Americani Joutrnal of Pathologyq. Vol. 149, No. 5, November 1996

Copyright © Americani Society for Investigative Pathology

Scatter Factor Protein Levels in Human BreastCancers

Clinicopathological and Biological Correlations

Yan Yao,* Liang Jin,* Alexander Fuchs,tAnsamma Joseph,* Harold M. Hastings,tItzhak D. Goldberg,* and Eliot M. Rosen*From the Departments ofRadiation Oncology * andPathology,t Long Island Jewish Medical Center, The LongIsland Campus for Albert Einstein College ofMedicine, New

Hyde Park, and the Department ofMathematics,* HofstraUJniversity, Hempstead, New York

Scatterfactor (SF) is an invasogenic and angio-genic cytokine the cellular receptor of which isencoded by a proto-oncogene (c-met). We mea-sured the immunoreactive SF content (nano-grams of SF per miligram ofprotein) in tissueextractsfrom 166 breast cancers and correlatedthe values with various known prognostic pa-rameters. Invasive cancers had nearlyfour timesgreater SF content than did ductal carcinoma insitu, and the difference was statisticaly signifi-cant (P < 0.02, two-tailed t-test). However, therewere no significant differences in SF contentamong different histological types of invasivecancer. Invasive cancers that had spread to ax-illary lymph nodes exhibited higher SF contentthan did invasive cancers without regionalspread (P < 0.02), but the difference in SF con-tent between node-positive and node-negative tu-mors was not as great as that between invasiveand ductal carcinoma in situ tumors. There wasa trend toward increased SF content in largerprimary tumors as compared with smaUer tu-mors, but statistical comparison revealed bor-derline significance (0.05 < P < 1.0). There wasno significant correlation between SF content andother parameters, including estrogen receptor,progesterone receptor, DNA ploidy, S phase, orScarff-Bloom-Richardson score. We also mea-sured the content of von Willebrand factor (amarker of blood vessels) and interleukin-1 ,( (apro-infZammatory cytokine) in the same tumor

extracts. SF content showed a strong positivecorrelation with von Willebrandfactor content

(P < 0.001) but did not appear to be correlatedwith interleukin-l (. These findings suggestthat SF is correlated with several other clinico-pathological indicators of aggressive tumor

behavior, consistent with the hypothesis thatSF is a biologicalfactor that may play a role inbreast cancer pathogenesis. (Am J Pathol1996, 149:1707-1717)

Scatter factor (SF; also known as hepatocyte growthfactor) is a soluble fibroblast-derived cytokine capa-ble of stimulating motility, morphogenesis, and pro-liferation of epithelia.1`3 Structurally, SF is distinctfrom other classical growth factors. SF is a memberof the family of kringle proteins, so named becauseof the presence of triple-disulfide looped structures(kringles) that mediate protein-protein interactions.Within this family, SF is most closely related to theserum zymogen plasminogen (38% identity) and to arecently identified macrophage-stimulating protein(MSP; 50% identity).2'4 These proteins are charac-terized by an af3 heterodimeric structure generatedby an arginine-valine cleavage, with the a-chain con-taining four or five kringle domains and the 3-chaincontaining a trypsin-like serine protease homologydomain.24 In contrast to plasminogen, SF and MSP

Supported in part by grants from the United States Public HealthService (CA-64869) and the American Cancer Society (EDT-102).Dr. Rosen was an Established Investigator (award 90-195) of theAmerican Heart Association during the performance of these stud-ies.

Accepted for publication June 17, 1996.

Address reprint requests to Dr. Eliot Rosen, Department of Radi-ation Oncology, Long Island Jewish Medical Center, The LongIsland Campus for Albert Einstein College of Medicine, 270-05 76thAvenue, New Hyde Park, New York 11040.

Drs. Yao and Jin contributed equally to the studies contained inthis report.

1707

1708 Yao et alAJP November 1996, Vol. 149, No. 5

lack protease activity due to several amino acid re-placements at the catalytic triad of the ,-chain.

Various observations, including in vitro, experi-mental animal, and clinical studies support a possi-ble role for SF in the development and progression ofmalignant tumors. First, SF induces various carci-noma cell types, including mammary carcinoma, todissociate from their neighbors and stimulates theirmotility and invasiveness.1' 35-8 Second, the SF re-ceptor is encoded by a proto-oncogene (c-met).9Third, SF is a potent inducer of angiogenesis, aprocess necessary for the continued growth of solidtumors.10'11 And fourth, several experimental andclinical studies indicate that high level expression ofSF and c-met are associated with a biologically ag-gressive tumor phenotype. Transfection and overex-pression of human SF or c-met in mouse 3T3 cellsresult in a tumorigenic phenotype.12 Transfectionwith both SF and c-met induces maximal tumorige-nicity, via an autocrine loop.12 SF enhances the met-astatic phenotype of EMT6 mouse mammary carci-noma cells in an in vivo murine metastasis model.8 SFmRNA and immunoreactive protein are expressed inhuman breast cancers, and high SF content in pri-mary invasive human breast cancers was reported tobe a powerful independent predictor of relapse anddeath13 (see Discussion). Similarly, SF is present inurine and tissue from human transitional carcinomaof bladder, with the highest titers found in muscle-invasive tumors.14To further elucidate the significance of SF for hu-

