ICANCERRESEARCH56.325-329.January15.19961 Expression of a-1,3-Fucosyltransferase Type IV and VII Genes Is Related to Poor Prognosis in Lung Cancer Jun-ichi Ogawa,1 Hiroshi Inoue, and Shirosaku Koide First Department ofSurgerv. School ofMedicine, Tokai University, Bohseidai, Isehara, Kanagawa, 259-11, Japan ABSTRACT MATERIALS AND METHODS To date, five a-1,3-fucosyltransferasegenes(Fuc-Till, IV, V, VI, and Materials. Three hundred thirty-three consecutive patients with lung can VI!) have been cloned. To examine the role of a-1,3-fucosyltransferase in cer who hadundergonecurativetumor resectionandlymph nodedissection the synthesisofsialyl Lewisx and the prognosisoflung cancer,PCR from 1980 to 1993 were included in this study. Staging was according to the amplificationof five fucosyltransferasegenesandimmunohistochemical AmericanJoint Committeeon CancerThM classification(17). Patientswith staining of sialyl Lewis x wereperformed in 333patientswith lung cancer positive surgical margins were excluded. Procedures at the time of surgery whounderwentsurgicalresectionfrom1980to 1993.The frequenciesof were the same for all patients. Of the patients with non-small cell carcinoma, Fuc-TJIIIV, Fuc-TVI, Fuc-TIV, and Fuc-TVJI expression were 9%, 26%, none with stage I or II disease receivedadjuvanttherapy. Patientswith stage ifi 75%, and 66%, respectively. The frequency of sialyl Lewis x expression disease received postoperative chemotherapy or radiation therapy. Those with (75%) was comparable to Fuc-TIV and Fuc-TVII expression.However, smallcell carcinomareceivedpreoperativeandpostoperativechemotherapy. the grading of sialyl Lewis x staining correlated only with the grading of DNA Extraction. DNA was extracted from formaldehyde-fixed, paraffin Fuc-TVII gene amplification. Survival of the patients whose tumors embedded blocks (18). Three 3(L@m-thick sections were prepared from each blockaftercuttingawayasmuchof theparaffinaspossible.Thesectionswere showed strong expression of Fuc-TIV and/or FucT-Vil was significantly immersed in 1 ml xylene at room temperature for 30 mm and centrifuged for shorter than that of the patients whose tumors did not express either 5 mm at 15,000x g twice. After discardingthe supernatant,thepellet was Fuc-TIV or Fuc-TVII. These results suggest that Fuc-TIV and Fuc-T VII resuspendedin1 ml 100%ethanolandcentrifugedfor 5 mm at 15,000X g. expression may be of prognostic value among patients with lung cancer by Thepelletwaswashedagainwith 100%ethanol,driedthoroughly,and500 @l participating in the biosynthesisof sialyl Lewis x. buffer (50 mM Tris-HC1,1 mi@iEDTA,and 0.5% Tween20, pH 8.5) with proteinase K (200 @g/ml;WaKO Pure Chemicals, Osaka, Japan) were added. Incubationwasovernightat 48°C.Nucleicacid wasextractedtwice using 1 INTRODUCTION volumephenol:chloroform:isoamylalcohol(25:24:1)andthenextractedonce with 1 volume chloroform. The DNA was precipitated by the addition of 2.5 The SLex2 carbohydrate structure has been found on the surface of volumes 100%cold ethanol,incubationat —80°Cforat least30 mm, and various human cancer cells. Its expression plays an important role in centrifugation for 20 mm at 15,000 X g. The pellet was washed with 75% E-selectin-mediated adhesion to activated endothelium (1—3),leading ethanol,dried thoroughly,andresuspendedin20 mrsiTris-HC1solution(pH @ to hematogenous metastasis of cancers (4). Fuc-Ts are thought to be 7.5) to a final DNA concentrationof 1 The DNA concentrationwas key enzymes which regulate synthesis of SLex. Up to now, at least determinedspectrophotometrically. three distinct types of human Fuc-Ts can be distinguished by their PCR Analysis of Fuc-T. The primersequencespreparedfor PCR ampli enzymatic specificity (5), and some of their genes have been cloned fication of Fuc-T were as follows: Fuc-TllhIFuc-TV sense, 5'-CTGCTGGT GQCTGTGTGmCTfCTCCFAC-3' (positions 70-99/70-99) and anti (6—12).TheLewis type a-l,3/l,4-fucosyltransferase, found in kidney, sense, 5'-CAGCCAGCCGTAGGGCGTGAAGATGTCGGA-3' (positions gallbladder, and milk, is known to catalyze the transfer of fucose to 487—516/526—555). The size of the fragments amplified from Fuc-Till and both type 1 and type 2 chain-based structures. Fuc-TIII corresponds to Fuc-TV were 447 and 486 bp, respectively. Fuc-TIV sense, 5'-GCITGC this Lewis type. The myeloid type Fuc-T, found in leukocytes and CCGAAATI'GGQcTCCTGCACAC-3' (positions 807—833)and antisense, brain, catalyze the transfer of fucose to type 2 chain-based structures, 5'-CCAGAAGGAGGTGATGTGGACAGCGTA-3' (positions 1099-1125). but is much less active on sialylated substrates. Fuc-TIV corresponds The sizeof the fragmentamplifiedwas319 bp. Fuc-TVI sense,5'-CTCAA to this myeloid type. The plasma-type Fuc-T, found in plasma and