RCM-1

PROGNOSTIC SIGNIFICANCE OF ALPHA-1-ANTITRYPSIN IN EARLY STAGE OF COLORECTAL CARCINOMAS
Seiichiro KARASHIMA,Hiroaki KATAOKA,Hiroshi ITOH, Riruke MARUYAMA and Masashi KooNO
Department of Pathology,Miyazaki Medical College,5200 Kihara,Kiyotake, Miyazaki 889-16,Japan.
We have previously shown that human colorectal carci-noma cell lines,RCM-I and CoCM-I,synthesize alpha-l-antitrypsin (α,-AT) in culture. We have studied immunohis-tochemically the incidence of α,-AT on histologic sections from paraffin-embedded tissues of surgically resected colo-rectal carcinomas and their metastatic foci, polypectomized adenomas, and normal mucosae. α,-AT was detected in 89 (61%) of 145 carcinomas (including 14 carcinomas in adeno-ma), and 12(39%) of 31 ‘adenomas. But only 2 (4%) of 55 normal colorectal mucosae were positive for αj-AT. In met-astatic tumor cells of colorectal carcinomas in lymph nodes and other organs, α,-AT positivity was 60% and 82%, respec-tively. The incidence of α,-AT was markedly higher in ad-vanced adenocarcinomas than in early ones and more fre-quent in adenocarcinomas of right side (including transverse colon) than those of left side and rectum, regardless of their histological malignancy grades. In mucinous carcinomas the frequency was greater (8 of 9 cases) than in conventional ad-enocarcinomas.
Clinical follow-up of the patients with colorectal carcino-mas suggested that α,-AT positivity in Dukes’ stage A/B tends to correlate with unfavorable prognosis irrespective of the grade of histologic differentiation of carcinoma, but there is no significant relation in Dukes’ stage C/D. Our findings sug-gest that αj-AT in colorectal carcinoma is related to the in-vasive and metastatic capacity. It may thus serve as a biologic marker for prognosis of colorectal carcinomas at relatively early stages(Dukes’ stage A/B).
Alpha-l-antitrypsin (α,-AT) is a glycoprotein with a molec-ular weight of 47-52 kDa and is classically considered to be synthesized by liver cells, released in the blood plasma,and distributed to all partsof the body (Laurell and Jeppsson, 1975).Recently,α1-AT has been used as an immunohisto-chemical marker for various tumor cells (Palmer et al., 1976; Ordonez et al., 1983; Aroni et al., 1984;Ordonez and Man-ning, 1984; Wittekind et al., 1986;Zuccarello et al., 1987; Soini and Miettinen, 1989). It has been observed by immuno-histochemical or immunofluorescent techniques to be present not only in normal epithelial cells of the stomach (Kittas et al., 1982b) and the small intestine (Geboes et al., 1982), but also in the gastric cancer cells (Ray et al., 1980; Tahara et al., 1984;Kittas et al., 1982a; Wittekind et al., 1986).However, absence of α1-AT in normal epithelial cells and epithelial tu-mors of the large intestine using similar immunohistochemical method has also been reported (Kittas et al., 1982a).
Conversely,Yoshimura et al.(1978) demonstrated human α1-AT in the sera of nude mice bearing human colon adeno-carcinoma by a double immunodiffusion method.Cheung and Lau(1986)isolated a trypsin inhibitor immunologically related to a1-AT from the extract of human colorectal carcinomas. Ganjei et al. (1988) demonstrated immunohistochemically the presence of αj-AT in tumor cells in 3 of 6 metastatic colonic carcinomas of the liver. Elevated levels of α1-AT were also observed in sera of patients with colorectal carcinoma(Ber-nacka et al., 1988).
Although a few human cell lines have been reported to syn-thesize functionally active α1-AT in vitro, all previous reports concern hepatoma cell lines (Glasgow et al., 1982; Carlson et al., 1984). We have previously shown that human colorectal carcinoma cell lines, RCM-1 and CoCM-1, established in our laboratory synthesize and release 2 or more serine proteinase

inhibitors in serum-free conditioned media. One of them has been isolated and identified as functionally active α1-AT by the double immunodiffusion method. The presence of α1-AT in the cytoplasm of such cultured cells and in tumors transplanted into nude mice has also been proved immunohistochemically (Kataoka et al., 1989b). We have now investigated the inci-dence of αy-AT in normal and neoplastic lesions of colorectal epithelium and clarified the relation between the expression of α1-AT in carcinomas and patient prognosis.