man breast cancer, we measured the SF content of166 consecutively accrued samples of breast carci-noma tissue and we correlated the SF titers withother clinicopathological variables used as prognos-tic indicators and with several additional biochemicalparameters. We herein report the results of thesestudies.

Materials and Methods

Acquisition of Tissue SamplesBreast carcinoma tissue samples were obtainedfrom the Long Island Jewish Medical Center FrozenTumor Bank maintained by the Department of Pathol-ogy. One of us (A. Fuchs) serves as director of theFrozen Tumor Bank. Tissue samples from breastbiopsies were frozen in OCT compound, maintainedin liquid nitrogen storage, and listed in a computer-ized tumor registry. For each tumor accrued into theFrozen Tumor Bank, a diagnosis of cancer wasmade on a hematoxylin and eosin (H&E)-stainedfrozen section taken from that piece of tumor. The

diagnosis was subsequently confirmed on a perma-nent section. Frozen sections of tumors in the tumorbank contained at least 80 to 90% tumor. In thisstudy, consecutive samples from biopsies per-formed in 1993 and 1994 were accessed in batchesof approximately 12 to 15 each. The amount of tissueavailable varied from approximately thirty to severalhundred milligrams.

Preparation of ExtractsTissue extracts were prepared as previously de-scribed.8' 14 Samples were thawed in protein-free cellculture medium (Dulbecco's modified Eagle's me-dium) to dissolve the OCT. Tissues were thenwashed with buffered saline, cut into small pieces,and homogenized in tissue extraction buffer (20mmol/L Tris, pH 7.5, 0.5 mol/L NaCI, 0.1 mmol/Lphenylmethylsulfonyl fluoride, 5 ,ug/ml leupeptin; 4 to6 ml/g of tissue). The homogenized tissue was son-icated in an ice-water bath and then clarified bymicrofuging several times. The precipitate was re-extracted using the same buffer containing a highersalt concentration (1 mol/L NaCI). The purpose of thehigh salt extractions was to extract SF bound to cellmembrane and extracellular matrix proteins such asheparan sulfate proteoglycans. Pilot studies re-vealed that a third extraction did not yield additionalSF. The two clarified supernatants were pooled, andthe excess salt was removed by dialysis using a 12-to 1 4-kd membrane. Dialyzed extracts were assayedfor SF by enzyme-linked immunosorbent assay(ELISA) and for protein using the Pierce Micro BCAprotein assay reagent kit (Pierce, Rockland, IL). TheSF content was expressed as nanograms of SF permilligram of protein.

SF ELISAImmunoreactive SF was measured using a two-anti-body ELISA, as we previously described.14'15 Immu-Ion 11 96-well plates (Dynatech, Chantilly, VA) werecoated with anti-human SF mouse monoclonal 23C2ascites16 (1:500) in 0.1 mol/L Na2CO3 buffer, pH 9.6,overnight at 40C (100 ,tl per well). Wells werewashed four times with Tris-buffered saline (TBS; 20mmol/L Tris, 150 mmol/L NaCI, 0.05% Tween-20, pH7.5), blocked with 0.5% gelatin in TBS for 1 hour at250C, washed four times, incubated for 1 hour at250C with 100 ,l of test sample or standard (two-chain recombinant human SF (rhSF), Genentech,South San Francisco, CA), and rewashed. Wellswere then incubated with sheep anti-SF antiserum(Genentech; 1:2000) for 1 hour at 250C in 0.25%

Scatter Factor Protein in Breast Cancer 17094/P November 1996, Vol. 149, No. S

gelatin in TBS, washed four times, incubated withanti-sheep IgG conjugated to alkaline phosphatase(50 ,ug/ml; Sigma Chemical Co., St. Louis, MO) for 1hour at 250C to recognize bound anti-SF antibody,and washed again. Color was developed by incuba-tion with 100 ,lI of substrate solution (1 mg/ml p-nitrophenyl phosphate, 1 mol/L diethanolamine HCI, 2mmol/L MgCI2, pH 9.8). The reaction was stoppedby adding 50 ,tl of 3 mol/L NaOH, and absorbancewas read at 410 nm using a Dynatech multiwellspectrophotometer. Samples were serially diluted sothat some of the measurements fell within the effec-tive linear measuring range (approximately 0.2 to 4.0ng/ml rhSF).

