Anatomic Pathology / PGP9.5 Expression in Cancer-Associated Fibroblasts

Significance of PGP9.5 Expression in Cancer-Associated Fibroblasts for Prognosis of Colorectal Carcinoma Yuri Akishima-Fukasawa, MD, PhD,1 Yoshinori Ino,1 Yukihiro Nakanishi, MD, PhD,1 Ayaka Miura,1 Yoshihiro Moriya, MD, PhD,2 Tadashi Kondo, MD, PhD,3 Yae Kanai, MD, PhD,1 and Setsuo Hirohashi, MD, PhD1 Key Words: Colorectal cancer; Cancer-associated fibroblast; PGP9.5; Prognosis; Immunohistochemistry DOI: 10.1309/AJCPRJP39MIDSGBH

Abstract To assess the expression of a cancer-associated fibroblasts (CAFs) marker as an indicator of prognosis, we raised anti–protein gene product 9.5 (PGP9.5) monoclonal antibody against cultured fibroblasts. PGP9.5 expression in cultured normal fibroblasts was increased by transforming growth factor β stimulation, indicating the phenotypic alteration to activated fibroblast. We immunohistochemically evaluated PGP9.5 expression with the CAFs of 110 colorectal cancer cases under T3 stage. PGP9.5 immunoreactivity in 30% or more of CAFs was defined as high PGP9.5 expression, and the other cases were considered as having low PGP9.5 expression. Patients with high PGP9.5 expression (42.7%) had significantly shorter survival and a higher incidence of recurrence than the low PGP9.5 expression group (P = .002 and P < .001, respectively). Multivariate analysis indicated PGP9.5 expression as an independent prognostic factor for overall and recurrence-free survival partly as well as lymph node metastasis. These results indicate that PGP9.5 expression in CAFs is a helpful finding to represent the overall biologic behavior of advanced colorectal cancer.

Malignant tumors consist of cancer cells and cancerassociated host cells, and the latter have been considered important for their participation in tumor development, as well as in invasion and metastasis.1-5 As cancer-associated cells, fibroblasts acquire a modified phenotype and have a pivotal role in the process of cancer spread.6 Fibroblasts in the cancer stroma, showing characteristics of myofibroblasts, are frequently called cancer-associated fibroblasts (CAFs).7,8 CAFs can produce cytokines, growth factors, and extracellular matrix–degrading proteases and, in turn, regulate the growth and progression of carcinoma cells.7,9,10 One of the well-appreciated immunohistochemical markers for CAFs is α-smooth muscle actin (α-SMA) that has been used for studies of CAFs of some cancers.8,11,12 However, the signals that mediate the alteration of fibroblast phenotype are not fully understood because there is no reliable marker detecting CAFs, except α-SMA. Protein gene product 9.5 (PGP9.5), known as ubiquitin carboxyl-terminal hydrolase L1, that catalyzes the hydrolysis of C-terminal ubiquitin esters and amides has an important role in protein degradation through the recycling of free ubiquitin by cleaving ubiquitinylated peptides.13 PGP9.5 expression is found in fibroblasts in vivo and in vitro. PGP9.5 expression takes place in cultured dermal fibroblasts and in human cutaneous wounds at the later phase of repair.14,15 As for the relation of PGP9.5 to cancer cells, its up-regulation is demonstrated in various tumors, including leukemia; some types of carcinomas such as esophageal, colorectal, pancreatic, lung, and breast; and various mesenchymal neoplasms such as nerve sheath, (myo)fibroblastic, and vascular tumors.16-19 However, there has been no attempt to focus on PGP9.5 expression specifically in fibroblasts of cancer stroma

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and its role in the biologic behavior of colorectal cancer, while paying attention to prognosis. In this study, we assessed PGP9.5 expression in CAFs in colorectal cancer by immunohistochemical analysis using the antibody we raised, and explored the alterations in PGP9.5 expression in cultured dermal fibroblasts with transforming growth factor (TGF)-β stimulation. Moreover, we investigated the relationship between PGP9.5+ fibroblasts in cancer stroma and the biologic behavior of advanced colorectal cancer.