MATERIAL AND METHODS
Tissues
Formalin-fixed and paraffin-embedded tissue blocks from 131 cases with colorectal carcinomas (the rectum in 63,the sigmoid colon in 35, the cecum in 12,the ascending colon in 7,the transverse colon in 7, and the descending colon in 7),26 cases with metastatic foci (15 lymph nodes, liver, lung,stom-ach, kidney, omentum, 2 peritoneum,2 retroperitoneal soft tissues, urethra and seminal vesicle), 14 cases with carcinomas in adenoma, 31 cases with colorectal adenomas, and 55 cases of the normal colorectal mucosae removed far enough from the carcinomas,were studied. Of the patients with carcinoma and adenoma, 84 and 23 were men,and 47 and 8 were women, respectively.Their ages ranged from 33 to 93 years (mean,65) in carcinoma and from 27 to 78 years (mean, 58) in adenoma.
In all cases, α-AT immunoreactivity was examined and in some cases chosen at random, alpha-2-macroglobulin (α2-MG) was also tested. Albumin immunoreactivity was tested as a marker for non-specific uptake of plasma proteins.
Histology, immunohistochemistry and antibodies
The histological diagnosis of tumors was made on hematox-ylin-eosin (H-E) stained sections and adenocarcinomas were divided into 3 histologic grades following the criteria of Jass et al.(1986): well differentiated,moderately differentiated,and poorly differentiated type. The staging of carcinoma followed the criteria of the staging system of UICC (Hutter and Sobin, 1986) and we classified into 2 groups: early (Tis,T1,and T2) and advanced carcinomas (T3 and T4).
After routine histologic examination, serial sections 4 μm thick were obtained. For immunohistochemical evaluation, de-paraffinized sections were rehydrated,and pre-treated with 0.05% pepsin in HC1, pH 1.8, for 20 min at 37C,to restore immunoreactivity to αj-AT, α2-MG, and albumin (Soini and Miettinen, 1988).After blocking of the endogenous peroxidase with 0.3% H2O2 in methanol for 30 min, washing in phos-phate-buffered saline (PBS) and incubating with normal goat serum (1:75, 20 min, Vector,Burlingame, CA), sections were incubated with the primary antibody (1:400 anti-α1-AT;1:400 anti-α2-MG; 1:400 anti-albumin, rabbit gammaglobulin, DAKO,Glostrup, Denmark) for 30 min at room temperature in a moist chamber, and were followed by biotin-labelled second-
To whom reprint requests should be sent.
Received:August 8,1989.

FIGURE 1-SDS-PAGE/Western blot analysis (a). a: human α,-AT (2 μg); b and c: extracts of 2 carcinomas(68 μg).Proteins were immunochemically stained with anti-human α,-AT rabbit IgG by ABC method; d: extracts of carcinoma in b were stained with normal rabbit IgG by ABC method as a control. The positions and molecular weights (kDa) of the reference proteins are indicated on the left.Absorption test with antigen in α,-AT positive tubular adenoma. H-E stain (b); α,-AT immunostain (c); and αj-AT immunostain after treatment of anti-α1-AT antibody with antigen of 10-fold titer (d). Immunoreactivity was completely eliminated.Bar:100 μm.
ary antibody(1:200,Vector) for 30 min at room temperature. After washing, avidin-biotin complex (ABC)(Vector) was ap-plied for 45 min. Finally,0.02% diaminobenzidine in PBS containing 0.02% H2O2 was used to visualize immunoreactiv-ity.Counter-staining was performed with methylgreen.In all specimens, serum in blood vessels and leukocytes was used as internal positive controls. Immunoreactivity and specificity of these antibodies were ensured by absorption tests which was done without antigens and with equal to massive antigens (α1-AT, α2-MG, albumin; Sigma, St. Louis, MO).
