Certai n Stains Tetsuya Inoue, M.D., Masaki Mori, M.D., Reishi Shimono, M.D., Hiroyuki Kuwano, M.D., Keizo Sugimachi, M.D. From the Department of Surgery II, Faculty of Medicine, Kyushu University, Fukuoka, Japan We made use of hematoxylin and eosin (H&E) stain, Verhoeff van-Gieson stain for elastic tissue (EVG), and factor VIII-related antigen (FVIII-RA) to stain tissues ex- cised from 94 patients wi t h colorectal carcinoma. Of these 94, 49 died of disease within t wo years (Group I), and 45 survived for five years or longer (Group II) after surgery. In the tissues from bot h groups, the use of EVG stain revealed a higher incidence of vascular invasion than was seen wi t h H&E stain. In Group I, the rates were 28. 6 percent and 61. 2 percent wi t h H&E and EVG, re- spectively, and those in Group II were 4.4 percent and 31.1 percent, respectively. Conversely, the FVIII-RA stain showed a decrease in the incidence of vascular invasion in bot h groups. In Group I, when vascular invasion was examined in EVG-stained tissues, the incidence was 81. 3 percent in cases of hematogenous metastases and 23.5 percent in those wi t hout hematogenous metastases ( P < 0. 01). These differences were not evident wi t h H&E. When observing the site of vascular invasion in tissues of the colorectal wall stained wi t h EVG, intramural and extramural types of vascular invasion were seen in 20 percent and 80 percent of cases in Group I and in 93 percent and 7 percent of those in Group II, respectively. Thus, not onl y the frequency, but also the site, of vascular invasion into the colorectal wall evidenced wi t h EgG stain provides a more precise prediction of the recurrence of hematogenous metastases. [Key words: Vascular inva- sion; Colorectal cancer; Metastasis; Elastic stain; Factor VIII-related antigen] Inoue T, Mori M, Shimono R, Kuwano H, Sugimachi K. Vascular invasion of colorectal carcinoma readily visible wi t h certain stains. Dis Colon Rectum 1992; 35: 34- 39. I n pat i ent s wi t h col or ect al car ci noma, t he ma i n caus e of deat h is l i ver met ast asi s. Vascul ar inva- si on, whi ch is c l os e l y as s oci at ed wi t h l i ver met as - tasis, is an i mpor t a nt pr ognos t i c fact or. >4 Th e r e ar e r out i ne p a t h o l o g y r e por t s s howi ng t he l ack of vas- cul ar i nvasi on e ve n in pat i ent s wi t h a s ync hr onous l i ver met ast asi s. The s e r out i ne s t udi es we r e d o n e o n l y wi t h he ma t oxyl i n a nd eos i n ( H&E) st ai n. Al- t hough it is wel l k n o wn t hat st ai ns f or el ast i c f i ber s ar e usef ul in r eveal i ng vascul ar i nvasi on, 5-7 t her e ar e f e w dat a on pr e c i s e c ompa r a t i ve r esul t s us i ng Address reprint requests to Dr. Sugimachi: Department of Sur- gery II, Faculty of Medicine, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812, Japan. 34 ser i al s ect i ons s t ai ned by H&E and Ve r hoe f f van- Gi e s on st ai n f or el ast i c t i ssue ( EVG) . I n addi t i on, t her e ar e f e w r e por t s c o n c e r n i n g i mmunohi s t o- c he mi c a l s t udi es of vas cul ar i nvas i on of col or ect al c a r c i noma us i ng f act or VI I I - r e l a t e d ant i gen ( FVI I I - RA) c o mp o u n d s t hat s h o w t he e n d o t h e l i u m of t he vascul ar vessel s. 8 The r e f or e , we di d a r e t r os pe c t i ve r e vi e w of dat a on 94 pat i ent s wi t h col or ect al car ci noma, t he ob- j ect i ve b e i n g t o c o mp a r e t he i nc i de nc e s of vascul ar i nvas i on in t hr ee ser i al s ect i ons s t ai ned by H&E, EVG, and FVIII-RA. PATIENTS AND METHODS Fi ndi ngs in 94 pat i ent s we r e e x a mi n e d r et r o- spect i vel y. All had u n d e r g o n e sur gi cal r e s e c t i on f or col or ect al c a r c i noma i n t he De p a r t me n t of