Cancer Res 1989 Schiffman 1322 6

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1989;49:1322-1326.Cancer Res

Mark H. Schiffman, Roger L. Van Tassell, Adam Robinson, et al.

ExcretionCase-Control Study of Colorectal Cancer and Fecapentaene

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[ CA N CE R R ES EA R CH 4 9, 1 32 2- 13 26 , M ar ch 1 , 1 98 9|C ase-C ontrol Study of C olorectal C ancer and Fecapentaene Excretion1M ark H . S ch iffm an,2 R og er L . V an T asseti, A dam R obinson, L ee S mith, Ja mes D anie l, R ob ert N . H oover,R oselyn W eil, Jean ne R osenthal, P adm anabhan P . N air, S am uel S chw artz, H ugh P ettigrew , S teph en C uriale ,G erald B atist, G lad ys B lock, and T racy D . W ilkinsE pid em io lo gy a nd B io sta tis tics P ro gra m, D ivisio n o f C an cer E tio lo gy, N atio na l C an cer In stitu te, B eth esd a, M aryla nd 2 08 92 [M . H . S ., R . N . H ., R . W ., H . P .];D e pa rtme nt o f A n ae ro bi c M i cr ob io lo gy , V ir gi ni a Po ly te ch ni c I ns ti tu te a nd S ta te Un iv er si ty , B la ck sb ur g, V ir gi ni a 2 4 06 1 [R. L . V ., T . D . W ] ; N a ti on al N a va l Me di ca lC en te r, B eth es da , M ary la nd 2 08 14 A .R ., S . C J; G eo rg e W ash in gto n U nivers ity H osp ita l, W as hin gto n, D C 2 00 37 [ L. S .] ; W alter R ee d A rm y M ed ica l C en ter,Wa sh in gt on , DC 20307 [ J. D .] ; We st at , I nc ., R o ck vi ll e, Ma ry la nd 2 08 50 [ J. R .] ; L ip id N u tr it io n L ab , Un it ed S ta te s D e pa rtme nt o f A g ri cu lt ur e, B el ts vi ll e, Ma ry la nd2 07 05 [P. P . N .] ;M i n ne ap o li s Me di ca l R es ea rc h Founda ti on , M i nn ea po li s, M i nn es ot a, 5 54 04 [ S. S ./ ; D iv is io n o f C anc er T re atme nt , N at io na l C anc er I ns ti tu te , B et he sd a,Ma ry la nd 2 08 92 [G . Ba j; a nd D iv is io n o f C anc er P re ve nt io n a nd C ont ro l, N at io n al C an ce r I ns ti tu te , B et he sd a, Ma ry la nd 2 08 92 [G . B I. JABSTRACT

T he f ec ap en ta en es a re p ote nt m u ta ge ns fo un d i n h ig h c on ce ntr ati on si n th e s to ols o f s om e i nd iv id ua ls . T he se c om po un ds a re p ro du ce d i n v iv ob y c om m o n s pe ci es o f t he c ol on ie m i cr of lo ra , f ro m p re cu rs or s o f u nk no w no rig in . T he fec ap entae nes h av e b ee n p ostu lated to in cre ase th e ris k o fc ol or ec ta l c an ce r. T o te st t hi s h yp ot he si s, w e m ea su re d f ec ap en ta en eex cre tio n in 6 9 p atien ts w ith ad eno carcin om a o f th e co lon o r re ctum ,n ew l y d ia gn os ed a t t hr ee W a sh in gt on , D C a re a h os pi ta ls . T h e c as es w e rec om pa re d w ith 1 14 s urg ic al c on tr ols , f re qu en cy m atc he d to t he c as es o na ge , s ex , a nd h os pit al . W e a tte mp te d t o m ea su re f ec ap en ta en e e xc re ti on4 tim es for each subject: before surgery; and at 1 m o; 3 m o; and 6 m of ol lo wi ng s ur ge ry . C on tr ar y to o ur s tu dy h yp ot he si s, w e f ou nd f ec ap enta en e e xc re tio n d ur in g th e f ou r s tu dy p er io ds to b e s im ila r o r e ve n lo we ri n c as es c om p ar ed t o c on tr ol s. A n i nd ir ec t m e as ur em e nt o f f ec ap en ta en ep re cu rs or s a ls o t en de d t o b e l ow e r i n c as es . T h e c as e- co nt ro l d if fe re nc esc ou ld n ot b e e xp la in ed a s e ff ec ts o f b le ed in g o r o f t he c ol or ec ta l d ia gn os ti cw orkup, w hich w as assessed in a separate group of 86 patients. W eco nc lu de from the se d ata tha t th e e xcretion o f fec ap entae nes d oes n otin cre as e th e risk o f c olo re ctal can cer, a t le as t w hen m ea su red n ear th et ime o f d iag n os is .INTRODUCTION

