Prognostic significance of cancer cells in the circulating blood

Prognostic Significance of Cancer Cells in the Circulating Blood A Ten Year Evaluation S.

STUART

M.D.,*~

KOBERTS,

JONAS VALAITIS,

JOHN W.

M.D., ELIZABETH

HENGESH,

A. MCGREW,

Chicago,

LTHOUGH RESECTION Of metaStatiC the lung

or liver

cure in some cases are still a major

has resulted

[I],

down despite improvements primary

tumors.

semination The

deserves

channels

may

sequence

of four phases:

in the blood bolic

cancer

followed survival That

by

(3)

(4)

fate

of the embolic phase

with

and

than

capillaries,

either

with

experience the positive

and

report

presents

material

or

the transporta-

in our laboratories This

death

cancer cells.

concerned

a decade.

in a

of the em-

tion of cancer cells in the circulating been investigated

vascular

(2) transportation

lodgment

cells in arterioles the

by

as occurring

(1) entrance of cancer

channels,

stream,

of

study.

cancer

be considered

cells into vascular

rates

this route of dis-

intensive of

metastases

in the treatment

Accordingly,

dissemination

lesions in

survival

blood

a cytologic

Nethods of Blood. Blood

has that

review

Isolating

Cancer

of

and the five to ten year

survivals. MATERIAL AND METHODS Patient Xaterial. Since July 1956, more than 1,700 patients at the University of Illinois Research and Educational Hospitals have been studied for the presence or absence of cancer cells in their blood samples. Almost half, ifiT of these patients, were

” Markle Scholar in Academic Medicine. i PRESENT ADDRESS: Department of Surgery, 1’01 11.3, June

1967

Cells

from

Ll’hole

samples from the patients in this study were processed by one of four methods of isolating cancer cells (reported in detail elsewhere) : (1) albumin flotation method (Roberts and associates [a]), (2) streptolysin method (Long and associates [3]), (3) dextran sedimentation method (Romsdahl and associates 141). and (4) differential filtration method (McGrath and associates [j]). Eight to twelve slides were made from each 10 cc. blood sample in each of the methods. All slides were stained by Cytologic Identijcatiorr. the Papanicolaou technic 161. The slides were systematically screened and all large nucleated cells and

for more

summarizes

B.S.,

studied more than five years, and many as long as ten years. They were classified as “curable” or “incurable” at the time the blood samples were ob tained. The term “curable” implies only a theoretic possibility based on radiographic. gross, and microscopic exclusion of all evidence of tumor extension beyond the confines of surgical resection. Of the T(i; patients studied five to ten years ago, 24” were classified as “curable.” A total of 20.5 patients were studied before, during, and after operation. The iti7 patients represent a cross section of all types of malignancies, although it should be emphasized that the patient material does not, until recently, reprcsent consecutive admissions to the hospital. Collection of Blood Samples. Xliquots of IO cc. heparinized blood samples were collected from various sites, including the antecubital vein and veins draining the tumor. Sometimes the sample was obtained by inserting a catheter into the superior or inferior vena cava, depending on the tumor area 11 1. Aksfew as one or as manv as fifty-live blood samples were collected from individual patients.

in apparent

blood-borne

factor in holding

MCGRATH,

Illinois

From the Departments oJ‘ Surgery and Pathology, Univeuszty of Illinois College of Medicine, Chicago, Illinois. This amork was supported by l’.S.P.H.S. Grant CS 9592.

A

M.D., RUTH G.

M.D., AND WARREN H. COLE, M.D.,

Ohio State 7.57

Urliwrsity,

Cdurt~bu~,

Ohio.