Staining results were evaluated semi-quantitatively,taking into account the number of the stained cells (<10%=-, 10-50%=+,>50%=2+) and the staining intensity (negative=-,weakly stained = +,moderately to strongly stained=2+). Total point over 2+ was considered as pos-itive.Albumin-positive cases were excluded to rule out non-specific uptake of plasma protein by tumor cells.
Immunoblotting
To check the specificity of anti-human α1-AT antibody im-munoblotting technique on nitrocellulose after SDS-PAGÉ was performed(Kataoka et al., 1989a). Briefly, one part of 2, surgically resected,primary colorectal carcinomas before for-malin-fixation were minced,homogenized and extracted with
TABLE I-IMMUNOREACTIVITY FOR a,-AT AND α2-MG IN NORMAL MUCOSA AND IN COLORECTAL TUMORS
Histopathologic -AT a 2-MG
diagnosis Positive/total(%) Positiv e/total(%)
Normal mucosa 2/55 (3.6) 0/24 (0.0)
Adenoma 12/31 (38.7) 3/16 (18.8)
Carcinoma in adenoma 7/14 (50.0) 1/4 (20.0)
Adenocarcinoma 74/122(60.7) 6/29 (20.0)
Well differentiated 24/40 (60.0) 6/0 (0)
Moderately differentiated 47/77 (61.0) 4/18 (22.2)
Poorly differentiated 3/5 (60.0) 1/1 (100)
Mucinous carcinoma 8/9 (88.8) 1/1 (100)
Metastatic lesion 18/26 (69.2) 1/11 (9.1)
Lymph nodes
Other organs 9/15 (60.0)
9/11 (81.8) 1/8
0/3 (12.5)
(0.0)

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FIGURE 2 – Variation in αj-AT immunoreactivity. α,-AT positive adenocarcinomas by immunostain:well differentiated (a);moderately differentiated (b); poorly differentiated type (c); and mucinous carcinoma (d). In poorly differentiated adenocarcinoma,weakly positive cells distributed diffusely and several scattered, strongly positive tumor cells are seen.Bar:50 μm.
100 mM Tris-HCI(pH 6.8)/1% 2-mercaptoethanol/1% SDS. After centrifugation of the extracts,supernatants were sepa-rated by SDS-PAGE and transferred on nitrocellulose mem-brane.The membrane was treated with anti-α1-AT antibody (20 μg/ml) and stained using Vectastain ABC Kit.
Clinical follow-up
Clinical data were available from 74 cases in 131 of the colorectal carcinomas. Patients who died within 1 month after operation were excluded to rule out operative death.Staging of the carcinomas was performed according to the Dukes’ classi-fication modified by Turnbull et al. (1967). Stage A tumors

were confined to the bowel wall; stage B extended into the peri-colic fat;stage C had regional lymph node metastasis;and stage D either invaded adjacent organs or had distant metasta-sis.The Kaplan-Meier method was used to calculate the post-operative survival rate and to determine the statistical differ-ence.The log-rank test was used to compare prognosis.
RESULTS
Initially, 2 methods were performed to check the specificity of anti-human α1-AT antibody used in our experiment,i.e., immunoblotting technique and absorption test. SDS-
TABLE II-DISTRIBUTION OF COLORECTAL CARCINOMAS,HISTOLOGIC TYPES AND a-AT IMMUNOREACTIVITY
Histological ite,aj-AT(+)cases/total,(a,AT(+)% )
type Rectum 35/63(55.6) Sigmoid 21/35(60.0) Descending 4/7(57.1)
Left side 60/105 (57.1)
Well 20/36 (55.6) 11/17 8/15 1/4
Moderate 33/61 (54.1) 18/39 12/19 3/3
Poor 1/2 (50.0) 1/2 –
Mucinous 5/5 (100.0) 5/5 1/1 2/2
Transverse 6/7(85.7) Ascending 6/7(85.7) Cecum 10/12(83.3)
Right side 22/26 (84.6)
Well 4/4 (100.0) 1/1 1/1 2/2
Moderate 14/16 (87.5) 2/3 5/6 7/7
Poor 1/2 (50.0) 1/1 – 0/1
Mucinous 3/4 (75.0) 2/2 1/1 1/2

a1-ANTITRYPSIN IN COLORECTAL CARCINOMA

247
PAGE/Western blot analysis of commercial human α1-AT and each tissue extract from 2 colorectal carcinomas were per-formed. One band 56 kDa is present for human α1-AT.Two extracts also showed only one band at the same molecular weight(Fig. la). It is clear that α1-AT reacts specifically with anti-human α1-AT antibody. One absorption test is shown in the case of tubular adenoma. Tumor cells showed moderate to strong α,-AT immunoreactivity in their cytoplasm and staining was eliminated by pre-treatment of the antibody with antigen at 10-fold titer (Fig.1b-1d).