T he fec ape ntaene s are p oten t m utag en s ex creted in th e fecesof hum ans and som e anim als (1, 2). T hese ether-linked lipidsare p rod uced by co lo nie B actero id es sp ecies fro m a n u nide ntified g ro up of p recurso r co mp oun ds (3 ). T he fecap en taen es a rehighly genotoxic in a variety of bacterial and m am malian inv itr o a ss ay s, a cc ou ntin g fo r m uc h o f th e m uta ge nic ity d ete cte dby Salm onella typhim urium tester strains T A98 and T A 100 ino rgan ic ex tracts o f feces fro m N orth A merican su bjects (4 -9 ).Since fecal m utagenicity has been observed to be increased inp op ula tio ns a t e le va te d ris k o f c olo re cta l c an ce r, th e d is co ve ryof the fecapentaenes has raised the possibility of identifyingc olo re cta l c ar cin og en s o f e tio lo gic al a nd s cr ee nin g im p or ta nc e(10-12).T o te st th e h yp oth es is th at in cre ase d fe ca pe nta en e e xc re tio nca uses co lo rectal can cer, w e co nd ucted a case-con tro l stud y inW ashington, D C. A s part of the investigation, w e addressedtw o m ethodological questions view ed as critical to the case-control com parison of fecapentaene excretion: (a) Does thecolorectal diagnostic w orkup (undergone by cases but not byc on tro ls ) a ffe ct fe ca pe nta en e e xc re tio n a nd th us in va lid ate th ecom parison? and (b) Does fecapentaene excretion in caseschange as a result of hospitalization, surgery, and recovery?T he results of both the m ethodological investigation and thec ase -c on tro l c om pa riso n a re p re se nte d h ere .R e ce iv e d 6 /2 4/ 88 ; r ev is ed 1 1/ 3/ 88 ; a c ce pt ed 1 1/ 14 /8 8.T he c os ts o f p ub lic atio n o f th is a rtic le w ere d ef ra ye d in p an b y th e p ay me nto f p ag e c ha rg es. T his a rtic le m ust th ere fo re b e h ere by m ark ed a dv er tise me nt ina cc or da nc e w ith 1 8 U .S .C . S ec tio n 1 73 4 s ole ly t o i nd ic at e th is f ac t.' T his re se arc h w as s up po rte d in p art b y N atio na l C an ce r In stitu te C on tra ctFOD-0653.2 T o w ho m r eq ue sts f or r ep ri nts s ho uld b e a dd re ss ed .

SU BJECTS AN D M ETHOD SS tu dy P op ula ti on . W e re cr ui te d p ati en ts n ew ly d ia gn os ed w it h a den oc arc in om a o f t he c ol on o r r ec tu m, s ee n d ur in g th e s tu dy p er io d ( Ap ril1 98 5 t o J un e 1 98 7) b y t he c lin ic al w or ke rst t hr ee W a sh in gt on , D C ,area hospitals: N ational N aval M edical C enter; W ater R eed A rm yM ed ica l C ente r; a nd G eo rge W ash in gto n U niv ersity H osp ita l. S in cew e p la nn ed t o m ea su re f ec ap en ta en e e xc re tio n b ot h p rio r t o a nd f ol lo win g t re atm en t, c as es s ch ed ule d f or i mm ed ia te s urg er y f ol lo wi ng d ia gn osis an d th ose res id ing o utsid e th e W as hin gto n, D C, m etro po lita narea w ere not eligible (n = 26). O f the 162 presum ably eligible cases,21 (13% ) w ere not reached before surgery, and 49 (30% ) refused top ar tic ip ate d ue t o t he i nte ns iv e s tu dy d em an ds . A n a dd it io na l 1 4 c as esd ro pp ed o ut b ef or e c oll ec ti ng a s to ol s am p le , l ea vi ng 7 8 p ar tic ip ati ngc as es ( 48 % ). N i ne o f t he se p ar ti ci pa nt s w e re f ou nd t o h av e a de no m at ou sp oly ps o r n on ne op la sti c b ow el c on dit io ns f ol lo wi ng s ur ge ry o r p at ho logy review and w ere excluded from analysis. A s a result, 69 casesc om pr is ed t he a na ly tic al s tu dy g ro up . F or c on tr ol s, w e r ec ru it ed W a shi ng to n, D C , a re a r es id en ts a w ai ti ng e le ct iv e s ur ge ry f or n on on co lo gi ca l,n on ga st ro in te st in al c on di ti on s a t th e th re e s tu dy h os pi ta ls . C on tr ol s a teach hospital w ere frequency m atched to cases on age and sex. W ea pp ro ac hed 3 15 p ote ntial c ontro ls a nd fou nd 2 77 to b e elig ib le b ase don the additional criteria of W ashington, D C, area residency andavailability for follow -up. O f the 277 eligibles, 55 (20% ) w ere notre ac he d b efore su rg ery, 95 (3 4% ) refu se d to p artic ip ate , a nd 1 3 (5 %)d ro pp ed o ut b ef or e c oll ec tin g a s to ol, l ea vi ng 1 14 p ar tic ip an ts (4 1% ).T he d istrib utio n of c on trol su rg eries w as a s fo llo ws: 6 2 o rth op ed ic o rn eu ro su rg ic al p ro ce du re s ( 54 % ); 3 1 h er nia re pa ir s ( 27 % ); 1 6 v as cu la r

