T U M O R AND HOST CALCIUM IN HUMAN COLON CARCINOMA

BERNARD FISHER, MD,* ELIZABETH SAFFER, BA, AND EDWIN R. FISHER, M D ~

The popular hypothesis that local invasiveness and distant spread of tumors is the result of decreased adhesiveness of cancer cells due to the lowered calcium content has recently been subjected to critical reappraisal. Because of the paucity of information concerning the calcium content of human tu- mors relative to normal tissues, the present investigation was carried out to determine the calcium content of 25 adenocarcinomas of the colon, together with normal adjacent colonic mucosa, utilizing the highly sensitive technique of atomic absorption spectrophotometry. Also, calcium content of tumors was related to lymph node involvement and tumor invasiveness as characterized by Dukes’ classification. Findings failed to demonstrate a significant differ- ence between tumor and mucosal calcium. Likewise, no correlation could be demonstrated between tumor calcium, lymph node involvement, or degree of tumor invasiveness. Such results provide further reason to question the pre- cise role of calcium in the invasiveness of human tumors.

INFORMATION WAS OBTAINED SEVERAL DECADES I ago by Comano, 7 and his associatesl3~ 15

relative to differences in force required by micromanipulation to separate normal cells and tumor cells in vitro. They observed that cells from carcinoma of the lip and cervix showed far less adhesiveness than did normal cells. These findings, and reports by others,=, 8.10 that tumors possess less calcium than do normal tissues has resulted in the hypothesis that local invasiveness and distant spread of tumors may be the result of decreased adhe- siveness of cancer cells due to their lowered calcium content.

It was recently observed by us11 in a series of in vivo investigations that neither levels of host serum calcium nor of tumor calcium influenced the incidence of metastases. More-

From the Departments of Surgery and Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pa. 15213.

Aided by U.S.P.H.S. Grants CA-05949 and CA-05716. * Professor of Surgery, University of Pittsburgh

School of Medicine. t Professor of Pathology, University of Pittsburgh

School of Medicine. The authors are grateful to Eugene Corriden, Ph.D.

and Miss Barbara Stewart of the Veterans Administra- tion Hospital, Leech Farm Road, for determination of calciums and to the Department of Pathology, Presby- terian-University Hospital, for aiding in obtaining the surgical specimens.

Address for reprints: Bernard Fisher, MD, Depart- ment of Surgery, University of Pittsburgh, Pittsburgh, Pa. 15213.

Received for publication July 25, 1969.

over, there was no difference in the invasive characteristics of cells obtained from low or high calcium-containing tumors. Also, re- cently Hickie and Kalantlz found by means of atomic absorption spectrophotometry that Morris Hepatomas contained at least 100% more calcium than did normal rat livers. These findings, as well as our concurrence with the statement by Weiss and Mayhewl4 in their critical review of the cell periphery that “there are not nearly enough data to make sweeping generalizations about the calcium content of tumors” and that old observations should be confirmed and ex- tended using the more sensitive technique of atomic absorption spectrophotometry, have prompted the present investigation. This paper records findings relative to the calcium content of 25 adenocarcinomas of the colon and of the normal mucosa adjacent to each obtained by utilizing this technique. I t also relates tumor calcium to lymph node in- volvement and to tumor invasiveness as characterized by Dukes’g classification.

MATERIALS AND METHODS

Data presented was obtained from speci- mens resected from 25 patients at the Uni- versity of Pittsburgh Medical Center having curable adenocarcinomas of the colon. Tissues were prepared for analysis immedi- ately following their removal. All mucosal

41

42 CANCER January 1970 Vol. 25

samples were removed from colon which was a t least 6 cm from the tumor edge. Segments of tumor and mucosa were trimmed free of all necrotic tissue, and all blood was eliminated in so far as it was possible. Specimens were neither washed, blotted with paper, nor ex- posed to any extraneous source of calcium. Tumor and mucosa obtained from each specimen were weighed to obtain the wet weight of the sample, wrapped in aluminum foil, and frozen until calcium determina- tions were made.