Immunohistochemistry
The results of immunohistochemistry of α1-AT and α2-MG in primary carcinomas,their metastatic foci, adenomas,and normal mucosae of colon and rectum are summarized in Tale I. αj-AT was positive in only 2 cases (3.6%) of normal mu-cosae in which luminal surfaces of crypts were weakly stained but cytoplasms of epithelium were seldom stained. Positive results were obtained in 39% adenomas, in 62% carcinomas including carcinomas in adenoma, and in 69% metastatic le-sions.Positive tumor cells showed diffuse cytoplasmic and intra-cytoplasmic vacuolar staining with variable intensities (Fig. 2). α2-MG was also positive in adenomas, carcinomas and their metastatic lesions; 19%, 21%, and 9%,respectively, but the positive cases were less than for a1-AT.
There was no relationship between a,-AT immunoreactivity and histological grade of carcinomas (Fig. 2a-2c): the inci-dence of well differentiated,moderately differentiated and poorly differentiated types of adenocarcinomas were 24/40, 47/77,and 3/5,respectively. There was no significant differ-ence of positivity (about 60%). But a high incidence of α-AT positivity, 88% (8/9), was found in mucinous carcinomas (Fig. 2d).Concerning the site, positive cases were significantly more frequent in the right side of the colon (including trans-verse colon) than in the left side and rectum(p<0.05)as shown in Table II. A correlation between the depth of invasion and α1-AT im-munoreactivity was observed. Infiltrative cancer cells in the invasive margin tended to increase α,-AT positivity (Fig. 3). The number of positive cases in early carcinomas(Tis,TI,and T2) and advanced carcinomas (T3 and T4)were 41%(7/17) and 66% (75/114), respectively, (p < 0.05) (Fig. 4). Metastatic lesions in lymph nodes were positive in 60% of cases, and in organs other than lymph nodes, such as liver, lung, kidney, peritoneum, seminal vesicles,urethra and retro-peritoneal soft tissue, were positive in 82%. Staging FIGURE 4-Relationship between α1-AT immunoreactivity and depth of invasion. αj-AT positive （口） and negative carcinoma（口）. Tis: carcinoma in adenoma;T1,2,3, and 4:tumor invades into the submucosa,muscularis propria,subserosa,or into non-peritonealized peri-colic or peri-rectal tissue, and visceral peritoneum or invades into other organs or tissue,respectively. In the cases of carcinomas in adenomas, 7 (50%) of 14 cases were α1-AT positive, as detected in carcinoma cells only (Fig. 5), or in both carcinoma and surrounding adenoma cells. In no case were a1-AT-positive cells detected in the adenomatous FIGURE 3-α1-AT immunoreactivity in invasive zone of moderately differentiated adenocarcinoma in stage B:H-E stain (a);and immu-nostain (b). Bar:200 μm. 248 KARASHIMA ET AL. FIGURE 5-a1-AT immunoreactivity in a carcinoma(right) in adenoma (left): H-E stain (a); and immunostain (b). α,-AT positive cells were seen in the part of carcinomatous region.Bar:500 μm. region only. In adenomas,α1-AT immunoreactivity showed a tendency to be negative in well-characterized types and posi-tive in more atypical cells with poor differentiation toward goblet cells(Fig.1b,1c). Clinical follow-up We also conducted a follow-up study in 74 patients with known disease stage who had been operated for primary colo-rectal cancer at the Second Department of Surgery of Miyazaki Medical College(Table III). The average 5-year survival rates in patients were 78% in stage A, 63% in stage B,54% in stage C,and 0% in stage D.There was no statistical difference inthe survival rates between stages A and B. We thus grouped the patients into 2 populations: non-metastatic group,stage A/B, and metastatic group, stage C/D (Fig. 6a). In patients with stage A/B carcinomas,the 5-year survival rates were 61% in α,-AT-positive and 100% in α1-AT-negative cases.Patients with α-AT-positive colorectal carcinomas had significantly