procedures (14% ); and 5 other general surgeries, prim arily of thet hy ro id ( 5% ) .W e w is he d t o s tu dy t he e ff ec ts o f t he d ia gn os ti c w o rk up f or c ol or ec ta lc an ce r o n f ec ap en ta en e e xc re ti on , b ut it p ro ve d i mp os si ble to i de nti fye ff ic ie nt ly t he c as es p ri or t o t he ir d ia gn os ti c p ro ce du re s, w h ic h i nc lu de dsigm oidoscopy, barium enem a, and colonoscopy. T herefore, w e rec ru ite d a s ep ara te g ro up o f 8 6 s ub je ct s u nd erg oi ng o ne o r m or e o f th es eprocedures as routine screening or for sym ptom s com patible w ithc ol or ec ta l c an ce r ( su ch a s r ec ta l b le ed in g, c on st ip at io n/ di ar rh ea , u ne xp la in ed a ne mi a). T hi s i nv es tig at io n w as c al le d t he m e th od s s tu dy t od is tin gu is h it f ro m t he c as e- co nt ro l c om pa ri so n. F or ty o f t he s ub je ct si n t he m e th od s s tu dy u nd er w en t c le an si ng e ne m as p lu s s ig m oi do sc op y,21 h ad b ariu m e nem as, an d 32 h ad p .o . b ow el p re pa ratio n fo llo we d b yco lo no sc op y. (S ev en of th e ind iv id uals h ad m ore th an o ne d ia gn os ticp roc ed ure.) T wo o f th e 8 6 su bje cts in th e m eth od s stu dy p rov ed to ha vec ol ore ct al c an ce r a nd w ere s ub se qu en tl y i nc lu de d i n t he c as e- co nt ro lc om pa ris on a s w el l.C olle ctio n P ro ced ure s. S ub je cts in th e m etho ds stu dy w ere a sk ed toco lle ct tw o 2 -d ay sto ol sp ec im en s at h om e, o ne b efo re an d o ne sh ortlya ft er t he d ia gn os ti c p ro ce du re . T he f oll ow -u p c oll ec ti on r an ge d f ro m 1to 7 4 d ay s after th e p ro ced ure (m ed ia n, 1 2 d ays ). In th e ca se -co ntro ls tu dy , s ub je cts w er e a sk ed t o c oll ec t f ou r 2 -d ay s to ol s am ple s a t h om e.T he tirsi sa mp le w as c ollec te d b efo re ho sp ita liz atio n an d tre atm en t,w hi ch u su al ly in vo lv ed s ur ge ry . T he th re e fo llo w- up c ol le cti on s w er escheduled at 1,3, and 6 m o follow ing surgery. C ases not undergoingsurgery w ere asked to donate a single pretreatm ent sam ple. S ubjectp ar tic ip ati on v ar ie d fo r e ac h c oll ec ti on p er io d, s o th at t he f ul l g ro up o f6 9 c as es a nd 1 14 c on tr ols d id n ot p ar tic ip ate i n e ac h c oll ec ti on .F or ea ch 2 -d ay co lle ctio n, th e s ub jec ts re ce iv ed a styro fo am c hestfilled w ith d ry ic e. T hey co lle cted stoo l in to a p lastic c on tain er he ld b y1322

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C SE C ONT RO L ST UDY O F C OL OR EC T L C NC ER ND F P E XC RE TIO N

