFBP-1 protein and mRNA. No correlation was foundbetween the rate of IGFBP-1 expression and patholog-ical T and N factors of the tumour–node–metastasis(TNM) classification. All CCA cases except for oneexhibited expression of HNF-1b protein, whereas only15.9% of non-CCAs did so.Conclusion: The expression of IGFBP-1 in CCA is morespecific than that of HNF-1b. IGFBP-1 shows expres-sion by decidual endometrium and Arias-Stella glands,and CCA also exhibits characteristic expression. Theseresults indicate that IGFBP-1 is a immunohistochem-ical marker for CCA.

Keywords: clear cell adenocarcinoma, HNF-1b, IGFBP-1, immunohistochemistry, in situ hybridization

Abbreviations: CCA, clear cell adenocarcinoma; EA, endometrioid adenocarcinoma; HNF-1b, hepatocyte nuclearfactor-1beta; IGFBP-1, insulin-like growth factor binding protein-1; MA, mucinous adenucarcinoma; SA, serousadenocarcinoma

Introduction

Clear cell adenocarcinoma (CCA) has the worst prog-nosis among the various subtypes of ovarian surfaceepithelial cancers, and is characterized histologically byglandular, papillary and solid proliferation of a largeamount of glycogen-containing clear and hobnailtumour cells with a hyalinous fibrotic stroma. Middle-

aged and elderly women are affected predominantly,and the pathogenesis of CCA is associated closely withovarian endometriosis.

Using the suppression subtractive hybridizationmethod, we have demonstrated previously that thegene expression of CS0DI066YJ10, as a full-lengthplacental cDNA clone, is reduced in cases of endome-trial stromal sarcoma in comparison with stromal cellsof endometrium in the normal secretory phase.1

Homology retrieval revealed that CS0DI066YJ10 isidentical to insulin-like growth factor binding protein 1(IGFBP-1), which is one of the key members of theinsulin-like growth factor (IGF) system. IGFBP regu-lates the biological effects of IGFs by binding to and

Address for correspondence: M Noguchi, MD, Department of

Pathology, Institute of Basic Medical Science, Graduate School of

Comprehensive Human Science, University of Tsukuba,

1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.

e-mail: [email protected]

� 2011 Blackwell Publishing Limited.

Histopathology 2011, 58, 729–738. DOI: 10.1111/j.1365-2559.2011.03817.x

then being released from them at various sites inhuman organs.2 IGFBP includes six members, IGFBP-1–6, of which IGFBP-1 plays a particularly importantrole in gynaecological physiology.3 IGFBP-1 closelyregulates the menstrual cycle, decidualization of theendometrium, implantation, trophoblast invasion andfetal growth.4 Strong and specific expression of IGFBP-1 is observed ordinarily in the secretory endometrium,decidualized endometrial stromal cells and the deciduaof the placenta. The expression of IGFBP-1 in suchtissues has already been confirmed by immunohisto-chemistry and mRNA in situ hybridization.1

While the family of molecules of the IGF system areassociated significantly with development of the organ-ism and maintenance of a normal physiological state,they also play a role in tumour development in severalorgans including the lung, intestine, breast, prostateand gynaecological organs.5,6 In the gynaecologicalfield, they are involved in the development and progres-sion of ovarian and uterine endometrial carcinomas.7

Some studies have demonstrated a relationship betweenIGFBP-2,5 and the development and grade of ovarianepithelial cancers, including serous adenocarcinoma(SA), endometrioid adenocarcinoma (EA) and CCA.8,9

However, no reported study has focused in detail uponthe connection between IGFBP-1 and ovarian CCA.

Hepatocyte nuclear factor-1 (HNF-1b) was recog-nized recently as a specific marker for ovarian CCA.10

HNF-1b is one of the isoforms of HNF-1, which is atranscription factor expressed mainly in liver andkidney, where it regulates the promoters of severalgenes. On the other hand, HNF-1 has been reported toactivate transcription of human IGFBP-1, since abinding site for HNF-1 is located upstream of thepromoter region of IGFBP-1. These characteristicssuggest that IGFBP-1 located downstream of HNF-1would also be a good marker of CCA.11,12

In the present study, the expression of IGFBP-1 wasinvestigated by both immunohistochemistry andmRNA in situ hybridization in the four major histolog-ical subtypes of ovarian surface epithelial cancers –CCA, SA, EA and mucinous adenocarcinoma (MA) – todetermine whether or not the expression of IGFBP-1 isspecific to CCA. We also addressed the role andimportance of IGFBP-1 in the development of ovariansurface epithelial cancers, particularly CCA.