Samples were dried at 100°C for approxi- mately 12 hours and were then washed (6OO0C for 8 hours). Following cooling, samples were taken u p in 2 N HCl, and lanthanum oxide was added to protect against interference from phosphorous and aluminum. Solutions were brought to volume so that there was a final concentration of 1% lanthanum oxide in 5% v/v HCl. T h e calcium content of the samples was determined on a Perkin-Elmer Atomic Absorption Spectrophotometer (Model 303). A standard curve was estab-

lished using Fisher Scientific Company Cal- cium Reference Solution.

T h e parts/million of each sample was determined from this standard curve. T h e mg Ca/100 g of sample was calculated using the dilution and the original wet weight of the samples. Each determination was per- formed in duplicate. All tissue calcium values are expressed as mg/100 g wet tissues.

Information relative to lymph node invol- vement and tumor invasiveness was obtained from pathologic study of the specimen.

RESULTS

T h e average calcium content of 25 adeno- carcinomas of the colon was 11.96 e 5.82 mg/100 ml and that of the normal mucosa adjacent to the tumors 12.03 -C 2.85 mg/100 ml (P, > 0.9), Table 1. In 13, the tumor calcium was similar to, or greater than, the mucosal calcium. No statistically significant difference existed between the tumor and mucosal calciums of patients with tumor-

TABLE 1. Calcium Content of Colon Carcinomas and of Adjacent Normal Mucosa

# Positive nodes

Pt # Sex Age grade removed Tumor Mucosa 1 M 71 C 5/13 19.94 11.30 2 M 61 B 0/16 14.40 13.13 3 M 64 B 0/17 27.63 13.36 4 F 62 A 0/13 8.91 16.40 5 F 70 B 0/20 11.39 12.27 6 F 60 A 0/11 7.26 12.48 7 M 66 C 1 /7 6.98 14.03 8 M 76 C 5/16 9.18 7.87 9 M 41 C 12/17 6.78 10.25 10 M 79 C 1 /6 7.13 13.77 11 F 53 C 0/2 16.17 12.27 12 F 42 B 0/5 8.34 10.40

Calcium Content Dukes' # Nodes (mg Ca/100 g wet tissue)

13 F 71 B 0/6 7.06 9.53 14 M 58 B 0/8 10.40 21.10 15 F 80 B 0/11 24.60 8.10 16 F 48 C 6/17 11.30 9.70 17 M 48 C 0/17 7.44 11.47 18 hl 61 C 0/5 6.19 9.78 19 M 74 C 1 /6 11.90 12.60 20 M 87 C 8/10 8.58 12.91 21 M 65 C 17/18 16.77 15.60 22 M 64 C 1/17 9.69 8.68 23 M 58 B 0/17 7.15 11.83 24 M 45 B 0/13 17.07 10.59 25 M 66 C 1 /5 16.78 11.31

- - 11.96 12.03* - - Avg. f5.82 f2.85

* Tumor vs. mucosa: P value > 0.9.

No. 1 CALCIUM IN HUMAN COLON CARCINOMA - Fisher et al. 43

positive or tumor-negative lymph nodes in the resected specimen, nor was there a sig- nificant difference in the calcium content be- tween tumors and mucosa in patients with less or more invasive tumors (i.e., Dukes' A and B vs. C), Table 2. In the 12 patients whose tumor calciums were less than their mucosal calciums, 8 had negative lymph nodes.

DISCUSSION

The popular belief that human tumors contain less calcium than normal tissues is primarily the result of information from 3 published reports. I n 1946, Brunschwig et al.3 noted that an analysis of calcium in gas- tric carcinomas and adjacent mucosa in 14 patients revealed an average calcium of 7.2 mg/100 ml in the former and 9.3 mgJlO0 ml in the latter. I t was concluded that such tumors contained less calcium than did nor- mal tissue. It is of interest, however, that in the same paper it was also reported that the calcium content of gastric mucosa from pa- tients not having a gastric carcinoma was 7.1 mg/100 ml. It might as easily have been con- cluded that the gastric mucosa of patients with stomach cancer contained a greater amount of calcium than did normal gastric mucosa.