a co lle ctio n b on ne t p lace d on th e to ilet rim , th en p lace d th e c on tain er co ntrols), the M an n-W h itn ey tw o-s am p le te st w as us ed . For all s tatis-im m e diate ly in to th e d ry ic e c hes t. S ubjec ts w e re s ho w n h ow to u se thec ollec tio n k its to av oid u rin e co ntam in atio n, an d th is p ro ved n ot to b ea p ro bl em . S to ol s n ot c ol le ct ed d uri ng th e 2 -d ay p eri od w e re re co rd ed ,an d t his li st w as tran sp ort ed w i th th e f ro ze n s to ol s to th e l yo ph ili zati onl ab orat ory ( U ni te d S t at es D e part m e nt o f A g r ic ul tu re ).S to ols w e re f re ez e- dri ed i n th ei r in div id ual c on tai ne rs w i th ou t e v erth aw in g. T he ly op hilate w as p ooled , m ix ed f or th e en tire 2 -d ay c ollecti on , an d t he n s to re d at 0 Cor co lder in sealed , ai r- tigh t containers .A liquots of the sam ples w ere sent in screw -top v ials in batches on dryi ce t o th e t es ti ng lab orato ry at V i rg in ia Po ly te ch ni c I ns tit ute (B l ac k s-b urg , V A ).W e c on du cte d t w o d if f ere nt f ec ap en tae ne as say s. D up li cat e 1 -g s amples of the ly ophiliz ed stool w ere placed in 25- x 150-m m tubes ands to pp ere d u nd er arg on . O ne s am p le w as s to re d at - 80 C to b e as say eddirectly for f ecapentaenes. W e incubated the other sam ple w ith af e cap en tae ne -p ro du ci ng s trai n o f B . t he tai ot ao m i cro n ( V I P s trai n 5 48 2)and m easured the am ount of f ecapentaenes produced f rom alreadyf orm e d p rec urso rs o ver 4 to 5 day s. T his le ngth of in cu bation is k no w nto b e su ff ic ie nt to e xh au st th e av ailab le pre cu rso rs in th e sam p le . T hei nc ub at ed m e as ure m e nt t hu s re pre se nt s al re ad y f o rm e d f e cap en tae ne sas w e ll as th ose n ew ly p roc ess ed f ro m p rec urso rs , and it is co nside redto b e an in dire ct ass ay o f the pre cu rso rs (1 3). S pec if ically , th e sam p leincubated to m easure precursors w as (a) inoculated w ith 5 m l of s tan dard i no cu lu m as p re v io us ly d es cri be d ( 2) , ( b) i nc ub at ed at 3 7 Cf or 4 to 5 day s, (c) f roz en at 0 C, d) re ly op hi li ze d, an d ( e) s to re d at80 C.l l i no cu lati on s, m i x in g, an d in cu bati on s w e re d on e an ae ro bi-c all y u nd er C O 2. T h e u ni nc ub at ed an d in cu bate d f re ez e- drie d s am p le sw e re e xt rac te d an d an al yz ed f or f ec ap en tae ne s b y H PL C 3 as p re v io us lyd es crib ed . B ri ef ly , f re ez e- drie d s am p le s w e re e xt rac te d w i th ac et on esupplem ented w ith the antiox idant B H T. E ach ex tract w as v acuumf i lt ere d, e v ap orat ed u nd er v ac uu m at 6 0 C , t op pe re d u nd er arg on , an dp lac ed in ice . T he ev ap orate d e xtrac ts w ere (a) res us pe nd ed in 0 .5 m lof prechilled H PL C solv ent, (b) f iltered through an A c rodisc L C-13f ilter (0.5 M m ) into l-m ' septum -capped v ials, and (c) sealed underargon.W e determ ined the concentration of total f ecapentaenes in eache x trac t o n a W a te rs l iq uid C hro m at og rap h u si ng a rad ial c om p re ss io nm o d ul e an d 8 -m m ( 5 ^ m ) s il ic a c art ri dg es , w i th c hl oro f orm : is op ro pan ol(9 5:5 ) c on tain in g 50 g /m l o f B H T as an an tio xidan t. T he f lo w rate w as2 m l/m in . T he H PL C w as c alib rate d us in g d ilu tio ns o f k no w n co nc ent rat io ns o f s y nt he ti c f e cap en tae ne -1 2; t he p eak are as o f t he se s tan dard sw e re th en co rrelate d w ith th ose o f the ex perim e ntal sam p le s. A b so rb -ance w as m onitored at 365 nm on a W aters 440 U V detector (A UFS0.05), and peak areas w ere integrated on a H ew lett Pack ard 3390Arecording integrator. T he reading corresponded to total f ecapent ae ne s, w i th ou t s ep arat io n o f f e cap en tae ne -1 2 an d f e cap en tae ne -1 4( 14 ). A s say s w e re p erf orm e d i n b atc he s as th e s am p le s w e re c ol le ct ed ,w i th ou t k n ow l ed ge o f w h ic h s am p le d eri ve d f ro m w h ic h in di vi du al .In ad ditio nal lab orato ry m e asu re m en ts, s elec te d case (n = 3 8) andc on trol (n = 5 0) p resu rg ic al sp ecim en s w ere an aly z ed b y H em o Qu an tf or f ec al h em o glo bin ( 1 5), to asse ss th e ef fec t o f blee din g, w h ich w ascom m on in cases, on f ecapentaene lev els. T o v erif y that no sam pled eg rad at io n o r l ab orat ory d ri ft h ad o cc urre d o v er t he 2 -y r s tu dy p eri od ,w e s ele ct ed e ve ry 2 0th s am p le s ub m it te d t hro ug ho ut t he in v es ti gat io nand resubm itted these in a m ask ed batch at the end. T he correlationb etw ee n f irst an d re plicate m e asu re m en ts w as 0 .9 1 f or th e d ire ct an d0 .94 f or the in cu bated f ecap en taen e m e asu re m en ts, and th ere w as n ot re nd t o i nd ic at e l ab orat ory o r s to rag e p ro bl em s .S t at is ti cal A n a ly s es . F ec ap en tae ne v al ue s w e re n on no rm a ll y d is tri bu te d. S im p le s tati st ic al t ran sf orm at io n w as n ot p os sib le , s o n on para-m etric statistical m eth ods w ere u sed (1 6). C orre sp ond in gly , m e dianv alues are presented in the tables, since the m eans w ere ex cessiv elyinf luenced by a f ew v ery high m easurem ents at the tails of the distrib ut io ns . F or c orre lat io nal an al y se s, t he S p earm a n ran k c orre lat io n w a su se d. F or p ai re d an al ys es ( e.g ., m e as ure m en ts f or t he s am e i nd iv id ual ,bef ore v ersus af ter barium enem a or surgery ), the W i lcox on paired-sam p le test w as c ho sen . For u npaire d co m paris on s (e.g ., c as es v ersu s

tical m an ipu latio ns, sam p le s w ith no d ete ctab le f ecape ntaen es w e reassigned a v alue of 10 ng/g (the threshold of detection is 20 ng/m g).S am p le s w i th d ete ct ab le f ec ap en tae ne s b ut n on in te grab le p eak s ( le ssthan 100 ng/g) w ere assigned a v alue of 60 ng/g, the m idpoint of thenonintegrable range.R ESU LTS