Prior studies established the specificity of theELISA for SF. Plasminogen (which is homologousto SF), albumin, and a variety of growth factors andcytokines were not detected. In pilot tests of assayreproducibility, the interassay variability in two tothree measurements of a single extract was within5 to 10% of the mean, and the interassay variabilitywas within 15 to 20% of the mean. These findingsare consistent with the usual limitations of theELISA.

von Willebrand Factor (vWF) ELISA

We developed a two-antibody sandwich ELISA tomeasure vWF similar to that used to measure SF.Plates were coated with mouse anti-human vWFmonoclonal antibody (1:100; Dako M0616 F8/86), asdescribed above, washed four times with TBS, andblocked with TBS containing 2% bovine serum albu-min (2% BSA-TBS). Wells were then incubated with100-ul aliquots of serial dilutions of purified humanvWF standard (Dako) or test sample in 0.2% BSA-TBS for 2 hours at 25°C. After sample incubation,wells were washed four times and incubated with thesecond antibody (Dako A0082 rabbit anti-humanvWF, 1:200, in 0.2% BSA-TBS) for 1 hour at 250C,washed four times, and incubated with goat anti-rabbit IgG conjugated to alkaline phosphatase (Sig-ma; 1:1000 in 0.2% BSA-TBS) for 1 hour at 250C torecognize bound vWF. The rest of the procedure wasthe same as described above for SF. The sensitivityof the vWF ELISA was approximately 10 to 100 ng/mlvWF standard, and the linear measurement rangewas usually up to approximately 2500 ng/ml vWF.Pilot experiments revealed that vWF quantitation oftumor extracts agreed fairly well with assessment ofthe 250-kd vWF monomer by Western blotting.

Interleukin (IL)- 1(3 ELISA

IL-108 was quantitated using a sensitive and specificcommercial human IL-1f, ELISA kit (Sigma CKH-101B) according to the manufacturer's instructions.The sensitivity of the assay was approximately 4pg/ml, and the linear measurement range was usu-ally up to approximately 250 pg/ml. When a limitedquantity of tumor extract was available, the priorityfor its use was SF first, vWF second, and IL-1,B third.Therefore, slightly fewer extracts had vWF and IL-1Bassays than had SF assays.

Clinicopathological Data on PatientsFor each tissue sample studied, the following clini-copathological data were obtained from the LongIsland Jewish Medical Center Pathology Departmentreports: histological type of tumor, primary tumorsize, pathological axillary lymph node status, hor-mone receptor values (estrogen receptor (ER) andprogesterone receptor (PR) immunochemical analy-sis score (ERICA and PRICA)), DNA flow cytometry(ploidy and S-phase), and Scarf-Bloom-Richardson(SBR) score. Some of these tumor characteristicshad been determined for all or nearly all lesions (eg,ERICA and PRICA), whereas some had been mea-sured for only some of the tumors (eg, DNA flowcytometry, SBR score). The latter parameters weremost commonly determined for the tumors accruedduring the later part of the study period. Approxi-mately two-thirds of patients with invasive cancersunderwent axillary lymph node dissections. For theremaining third of invasive cancer patients and formost intraductal cancer patients, the axillary lymphnode status was not known. Because the maximalavailable follow-up is less than 3 years, we considerit to be far too early to analyze probability of survivalor probability of relapse as a function of tumor SFcontent.

Method of Data AnalysisFor each group and subgroup of patients, SF contentvalues (means and SEMs) were calculated. Thesevalues were compared using two-tailed Student'st-tests and, in some cases, with the X2 test. Whereindicated, analysis of variance was performed usingthe F-distribution.17 The appropriate use of statisticalmethodology and interpretation of the results of sta-tistical tests was overseen by one of us (H. M. Hast-ings).

1710 Yao et alA/P November 1996, Vol. 149, Vo. 5

Table 1. Scatler Factor Conlent of BFreast Cancers as a Fuinction oJ Invasion aiicl Histological Stubtipe

SF contentTumor type (ng/mg protein) n Comparison

1 DCIS2 Invasive carcinoma (all types)3 Infiltrating ductal carcinoma (IDC)4 Other invasive histologies (non-IDC)

Infiltrating lobular carcinoma (ILC)Infiltrating medullary carcinoma (IMC)Miscellaneous histologies

TubulolobularTubularMucinousLocal recurrenceMixed lobular-ductalAxillary node (IDC)

0.42 + 0.101.50 + 0.161.56 ± 0.191.29 + 0.261.67 + 0.420.77 + 0.160.73 + 0.23

1.6200

0.69 + 0.690.92 + 0.38

0.79

1914710839237911

231

1 versus 2; P = 0.017

3 versus 4; P = NSNon-IDC versus IDC; P = NS

IMC versus IDC; P = NS

n, number of tumors; NS, not significant (P > 0.1). Values listed are means + SEMs. Statistical comparisons were made using the two-tailed t-tests.