Materials and Methods Cell Culture CAFs were obtained from surgically resected colorectal cancer tissues at the National Cancer Center Hospital, Tokyo, Japan. The tissue was minced into 2- to 3-mm fragments and seeded into Dulbecco modified Eagle medium (Sigma, Tokyo, Japan) containing 10% fetal bovine serum and penicillin/ streptomycin (Invitrogen, Carlsbad, CA).11 Specifically, the procedure, which is based on the selective growth advantage of fibroblasts under the culture conditions used, allowed for a 100% pure fibroblast population, as confirmed by positive staining for vimentin and α-SMA and negative staining for cytokeratin (data not shown). The fibroblasts were then cultured in the same medium. Fibroblast cultures were used until passage 10. This study was approved by the Ethics Committee of the National Cancer Center, Tokyo. Production of the Monoclonal Antibody Membrane/organelle fractionation was performed on whole-cell lysates of 4 × 10-cm dishes of subconfluent fibroblasts obtained by using a ProteoExtract Subcellular Proteome Extraction kit (Calbiochem, Darmstadt, Germany) according to the manufacturer’s instructions. After electrophoresis, whole gel elution was performed using a whole gel eluter (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions, and the membrane/organelle fraction was separated into 10 fractions. Each fibroblast fraction was used to immunize a BALB/c mouse, and hybridomas were produced as described previously.20 The obtained monoclonal antibodies (mAbs) were selected on the basis of fibroblast staining of formalin-fixed, paraffin-embedded sections of several cancer tissues. Some of these established antibodies were immunopositive with smooth muscle cells and CAFs or with fibroblasts weakly. Of 9 antibodies, 1, an NCC-2711 antibody, was immunopositive in CAFs clearly and negative for smooth muscle cells. Western Blot Analysis CAFs obtained from colorectal cancer tissues were lysed on ice for 30 minutes with lysis buffer (RIPA buffer, 72 72

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1 mmol/L calcium chloride, 1 mmol/L magnesium chloride, and protease inhibitor cocktail tablets; Roche Diagnostics, Mannheim, Germany). After centrifugation (15,000 rpm for 30 minutes), the supernatant was collected as the soluble fraction. Samples of 10 μg of protein per lane were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to an Immobilon-P transfer membrane (Millipore, Billerica, MA) as described previously.20 After blocking, the membranes were incubated with NCC-2711 antibody followed by incubation with peroxidase-conjugated secondary antibody (GE Healthcare, Little Chalfont, England). Peroxidaselabeled bands were visualized by using an ECL kit (GE Healthcare). As a loading control, the same membrane was reprobed with an anti–β-actin mAb (dilution 1:500; SigmaAldrich, St Louis, MO). Protein Identification by Mass Spectrometry The proteins immunoprecipitated by the NCC-2711 antibody raised from fibroblast lysate were subjected to SDS-PAGE. Each protein was visualized using a negative gel stain kit (Wako Pure Chemical Industries, Osaka, Japan), and its band was excised from the gel. In-gel digestion was then carried out with trypsin (Promega, Madison, WI), as described previously.21 Mass spectrometric analyses of trypsin digests were done using LTQ (Thermo Electron, Waltham, MA) and the Mascot program (Matrix Science, Boston, MA), using the Swiss-Prot database for protein identification. The Mascot score for the identified proteins was based on the peptide ions score (P < .05) (http://www.matrixscience.com). The Mascot score for PGP9.5 was 446, and the other significant proteins were not identified. Then, the antigen of NCC-2711 antibody was identified as PGP9.5. TGF-β Stimulation of Dermal Fibroblasts Normal human dermal fibroblasts (Takara Holdings, Kyoto, Japan) were cultured in an FGM-2 Bullet Kit (Takara Holdings) containing a special medium for fibroblasts. Experiments were performed on these cell cultures between passages 3 and 10. When subconfluent, normal human dermal fibroblasts were washed and incubated in culture medium without supplement for 24 hours. TGF-β (2 ng/mL; R&D Systems, Minneapolis, MN) was added, and, after 3 days, the cells were subjected to immunofluorescence staining and Western blotting. For neutralization of TGF-β bioactivity, anti–TGF-β antibody (0.1 μg/mL; R&D Systems) was used according to the manufacturer’s instructions. Western blotting studies were performed by using the anti-PGP9.5 mAb (established antibody, NCC-F2711; dilution 1:1,000), α-SMA mAb (clone 1A4, dilution 1:500; DAKO, Tokyo, Japan), and β-actin (Sigma-Aldrich). © American Society for Clinical Pathology