shorter survival times compared with those with α1-AT-negative carcinomas,the result being statistically significant (p<0.05) (Fig. 6b). On the other hand, no significant differ-ence in survival rates in stage C/D was observed between pa-tients with aj-AT-positive and -negative cancer cells,42.4% and 50.8%,respectively(Fig.6c). DISCUSSION We have demonstrated the presence of α1-AT by immuno-histochemistry in 63% of colorectal carcinomas and in 39% of colonic adenomas.Failure to demonstrate the presence of a1-AT in colorectal carcinoma cells (Kittas et 'al.,1982a) might result from the absence of pepsin pre-treatment.Pro-teinase pre-treatment indeed improves immunoreactivity for many antigens by unmasking hidden antigenic sites in forma-lin-fixed tissues. Although quantitative analysis was not performed,the in-tensity of staining and the number of α,-AT-positive cells were increased in invasive zones. A higher incidence of cancer cells in advanced stages and metastatic foci than in early stages of carcinoma were also observed. Such association suggests that α1-AT-positive tumor cells which might correspond to more highly malignant clones tend to emerge and become dominant as the tumor spreads. The higher incidence of α,-AT in mu-cinous carcinomas may be related to poor prognosis (Minsky et al.,1987). An interesting finding was the correlation of α1-AT immu-noreactivity with prognosis. Following the classic study of Dukes (1932), the prognosis of carcinoma patients is closely related to the stage of spread at the time of resection. Patients' prognosis was compared with a1-AT immunoreactivity in stage A/B.This showed that α1-AT-positive patients had a significantly worse prognosis than negative ones. Survival of positive patients in stage A/B approached that in stage C.Our results suggest that a-AT in colorectal carcinoma is related to invasive growth,and that a1-AT immunohistochemistry is probably a useful tool to differentiate colorectal carcinoma cases at an early stage according to low or high invasion and metastasis capacities. Relationships between α1-AT positivity and survival of pa-tients with gastric carcinomas are contradictory: Tahara et al. (1984)suggested a worse prognosis, whereas Wittekind et al. (1986) suggested better survival probability in patients with α1-AT-positive tumors. Our results are in agreement with the former observations. Bernacka et al. (1988) also described an increase of αj-AT in the serum correlating with the severity of symptoms of colorectal carcinomas at initial diagnosis. The role and significance of tumor-derived α1-AT and the reasons for the poor prognosis of α1-AT-positive colorectal carcinoma cases are not established.Three possibilities may be TABLE III-RELATIONSHIP BETWEEN DEATH RATE AND a,-AT IMMUNOREACTIVITY IN STAGES A/B AND C/D COLORECTAL CARCINOMAS -AT Well Histological typ e(death/survival) Mucinous Total(death%) Moderately Poorly Stage A/B:death/total=7/39(17.9%) Positive 4/6 27 二 1/2 7/22(31.8) Negative 6/0 0/8 - 0/17 (0) Stage C/D:death/total=18/35(51.4 %) Positive Negative 2/3 0/2 77 5/5 - 1/0 3/0 - 12/22(54.5) 6/13(46.1) a1-ANTITRYPSIN IN COLORECTAL CARCINOMA 249 Survival Rate (%) Months FIGURE 6-Survival of patients with colorectal carcinoma.Stages A/B and C/D (a); survival of patients of α,-AT-positive and-negative groups in stages A/B (b) and C/D (c).The difference is significant between the α1-AT-positive and -negative groups in stage A/B and not significant in stage C/D. considered to explain our results.First,host-tumor immune responses may be modulated by tumor-derived α1-AT.There is general agreement that better prognosis is associated with stro-mal lymphocyte infiltration (Pihl et al., 1977;Watt and House, 1978; Nacopoulou et al., 1981; Jass et al., 1986; Ambe et al., 1989). Several lines of evidence suggest that α1-AT may mod-ulate the