T o e stim a te n orm a l f ec ap en tae ne e x cre tio n am o n g s ub je ctsw ith ou t ap parent g astro in testin al ab no rm alities, w e f irst exam ined fecapentaene m easurem ents in the control group,am ong the 113 m en and w om en w ho collected a stool sam pleb ef ore th ei r s urg erie s f or n on gas tro in te stin al, n on on co lo gic alconditions. A s show n in T able 1, about 33% of the controlsex creted v ery low lev els of f ecapentaenes, less than the integrab le m e asu rem e nt thresh old o f 1 00 n g of FP/g o f ly op hiliz edsto ol. A n oth er 4 1% o f th e co ntro ls ex creted m o derate f ecap entaene lev els, f rom 100 to 999 ng of FP/g. T he upper 26% ofthe co ntrols ex creted o ver 1 ,0 00 n g o f FP/g, w ith th e u pperm o st5% of the group ov er 5,000 ng of FP/g. A s ex pected, incubatedv alues, representing the sum of f ecapentaene and precursorcon cen tration s, w e re alw ay s eq ual to o r g reater than the dire ctm e as ure m e nt s. O n av e rag e, i nc ub atio n in cre as ed f ec ap en tae neconcentration about 10- to 20-fold. A s a result, only 12% of thecontrols had incubated m easurem ents less than 100 ng of FP/g. Eighty % had v alues ov er 1,000 ng of FP/g, and 31% hadlev els o ve r 1 0,0 00 n g o f FP/g.T he direct and incubated f ecapentaene m easurem ents w erehighly correlated in the 730 stools tested (S pearm an r = 0.69,P =


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CA SE -C ON TR OL S TU DY O F C OL OR EC TA L C AN CE R A ND FP E XC RE TIO Nb y an y o f th e diagn ostic p ro ced ures. Incu bated fec apen ta enev alu es d id s ee m to i nc re as e f ol lo w in g c le an sin g e ne m a/s ig m oi d-oscopy (m edian rise of 1206 ng/g, P = 0.04). T his increase w asapparent even in the few subjects w hose postprocedure m easurem ents w ere delayed up to 2 m o follow ing sigm oidoscopy.H owever, the m ore invasive procedures, barium enem a andco lonos copy, d id no t s ign if ican tl y a ff ec t i ncuba ted f ecapent aenemeasurements.C as e- Co ntr ol C o mp ar is on . C as es a nd c on tr ols d if fe re d s lig htlyw ith re ga rd to d em og ra ph ic fe atu re s, in dic atin g th at fre qu en cy -m atching was im perfect. The m edian age of the cases was 65yr com pared w ith 60 yr in controls; 67% of the cases w erem ales, com pared with 54% m ales am ong the controls. Race,w hich w as not a m atching factor, also varied slightly betw eenthe tw o groups. Seventy-seven % of the cases were white,co mp ared w ith 8 8% o f the co ntrols.O f the fo ur req uested sto ol collectio ns, th e cases co mp leteda m ean of 2.6 collections, and the controls com pleted a m eanof 2.8. Other param eters of stool sam pling were also quitesim ilar in th e tw o g ro up s, in clu ding n um bers of sto ols co ntribu tin g to each co llec tio n, as w ell as tim e in terv als fro m p resu rg -ical c ollectio n to su rgery , an d fro m su rg ery to each o f the th reefo llo w-u p collectio ns, w hich so metim es v aried con sid erablyfrom the planned 1-3-6-mo schedule (ranges are given in thetables).T h e e ffe cts o f s ur ge ry a nd r ec ov er y o n f ec ap en ta en e e xc re ti onare sh ow n in pa ired an aly ses fo r cases an d co ntro ls sepa ratelyin Table 3. In both groups, when presurgical m easurem entsw ere co nside red as b aselin e, fecap en taen e ex cretio n ten ded toin crease d uring fo llo w-u p. T he rise in p ostsu rg ical va lue s w asesp ecially p ro no un ced in cases, reach in g m arg in al statistic alsignificance at 3 m o postsurgery (P = 0.04) and 6 m o postsur-gery (P = 0.06).W hen w e d iv id ed th e cases into th ree cru de stag es, in situ ,lo ca lly in va siv e, a nd m eta sta tic , w e o bse rv ed th e sa me p os ts urgical pattern of increasing FP excretion in all three stages.S eparating the cases into right-sided, left-sided, and rectallesions revealed the sam e pattern, w hich w as also persistentw hen the cases were divided by age group (55 or less, 56-65,an d o ver 6 5), sex , race, an d h osp ital.F or inc ub ated fecap en taen e va lu es, th e g en eral p attern w asthe sam e, but the rise at 3 and 6 m o in cases was weaker, whileth e in cre as e a mo ng c on tro ls w as s tro ng er, re ac hin g s ta tistic als ig nific an ce w he n th e v alu es fo r c on tro ls a t 3 m o w ere c om pa re dw ith th eir b aseline (P = 0 .0 3).In Table 4, the results of the case-control com parison arepresented. T hese indicate that cases excreted low er fecapentaene levels than controls. T he tim e trends w ithin the case andco ntrol g rou ps sho wn in T ab le 3 w ere also re flected in th e case-

T a bl e 3 E ff ec ts o f s ur ge ry a nd r ec ov e ry o n f e ca pe nt ae ne e x cr et io n i n c ol or ec ta lcanc er pa ti en ts and cont ro l sC h an ge c om p ar ed t o p re su rg ic al v al ue s

T ab le 4 F P e xc re ti on i n c olo re ct al c an ce r p ati en ts v er su s c o ntr ols , a t f o ur t im e saround surgery

Cases ControlsSampling period Mediann change Medianchange Pb

Presurgery Baseline BaselineOne m o follow-up (13-69 days 35 0 0.87 68 0 0.33postsurgery)Three mo follow-up ( 78-150 36 +234 0.04 67 0 0.43days postsurgery)Six m o follow-up ( 153-223 35 +50 0.06 65 +50 0.17days postsurgery) F ec ap en ta en e m ea su re m en t a t f ol lo w- up m in us f ec ap en ta en e m e as ur em en tbefo re su rgery .* P v alu es f ro m W ilc ox on p ai re d- sa m ple t es t.