Sur- ge r y II, Kyus hu Uni ver s i t y Hos pi t al , b e t we e n 1978 a nd 1983. Of t hes e 94 pat i ent s, 49 di e d of di s eas e ( r e c ur r e nc e a n d / o r met as t as i s ) wi t hi n t wo year s af t er t he s ur ge r y ( Gr o u p I ) . For t y- f i ve pat i ent s sur- vi ved mo r e t han fi ve year s af t er s ur ge r y wi t hout e v i d e n c e of di s e a s e ( Gr o u p I I ) . Gr o u p I was di- v i d e d i nt o t wo gr oups : Gr o u p IA (32 pat i ent s ) d e v e l o p e d h e ma t o g e n o u s di st ant met as t as es t o t he l i ver, l ung, br ai n, a nd b o n e , as n o t e d at t he t i me of deat h, and Gr o u p I B (17 pat i ent s ) had no di st i nct e v i d e n c e of h e ma t o g e n o u s di st ant met as t as es . The l at t er di e d of l ocal r e c ur r e nc e s , l y mp h n o d e met as - t ases, a n d / o r pe r i t one a l di s s e mi na t i on. All di st ant met as t as es or r e c ur r e nc e s we r e i nves t i gat ed us i ng c o mp u t e d t o mo g r a p h y , e c hogr a phy, b o n e sci nt i g- r aphy, a nd ches t x-ray. For all t hes e pat i ent s , ade- quat e pa t hol ogi c mat er i al s and f ol l ow- up dat a we r e avai l abl e. All sur gi cal s p e c i me n s had b e e n f i xed in 10 p e r c e n t neut r al f or ma l i n and r out i ne l y p r o c e s s e d f or par af f i n e mb e d d i n g . I n t hi s st udy, o n e t o t hr e e bl oc ks f or each p r i ma r y t u mo r we r e used. Th r e e Vol. 35, No. 1 VASCULAR INVASION IN COLORECTAL CANCER 35 serial 5-micro-thick sections were cut from each block and included the center of the tumor, show- ing the deepest penetration. One section was stained with H&E, one was stained with EVG, and the other was mount ed on poly-lAysin-coated slides for immunohistochemical stainings for FVIII-RA. Immunohistochemical Techni que In the case of the immunoperoxidase method, 9 the tissue sections were deparaffinized in xylene and hydrated through a graded series of ethanol. Sections were digested with 0.4 percent pepsin (Sigma, P-7000; St. Louis, MO) in 0.01 N HC1 solution for two hours at 37~ and washed after blocking the endogenous peroxidase activity with 3 percent hydrogen peroxidase in 100 percent methanol for 30 minutes. After rinsing in phos- phate-buffered saline and incubating with normal goat serum for 30 minutes, the sections were in- cubated in humid chambers with primary antibody, rabbit anti-F VIII-RA (Dakopatts, Glostrup, Den- mark), 1:200, for 24 hours at room temperature. This was followed by immunoperoxidase staining using the avidin-biotin-peroxidase complex (ABC) kits (Vector Laboratories, Burlingame, CA). Im- munostaining was developed by 0.01 percent H202 and 0.05 percent diaminobenzidine tetrahydro- chloride for two minutes. After a light counterstain- ing with methylgreen, the sections were mounted. Control stainings were always negative. Identification of Vascular Invasion Routine examinations for vascular invasion and other histologic features were done in H&E sec- tions by one of the authors (T.I.), who had no knowledge of any clinical information on the pa- tients. Vascular invasion was strictly defined as tumor cells within a round or ovoid endothelium- lined, or often endothelium-lacking, space with a smooth muscle wall. The presence of the adjacent artery aided in detecting the vascular invasion. Lymphatic involvement was strictly distinguished from vascular invasions. Lymphatic vessels lacked the thick walls composed of smooth muscles and were often filled with fluid. There were no red blood cells. Vascular invasion was then examined in the EVG- stained sections; detection was made by the pres- ence of carcinoma ceils surrounded by a rim of black-stained elastic fibers, usually together with an artery. In sections stained for