Materials and methods

sample selection

The archival pathology files at the Department ofPathology, University Hospital of Tsukuba, Ibaraki,

Japan, were searched for patients who had beendiagnosed as having the various histological subtypesof ovarian surface epithelial carcinoma. Comprehensiveinformed consent from the patients was obtained beforecollection of the specimens. One hundred cases ofprimary ovarian carcinoma were selected for the study,comprising 31 cases of clear cell adenocarcinoma(CCA), 27 cases of serous adenocarcinoma (SA), 22cases of endometrioid adenocarcinoma (EA) and 20cases of mucinous adenocarcinoma (MA). Some non-neoplastic endometrial tissues were also selected forimmunohistochemistry. These tissues included 10cases of proliferative endometrium, secretory endome-trium, decidua of the placenta and Arias-Stella glandsin the endometrium of dilatation and curettage mate-rial following miscarriage, respectively. Five cases ofendometrium with marked decidual change were alsoexamined.

pathological evaluation

All haematoxylin and eosin (H&E) sections containingof the tumour were reviewed and the primary diagnosisof all cases was confirmed by two pathologists, S.S. andY.M. Several tumours having a minor component ofthe other histological subtypes were reclassified intoone of the four histological subtypes according to thepredominant histology. Sections showing a represen-tative histological picture were selected for immuno-histochemistry and in situ hybridization in each case.

immunohistochemistry

Immunohistochemistry was performed using anti-bodies to IGFBP-1 and HNF-1b in accordance withthe manufacturer’s instructions and the procedurereported previously.13 The antibodies used were amouse monoclonal antibody to IGFBP-1 (sc-25257; lotno. G1508, E2907; dilution 1:100; Santa Cruz Bio-technology, Santa Cruz, CA, USA) and a goat poly-clonal antibody to HNF-1b (sc-7411; lot no. H1808;dilution 1:200; Santa Cruz). All formalin-fixed, paraf-fin-embedded specimens were cut into sections 3 lmthick. The slides were deparaffinized and rehydrated,and then for antigen retrieval they were pretreated bymicrowave heating at 98�C for 15 min (IGFBP-1) in TEbuffer [10 mm Tris–HCl with 1 mm ethylenediaminetetraacetic acid (EDTA), pH 8.0] and at 95�C for20 min (HNF-1b) in sodium citrate buffer (10 mm

sodium citrate monohydrate, pH 6.0). After the antigenretrieval procedure, endogenous peroxidase activitywas blocked and then the slides were incubated withthe primary antibodies at room temperature (RT) for

730 S Sugita et al.

� 2011 Blackwell Publishing Ltd, Histopathology, 58, 729–738.

30 min (IGFBP-1) or at 4�C overnight (HNF-1b). Theimmunohistochemical reaction was visualized usingEnvision (IGFBP-1) or the ABC (HNF-1b) method withdiaminobenzidine as the chromogen. Normal decidualcells of human placenta and a typical case of CCA wereused as the positive control for IGFBP-1 and HNF-1b,respectively. Expression of IGFBP-1 was defined asnegative (score 0; no staining), weakly positive (score1+; staining in <5% of tumour cells), moderatelypositive (score 2+; staining in 5–20% of tumour cells)and strongly positive (score 3+; staining in >20% oftumour cells). Expression of HNF-1b was assessed asnegative (score 0; no staining), weakly positive (score1+; staining in <10% of tumour cells), moderatelypositive (score 2+; staining in 10–50% of tumor cells)and strongly positive (score 3+; staining in >50% oftumour cells).