758

Roberts

et al. clumps of cells were marked by cytotechnologists. The final interpretation of all marked slides was made by one of us (E. A. M.), a cytopathologist. Whenever possible, a direct smear of the resected tumor was prepared to allow comparison with cells isolated from the blood stream. (Fig. 1.) In a few cases, suspensions of bone marrow cells were prepared and stained by the Papanicolaou technic for comparison of the morphologic features of immature hematopoietic cells. The criteria of malignancy have been listed in detail elsewhere [7]. With the experience of the immature hematopoietic cells [8] and endothelial cells [9], all slides originally reported as positive have recently been reviewed by two of us (E. A. M. and J. V.). RESULTS

FIG. 1. Malignant melanoma of the rectum; Papanicolaou stain, original magnification X 1300. Top. clump of malignant cells obtained from a direct smear of the resected tumor. Bottom, clump of malignant cells isolated from the inferior mesenteric vein during opera-

tion.

Incidence of Cancer Cells in Blood Samples. Only those patients who were studied more than five years ago and available for follow-up study are included under this heading, that is, 767 patients. Not included are twenty-three patients with cancer lost to follow-up study. These patients have been categorized under three headings: (1) all cancer patients (Fig. 2), (2) all “curable” patients (Fig. 3), and (3) all patients during operation (Fig. 4). No distinction is made with regard to the source or number of blood samples. In other words, a patient is recorded as having a positive blood sample only once, regardless of whether the blood sample was obtained from the vena cava or antecubital vein or whether only one of fifty blood samples was positive. The original cytologic diagnosis was positive for cancer cells in 279 (36 per cent) of the 767 cancer patients. However, with the recent review, only 52 (7 per cent) of the 767 cancer patients had blood samples which were upheld as positive for cancer cells. All cases with only suspicious findings are included in the negative classification. Approximately the same reduction rate that was found in the review of the positive cases obtains when the “curable” patients are considered, or the patients studied during operation. Hereafter, all results in this report will refer to data resulting from the recent review. As seen in Figures 2, 3, and 4, there was no significant difference in the incidence of positive blood samples in the various categories, including 7 per cent of all cancer patients (including “curable” and “incurable”), 8 per cent of all “curable” patients, and 9 per cent of all patients studied before, during, and after operation. A positive case in this American

Journal

of Surwy

Cancer

Cells in Circulating

latter category refers only to patients who had neptive blood samples prior to operation but a positive blood sample either during or after operation. l%e to Ten Year Survival. Of all 767 cancer patients studied more than five years ago, only ninety-four are alive, with no evidence of disease (NED). In this surviving group, only 7 per cent had positive blood samples whereas 93 per cent had negative blood samples. Again, considering all cancer patients as seen in Figure 2, there was no difference in the five to ten year survival when considered in terms of positive or negative blood samples, that is, 13 versus 12 per cent. (Fig. 2.) Likewise, in the 242 patients classified as “curable,” there was no difference in the five to ten year survival in terms of the positive or negative blood samples; the rate was 32 per cent in both categories. (Fig. 3.) Although there was no apparent difference in the five to ten year survival of the entire group of patients with positive or negative blood samples, a shower of cancer cells during or after an operative procedure was associated with a survival rate that was only a half (8 versus 16 per cent) of what it was for patients with negative blood samples before, during, and after operation. (Fig. 4.) The development of vascular metastases more than doubled (39 per cent) in the patients with showers of cancer cells during or after operation as compared with those (18 per cent) with negative blood samples at all times. (Fig. 4.) This same increase in the incidence of vascular metastases in patients with positive blood samples is seen in all “curable” cancer patients, that is, 32 versus 16 per cent. (Fig. 3.) No difference is seen in the entire group of cancer patients (Fig. 2) with regard to the development of vascular metastases.

7.X

Blood

87pts

Negafive ,S(q

30% 212 pts

FIG. 2. All cancer

5-/o reor Surnval -

Blood sampies

-

‘~~~

patients

VOSC’hX MetOStOSeS

d3,F20 monthr

/

00

242 Patients

32% NED

/

OY 64 mo”,h~

\

-‘A

16% dead 27 months

FIG. 3. Only “curable”

patients.