FVIII-RA, vascular invasion was defined as tumor cells within the positive endothelium-lined circles. All observa- tions were repeated three times every two weeks until a consensus was reached between two ob- servers (T.I. and M.M.). For statistical analyses, we used Student' s /-test, chi-squared test, and Fisher's exact probability test. RESULTS Clinicopathologic findings in the 94 patients studied are shown in Table 1. There were no sta- tistical differences between Groups I and II with regard to mean age, sex ratio, and tumor site. Significant differences were seen in histologic type, depth of invasion, and Dukes' stage (P < 0.01). Ta bl e 1. Clinicopathologic Dat a Group I (n = 49) Group II Total Group IA Group IB (n = 45) ( n=32) ( n=17) Mean age 58.3 57.4 59.8 60.9 Male:female 1:0.81 1:0.68 1:1.13 1:1.14 Tumor site Right colon 9 3 6 9 Transverse 0 0 0 2 colon Left colon 15 13 2 18 Rectum 25 16 9 16 Histologic type Well 12 10 2 28 Moderate 26 17 9 10 Poor 9 5 4 3 Others 2 0 2 4 Depth of invasion Mucosa 0 0 0 0 Submucosa 0 0 0 3 Muscle layer 1 0 1 6 Subserosa or 48 32 16 36 adventitia Dukes' stage A 0 0 0 8 B 10 6 4 18 C 39 26 13 19 Group I: patients died of disease within two years after surgery. Group IA: patients died mainly of hematogenous metastasis. Group IB: patients died mainly of local recur- rences, lymph node metastases, or peritoneal dissemina- tion. Group I1: patients survived five years or more after surgery. Well: well-differentiated adenocarcinoma. Moderate: moderately differentiated adenocarcinoma. Poor: poorly differentiated adenocarcinoma. 36 INOUE ET AL Table 2. Incidence of Vascular Invasion Dis Colon Rectum, January 1992 Stainings H & E EVG FVIII-RA Group I(n = 49) 28.6% (14/49) 61.2% (30/49) 8.0% (4/49) Group IA (n = 32) 31.3% (10/32) 81.3% (26/32)* 9.4% (3/32) Group IB (n = 17) 23.5% (4/17) 23,5% (4/17)* 5.9% (1/17) Group II (n = 45) 4.4% (2/45) 31.1 % (14/45) 2.0% (1/45) Group I: patients died of disease within two years after surgery. Group IA: patients died mainly of hematogenous metastasis. Group IB: patients died mainly of local recurrences, lymph node metastases, or peritoneal dissemination. Group I1: patients survived five years or more after surgery. * The correlation between Groups IA and IB showed a statistical difference (P < 0.01). The i nci dence of vascular i nvasi on in Groups I and II is gi ven in Tabl e 2. In bot h groups, t he i nci dence of vascular invasion wi t h the use of EVG stain was hi gher t han that seen wi t h H&E stain. In contrast, the i nci dence of vascular invasion wi t h t he use of FVIII-RA stain was remarkabl y l ower t han that seen with H&E stain. In the Gr oup I pat i ent s wi t h a poor prognosi s, t he i nci dence of vascular invasion wi t h EVG stain was about t wi ce that seen wi t h H&E stain. In t he Gr oup IA patients, in whom a hemat ogenous metastasis occurred, t he i nci dence of vascular invasion was the highest, whi l e, in Gr oup IB patients, who showed no evi- dence of any distant metastases, t he i nci dence de- t ect ed by EVG stain was the same as that seen wi t h H&E stain. When the EVG sect i ons wer e used for t he i dent i fi cat i on of vascular invasion, t here wer e significant di fferences bet ween Groups IA and IB ( P < 0.01); however, when rout i ne H&E sect i ons onl y wer e used, t here wer e no significant differ- ences. In the H&E sections, det ect i on of vascular inva- sion was most difficult in areas wher e t he t umor cells grew compact l y and densel y wi t h little st roma (Fig. 1). Many of t he col orect al carci nomas wer e well- or moderat el y-di fferent i at ed adenocarci no- mas, showi ng compact and densel y growi ng cancer cells. In contrast, in t he EVG sections, a black- st ai ned rim of elastic fibers was cl earl y visible, maki ng