In the same year (1946), Dunham, Nichols, and Brunschwiglo reported that colon carci- nomas also contained less calcium than did the adjacent normal colonic mucosa (7.9 mg/100 ml vs. 12.1 mg/100 ml for 10 patients). A chemical method was employed for calcium determinations in this study as well as the one concerned with stomach carcinoma. Using

flame photometry for calcium measurement, delong, Coman, and Zeidman,8 in 1950, re- ported that the calcium content of 10 colon carcinomas was lower than that in nearby mucosa. The values were almost identical with those reported by Dunham et al. In none of the studies was there an attempt to relate tumor calcium to tumor invasiveness or metastases.

The present study, employing the highly accurate and reproducible technique of atomic absorption spectrophotometry for calcium determination, has produced find- ings which fail to confirm those obtained using other analytic methods. While a certain num- ber of tumors did contain less calcium than did associated normal colonic mucosa, in an equal number there was either no difference, or tumor calciums were higher. No trend was evident to permit the conclusion that adenocarcinomas contain less calcium than colonic mucosa, nor could the tumor calcium content be related to either tumor invasive- ness or lymph node involvement. It is to be emphasized that nonnecrotic tumor tissue was selected for analysis from all specimens, for it has been demonstrated that necrotic tumor has a higher calcium content than viable tumor cells.1 It is, thus, not likely that this is the reason for the difference in find- ings.

The results of this study agree with our experimental findings that, in the model sys- tems employed, cell adhesiveness was not found to be sufficiently altered, despite a wide range of tumor calcium, to influence cell separation and consequently the inci- dence of metastases. In the present study, the presence or absence of lymph node in-

TABLE 2. Comparison of Tumor and Mucosal Calcium in Patients Having Adenocarcinomas of Colon With and Without Lymph Node Involvement and With Varying Invasiveness

P Values

Calcium Content Tumor (mg Ca/100 g wet tissue) vs. 1 vs. 2 3 v s . 4

# Tumor Mucosa Mucosa Tumor Mucosa

1 . Negative 12.43 nodes 14 f 6 . 7 9

2 . Positive 11.37 nodes 11 1 4 . 5 3

3. Dukes' 13.11 A & B 11 f 7 . 1 9

4 . Dukes' 11.05 C 14 f 4 . 5 5

12.34 f 3 . 2 2 > 0 . 9

0 .8 0 . 5 11.64

f 2 . 3 9 12.65

=k3.56

11.55 f 2 . 2 5

0 . 9

0 . 9 0 . 3 0.4

0.7

44 CANCER January 1970 1’01. 25

volvement with tumor was unrelated to the tumor calcium content.

T h e findings do not specifically refute Co- man’s hypothesis relating decreased mutual adhesiveness of neoplastic cells to their de- creased calcium content, or more precisely to a decrease in binding by the cell mem- brane. For it is possible that, despite a normal or elevated tumor calcium, there might be defective calcium binding at the cell peri- phery. How much of the tumor calcium de- termined was due to bound or diffusible (intracellular) calcium is unknown. Since,

however, Coman likewise did not have this information but based his conclusions on the presumed decrease of total tumor calcium, and if such a decrease fails to exist as is suggested in this study, then it would seem that there is little reason to continue to emphasize his conclusion as a factor in tumor invasion until methodology becomes avail- able which can quantitate calcium in the cell periphery or within the cell. Only then can definitive evaluation of this concept become possible.

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3. Brunschwig, A., Dunham, L. J., and Nichols, S.: Potassium and calcium content of gastric carcinoma. Cancer Res. 6:23&232, 1946.

4. Carruthers, C., and Suntzeff, V.: Calcium, copper and zinc in the epidermal carcinogenesis of mouse and man. Cancer Res. 6296-297, 1946.

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10. Dunham, L. J., Nichols, S., and Brunschwig, A.: Potassium and calcium content of carcinomas and papillomas of the colon. Cnncer Res. 6:233-234, 1946.

11. Fisher, B., and Fisher, E. R.: Role of host and tumor calcium in metastases. Cnncer Res. 28: 1753-1758. 1968.

12. Hickie, R. A., and Kalant, H.: Calcium and magnesium content of rat liver and Morris Hepatonia 5123tc. Cancer Res. 27:1053-1057, 1967.

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14. Weiss, L., and Mayhew, E.: The cell periphery. New Eng. J. Med. 276:1354-1361, 1967.

15. Zeidman, I.: Chemical factors in the mutual ad- hesiveness of epithelial cells. Cancer Res. 7: 386-389, 1947.