COMMENTS

Cells in Blood Samples. The reports in the literature on this subject have varied widely. More than 5,000 patients have been reported on during the past ten years by more than forty different investigative teams in various parts of the world [IO]. As summarized by Goldblatt and Nadel in 1965 [lo], approximately 20 per cent of “curable” cancer patients have cancer cells in the blood stream, and 30 per cent of “incurable” patients have positive blood samples. As indicated in Incidence

of

Cancer

Vol. 113, June 1967

iE-te

Posi,,“e/

months

9%\

23pts.

205 Patients

/

8% NED 0” 54 n?O”,bS

\

dead 24 r”o”ths

FIG. 4. All patients during operation.

760

Roberts

our material, recent reports on the frequency of positive blood samples tend to be lower than they were ten years ago. The wide variation in the frequency of positive blood samples in the literature is to be expected when one considers the widely different approaches to the methods of isolation, collection of blood samples, and cytologic criteria of malignancy. Despite these variations, there are several areas of agreement with regard to the many factors which govern the frequency of positive blood samples. The site of the blood samples appears to be of great importance. About three times as many positive blood samples are obtained from the local venous blood draining the tumor area as are obtained from the antecubital vein. Clumps of cancer cells are far more common in local venous blood samples than in peripheral blood samples, where they are rare indeed. The timing of these blood samples is of equal importance; they must be drawn at frequent intervals before, during, and after the various forms of any manipulation of the primary tumor in order to detect an isolated shower of cancer cells in the blood stream which may be undetectable in a matter of minutes after a positive blood sample [I]. Although cancer cells have occasionally been seen on routine blood smears, such a chance finding is so rare that some method must be used to isolate or concentrate the relatively few cancer cells from the numerous formed elements of whole blood. The fact that twenty different methods have been reported in the literature shows that none of these methods is ideal in terms of ease of performance, quantitative recovery, good cytologic quality of nucleated elements, and so forth. Most methods are either too complicated or time-consuming for general usage. Some methods, such as the albumin flotation and dextran sedimentation methods, preserve the quality of the cytologic preparation of the isolated cancer cells at the expense of the quantitative recovery. Other methods, such as the streptolysin method, preserve the quantitative recovery at the expense of the quality of the cytologic preparation. At present we have settled on the differential filtration method using the Millipore filter as the quantitative recovery with maintenance of a good cytologic preparation [5]. Probably the most important single factor which influences the incidence of cancer cells in the blood stream is the cytologic criteria

et al. used for identifying a cell in a peripheral blood sample as being malignant. During the past ten years, more than 40,000 slides have been scanned in our laboratory for the presence or absence of abnormal nucleated cells in blood. With our recent review of the original positive cytologic diagnosis rendered five to ten years ago, the decrease rate in positive cases is Sl per cent (from 279 cases to only 52 cases remaining positive). During the first two to three years of our study, the megakaryocytes and immature cells of the blood series were the major problem areas of differential diagnosis [8]. With a refined technic of double filtration [5] during the past two years, endothelial cells or clusters of capillaries have been a problem area of differential diagnosis [9]. Experience in our laboratory and in others has clearly shown that problems in blood studies are different from those in other areas of diagnostic cancer cytology [II 1. Five to Ten Year Survival. Although considerable data concerning cancer cells in the blood have been accumulated in the past ten years, few studies of clinical survival have been reported [IZ-141. Engell [12] in 1959 reported the five to nine year follow-up study of 125 cancer patients. Cancer cells were found in either the peripheral or regional blood samples in 61 per cent of these patients, most of whom had had an operation for malignancy of the intestinal tract. Of the fifty-five patients who survived more than five years, half (51 per cent) had cancer cells in the blood samples at operation. Engell therefore concluded that the demonstration of cancer cells in the blood had very little prognostic significance. Watne, Sandberg, and Moore [13] reported an eighteen month follow-up study of 817 patients with various stages of malignancy after palliative or curative types of surgery. The survival rate was 54 per cent for the fiftyseven patients who had cancer cells in the circulating blood as compared with 71 per cent for those patients with negative blood samples. There are several possible explanations for the finding that our entire group of 767 cancer patients had no difference in survival with positive or negative blood samples. As mentioned previously, the patient material in this report does not represent consecutive admissions to the hospital but rather a highly selected group. Because of the time (four to six hours) and American