Results

SF Content as a Function ofHistopathological and Other Parameters

Tumor Invasion

Non-invasive breast cancer (ductal carcinoma insitu (DCIS)) is biologically less aggressive than inva-sive breast cancer and is associated with a very highprobability of cure.18 Among 166 cases of breastcancer in the Long Island Jewish Medical CenterFrozen Tumor Bank, 19 were non-invasive cancers(DCIS) and 147 were invasive cancers. The SF con-tent of the invasive cancer group (1.50 + 0.16 ng/mgprotein) was 3.6-fold greater than that of the DCISgroup (0.42 + 0.10 ng/mg; P < 0.02 by two-tailedt-test; see Table 1). Calculation of this P value isbased on use of the pooled variance of the DCIS andinvasive cancer groups, an assumption that biasesthe calculations toward the variance of the largerinvasive cancer group. As the variance of the inva-sive cancer group was significantly greater than thatof the DCIS group (variance ratio F = 20, corre-sponding to P << 0.05 for the F-test), the use ofpooled variance in t-test calculations would under-estimate the significance of the comparison. Whenthe SF content of invasive cancer versus DCIS wascompared using the more realistic assumption ofindependent variances, the P value was less than0.001. The latter assumption was not required in thevarious comparisons of different subgroups of inva-sive cancer presented below, as these subgroupshad similar variances.The frequency distribution of SF content values in

invasive cancer versus DCIS is shown in Figure 1. ForDCIS cases, all SF values were less than 1.5 ng/mgprotein, and more than two-thirds of the values were

less than or equal to 0.5 ng/mg. In contrast, a signif-icant proportion of invasive cancers (approximatelyone-third) had SF content values greater than 1.5ng/mg. A subgroup consisting of 11% of invasivecancers had very high SF values, ranging from 3 to14.6 ng/mg. Thus, high tumor SF content appears tobe associated with the invasive phenotype of humanbreast cancer as compared with the non-invasivephenotype.

Histological Type

Within the invasive cancer group, 108 tumors (73%)were infiltrating ductal carcinomas (IDCs) and 39 tu-mors (27%) were of other histologies (non-IDC). Theaverage SF content of the IDC group was approxi-mately 21% higher than that of the non-IDC group, butthe difference was not statistically significant (P > 0.1;Table 1). Among the non-IDC invasive histologies, in-filtrating lobular carcinomas had an approximately 2.2-fold higher average SF content than did infiltrating

0 0.01-0.5 0.51-1.0 1.01-1.5 1.51-2.0 2.01-2.5 2.51-3.0 3.01-upSF CONTENT RANGE (ng/mg)

Figure 1. DisJittihlon of tlumor SF cooiteot in invsiv'e breast ccLancerversus X(IS. Valhes are plolled/for 14 7 invasie canceC s and 19 DCIS111111ors,.

Scatter Factor Protein in Breast Cancer 1711AJP November 1996, Vol. 149, No. 5

Table 2. SF Content of Invasive Breast Cancers as a Function of Pathological Axillary Node Statuis

Lymph node status

Node negative (N-)

Node positive (N+)Total

SF (ng/mg protein)

1.23 ± 0.15 (n = 75)

2.10 + 0.44 (n = 39)1.53 0.18 (n = 114)

Comparison

N- versus N+,P= 0.018(two-tailed t-test)

medullary carcinomas. However, the numbers of tu-mors in these subgroups was relatively small, and thedifference in SF content was not significant. Thus, ifdifferences exist in the SF content as a function ofinvasive histological type, larger numbers of sampleswill be necessary to demonstrate them.

Lymph Node Status

values were 31 and 8, respectively, whereas thecorresponding percentages of node-positive tumorswere 12 and 16, respectively. These findings sug-gest that involvement of regional lymph nodes issignificantly correlated with increased SF content ofthe primary tumor in invasive breast carcinoma.

Primary Tumor Size

Pathological axillary lymph node status is the mostpowerful known prognostic indicator for invasivebreast cancer.19 Among invasive cancers, 114 pa-tients (78%) had undergone axillary lymph node dis-sections. The SF content of these patients (1.53 +0. 18) was similar to that of the entire invasive cancergroup, suggesting that they were representative ofthe larger group (Table 2). Of the 114 patients whounderwent lymph node dissection, approximatelytwo-thirds had pathologically negative nodes andone-third had positive nodes. Primary tumors fromnode-positive patients had an approximately 1.7-foldhigher SF content than primary tumors from node-negative patients, and the difference was significant(P < 0.02 by two-tailed t-test; Table 2). In addition,when SF content values were divided into low versushigh ranges (0 to 1.5 versus >1.5 ng/mg), signifi-cantly more node-positive patients than node-nega-tive patients had high SF content values (P = 0.04 byx2 test).