igfbp- 1 in situ hybridization

In situ hybridization was performed as reported previ-ously.14 To prepare the cRNA probes for in situhybridization of IGFBP-1, total RNA was extractedfrom frozen decidual tissue of placenta material withoutany maternal disease. T7 RNA polymerase promoter-attached primers for IGFBP-1 were designed using theprimer designing tool of Primer 3 (http://frodo.wi.mit.edu/primer3/) on the basis of the sequence in theGenBank database (Accession no. NM000596.2).IGFBP-1 mRNA was amplified by reverse transcrip-tase–polymerase chain reaction (RT–PCR) with T7RNA polymerase promoter-attached primers (forward,5¢-CTTAATACGACTCACTATAGGGCTATGATGGCTCG-AAGGCTC-3¢; and reverse, 5¢-CTTAATACGACTCACT-ATAGGGGAGACCCAGGGATCCTCTTC-3¢), and thenthe PCR products were transcribed to antisense orsense cRNA probes labelled with digoxigenin (DIG)with a DIG RNA Labeling kit (Roche Diagnostic GmbH,Penzberg, Germany) in accordance with the manufac-turer’s instructions. Through this process, we estab-lished antisense and sense cRNA probes of about 260base pairs (bp) for in situ hybridization (Figure 1).Formalin-fixed, paraffin-embedded ovarian cancer tis-sues were cut into sections 3 lm thick and mounted onsilane-coated slides (Matsunami Glass Ind., Osaka,Japan). Hybridization was performed at 50�C for 16 hwith a hybridization mixture containing DIG-labelledantisense and sense probes. Detection of the hybridizedcRNA probes was performed with horseradish perox-idase-conjugated rabbit anti-DIG antibody (DakoCyto-mation, Kyoto, Japan), and the signals were amplifiedusing a GenPointTM Tyramide Signal AmplificationSystem (Dako North America, Carpinteria, CA, USA).

Signal detection was performed by immunohistochem-ical reaction with diaminobenzidine as a chromogen.Expression of IGFBP-1 mRNA was defined as negative(score 0; no staining), weakly positive (score 1+;staining in <5% of tumour cells), moderately positive(score 2+; staining in 5–20% of tumour cells) andstrongly positive (score 3+; staining in >20% oftumour cells).

statistical analysis

All statistical analysis was carried out by v2 test usingDr SSPS II software (SSPS, Chicago, IL, USA). For allanalysis, differences at P < 0.05 were consideredstatistically significant.

Results

clinicopathological findings in 3 1 cases

of cca

The patients ranged in age from 25 to 83 years (mean,50.4 years). In patients with CCA, the right ovary wasaffected in 18 cases (58.1%), the left ovary in 11(35.5%) and the bilateral ovaries in two (6.5%).Twenty-four of the 31 CCA cases were classified aspT1 (77.4%) by the pathological tumour–node–metas-tasis (TNM) classification, and seven as pT2 or 3(22.6%). Dissection of regional lymph nodes wasperformed in 25 patients with CCA, of whom threeexhibited regional lymph node metastasis (12%).

immunohistochemistry of non-neoplastic

endometrial tissue

First, we examined the expression of IGFBP-1 proteinin normal gynaecological materials including endo-metrial tissue in the proliferative and secretoryphases and in miscarriage material and placenta.Endometrial tissues showing marked decidual changewere also examined. All samples of endometrialstromal cells in the proliferative phase were negativefor IGFBP-1. Strong expression of IGFBP-1 wasobserved in decidualized stromal cells in secretoryendometrium and markedly decidualized endome-trium. Secretory endometrial epithelium, especiallyin mid- to late secretory phase, expressed IGFBP-1weakly, but the intensity was lower than that ofArias-Stella glands. Decidua of the placenta alsoexpressed IGFBP-1 consistently in all cases. Interest-ingly, all samples of Arias-Stella glands exhibitedboth cytoplasmic IGFBP-1 and nuclear HNF-1bexpression (Figure 2).