Anatomic Pathology / Original Article

Immunofluorescence Double Staining

Immunohistochemical Analysis

For immunofluorescence staining, normal human dermal fibroblasts were grown to subconfluence on glass coverslips and then fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) on ice for 30 minutes and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS solution for 1 minute. Coverslips were washed briefly in PBS and incubated with primary antibody. All antibodies were diluted in 2% normal swine serum. Fluorescein isothiocyanate–labeled anti–α-SMA mAb (1A4, dilution 1:50; GeneTex, San Antonio, TX) and biotinylated antiPGP9.5 mAb (NCC-F2711, dilution 1:100) followed by streptavidin-Alexa Fluor 594 (Nanoprobes, Stonybrook, NY) were used for double staining.20 The samples were washed extensively in PBS and mounted with Perma Fluor (Lipshaw Immunon, Pittsburgh, PA). Samples were examined with a confocal microscope (LSM5 PASCAL; Carl Zeiss Jena, Jena, Germany) equipped with a 15-mW krypton/argon laser.

After deparaffinization, all sections were subjected to antigen retrieval in citrate buffer (10 mmol/L, pH 6.0) at 121°C for 10 minutes. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol for 20 minutes. The sections were then incubated with mouse anti-PGP9.5 (NCC-F2711, dilution 1:1,000) antibody at 4°C overnight. The sections were washed in PBS and incubated with biotin-labeled antimouse IgG antibody and avidin-biotin complex (ABC kit, Vector Laboratories, Burlingame, CA). Immunoproducts were visualized using diaminobenzidine tetrahydrochloride, and the sections were counterstained with hematoxylin. As an internal positive control for PGP9.5 staining, the immunopositivity of normal neural cells and nerve fibers in neuroplexus was used. Sections from each paraffin block were used as negative control samples by replacing the primary antibody with normal mouse immunoglobulin. All immunostained sections were evaluated by 2 observers (Y.A.-F. and Y.I.) who were blinded to any patient information. PGP9.5 positivity was assessed according to the following criteria: Staining was designated as positive when almost equal staining to that of neural cells was observed, and negative for that showing absent or weaker staining than that of neural cells. The tumors were categorized by the ratio and localization of immunopositive fibroblasts in the sections of maximum tumor diameter. When the proportion of fibroblasts positive for PGP9.5 was 30% or more, the tumor was defined as showing high PGP9.5 expression, whereas tumors with a lower proportion of immunopositivity were defined as showing low PGP9.5 expression. The cutoff index was decided on the basis of a level that divided the entire group into approximately half.

Patients and Samples Between 1996 and 1997 at the National Cancer Center Hospital, 110 patients underwent surgery for primary colorectal carcinomas, including 63 colon (57.3%) and 47 rectal (42.7%) cancers. Sample selection was restricted to consecutive cases of only well- and moderately differentiated adenocarcinomas diagnosed as T3 without distant metastasis according to the International Union Against Cancer TNM classification.22 The reason for the restriction to only cases of well- and moderately differentiated adenocarcinoma was based on the difficulty evaluating the stroma around the cancer cells in cases of poorly differentiated adenocarcinoma and mucinous carcinoma (about 3%). Of the 110 cases, 55 (50.0%) were classified as stage II and 55 (50.0%) as stage III. All patients underwent curative resection, defined as the removal of gross cancer and the demonstration of tumor-negative surgical margins by histopathologic examination of the total circumference. No patients received preoperative chemotherapy, and all patients were free of distant visceral metastases. The patients comprised 67 men and 43 women, ranging in age from 32 to 93 years (mean ± SD, 61.6 ± 11.3 years). Postsurgical follow-up studies were completed for all patients, with follow-up periods ranging from 126 to 2,452 days (median, 1,999 days). Postsurgical recurrence was diagnosed by ultrasonography and computed tomography. All of the available routinely processed, formalin-fixed, paraffin-embedded blocks of colorectal carcinoma were obtained. Sections containing the maximum diameter of the tumor were used in the present study. This study was approved by the Ethics Committee of the National Cancer Center, Tokyo.