SamplingeriodPresurgeryO ne m o f ollo w- up (1 3-6 9 d ay spostsurgery)T h re e m o f ol lo w -u p ( 78 -1 50days postsurgery)S ix m o f ol lo w -u p ( 15 3- 22 3

days postsurgery)n64

393837CasesMedian

FP(ng/g)6060360

253ControlsH113

6968

66Median

FP(ng/g)276

184274

540f


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C SE C ON TR OL ST UDY O F C OL OR EC T L C NC ER ND F P E XC RE TIO N

T ab le 5 F P e x cre ti on i n 3 8 c ol ore ct al c an ce r p at ie nt s v e rs us 5 0 c on tro ls , b ybleeding status

Bleeding*CasesControlsNot

bleedingCasesControlsn2071843Median

FP(ng/g)6026782351f0.250.04

P v al ue s fr om Man n-Wh it ne y tw o- samp le te st.* B leeding def ined as greater than 7 m g of hem e per g of dried stool byHemoQuant.

tion do es no t app ear to p ose a risk o f co lo rectal can cer, at leastas m easured in a case-control study near the tim e of cancerd iag no sis . O u r c as es h ad g en eral ly lo w e r f ec ap en tae ne e x cre tio nthan controls, considering either m edian FP v alues or thep erc en tag e o f s ub je ct s w i th e sp ec ially e le v ate d le v els .O f concern in a case-control approach w as that colorectalcancer itself , or its diagnosis and treatm ent, could af fect them e as ure m en ts. In p artic ular, w e re co gn iz ed th at th e d iag no sticw o rk u p f or c olo re ctal c an ce r m i gh t in v alid ate th e c as e-c on tro lcom p ariso n, sin ce f ec ape ntaene s are pro duc ed by b ow el f loraw hich could be perturbed by barium enem as or cleansing regim ens preceding endoscopy . W e observ ed, how ev er, no strongef fec t o f th e d iag nostic w o rk up o n f ecap en taen e ex cretio n. T hef inding of an increase in f ecapentaene precursors f ollow ingclean sing en em a and sig m oid osco py is p ro bably a ch anc e f in ding, since the m ore inv asiv e diagnostic procedures show ed noeffect. W e conclude f rom this m ethods study that diagnosticw ork up cannot ex plain the low er fecapentaene lev els seen incolorectal cancer cases, although the statistical pow er of them ethods study w as not large enough to rule out the possibilityo f sm a ll e ff ec ts.O ur second concern w as that the tim ing of the case-controlco m parison m igh t inf lu en ce th e resu lts. In m o st case-con tro lstudies, cases are identif ied from m edical records f ollow ingtre atm e nt, w h ic h f or c olo re ctal c an ce r m ay in clu de b ow e l re se ction w ith p rep arato ry antib io tics, as w ell as ad ju nc tiv e ch em oth erap y o r rad iatio n. W e re aso ne d th at f ec ap en tae ne e xc re tio nm ig ht be pro fo un dly ch an ged by th ese p ro ced ures and , th us, w em e asu red cases b ef ore treatm e nt (w h ich usu ally w as lim ited tosurgery ) as w ell as af terw ard. W e also follow ed controls af terth eir n on gas tro in te stin al s urg erie s, to c he ck f or g en eral e ff ec tsof hospitaliz ation and recov ery . T he results indicate that thetim in g o f the case-c on tro l com p ariso n is im p ortant. B oth caseand control m easurem ents tended to rise f ollow ing surgery .C as es e x cre te d m u c h l ow e r l ev e ls o f f ec ap en tae ne s th an c on tro lsin the periods before hospitaliz ation and im m ediately af ters urg ery , b ut th e d if fe re nc e w as n ot sig nif ic an t late r in re co v ery .H o w ev e r, i nc ub ate d f ec ap en tae ne le v el s, re pre se ntin g f ec ap entae ne p re cu rso rs, w e re p ersiste ntly lo w er in c ase s, re gard le ss o fsam pling period. T hus, a general reduction w as seen in cases,in v olv in g f ec ap en tae ne p re cu rs ors as w e ll as f ec ap en tae ne p roduct ion i ts el f.T h e lo w ere d f ec ap en tae ne le ve ls in c ase s w e re c le arly n ot d ueto co lo rec tal b leed in g. A s an oth er p ossible ex plan atio n f or thec as e- co ntro l d if f ere nc e, w e are c urre ntly e x am i nin g d ie tary d ataco llected f rom all su bjects, to assess w h ethe r cance r-relatedd ietary chan ges m ig ht hav e lo w ered f ecap en taen e ex cretio n incases (17 ). W e h av e ruled ou t th e p ossibility th at alteratio ns inrecen t f at in tak e co uld pro du ce this ef fe ct (1 8).It could be argued that m easuring f ecapentaene ex cretionn ear th e tim e of d iagn osis, eith er b ef ore or af ter su rge ry , is to olate . Pe rh ap s o ur c olo re ctal c an ce r p atie nts o rig in ally h ad e le