immune responses in favor of tumor cells, i.e., αj-AT is important in inhibiting the blastogenic response of normal lymphocytes (Arora et al., 1978) and cytotoxic reactions of lymphocytes (Redelman et al., 1980; Ades et al.,1982),as well as lymphocyte-granule mediated cytolysis which requires serine proteinase activity (Hudig et al.,1987).The relationship between α1-AT positivity and the extent of lymphocytic infil-tration in colorectal carcinoma remains to be determined. Second, tumor-derived α1-AT may act as an endothelial cell growth factor or a survival factor. McKeehan et al.(1986) indeed showed that αj-AT exhibits growth-stimulatory activi-ties on endothelial cells. Nakamura et al. (1984) have also shown that trypsin inhibitors including α1-AT increase the sur-vival of rat hepatocytes in serum-free culture by inhibiting trypsin-like proteinase associated with plasma membrane. The tumor α1-AT may therefore act as an endothelial cell growth factor to maintain blood circulation in the tumor and/or as an autocrine cell survival factor by regulating the excess protein-ase activities of tumor cells themselves. Third, α1-AT may act as a proteinase “carrier” rather than a proteinase "inhibitor” (Beatty et al., 1982), particularly in the micro-environment of the invasion zone. The tumor-cell-derived proteinase-α1-AT complex may dissociate in the presence of suitable substrate and the released proteinase may play a role in degrading the extracellular matrix. This possi-bility is highly speculative and there is no evidence to support it. However, α1-AT-proteinase complex is present in the se-rum-free culture medium of the RCM-1 cell line (Kataoka et al., 1989a). On the other hand, in addition to the biological activities, expression of α1-AT may be an epiphenomena of dedifferentiation of colorectal carcinoma cells, since normal colonic epithelial cells appear almost negative for a1-AT. Thus, α1-AT immunohistochemistry may be an efficient tool for detecting potential dedifferentiation, which cannot be de-tected using routine histology. α,-AT-positive colorectal car-cinomas would then be more poorly differentiated tumors even though with routine histology they appear as well differentiated tumors.
It has been believed that increased α,-AT in cancer patients’ serum and/or tumor tissue cells derives from enhanced synthe-sis in liver cells as a result of increased release of proteinases by tumor-cell lysosomes or by mononuclear cells in the inflam-matory infiltrate that surrounds the tumor (Ikuta et al., 1982). According to Kittas et al. (1982a), Akatsuka et al. (1979)and Reintoft and Hagerstrand (1979),tumor cells themselves may be a third source of α,-AT. To ascertain the origin of αj-AT, we compared the immunoreactivity of α1-AT with that of al-bumin which is also a major plasma protein and has been used as a marker for non-specific uptake of plasma proteins. In a few cases,clear-cut similarities in the distribution of α1-AT and albumin have been observed. In most cases,however,such similarities were not observed.Previous work has shown that cultured colorectal carcinoma cell lines produce and release functionally active α1-AT in serum-free conditioned medium (Kataoka et al., 1989b). Our present findings indicate in ad-dition that in most cases α1-AT is produced by the tumor cells themselves.However,the origin of a-AT in the tumor cells cannot be established by immunohistology alone.
ACKNOWLEDGEMENTS
We thank Dr. I.Iwamoto and Dr. T.Shimayama,Miyazaki Shigun Ishikai Hospital, who generously gave us the chance to examine surgically resected colorectal specimens by immuno-blotting,and Mr. T. Miyamoto, Mr. S. Sato and Mrs. Y. Nomura for their excellent technical assistance. This work was supported in part by a grant from the Nippon Roche Institute, Tokyo,Japan.
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