the det ect i on of vascular i nvasi on easi er in areas with a compact growt h of cells (Fig. 1). In Gr oup IB, the i nci dence of vascular invasion was equal bet ween H&E and EVG. The f r equency of poor l y di fferent i at ed adenocar ci noma was rela- tively high in Gr oup IB. Det ect i on of vascular invasion in H&E sect i ons was not as difficult in cases of poor l y di fferent i at ed adenocar ci noma wi t h Figure 1. Left: detection of vascular invasion is difficult using H&E stain (x90). Right: detection of vascular inva- sion is easy in the same area in the tissue sample (EVG, x90). Arrows indicate vascular invasion. l oose cancerous tissue and a wi de stroma. The staining of FVIII-RA was speci fi c to the endot he- lium of capillaries, veins, and arteries. In vessels wi t h t umor i nvol vement , t he staining of FVIII-RA was ei t her lost or weak (Fig. 2). Presumably, this is due to dest ruct i on of t he vascular endot hel i um by t umor cells occupyi ng t he l umen (Fig. 3). Therefore, t he i nci dence det ect ed by FVIII-RA was l ow in each group. In Tabl e 3, vascular i nvasi on det ect ed in the EVG sect i ons was evi dent accordi ng to the site of t he ext ent of vascular i nvasi on into the col orect al wall. Thi r t een of 14 pat i ent s in Gr oup II had intramural vascular invasion, whi l e, in Gr oup I, 80 per cent of t he vascular i nvasi on was extramural. Ther e wer e significant di fferences bet ween Groups I and II wi t h regard to t hese fi ndi ngs ( / ' < 0.01). Ther e wer e no significant di fferences bet ween Groups IA and IB. Vol. 35, No. 1 VASCULAR INVASION IN COLORECTAL CANCER 37 DI SCUSSI ON When vascular i nvasi on by mal i gnant cells is present , liver metastases will occur t hr ee t i mes mor e f r equent l y t han in t he absence of t hese cells, l'z However, t he r epor t ed i nci dence of vas- cul ar invasion varies consi derabl y. >6'1~ 11 One rea- son for t he variability is the di f f er ence in the num- Figure 2. Note the weakly positive staining for FVIII-RA in the lumen of the vascular vessel involved with tumor cells (ABC method, x170). Figure 3. Vascular invasion is detected with EVG (left, xg0) but is not visible with FVlII-RA (right, x90). ber of exami ned sections; wi t h t hei r increase, the f r equency of vascular invasion increases. 2' 12 Whi l e di fferences in t echni ques used also need to be consi der ed, 2'13 in some institutions, special stain- ing for elastic fibers such as EVG is r out i nel y used to det er mi ne t he pat hol ogy, especi al l y for the de- t ect i on of vascular invasion. 14 The f r equency of vascular invasion may i ncrease wi t h t he combi ned use of special stains. The useful ness of t he elastic tissue stain is con- troversial. Minsky and Mies 15 stated that t he elastic tissue stain was i mport ant because t her e wer e de- creases in the fal se-negat i ve rates and because vas- cular i nvasi on coul d be di fferent i at ed from lym- phatic vessel invasion. 6 On t he ot her hand, Burns and Pfaff 16 stated that rout i ne H&E sect i ons wer e adequat e in most instances, al t hough t hey used elastic tissue stain when necessary. Tal bot e t al . 1~ consi der ed the elastic tissue stain to be rarely help- ful. The rectal wall cont ai ns a large amount of backgr ound fi ber wi t h staining characteristics of elastic tissue; thus, the useful ness of t hese stains was limited. In our study, the rectal wall di d con- tain a large amount of bl ack-st ai ned fi ber in some cases, but it was not difficult to di fferent i at e a black- st ai ned rim of vascular invasion f r om a large amount of backgr ound fi ber if at t ent i on was di- r ect ed to t he form or di rect i on of t he bl ack-st ai ned fibers. In t he current study, t he addi t i onal use of elastic