Statistical Analysis The relationships between clinicopathologic characteristics and the proportion of PGP9.5-immunopositive fibroblasts were analyzed by variance test as appropriate. The χ2 test was used to analyze variables such as sex, tumor location, lymphatic vessel invasion, blood vessel invasion, and lymph node metastasis. The Student t test was used for statistical comparisons of variables such as age and tumor size, and the Mann-Whitney U test was applied to compare the TNM stage. Deaths of causes other than colorectal cancer were treated as censored cases. Overall survival, recurrence-free survival, liver metastasis-free survival, and lung metastasis-free survival were determined from the date of surgery to the end of follow-up, death, recurrence, liver metastasis, and lung metastasis, respectively. Survival curves were obtained by using the Kaplan-Meier method and compared by using the log-rank test. Univariate and multivariate survival analyses were performed by using the Cox proportional hazards regression model with the StatView, version 5.0 (SAS, Cary, NC) software package in a stepwise manner.

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Results PGP9.5 Expression Is Mediated by TGF-β By immunofluorescence microscopy, a small population of human dermal fibroblasts expressed PGP9.5 and/or α-SMA under standard culture conditions. By TGF-β (2 ng/ mL) treatment, there was a pronounced increase in the number of PGP9.5+ and/or α-SMA+ cells. It was furthermore accompanied by a morphologic change from a typical spindle shape ❚Image 1A❚, ❚Image 1B❚, and ❚Image 1C❚ to an irregular shape, nuclear swelling, and cytoplasm with abundant stress fibers, resembling the characteristics of myofibroblasts ❚Image 1D❚, ❚Image 1E❚, and ❚Image 1F❚. Expression of PGP9.5 and α-SMA was colocalized in stress fibers of TGF-β-stimulated fibroblasts (Images 1D-1F). The results of immunofluorescence were confirmed by Western blotting. Expression of PGP9.5 and α-SMA was considerably increased by TGF-β stimulation and then blocked by neutralization of TGF-β with anti-TGF-β antibody ❚Image 1G❚. PGP9.5 Expression in Colorectal Cancer Immunoreactivity for PGP9.5 was localized in the cytoplasm and nucleus of fibroblasts around cancer cells. The PGP9.5+ fibroblasts were large and contained a large oval nucleus ❚Image 2A❚. As an internal positive control for PGP9.5 staining, normal neural cells and nerve fibers were strongly positive for PGP9.5, whereas PGP9.5 expression was absent in smooth muscle cells and endothelia ❚Image 2B❚. PGP9.5 was immunopositive to some extent for cancer cells ❚Image 2C❚. PGP9.5 expression in cancer cells was also localized in the cytoplasm and nucleus. Fibroblasts expressed PGP9.5 strongly in densely fibrotic areas with scant invasion of cancer cells or those in the stroma with abundant extracellular matrix ❚Image 2D❚. On the other hand, stroma with fine and elongated collagen fibers and thin spindle-shaped fibroblasts that were stratified into multiple layers in and around cancer showed weak or sparse PGP9.5 expression ❚Image 2E❚. Of the 110 cases, 47 (42.7%) exhibited immunopositivity in 30% or more of the spindle cells in the cancer stroma. Patients with tumors exhibiting high PGP9.5 expression showed significantly shorter survival than patients with tumors exhibiting low PGP9.5 expression (P = .002 and P < .001, respectively; log-rank test for overall and recurrence-free survival rates) ❚Figure 1❚. Also, patients whose tumors showed high PGP9.5 expression had a significantly higher incidence of liver metastasis than patients whose tumors showed low PGP9.5 expression (P = .028; log-rank test for liver metastasisfree survival rate), but the degree of PGP9.5 immunopositivity had no significant prognostic value for lung metastasis (P = .112; log-rank test for lung metastasis-free survival rate). The association of the degree of PGP9.5 immunopositivity in the cancer stroma with clinicopathologic findings is shown in 74 74

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❚Table 1❚. PGP9.5 expression in the cancer stroma was also associated with TNM stage (P = .019) and tended to also associate with lymph node metastasis (P = .054). The results of univariate analysis using the Cox proportional hazards model are shown in ❚Table 2❚. PGP9.5 expression in the cancer stroma was found to be a significant prognostic factor for overall and recurrence-free survival together with lymph node metastasis. In addition, by multivariate analysis, the percentages of PGP9.5 immunopositivity in the cancer stroma and lymph node metastasis were independent prognostic factors for overall survival. PGP9.5 immunopositivity was an independent prognostic factor for recurrence-free survival ❚Table 3❚.