IGFBP-1 expression in ovarian CCA 731


expression of igfbp- 1 in ovarian cancers

demonstrated by immunohistochemistry and

in situ hybridization

Twenty-eight of the 31 cases of CCA (90.3%) wereimmunoreactive for IGFBP-1 by immunohistochemis-try (Table 1 and Figure 3). The immunoreactive casescomprised five cases with a score of 3+ (17.9%), sixwith a score of 2+ (21.4%) and 17 with a score of 1+(60.7%). Three cases of CCA were negative for IGFBP-1protein expression. In contrast, four of the 69 non-CCAcases (5.8%) showed expression of IGFBP-1 proteinwith a score of 1+; these comprised two cases of SA andEA, respectively; the two cases of EA did not showtypical secretory change (Figure 4). Expression of

IGFBP-1 mRNA was also observed in 28 of 31 CCAcases (90.3%) by in situ hybridization (Table 2 andFigure 3). The cases positive for IGFBP-1 mRNAcomprised nine with a score of 3+ (32.1%), eight witha score of 2+ (28.6%) and 11 with a score of 1+(39.3%). Three cases of CCA demonstrated no expres-sion of IGFBP-1 mRNA, and these cases were the sameas those lacking expression of IGFBP-1 protein. Con-versely, two of 17 non-CCA cases (11.8%) expressedmRNA for IGFBP-1 in a minority of the tumour cellswith a score of 1+. These two cases were SA and EA,respectively. In summary, almost all cases of CCAexpressed both IGFBP-1 protein and mRNA at least inlimited areas, whereas the majority of non-CCAs lackedexpression of either IGFBP-1 protein or mRNA. In

A

C

B

D

Figure 1. Insulin-like growth factor binding protein-1 (IGFBP-1) protein and mRNA expression in decidua cells of human placenta tissue.

A, Decidua cells of placenta tissue (haematoxylin and eosin). B, Immunohistochemistry of IGFBP-1 protein. Decidua cells show expression of

IGFBP-1 protein. C,D, In situ hybridization of IGFBP-1 mRNA. C, Decidua cells exhibit reactivity for IGFBP-1 mRNA with anti-sense cRNA probe

of IGFBP-1. D, No reactivity was found with a sense-cRNA probe of IGFBP-1 mRNA.

732 S Sugita et al.


terms of the difference in expression score betweenIGFBP-1 protein and IGFBP-1 mRNA, 17 of the 31CCAs showed the same score, whereas in the other 14the scores obtained by immunohistochemistry andin situ hybridization differed (Table 3). Ten of these 14cases showed higher expression of IGFBP-1 mRNAthan the protein. However, four of the 14 cases showedlower expression of IGFBP-1 mRNA than the protein(Table 3). Statistically, a significant difference wasrecognized between CCA cases and non-CCA casesin the expression of IGFBP-1 protein and mRNA(P < 0.0001) (Tables 1 and 2). Clinicopathologically,

no significant difference was found, neither the sensi-tivity of IGFBP-1 expression and the extent of primarytumour (pT) (Table 4) nor regional lymph nodemetastasis (pN) (data not shown).

expression of hnf- 1b in ovarian cancers

demonstrated by immunohistochemistry

Thirty of the 31 CCA cases (96.8%) were positive forHNF-1b, comprising 15 cases with a score of 3+ (50%),eight with a score of 2+ (26.7%) and seven with a scoreof 1+ (23.3%) (Table 5 and Figure 3). Only one case

A

D E

H I

F

G

B C

Figure 2. Immunohistochemistry of insulin-like growth factor binding protein-1 (IGFBP-1) and hepatocyte nuclear factor-1beta (HNF-1b) in

non-neoplastic endometrial tissue. A, Proliferative endometrium. B, Stromal cells in proliferative endometrium were negative for IGFBP-1.

C, Decidualized secretory endometrium. D, Decidualized stromal cells in secretory endometrium express IGFBP-1 protein. E, Markedly

decidualized secretory endometrium. F, Decidualized stromal cells are positive for IGFBP-1. G, Arias-Stella endometrial glands. H, Cytoplasmic

reactivity of IGFBP-1 in Arias-Stella endometrial glands of dilatation and curettage (DC) material. I, Nuclear expression of HNF-1b in Arias-Stella

endometrial glands of DC material.