The distribution of SF content values in node-neg-ative versus node-positive patients are compared inFigure 2. Differences in these distributions were mostapparent at the extreme ranges of SF values (<0.5ng/mg and >3 ng/mg). Thus, the percentages ofnode-negative tumors with very low and very high SF

Primary tumor size is an independent prognosticindicator for breast cancer, although its prognosticvalue is not as great as that of axillary node status.20Table 3 shows an analysis of SF content as a functionof pathological tumor size for all 137 cases of pri-mary invasive breast cancer for which measure-ments of tumor size were available. There appearedto be a trend toward increasing SF content withincreasing tumor size, with the largest tumors (-3.0cm) having a twofold higher SF content than thesmallest tumors (<1.0 cm). Statistical comparisons

0 0.01-0.5 0.51-1.0 1.01-1.5 1.51-2.0 2.01-2.5 2.51-3.0 3.01-upSF CONTENT RANGE (ng/mg)

Figure 2. Distribution of tumor SF content in primary invasive breastcancers as afunction ofaxillary lymph node status. Values areplottedJbr 114 patients who had axillary node dissections ( 75 lymph nodenegative and 39 lymph node positive).

Table 3. SF Content as a Function of Primary Tumor Size

SF (ng/mg protein) n

0-1.9 1.33 ± 0.21 81>2.0 1.85 ± 0.27 560-0.9 1.03 + 0.23 191.0-1.9 1.42 ± 0.27 622.0-2.9 1.58 ± 0.29 23>3.0 2.03 + 0.40 33All tumors 1.54 + 0.17 137

All comparisons were made using two-tailed t-tests. NS, not significant (P > 0.1).

Comparison

0-1.9 versus >2.0; P = NS

>2.0 versus 0-0.9, P = 0.096>3.0 versus 0-0.9, P = 0.081

Tumor size (cm)

1712 Yao et alA/P Nov.ember 1996, Vol. 149, No. 5

Table 4. SF Content of Invasive Breast C'ancers as a Function of Hormnone Receptor Stattus

Hormone profile

ER+ (all)ER- (all)PR+ (all)PR- (all)ER+PR+ER+PR-ER--PR+ER-PR-

SF content(ng/mg protein)

1.46 + 0.181.59 + 0.231.61 ± 0.211.28 + 0.221.55 + 0.211.23 + 0.332.11 + 0.811.34 + 0.30

11334

1014390201 123

ComparisonER' versus ER-, P = NS

PR- versus PR+,P = NS

All subgroup comparisons; P = NS

All comparisons were made using two-tailed t-tests. NS, not significant (P > 0.1).

using two-tailed t-tests resulted in P values of bor-derline significance (between 0.05 and 0.1). Thisanalysis suggests that the relationship between SFand tumor size is not as strong as that between SFand nodal status. Although there is a suggestion thatSF content is related to tumor size, it will be neces-sary to analyze more tumors for a demonstration ofstatistical significance.

Hormone Receptor Profile

The hormone receptor profile of invasive breastcancers is both a prognostic indicator and an indi-cator of the likelihood of response of the tumor tohormonal manipulation.21 All patients with invasivetumors had ER measurements of their primary tu-mors, and all but three also had PR measurements.ER and PR were quantitated by ICA using the ERICAand PRICA programs of the cell analysis system fromBecton Dickinson (San Jose, CA). We divided the ERand PR scores into eight different categories of hor-mone receptor profiles based on whether ER and/orPR were detectable or not (+ or -; Table 4). NeitherER status nor PR status individually had any signifi-cant influence on SF content. Although ER-PR+ hadhigher than average SF content and ER+PR- pa-tients had lower than average SF content, the patientnumbers in these subgroups were relatively smalland the differences were not statistically significant.Thus, there were no apparent variations of SF levelsas a function of the hormone receptor profile.

significantly different from that of the entire invasivecancer group (Table 5). Diploid tumors had slightlyhigher SF content than aneuploid tumors, but thedifference was not significant. Very few patients hadtetraploid or multiploid tumors, so it is not possible toassess whether these ploidies are associated withalterations of SF content. Tumors with low S phasehad higher average SF content than did tumors withintermediate and high S phase, but again, the differ-ences were not significant. Finally, when SF wasanalyzed as a function of both ploidy and S phase,none of the observed differences were significant.Thus, SF content does not appear to be correlatedwith ploidy or S phase.