Discussion The present study demonstrates that PGP9.5 expressed in CAFs can be considered a new useful marker for the biologic behavior of colorectal cancer. By immunohistochemical analysis, PGP9.5 expression raised for this study was consistently detected in CAFs of colorectal cancer to various extents. The prognosis of patients with increased PGP9.5 expression in the cancer stroma significantly followed a more severe TNM stage and tended to have a higher incidence of lymph node metastasis. In addition, more frequent liver metastasis occurred in cases with high expression than in cases with low PGP9.5 expression. Furthermore, the degree of PGP9.5 expression was a distinct independent factor predictive of overall and recurrence-free survival by subsequent multivariate analysis. PGP9.5 expression in normal human dermal fibroblasts increases as a result of TGF-β stimulation. TGF-β is considered to have a central role in the alteration of fibroblastic phenotype to activated conditions.10 In our results, the fibroblasts stimulated by TGF-β altered not only PGP9.5 expression but also the phenotype of fibroblasts in which the stress fibers were colocalized by immunofluorescent microscopy. These findings indicated that the increase of PGP9.5 expression in fibroblasts showed the activated condition of fibroblasts, simultaneously exhibiting the characteristic of myofibroblasts. In the present study, we focused on CAFs expressing PGP9.5 because no investigation for the role of PGP9.5 had been attempted in CAFs. Immunohistochemical and in vitro studies have so far indicated that in the cancer cells of the pancreas,18 colon,17 lung,23 and breast,24,25 high PGP9.5 expression focused only on the cancer cells indicates a poorer prognosis and a more aggressive phenotype than in those with low PGP9.5 expression, ignoring the PGP9.5 simultaneously expressed in CAFs. On the other hand, PGP9.5 induces apoptosis,24,26 is hypermethylated in several types of cancers, and has tumor-suppressive activity.26-29 The roles of PGP9.5 in cancer cells are thus currently contradictory. In this study, we found © American Society for Clinical Pathology

Anatomic Pathology / Original Article

A

B

C

D

E

F

G

❚Image 1❚ Immunoreactivity of fibroblasts with PGP9.5 and α-smooth muscle actin (α-SMA) antibodies before (A, B, and C) and after (D, E, and F) treatment with transforming growth factor (TGF)-β. Normal human dermal fibroblasts were grown on coverslips and treated with TGF-β for 3 days. Subsequently, the cells were fixed and stained. Immunocytochemical studies with PGP9.5 (red) and α-SMA (green) staining are shown. A, PGP9.5 shows a punctate staining pattern in the nucleus and weak staining in the cytoplasm of untreated fibroblasts (×500). B, α-SMA expression is absent in untreated fibroblasts (×500). C, Overlay of A and B. D, Fibroblasts treated with TGF-β are larger than untreated fibroblasts and have abundant stress fibers in the cytoplasm. PGP9.5 exhibits a punctate staining pattern along the stress fibers and in nuclei (×500). E, α-SMA is present along the stress fibers (×500). F, Overlay of D and E. PGP9.5 and α-SMA are colocalized along the stress fibers. G, Western blotting of fibroblasts untreated or treated with TGF-β and neutralized TGF-β by anti–TGF-β monoclonal antibody. PGP9.5 and α-SMA expression is increased by TGF-β stimulation and not increased by neutralized TGF-β by anti– TGF-β monoclonal antibody.

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A

B

C

D

❚Image 2❚ Immunohistochemical studies of PGP9.5 in advanced colorectal carcinoma. A, PGP9.5 expression is positive in cancer-associated fibroblasts in and around cancer cells. PGP9.5 is positive in the cytoplasm and nucleus of fibroblasts whose cells and nuclei are larger than those of normal fibroblasts. Colorectal cancer cells are negative for PGP9.5 (asterisk) in this area (×600). B, PGP9.5 is strongly positive in the neuroplexus (arrow) and negative in smooth muscle cells (asterisks) in the muscularis propria. Note that the endothelia of the small artery below neuroplexus lack PGP9.5 expression (×250). C, PGP9.5 is positive not only in cancer-associated fibroblasts (arrows) but also in cancer cells (asterisks) in advanced colorectal carcinoma (×500). D, Numerous PGP9.5+ fibroblasts are observed in the cancer stroma of a case with high expression of PGP9.5 (×150). E, PGP9.5 is scarcely observed in the thin and spindle-shaped fibroblasts in the cancer stroma of case with low expression of PGP9.5 (×150).