Table 1. Expression of IGFBP-1 protein in ovarian adenocarcinoma by immunohistochemistry

Histological typeTotaln (%)

Negative(score 0)n (%)

Positive(score 1+ to 3+)n (%) P

Positive

Score 1+n (%)

Score 2+n (%)

Score 3+n (%)

CCA 31 (100) 3 (8.7) 28 (90.3) <0.0001 17 (60.7) 6 (21.4) 5 (17.9)

Non-CCA 69 (100) 65 (94.2) 4 (5.8) 4* (100) 0 (0) 0 (0)

IGFBP1, Insulin-like growth factor binding protein-1; CCA, clear cell adenocarcinoma.

Score 0, negative; score 1+, <5%; score 2+, 5–20%; score 3+, >20%.

Non-CCA includes serous adenocarcinoma, endometrioid adenocarcinoma and mucinous adenocarcinoma.

Sensitivity, 90.3%; specificity, 94.2%; positive predictive value, 87.5%; negative predictive value, 95.6%.

*Four score one cases of non-CCA were two serous adenocarcinomas and two endometrioid adenocarcinomas.

A B

DC

Figure 3. Clear cell adenocarcinoma (CCA) case showing high expression of insulin-like growth factor binding protein-1 (IGFBP-1) and

hepatocyte nuclear factor-1beta (HNF-1b). A, CCA consisting of papillary and glandular structures of clear tumour cells (haematoxylin and

eosin. B, Tumour cells express IGFBP-1 protein by immunohistochemistry. C, Tumour cells express IGFBP-1 mRNA by in situ hybridization.

D, Tumour cells are immunoreactive for HNF-1b.

734 S Sugita et al.


A B

C D

Figure 4. Non-clear cell adenocarcinoma (CCA) case [endometrioid adenocarcinoma (EA)] showing low expression of insulin-like growth

factor binding protein-1 (IGFBP-1). A, EA consists of papillary proliferation of columnar and polyhedral tumour cells [haematoxylin and

eosin (H&E)]. B, A few tumour cells are positive for IGFBP-1 protein by immunohistochemistry. C, The other EA case with glandular

structures (H&E). D, A few tumour cells are positive for IGFBP-1 protein by immunohistochemistry.

Table 2. Expression of IGFBP-1 mRNA in ovarian adenocarcinoma by in situ hybridization




Positive

Score 1+n (%)

Score 2+n (%)

Score 3+n (%)

CCA 31 (100) 3 (9.7) 28 (90.3) <0.0001 11 (39.3) 8 (28.6) 9 (32.1)

Non-CCA 17 (100) 15 (88.2) 2 (11.8) 2* (100) 0 (0) 0 (0)

Score 0, negative; score 1+, <5%; score 2+, 5–20%; score 3+, >20%.

CCA, Clear cell adenocarcinoma; IGFBP-1, insulin-like growth factor binding protein-1.

Non-CCA includes serous adenocarcinoma, endometrioid adenocarcinoma, and mucinous adenocarcinoma.


*IGFBP-1-positive cases of non-CCA were serous adenocarcinoma and endometrioid adenocarcinoma.



showed no expression of HNF-1b protein, and this casewas also negative for IGFBP-1 protein and mRNA. Incontrast, 11 of the 69 non-CCA cases (15.9%) wereimmunoreactive for HNF-1b, but the majority of thesepositive cases showed weak expression of HNF-1b witha score of 1+. A statistically significant difference wasrecognized between CCA cases and non-CCA caseswith regard to the expression of HNF-1b protein

(P < 0.0001) (Table 5). One non-CCA case positivefor IGFBP-1 protein was included among the 11 thatwere positive for HNF-1b.