SBR Score

The SBR score is a modified form of histologicalgrade that takes into account architectural grade (1, 2,or 3) plus nuclear grade (1, 2, or 3) plus mitotic count(indexed as 1, 2, or 3).23 SBR scores were assigned in101 (69%) of 147 cases of invasive cancer. For pur-poses of analysis, these scores were divided into threecategories: low (3 to 4.5 of 9), intermediate (5 to 6.5),and high (7 to 9). The patients with low SBR scores hadSF content that was 1.8-fold less than those with inter-mediate/high scores, whereas intermediate and highSF scores had very similar SF content. However, com-parisons between low and intermediate and low andintermediate plus high groups yielded P values greaterthan 0.1. Thus, a definite link between SF and SBRscore could not be established.

DNA Flow Cytometry

DNA ploidy and S-phase fraction are clinicallyutilized parameters for assessment of prognosis andtherapy in some categories of breast cancer pa-

tients.22 Of the 147 patients with invasive cancers,

86 (59%) had flow cytometric DNA analysis of theirtumors for assessment of ploidy and S phase.Among these patients, the mean SF content was not

vWF and IL- 138 Protein Levels in TumorExtractsvWF Content as a Function of Tumor Invasion

and Nodal Status

vWF (also known as factor VIII-related antigen) is aprotein expressed selectively by megakaryocytes

Scatter Factor Protein in Breast Cancer 1713AJP November 1996, Vol. 149, No. 5

Table 5. SF Content of Invasive Breast Cancers as a Function of DNA Flou' Cytometry Results

Ploidy

DiploidNondiploidAneuploidTetraploidMultiploid

TotalS PhaseLow SIntermediate plus high S

Intermediate SHigh S

Indeterminate STotal

Ploidy and S phaseDiploid low S

Diploid intermediate plus high SAneuploid low S

Aneuploid intermediate plus high STetraploid low STetraploid high S

SF content(ng/mg protein)

1.92 + 0.361.56 + 0.291.53 + 0.342.41 + 0.850.73 + 0.191.76 + 0.24

2.34 + 0.531.52 + 0.221.53 + 0.471.52 + 0.250.58 + 0.211.76 + 0.24

n

47393054

86

31509

415

86

Comparison

Diploid versus nondiploid; P = NS

Aneuploid versus Tetraploid; P = NS

Low versus intermediate plus high; P = NS

2.43 + 0.68 23 Diploid low versus Diploid intermediate plushigh, P = NS

1.50 + 0.26 281.26 ± 0.77 5

2532

1.59 ± 0.383.46 ± 1.030.85 ± 0.04

Aneuploid low versus Aneuploid intermediate plushigh; P = NS

All comparisons were made using two-tailed t-tests. NS, not significant (P > 0.1).

and vascular endothelial cells that is commonly usedto identify endothelium in vitro and in vivo.24 As arough measure of the vascularity of the tumor, wequantitated vWF levels in tumor extracts using aspecific two-antibody ELISA similar to that used forSF measurement. As for SF, invasive cancers hadmuch higher vWF levels (109 + 24 ng/mg; n = 139)than did DCIS tumors (14 + 10; n = 18). The stan-dard t-test using the pooled variance of the invasivecancer and DCIS groups did not yield a significant Pvalue (P = 0.17). However, as was the case for SF,the variance of the vWF content of the invasive can-cer group was significantly greater than that of theDCIS group (F = 44; P << 0.05 for the F-test). Com-parison of the vWF content of invasive cancer versusDCIS using the more realistic assumption of inde-pendent variances yields a highly significant P valueof <0.001. Unlike SF, the vWF content of node-neg-ative and node-positive invasive cancers was similar(114 + 40 ng/mg (n = 72) and 99 + 28 ng/mg (n =

38), respectively). Thus, it appears that the vWFcontent of the extract was correlated with tumor in-vasion but not with axillary node status.

vWF as a Function of SF Content

To determine whether the vWF content of the tu-mor was correlated with the SF content, SF wasdivided into four different ranges, and the vWF con-tent (mean + SEM) was calculated for each SF

range. There was a clear trend toward increasingvWF associated with increasing SF content, with thehighest SF range (>2 ng/mg SF) showing 12-foldhigher vWF content than the lowest SF range (0.5ng/mg SF; Figure 3). The trend toward increasingvWF content with increasing SF content was statisti-cally significant. Comparison of vWF contents in the

500

c- 400.00.0-

E 300CD

zW 200z00

3 100

o0-0.50 0.51-1.00 1.01-2.00 2.01-UP(n=33) (44) (38) (24)

SF CONTENT RANGE (ng/mg protein)

Figure 3. Relationship betu'een vWF and SF content in extracts ofinvasive breast cancers. Bars indicate mean + SEM of vWF contentcorresponding to the indicated SF content range. Values in parenthesesare the number of tumors in each SF content range. Statistical com-parisons ofvWFversus SF range using two-tailed Student's t-tests wereas follows: 0 to 1.00 versus > 1.01, P < 0.001; 0 to 0.50 versus >2.01,P < 0.001; 1.01 to 2.00 versus >2.01, P = 0.012.