E

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© American Society for Clinical Pathology

B Overall Survival Rate (%)

A Low PGP9.5 expression

100 80 60

High PGP9.5 expression

40 20 0

0

500

1,000

1,500

2,000

2,500

Follow-up Period (d)

Recurrence-Free Survival Rate (%)

Anatomic Pathology / Original Article

100

Low PGP9.5 expression

80 60

High PGP9.5 expression

40 20 0

0

500

1,000

1,500

2,000

2,500

Follow-up Period (d)

❚Figure 1❚ Kaplan-Meier survival curves for 110 patients with colorectal carcinoma, subdivided according to the proportion of cancer-associated fibroblasts expressing PGP9.5 in cancer stroma. Overall (A) and recurrence-free (B) survival of patients in relation to PGP9.5 expression (P = .002 and P < .001, respectively). ❚Table 1❚ Association of PGP9.5 Expression With Clinicopathologic Features in 110 Cases of Colorectal Carcinoma PGP9.5 Expression Variable

Low

High

P

Mean ± SD age (y) Sex Male Female Tumor location Colon Rectum Mean ± SD tumor diameter (cm) Lymphatic vessel invasion Present Absent Blood vessel invasion Present Absent Lymph node metastasis Present Absent TNM stage IIa IIIb IIIc

61.457 ± 10.720

61.851 ± 12.110

.857* >.999

38 25

29 18

36 27 4.883 ± 1.953

27 20 5.100 ± 1.718

49 14

38 9

41 22

32 15

26 37

29 18

37 23 3

18 22 7

* †

>.999 .545* .877 .900 .054 .019†

Student t test. Mann-Whitney U test; no mark χ2 test.

❚Table 2❚ Univariate Cox Proportional Hazards Analysis for the Candidate Variables Overall Survival

Recurrence-Free Survival

Prognostic Factor

HR

95% CI

P

HR

95% CI

P

PGP9.5 expression Age Sex Tumor location (colon vs rectum) Tumor size Lymphatic vessel invasion Blood vessel invasion Lymph node metastasis

5.123 1.010 0.430 0.865 0.930 0.463 0.565 8.768

1.669-15.724 0.967-1.054 0.140-1.320 0.334-2.243 0.714-1.213 0.106-2.027 0.184-1.733 2.003-38.368

.004 .650 .140 .766 .594 .307 .318 .004

4.495 0.984 0.945 0.846 0.921 0.320 0.486 4.122

1.769-11.421 0.949-1.021 0.409-2.184 0.373-1.918 0.733-1.158 0.008-1.365 0.180-1.309 1.529-11.115

.002 .984 .945 .846 .921 .124 .486 .005

CI, confidence interval; HR, hazards ratio.

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❚Table 3❚ Multivariate Cox Proportional Hazards Analysis for the Candidate Variables Overall Survival

Recurrence-Free Survival

Prognostic Factor

HR

95% CI

P

HR

95% CI

P

PGP9.5 expression Lymph node metastasis Tumor size Lymphatic vessel invasion Blood vessel invasion

4.851 8.289 0.846 0.933 1.380

1.494-15.756 1.641-41.884 0.618-1.159 0.184-4.723 0.394-4.831

.009 .011 .299 .933 .614

4.095 2.720 0.857 0.502 0.871

1.571-10.673 0.920-8.043 0.655-1.121 0.105-2.407 0.293-2.595

.004 .070 .261 .389 .805

CI, confidence interval; HR, hazards ratio.