Discussion

We have demonstrated that the immunohistochemicalexpression of IGFBP-1 protein is specific to cases ofCCA, although its expression is limited to relativelysmall areas in many cases. We have also confirmed thespecific expression of IGFBP-1 at the mRNA level.IGFBP-1 mRNA expression was detected specifically inCCA cases by in situ hybridization, although thedistribution of the sensitivity score differed betweenthe protein level and the mRNA level. Stronger andwider expression of IGFBP-1 was observed at themRNA level than at the protein level, although it isnot meaningful to compare the levels of protein andmRNA expression determined by these two methods.These results suggest that CCAs probably expressIGFBP-1 more strongly at the mRNA level than atthe protein level. We suspected that IGFBP-1 proteinmight show a tendency to disappear rapidly from thecytoplasm after its synthesis, because generally a largeproportion of IGFBP is secreted into the blood and

Table 3. Comparison of expression score between IGFBP-1protein and IGFBP-1 mRNA

IGFBP-1 mRNA expression

Score 0 Score 1+ Score 2+ Score 3+ Total

IGFBP-1 protein expressionScore 0 3 0 0 0 3

Score 1+ 0 8 4 5 17

Score 2+ 0 2 3 1 6

Score 3+ 0 1 1 3 5

Total 3 11 8 9 31

IGFBP-1, Insulin-like growth factor binding protein-1.

Table 4. Association of pathological T factor and sensitivity of IGFBP-1 protein expression in ovarian clear cell adenocarcinoma

Pathological T factor (pT)Totaln (%)

Sensitivity of IGFBP-1 protein expression

PLow (score 0 or 1+)n (%)

High (score 2+ or 3+)n (%)

pT1 24 (100) 15 (62.5) 9 (37.5) >0.05

pT2 or 3 7 (100) 5 (71.4) 2 (28.6)

pT1, pathological T factor of tumour–node–metastasis (TNM) classification.

IGFBP-1, insulin-like growth factor binding protein-1.

Table 5. Expression of HNF-1b protein in ovarian adenocarcinoma by immunohistochemistry




Positive

Score 1+n (%)

Score 2+n (%)

Score 3+n (%)

CCA 31 (100) 1 (3.2) 30 (96.8) <0.0001 7 (23.3) 8 (26.7) 15 (50)

Non-CCA 69 (100) 58 (84.1) 11 (15.9) 10 (90.9) 1 (9.1) 0 (0)

Score 0, negative; score 1+, <10%; score 2+, 10–50%; score 3+, >50%

CCA, Clear cell adenocarcinoma; HNF-1b, hepatocyte nuclear factor-1beta.

Non-CCA includes serous adenocarcinoma, endometrioid adenocarcinoma, and mucinous adenocarcinoma.


736 S Sugita et al.


circulates throughout the body by binding to insulationgrowth factors (IGFs).2 Conversely, the localization ofIGFBP-1 protein expression sometimes differed fromthat of IGFBP-1 mRNA expression in the same speci-men. As the preservation of the specimens was similarin each case, we concluded that mRNA expression andprotein expression were not usually simultaneous inindividual tumour cells, and showed heterogeneity ofdistribution in individual tumours. In contrast, non-CCA cases hardly expressed IGFBP-1 at either themRNA or protein level. These results indicated thatIGFBP-1 expression is a feature of ovarian CCA andmight represent a characteristic phenotype.

No previously reported study has focused in detailupon IGFBP-1 expression in ovarian adenocarcinomausing human histological specimens. In general,IGFBP-1 plays an important role in human femalereproductive physiology,3,4 and we have confirmed itsspecific expression in secretory endometrial stromalcells and decidua of the placenta by in situ hybrid-ization.1 IGFBP-1 was recognized formerly as pla-centa-specific tissue protein 12 (PP12), and Inabaet al.15 have investigated PP12 expression in speci-mens of ovarian cancer. They discussed the expres-sion of pregnancy-specific protein 1 (SP1), PP5,PP10, PP11 and PP12 in cases of mucinous andserous cystadenocarcinoma by immunohistochemis-try, and concluded that these proteins, especially PP5and PP11, might be useful markers for monitoringpatients with ovarian adenocarcinoma and for earlydiagnosis. However, no CCA cases were included intheir study, and they did not mention PP12 expres-sion in cases of CCA.