tissue stain for represent at i ve sect i ons pr oved to be practical and useful. The use of EVG stain in- creased t he i nci dence of vascular invasion, partic- ularly in pat i ent s wi t h a distant hemat ogenous me- tastasis ( Gr oup IA), wher e t he i nci dence i ncreased from 31.3 per cent by H&E to 81.3 per cent by EVG. Using onl y H&E stain for i dent i fi cat i on of vascular invasion, t he correl at i on bet ween distant hemat og- enous metastasis and vascular invasion was not Table 3. Site of the Extent of Vascular Invasion into the Colorectal Wall* Intramural Mucosa Submucosa Extramural Incidence Muscle Subserosa or of Vascular Layer Adventitia Invasion Group I (n = 49) 0 2 4 24 30/49 Group IA (n = 32) 0 2 4 20 20/32 Group IB (n = 17) 0 0 0 4 4/17 Group II (n = 45) 3 9 1 1 14/45 There were significant differences between Groups I and II (P < 0.01) and between Groups IA and II (P < 0.01). * Vascular invasion was detected in EVG sections. 38 INOUE E T AL Dis Colon Rectum, January 1992 si gni f i cant , whi l e t her e we r e si gni f i cant di f f e r e nc e s b e t we e n t he t wo wi t h t he us e of EVG ( P < 0. 01). Appl i cat i on of FVIII-RA f or de t e c t i ng vas cul ar i nvas i on has b e e n ma d e f or t umor s in t he br east , 1v'18 t hyr oi d, 19 and gast ri c areas, a~ For br e a s t cancer , Mart i n e t al . 18 f ound t hat t hi s me t h o d facil- i t at ed t he i dent i f i cat i on of i nt r avascul ar t u mo r cel l s, whi l e Sai go a nd Ros en *v f o u n d t hat it mi ght b e usef ul in s e l e c t e d cases and t hat H&E st ai n was t he mos t r el i abl e me t h o d . I n t he case of gast r i c cancer , Noguc hi a~ f ound t hat us e of FVI I I - RA was l i mi t ed. St e phe ns on e t al . 19 e x a mi n e d f ol l i cul ar c a r c i noma of t he t hyr oi d a nd n o t e d t he l ack of FVIII-RA st ai ni ng in ar eas wh e r e t he t u mo r a d h e r e d t o t he e n d o t h e l i u m. I n our s t udy on col or ect al c a r c i noma , ma n y of t he vascul ar ves s el s i nvol ve d b y t u mo r cel l s l a c ke d r eact i vi t y f or FVIII-RA, pr ob- abl y be c a us e t he e n d o t h e l i u m had b e e n des t r oyed. The r e f or e , t he a ppl i c a t i on of FVIII-RA was not usef ul . Our s t udy cl ear l y s h o we d t hat e xt r a mur a l vascu- l ar i nvasi on was f r e que nt in pat i ent s wi t h a p o o r pr ognos i s or a di st ant met as t as i s but was r ar e i n t he f i ve- year sur vi vor s. Sunde r l a nd 21 s u g g e s t e d t he i m- por t a nc e of vascul ar i nvas i on i nt o t he s ubmuc os a , a nd Tal bot e t a L ~~ f o u n d a si gni f i cant c or r e l a t i on b e t we e n ext r amur al i nvas i on a nd hepat i c met as - t ases and survi val t i me. Our r esul t s agr ee wi t h t he l at t er. I n concl us i on, t he us e of el ast i c t i ssue st ai n can pr ovi de a mo r e pr e c i s e de t e c t i on of vas cul ar inva- s i on t han wh e n onl y r out i ne H&E st ai n is used. I n t he r out i ne pa t hol ogi c e xa mi na t i ons of col or ect al c a r c i noma cel l s, t he us e of el ast i c t i ssue st ai n com- b i n e d wi t h H&E st ai n is des i r abl e. I t s e e ms per t i - ne nt t o def i ne not onl y t he p r e s e n c e or a b s e n c e of vas cul ar i nvasi on but al so t he si t e of t hi s vas cul ar i nvas i on i nt o t he wal l of t he c ol or e c t um. The me nt s . ACKNOWL E DGME NT aut hor s t hank M. Ohar a f or cri t i cal corn- R E F E R E N C E S 1. Aldridge MC, Phillips RK, ~Iittinger R, FryJS, Field- ing LP. Infl uence of t umour site on presentation, management and subsequent out come in large bowel cancer. Br J Surg 1986;73:663-70. 2. Knudsen JB, Nilsson T, Sprechler M, Johansen A, Christensen N. 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