only a few apoptotic cells among abundant PGP9.5+ fibroblasts by the observations for the colocalization of PGP9.5 and apoptotic cells using the terminal deoxynucleotidyl transferase– mediated deoxyuridine triphosphate-biotin nick-end labeling method (data not shown). Besides these observations, there is also no relationship between apoptosis and PGP9.5 expression in wounds.15 It is thus suggested that PGP9.5 expression in fibroblasts is not related to apoptosis, at least in CAFs, but may be rather indicative of tumor aggressiveness. In our study, PGP9.5+ fibroblasts were observed in densely fibrotic areas with scant invasion of cancer cells or fibrotic areas in the stroma with abundant extracellular matrix. In these areas, cancer cells were mainly irregular or not glandforming, ie, an invasive growth pattern at the invasive front.30 On the other hand, stroma with fine and elongated fibers and thin spindle-shaped fibroblasts that were stratified into multiple layers in and around cancer, ie, an expanded growth pattern,30 showed weak or sparse PGP9.5 expression. Some markers of CAFs are already available,9,31 the most well-known being α-SMA. However, α-SMA expression was diffusely observed in the cancer stroma and recognized not only in the CAFs but also in smooth muscle cells of the muscularis mucosae and muscularis propria,12 suggesting limitations for interpretation of α-SMA stromal positivity. In our study, PGP9.5 expression was absent in smooth muscle cells but often present in CAFs that appeared to be “activated” morphologically. The mechanism whereby PGP9.5 expression in fibroblasts works as an independent prognostic factor is still unclear, but its expression practically predicts the tumor aggressiveness. Some mAbs or polyclonal antibodies against PGP9.5 are commercially available. We have confirmed that rabbit antiPGP9.5 polyclonal antibody (dilution 1:100, DakoCytomation, Carpinteria, CA) had the same immunoreactivity as our established anti-PGP9.5 mAb in cancer sections (data not shown). It has been so far reported that there are multiple processes involved in the desmoplastic reaction that exert opposing effects on cancer behavior.32 These different processes may independently control neoplastic growth or can occur simultaneously at different locations in the same tumor. 78 78

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To understand such complex interactions between cancer cells and the surrounding matrix, it is necessary to examine not only the amount of fibrosis but also its qualitative nature.32 In our results, we observed PGP9.5 expression in CAFs at the cancer stroma to various extents in the cases. As for human cutaneous tissue, Boraldi et al14 compared the proteome of cultured dermal fibroblasts from healthy subjects of different ages and found that expression of PGP9.5 protein was reduced with age. Olerud et al15 also showed by immunohistochemical analysis that in human cutaneous wounds, PGP9.5 staining was present in the ulcer bed and adjacent to the ulcer at the later phase of repair. These findings, taken together with the findings of our study, suggest that the degree and localization of PGP9.5 expression in CAFs may be considered some of the characteristic conditions of the cancer stroma such as promoting cancer invasion and metastasis in colorectal cancer. In this study, using PGP9.5 as a marker of CAFs, we found that high PGP9.5 expression in the cancer stroma was associated with a poorer prognosis than low PGP9.5 expression in terms of overall and recurrence-free survival. High PGP9.5 expression in the cancer stroma was significantly associated with a high risk of liver metastasis, suggesting certain roles in promotion of cancer metastasis. Moreover, by multivariate analysis, the percentage of cells expressing PGP9.5 in the stroma was an independent factor predictive of overall and recurrence-free survival. Previous studies of PGP9.5 expression have focused only on cancer cells,17,18,23-29 whereas in our study, we noted that PGP9.5 was found also in CAFs and showed the relationship between its expression and prognosis. The ability to predict for recurrence in stage II colorectal cancer is of particular clinical relevance for adjuvant chemotherapy because direct evidence does not support the routine use of adjuvant chemotherapy.33 Definitive markers, such as lymph node metastasis in stage III, have not been so far identified in stage II. In our study, PGP9.5 expression in the cancer stroma is an independent prognostic factor for recurrence; the evaluation of PGP9.5 expression in the cancer stroma may identify patients with the high-risk stage II disease required for adjuvant chemotherapy. © American Society for Clinical Pathology

Anatomic Pathology / Original Article

We have established an mAb of PGP9.5 by immunizing mice with membrane/organelle fractionation of primary fibroblasts obtained from patients with colorectal cancer. PGP9.5 expression in cultured normal dermal fibroblasts was induced by addition of TGF-β, simultaneously exhibiting α-SMA expression. By immunohistochemical analysis, we elucidated that PGP9.5 expression in CAFs was well associated with the prognosis of patients with advanced colorectal cancer. From the 1Pathology Division and 3Proteome Bioinformatics Project, National Cancer Center Research Institute, Tokyo, Japan; and 2Surgery Division, National Cancer Center Hospital, Tokyo. Address reprint requests to Dr Akishima-Fukasawa: Pathology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.

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© American Society for Clinical Pathology

Am J Clin Pathol 2010;134:71-79 79

DOI: 10.1309/AJCPRJP39MIDSGBH

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