GACGATCCCACTGTGTAC-3' (positions 110-132) and antisense,5'- liver, transfers fucose to type 2 chain-based structures whether they CAGCCAGCCGTAGGGCGTGAAGATGTCGGA-3' (positions 484-513). The sizeof the fragmentamplified was404 bp. Fuc-TVII sense,5'-CTCG are sialylated or not. Fuc-TV or Fuc-TVI, which are very similar in GACATC1TrGTGCCCTATG-3' (positions 438-460) and antisense,5'- their structures, correspond to this plasma type. In addition to the CGCCAGAA'1TFCTCCGTAATGTAG-3'(positions702-725). The size of aforementioned Fuc-T, a novel one designated Fuc-TVII recently has the fragmentamplifiedwas288bp. One @xggenomicDNA wasamplifiedin been cloned from human leukocytes (13, 14). This enzyme can a 50-pi reactionmixture containing25 pmol of primers, deoxynucleotide efficiently utilize sialylated type 2 chain-based acceptorsbut not those triphosphates (2.5 mM), 10 mM Tris-HC1 (pH 8.0), 50 mM KCI, 1.5 mM MgCI2, thatare not sialylated. 0.01%gelatin,and 1.5units Taq polymerase(TakaraSchuzo,Otsu,Japan). In a previous study, we have demonstrated that immunohistochem Forty cycles of amplification were performed with a thermal cycler (Iwaki Glass,Funabashi,Japan).Eachcycleconsistedof 1mmdenaturationat95°C, ical expression of SLex correlates with the prognosis in lung cancer 1mmannealingat60°C,and2 mmextensionat72°C.ElectrophoresisofPCR (15, 16). However, the question of which fucosyltransferase is related products (10 @tl)ona 2% agarosegel containing ethidium bromide (0.5 @g/ml) to the SLex expression and also to the correlation with prognosis wasdoneto evaluatethesizeof generatedfragments.Positivecontrolreactions remains to be answered. Therefore, we examined the expression of were performed with DNA template from the same patient whose DNA five Fuc-T genes in patients with lung cancer. templatestronglyexpressedallof the Fuc-Tgenes(Fig. 1,case1).Negative control reactions were done without DNA template. The grading of gene expression was as follows: (—),no expression; (+), weak expression in which Received 8/29/95; accepted I 1/7/95. The costsof publicationof this article weredefrayedin part by the paymentof page PCR productswere visible but much weakerthan the positive control; and charges. This article must therefore be hereby marked advertisement in accordance with (+ +), strongexpressioncomparabletothe positivecontrol. 18 U.S.C. Section 1734 solely to indicate this fact. InununohistochemicalExpressionof SLex. A streptavidin-biotinylper @ To whom requests for reprints should be addressed. Phone: 463-93-1 121; Fax: 463-95-7567. oxidase complex method using FH6 monoclonal antibody (kindly provided by 2 The abbreviations used are: SLex, sialyl Lewis x; Fuc-TIll—Vil, a-1,3-fucosyltrans OtsukaPharmaceutical,Tokyo,Japan)againstsialyl dimeric Lewis x were ferasetypesIll—VII. performed on thin sliced sections as previously reported (15). Immunohisto 325 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1996 American Association for Cancer Research. EXPRESSION OF Fuc-T IN LUNG CANCER Table I Patientcharacteristics guished (Fuc-TIII 447 bp versusV 486 bp, Fuc-TIV 319bp versusVII patientsNo. No. of 288 bp), they were in separatelanes. ofpatients333Age Relationship between Fucosyltransferaseand SLex Expression. 64)SexMale231Female102Histological(yr)32—84 (median, The frequencies of expression (+) and (+ +) of Fuc-T were 9% for Fuc-TIII/V, 26% for Fuc-TVI, 75% for Fuc-TIV, and 66% for Fuc TVH, respectively. The frequencies of Fuc-TIV and Fuc-TVII expres typesAdenocarcinoma172Squamous sion were significantly greater than Fuc-TIII/V and Fuc-TVI. The 18Large cell carcinomaI frequency of expression (+) and (+ +) of SLex was 75%, which was cellcarcinoma24Small comparable to that of Fuc-TIV and Fuc-TVII. However, the grading cellcarcinoma15Adenosquamous of SLex staining correlated only with the grading of Fuc-TVII (Table carcinoma4Stage―I162II40III131acell 2; P < 0.01). Survival According to SLex, Fuc-TIV, and Fuc-TVII Expres sion. Survival of patientswho had tumorswith SLex (+ +), Fuc-TIV (+ +), and Fuc-TVII (+ +) expression was significantly shorter than Stages were in accordance with the AJCC staging system (17). that in patients with SLex (—)or (+), Fuc-TIV (—)or (+), and Fuc-TVII (—)or (+) expression, respectively. Survival difference chemicalstainingwasexaminedinatleast1000cellsin five high-powerfields between the patients with (—)and (+) expression of SLex, Fuc-TIV, (magnification x 1000). The grading of SLex staining was as follows: (—), and Fuc-TVII was not significant (Fig. 2). The expression of Fuc <5% of cells had membraneousstaining;(+), 5—29%ofcells stained;and Till/V and Fuc-TVI did not significantly affect the survival of patients (+ +), 30%or morecellsstained.NegativecontrolsomittedtheFH6antibody. (data