v ate d f ec ap en tae ne e xc re tio n, w h ic h f ell as d is eas e p ro gre sse d.T w o pieces of ev idence argue against this objection, (a) W h enthe case group w as div ided by stage, the nine patients w ithm inim al, in situ cancers had low er lev els prior to surgeryco m pare d w ith co ntrols, (h) In div id uals w ith ade no m ato us p oly ps m ay also dem onstrate low ered f ecapentaene ex cretion, asseen in a cross-sectional autopsy study reported by C orrea etal. 19). They used Salm onella tester strain TA 100, a m utage-nicity assay k now n to detect f ecapentaenes, and found low erm u tag en ic ity in su bje cts w i th ad en om ato us p oly ps th an in th osew ith no poly ps. T hus, if f ecapentaene ex cretion is truly inc re as ed in s ub je cts p ro ne to c olo re ctal n eo plasia, th is as so ciation, opposite to w hat w e observ ed, m ight be seen only v eryearly in the natural history of the disease. A cohort studyspanning decades w ould be needed to assess this possibilityd ef in itiv e ly . C ro ss -s ec tio nal s tu die s o f f ec ap en tae ne e x cre tio nin p atie nts w i th p re in v asiv e c olo re ctal n eo plas ia o f in cre as in gs ev e rit y w o u ld b e u se fu l as a p rac tic al alt ern at iv e .If f ecapentaene ex cretion is truly low er in indiv iduals w hod ev e lo p c olo re ctal c an ce r, as s ug ge ste d b y o ur c as e- co ntro l d ata,the biological im plications are unclear. W e hav e show n in anau to psy stu dy th at e xc re te d f ec ap en tae ne le ve ls are re pre se ntativ e of bow el contents throughout the colon (2), but v ery littleis k no w n ab ou t th e relation ship o f f ecap entaen es in th e co lo nielu m en to tiss ue -b ou nd le ve ls. Pe rh ap s f ec ap en tae ne e xc re tio nre pre se nts a p ro te ctiv e m e ch an ism ag ain st c olo re ctal d am ag e,b ut m o re in fo rm ation is need ed ab ou t the p ro ductio n, m e tabolism , tissue binding, and anim al carcinogenicity of f ecapentaen es b ef ore w e can ju dge th e plausib ility o f th is h yp othesis.T i ss ue -b in din g s tu di es an d an im a l c arc in og en ic ity e x pe rim e n tsare underw ay in sev eral laboratories. C ontinuing studies ofother fecal m utagens w ill also clarif y if f ecal m utagenicityg en erally d ec re ase s th e ris k o f c olo re ctal c an ce r, in cre as es riskas sug gested b y th e early c orrelatio nal stu dies, o r is u nim p ortant.T w o cav eats arise f ro m ou r in vestig ation . T he f irst co ncernsp atie nt p artic ip atio n in s tu die s re qu irin g sto ol c olle ctio n. It w asapparent f rom the low participation rates am ong our eligiblecases and con tro ls that rep eated sto ol c ollectio n is d if ficu lt toachiev e. R epeated m easurem ents appear to be usef ul to ty pif yc orre ctly th e e x cre tio n p atte rn s o f in div id ual s, b ut c on f irm a to rystudies should probably aim for increased participation bydecreasing the num ber of stool collections requested. W e areh op ef ul th at th e lo w p articip ation rates d id n ot seriou sly af fecto ur c as e-c on tro l c om p aris on , s in ce th e c on clu sio ns d id n ot v aryw h en particip an ts w ere stratif ied b y the n um b er o f sam p les the yc olle cte d, o r w h en s ub je cts w i th in co m ple te p artic ip atio n w e reexcluded.A s a final caution, it seem s that any case-control study off ec al c on st itu en ts an d c olo re ctal c an ce r m u s t c on sid er c are fu ll yth e p oss ib le e ff ec ts o f d iag no stic w o rk u p, b le ed in g, s urg ery , an drecov ery on the assay results. It is unclear w hy f ecapentaenelev els v aried during f ollow -up, ev en am ong controls, but ourin v estig atio n c le arly in dic ate s th at at le ast s om e c lin ic al p ro cedures can be im portant conf ounding inf luences on fecal m easurem ents. S ince it is laborious to accum ulate relev ant stoolsam p le s to ad dre ss th ese m e th od olo gic al c on ce rn s, w e o ff er o urs to re d s pe cim e n s to in v es tig ato rs e m b ark in g o n re lat ed p ro je cts .W e currently are ex am ining other f ecal m utagens, bile acids,and neutral sterols using the ly ophiliz ed stools and the sam eme t hodol og ic al approac h .R EFER EN C ES

1 . H irai, N ., K in gs to n, D . G . I ., V a n T as se ll, R . L ., an d W i lk in s, T . D . S tru ctu reelu cid atio n o f a p oten t m u tag en f ro m h um an f eces . J. A m . C hem . S ot., 1 04 :6 14 9- 61 50 , 1 98 2.1325