Conversely, some IGFBPs, except for IGFBP-1, havebeen discussed in relation to their association with thedevelopment of, and susceptibility to, ovarian can-cer.7,16 Using tissue microarray analysis, Wang et al.8

demonstrated that the expression of IGFBP-2 andIGFBP-5 differed among high-grade ovarian SA, CCAand MA, and concluded that both IGFBPs are impor-tant for the development of high-grade SA cases alone.Some studies have shown that serum IGFBP-2 levelsare elevated in early and advanced ovarian cancers,regardless of histological type, and that increasedexpression of IGFBP-2 mRNA can be identified inovarian cancer at any stage.9 A recent study by Martinet al. suggested for the first time that insulin-likegrowth factor 2 mRNA-binding protein 3 (IGF2BP3,also known as IMP3) could be a prognostic biomarkerof ovarian CCA.17 In the present study, there was norelationship between the sensitivity of IGFBP-1 expres-sion and pathological T and N factors of the TNMclassification in CCA patients.

HNF-1b has been established recently as a specifichistological marker of CCA, as first reported by Tsuchiyaet al., and its specific expression has already beenconfirmed by many researchers.10,13,18–20 HNF-1 is atranscription factor expressed mainly in the liver andkidney, and regulates the promoters of several genes.HNF-1 consists of two isoforms: HNF-1a and HNF-1b. Ithas been revealed that HNF-1a binds to the promoterregion of the IGFBP-1 gene and regulates its expres-sion.11,12 In the present study, we found that all theIGFBP-1-positive cases of CCA also expressed HNF-1b,another isoform of HNF-1, and we speculate that bothHNF-1b and IGFBP-1 might represent a characteristicphenotype of CCA. Indeed, the sensitivity of IGFBP-1expression was lower than that of HNF-1b in many CCAcases, but we conclude that IGFBP-1 might also be amore specific marker of CCA than HNF-1b.

We also performed immunohistochemistry for IG-FBP-1 and HNF-1b in Arias-Stella glands in miscar-riage material, as the histology of Arias-Stella glandsmimics that of CCA. The Arias-Stella reaction has beenrecognized classically as a frequent histological sign ofpregnancy in endometrial glands, and is often noticedin routine DC materials from abortion cases. The mostcharacteristic histological feature of Arias-Stella glandsis conspicuous clear cell change with hypersecretoryfeatures, and the nucleus sometimes exhibits notableatypia. Some studies have emphasized the importanceof differential diagnosis, including benign clear celllesions and malignant clear cell neoplasms, because ofthe histological similarity of gynaecological clear cellmalignancy and glands showing the Arias-Stella reac-tion.21,22 Such clear cell lesions can sometimes presentdiagnostic difficulty if the Arias-Stella reaction occursin the absence of pregnancy in older women. We havedemonstrated that Arias-Stella glands show both cyto-plasmic expression of IGFBP-1 and nuclear expressionof HNF-1b, and that the expression pattern of the twomarkers is similar to that in CCA. These results indicatethat CCA and Arias-Stella endometrial glands sharecommon features, not only morphologically but alsoimmunophenotypically. Placental decidua and decidu-alized endometrial stromal cells also express IGFBP-1protein strongly.

In some situations, the distinction between clear celladenocarcinoma and endometrioid adenocacinomashowing secretory change may be difficult becauseboth of the tumour cells have clear cytoplasm. Thesecretory variant of endometrioid adenocarcinomaoften consists entirely of tumour cells with clearsubnuclear or supranuclear vacuoles, similar toendometrial epithelium in the early secretory phase.Among 22 ovarian endometrioid adenocarcinoma



cases examined in this study, there was no endomet-rioid adenocarcinoma showing secretory change. Weperformed immunohistochemistry for IGFBP-1 in sev-eral cases of uterine endometrial adenocarcinoma withsecretory change, including the secretory variant ofendometrioid adenocarcinoma. The results showedthat no reactivity of IGFBP-1 was observed in thetumours (data not shown).

In conclusion, the expression of IGFBP-1 is morespecific in ovarian CCA than in other histological typesof ovarian cancer, in comparison with HNF-1b. Thesensitivity of IGFBP-1 is obviously lower than that ofHNF-1b in CCA cases, and no clinicopathologicalsignificance of IGFBP-1 expression in CCA patientswas shown. IGFBP-1 may represent a characteristicphenotypic component of CCA, and is a potentiallyspecific marker of this cancer.

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