1714 Yao et alAJP November 1996, Vol. 149, No. 5

Table 6. SF Content as a Function of Scarff-Bloom-Richardson (SBR) Score

SBR score range

3.0-4.5 (low)5.0-6.5 (intermediate)7.0-9.0 (high)5.0-9.0 (intermediate/high)All SBR scores

SF (ng/mg protein)

1.02 + 0.251.84 + 0.311.86 + 0.401.84 + 0.251.70 + 0.21

n

17562884

101

Comparison

3-4.5 versus 5-6.5; P = NS

3-4.5 versus 5-9; P = NS

All comparisons were made using two-tailed t-tests. NS, not significant (P > 0.1).

lowest versus highest SF range yielded a P value of<0.001 (two-tailed Student's t-test), whereas com-parison of vWF content in the two lower SF ranges (0to 1 ng/mg) versus the two higher SF ranges (>1ng/mg) also yielded a highly significant P value of<0.001 (two-tailed t-test). Intercomparison of themean vWF contents of the two higher SF ranges (1 to2 ng/mg versus >2 ng/mg) also yielded a significantP value (0.012). Thus, vWF content appears to varyas a function of SF content, with the highest vWFvalues associated with the highest SF values.

IL-1,3 Content as a Function of Tumor Invasion,Nodal Status, and SF Content

IL-1,8 is a pleiotropic cytokine that generally up-regulates the inflammatory response. We were inter-ested in IL-1,3 expression because 1) IL-1p stimu-lates SF expression by fibroblasts15,25 and 2) IL-1, isa pro-angiogenic factor.26'27 IL-1f, content washigher in invasive cancers (18.8 + 2.8 pg/mg; n -125) than in DCIS (10.3 + 3.9 pg/mg; n = 16), butthe difference was not significant (P > 0.1). Similarly,IL-1p3 content was higher in node-positive invasivecancers (25.3 + 7.4 pg/mg; n = 39) than in node-negative cancers (16.7 + 3.5; n = 67), but the dif-ference was not significant. When the SF contentwas divided into the same ranges as for analysis ofvWF, there was no significant variation of IL-18 as afunction of SF content.

DiscussionSF stimulates invasion of tumor cells in vitro,5 8 in-duces angiogenesis in vivo,10'11 and is present inbreast carcinomas. Therefore, SF is a particularlyinteresting molecule to study as a potential prognos-ticator for breast cancer. Yamashita and col-leagues13 assayed the SF content of 258 primaryinfiltrating ductal breast cancers that were main-tained in frozen storage, using an ELISA similar toours. They found a wide distribution of SF contentvalues, similar to that obtained in our study. Patientswith high SF content (defined as greater than 0.2

ng/mg protein) had significantly shorter relapse-freeand overall survival than did patients with low SFcontent (less than 0.2 ng/mg protein). The prognos-tic value of SF was found to be independent of otherknown indicators such as axillary lymph node status.In that study, there was no significant correlation ofSF content with other prognostic variables, includingaxillary lymph nodes, ER, tumor grade, tumor size,and other parameters. In our study, the average SFcontent values were slightly higher than that foundby Yamashita. These differences may reflect differ-ent protein extraction methods, antibodies, and stan-dards and are not uncommon with the use of ELISAs.In pilot studies, we compared a detergent extractionmethod with isotonic saline buffer (as employed byYamashita) with our high salt extraction method andfound that the latter often yields higher extracted SFper milligram of extracted protein. Another source ofvariability that might affect SF content is heteroge-neity of tumor composition (ie, proportion of viabletumor versus necrosis, fibrosis, and reactive chang-es). As described in Materials and Methods, frozensections of tumors suggested that the tissue con-tained at least 80 to 90% tumor. The problem oftissue heterogeneity is also overcome, in part, by useof large sample numbers so that real biological dif-ferences in tumor populations can be observed.

In our study, invasive breast cancers had signifi-cantly higher SF content than DCIS. Furthermore, thevariance of the SF values for the invasive cancergroup was considerably greater than that for DCIS,indicating that invasive cancers exhibit much greaterheterogeneity of SF protein values. Similarly, wehave found that muscle-invasive bladder cancershad significantly higher SF content than non-muscle-invasive tumors and that urine from patients withmuscle-invasive bladder cancers had higher SF con-tent than urine from patients with non-muscle-inva-sive cancers14 (Rosen et al, submitted for publica-tion). These findings suggest that the accumulationof SF within tumors is correlated with the invasivephenotype, and they raise the possibility that SFmight function as a tumor progression factor. Thepopulation of invasive cancers exhibited a spectrum

Scatter Factor Protein in Breast Cancer 17154/FP November 1996, Vol. 149, No. 5

of SF content values ranging from undetectable to ashigh as 14.6 ng/mg protein. The latter value is similarto the SF content of experimental mammary tumorsin nude mice generated from human breast carci-noma cells transfected with SF cDNA (unpublishedresults). Although these tumors grew much more

rapidly than control tumors generated from untrans-fected cells, we do not yet know what minimal level ofSF production is required for enhanced in vivo tumorgrowth.