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C ASE -C ON TRO L ST UD Y O F C OL OR EC TA L CA NC ER A ND F P E XC RE TIO N2. S chiffm an, M . H ., B itterm an, P .. V iciana, A . L ., S chairer. C . R ussell, L ..V an T asse ll, R . L ., a nd W ilk in s, T . D . F ec ap en ta en es a nd th eir p re cu rso rst hr ou gh ou t t he b ow e l re su lt s o f a n a ut op sy s tu dy . M u t t.R e s. , 2 08 : 9 -1 5,1988.3 . V an T asse ll, R . L ., a nd W ilk in s, T . D . T he p re cu rso rs o f fe ca pe nta en es ap re li m in ar y r ep or t. A n n. 1 st . S u p er . S a ni t , 2 2: 9 33 -9 42 , 1 98 6.4 . D io n, P .. a nd B ru ce , W . R . M uta ge nic ity o f d iff ere nt f ra ctio ns o f e xtr ac ts o fh um a n f c es .M u t t.R e s. . 1 19 : 1 51 -1 60 . 1 98 3.5. Goggelm ann. W ., M aier. F. K ., and Pfaendler, H . R. M utagenicity ofs yn th et ic r ac em i c f ec ap e nt ae ne -1 2. M u t t.R e s. . 1 74 : 1 6 5- 16 7, 1 98 6.6 . P lu mm er. S . M ., G ra fs tr om , R . C ., Y an g, L . L ., C urre n, R . D ., L in na in ma a,K ., a nd H arris, C . C . F ec ap en ta en e-1 2 c au se s D NA d am ag e a nd m uta tio nsi n h um an c el ls . C ar cin og en es is ( Lo nd .) , 7 : 1 60 7- 16 09 . 1 98 6.7. C urren, R . D ., Putm an. D. L., Yang, L. L., H aworth, S. R ., Lawlor, T. E.,Plum mer, S. M ., and Harris, C . C . Genotoxicity of fecapentaene-12 inb a ct er ia l a nd m a m m al ia n c el l a ss ay s ys te m s. C a rc in og e ne si s ( Lo nd .) , 8 :3 49 -3 52 . 1 98 7.8. Hinzman, M . J., Novotny, C., U llah, A., and Shamsuddin, A . M . Fecalm u ta ge n f ec ap en ta en e- 12 d am a ge s m a mm a li an c ol on e pi th el ia l D N A. C arc in og en e si s ( Lo nd .) . 8 : 1 4 75 -1 47 9. 1 98 7.9 . S ch mid , E ., B au ch in ge r, M ., B ra se lm an n, H .. P fa en dle r. H . R ., a nd G og ge lm an n, W . D os e r es po ns e r ela tio ns hip f or c hr om o so m e a be rr at io ns i nd uc edb y f ec ap en ta en e- 12 i n h um a n l ym p ho c yt es . M u t t.R e s. , 1 91 : 5 -7 , 1 98 7.10. E hrich, M ., A sw ell, J. E .. V an T assell, R . L ., and W ilkins, T . D . M utagensin the feces of 3 S outh-A frican populations at different levels o f risk forc ol on c an ce r. M u t t.R e s. , 6 4: 2 31 -2 4 0, 1 97 9.1 1. M ow er, H . F ., Ic hin ots ub o, D .. W an g, L . W ., M an de l. M ., S te mm erm an n.

G ., N om ur a, A ., H cilb ru n, L ., K am iy am a, S ., a nd S him ad a, A . F ca lmuta-g en s in tw o J ap an es e p op ula tio ns w it h d if fe re nt c ol on c an ce r r is ks . C an ce rR e s. . .- 1 I6 4 -1 16 9. 1 98 2.1 2. R ed dy , B . S . , S h ar ma , C ., D ar by , L ., L aa ks o, K ., a nd W y nd er , E . L . M e ta bo lieepide miology of large bow el ca ncer: fec al m utagens in high a nd low riskp op ula tio n f or c olo n c an ce r a p re lim in ar y r ep or t. M u t t. R es ., 7 2: 5 11 -5 22 . 1 98 0.1 3. V an T as se ll , R . L ., S ch r m .R . M ., a nd W ilk in s, T . D . M ic ro bi al b io sy nt he si so f fe ca pe nta en es. In : I . K nu dse n (e d.) . G en etic T ox ic olo gy o f th e D ie t, p p.1 99 -2 11 . N ew Y or k: L is s, 1 98 6.14. B aptista, J., B ruce , W . R ., G upta, I., K repinsky, J. J., V an T assell, R . L .,and W ilkins, T . D . O n distribution of different fecapentaenes, the fecalm ut ag en s, i n th e h um an p op ula tio n. C an ce r L ett ., 2 2: 2 99 -3 03 , 1 98 4.15. S chw artz, S ., D ahl, J., E llefson, M ., and A hlqu ist, D . T he H em oQ ua ntte st: a s pe cif ic a nd q ua nt ita ti ve d ete rm in ati on o f h em e ( he m og lo bin ) i n f ec esa nd o th er m a te ria ls . C li n. C he m ., 2 9: 2 06 1- 20 67 , 1 98 3.1 6. Z ar , J . H . B io sta tis ti ca l A na ly sis . E ng le wo od C lif fs , N J: P re nti ce -H al l, I nc .,1974.1 7. S ch iffm an , M . H . D ie t a nd fa ec al g en oto xic ity . C an ce r S urv ., 6 : 6 53 -6 72 ,1987.1 8. T ay lo r, P . R ., S ch if fm an , M . H ., J on es, D . Y ., Ju dd , J. , S ch atz kin , A ., N air,P . P ., V an T assell, R . L ., and B lock , G . R elation of chang es in am ount andty pe o f d ie ta ry f at to f ec ap en ta en es in p re m en op au sa l w om e n. M u t t .R es .,2 06 :3 -9 , 1 98 8.19. C orrea, P ., P ascha l, J., P izzola to, R ., P el n, W ., and Lesley, D . E . F ecalm uta ge ns a nd c olo re cta l p oly ps : p re lim in ary re po rt o f a n a uto psy stu dy .B an bu ry R ep ., 7 : 1 19 -1 27 , 1 98 1.

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