Within the group of invasive cancers, node-posi-tive tumors contained significantly higher SF contentthan node-negative tumors. Of all prognostic indica-tors for breast carcinoma, pathological nodal statusappears to be the most powerful, indicated by stud-ies from the National Surgical Adjuvant Breast Can-cer Project.19 Although follow-up in our study is tooshort to assess the prognostic significance of SFcontent in terms of clinical outcome, the linkage ofhigh SF content with nodal positivity is suggestive ofa potential linkage with poor prognosis. In addition toinvasion and axillary node status, our analysis re-

vealed a trend toward increasing SF content withincreasing primary tumor size (P = 0.08 for compar-ison between largest and smallest tumors). Patho-logical studies from the National Surgical AdjuvantBreast Cancer Project indicate that tumor size is an

independent prognostic indicator for both overallsurvival and disease-free survival in breast cancer,

although its impact is not as great as that of nodalstatus.20 Other prognostic variables that are com-

monly assessed in breast cancer patients and usedin clinical management decisions include ER/PR sta-tus, DNA content and S phase, architectural (pat-tern) grade, and nuclear grade. The latter two are

incorporated into the SBR score, which also includesthe mitotic count.23 There was no significant corre-

lation between SF content and these variables, al-though there was a tendency toward increasing SFcontent with increasing SBR score. The absence of asignificant correlation does not preclude the exis-tence of a relationship between SF and some ofthese variables. If the correlation is relatively weak,larger numbers of patients may be necessary toprove its existence.

Recent studies suggest that tumor angiogenesis,assessed by the peak density (hot spots) of stromalmicrovessels in regions of tumor, is an independentprognostic indicator for breast carcinoma,28-30 al-though some studies have failed to demonstrate a

linkage between the peak microvessel count andprognosis.31 Our results suggest a correlation be-tween the vWF and SF content of extracts of invasivebreast cancers. The vWF content of extracts was

intended as a rough index of the extent of tumorvascularity, and several cautions are indicated ininterpreting these values. This method does not dis-tinguish new angiogenic vessels from pre-existinghost vasculature. Furthermore, vWF may not be ex-pressed on small capillaries in all cases, a problemthat also occurs with vWF immunostaining to high-light endothelium for microvessel counts of paraffin-embedded tumor sections. We expect that the vWFcontent (normalized per milligram of tissue protein)would tend to reflect an average tissue vascularityrather than a peak degree of tumor-specific angio-genesis. Therefore, vWF content of extracted tissueand peak microvessel counts of paraffin sectionsmay not measure exactly the same thing. Neverthe-less, the correlation between vWF and SF content inthe same extract suggests a potential relationshipbetween the angiogenic growth factor SF and theoverall level of tumor vascularity. Additional studiesalong these lines might include investigation of thelevels of other angiogenic growth factors (eg, vas-cular endothelial growth factor) and of other markersof endothelium (eg, platelet-endothelial cell adhe-sion molecule or avf3, an integrin found on newlyformed blood vessels.32'33As IL-1,B has been reported to induce SF expres-

sion in various types of cultured fibroblasts15'25 andto induce angiogenesis in some in vivo assays,26'27we used residual extract after SF and vWF measure-ments to assay the IL-1,B content. Like SF, breastcancers exhibited a wide distribution of IL-1f3 con-tent values. However, these values were not signifi-cantly correlated with tumor invasion (invasive can-cer versus DCIS), axillary node status, or SF content.As invasion, node status, tumor size, and vascu-

larity are prognostic parameters in breast cancer,the finding of a relationship between SF and theseparameters suggests a role for SF in progression toan aggressive tumor phenotype. Based on our studyand the study cited above suggesting that SF is anindependent prognosticator for invasive breast can-cer,13 we now have prima facie evidence that SFpredicts an aggressive phenotype in a subset ofpatients. At this point, our priorities will include 1)obtaining corroborative evidence for the predictivevalue of SF in invasive breast cancer and 2) deter-mining whether the subset of tumors with high SFcontent overlaps with subsets of tumors with otherbiological indicators of adverse prognosis, such asHER2/neu oncoprotein and p53 gene mutations. Thisinformation may ultimately be useful in developmentof new targets for anti-cancer therapy.

1716 Yao et alAJP November 1996, Vol. 149, No. 5

AcknowledgmentWe are indebted to Dr. Ralph Schwall, Genentech,Inc. (South San Francisco, CA) for providing sheepantiserum against human SF as well as recombinanthuman SF used in the SF ELISA.

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