Journal

of Surgery

Cancer Cells in Circulating personnel required to process each blood sample completely, the study was limited at first to those patients who were thought to have a possibility of circulating cancer cells. The high degree of selectivity for patients in this series is reflected in the very low over-all five to ten year survival rate of 12 to 13 per cent. Although 242 patients were classified as having so-called “curable” lesions, very few of them had a small, early-staged tumor. It is also quite probable that many patients with blood samples interpreted as negative did in fact have cancer cells in the blood stream which were either too few to be isolated or were lost during processing of the blood sample. The fact that 212 (30 per cent) patients with negutiw blood smnples in the entire group of 767 patients had vascular metastatic disease supports this hypothesis. (Fig. 2.) The decreased survival rate of patients with previously negative blood samples which became positive during or after operation is to be expected from experimental data [I ,141. The number of tumor “takes” in the lungs of animals has been shown to be directly proportional to the number of cancer cells injected intravenously. Secondly, animals subjected to celiotomy followed by injection of cancer cells intravenously will have increased tumor “takes” in the lungs in comparison with control animals subjected only to anesthesia followed by injection of cancer cells intravenously [I]. Therefore, a shower of cancer cells during operation may well occur at a time when the natural host resistance of the patient may be reduced because of operative stress. SUMMARY

Seven per cent of 767 patients with cancer studied more than five and as many as ten years ago in our laboratory had positive blood samples. A similar frequency of positive blood samples was obtained when only those patients considered “curable” were tabulated, or those patients studied before, during, and after operation. The survival rate in the entire group of 767 patients was not different with regard to positive or negative blood samples, being 13 and 12 per cent, respectively. The survival rate for “curable” patients with positive blood samples was no different from the survival rate for those with negative blood samples; it was 32 per cent for each category. IYol. 113. June 1967

7(il

Blood

Although there is no apparent difference in the five to ten year survival rates of all FE cancer patients, regardless of whether the blood samples are positive or negative, a definite trend is apparent in the decreased survival of patients with a shower of circulating cancer cells during or after operation. Only two of twenty-three patients (8 per cent) with showers of cancer cells demonstrated during operation were alive and well five to ten years postoperatively as compared with a 16 per cent survival (26 of 182) of patients with negative blood samples at all times before, during, and after operation. Also, vascular metastases developed in 3!1 per cent of those patients with showers of circulating cancer cells during operation as compared with only 1X per cent of those patients with all negative blood samples. REFERENCES

Spread by the vascular system. In: Dissemination of Cancer and Preventive Measures, chapt. 5. Edited by Cole, W. H., McDonald, G. O., Roberts, S., and Southwick, H. Sew York. 1961. Appleton-Century-Crofts, Inc. ROBERTS, S., WATNE, A., MCGRATH, R., MCGREW, E. A., and COLE, W. H. Technic and results of isolation of cancer cells from the circulating blood. Arch. Surg., 76: 334, 1958. LONG, L.. ROBERTS, S., MCGRATH, R., and McGREW. E. Simplified technique for separation of cancer cells from blood. /.A.hl.A ., 170: 17X5, 1959. ROMSDAHL, M. M., CHU, E. W., HUME, R., and SOUTH, R. Method for cytological detection of tumor cells in vitro. J. ivat. Cancer Inst., 26: 19, 1961. MCGRATII, R., VALAITIS, J,, McGRE~, E., and ROBERTS, S. Separation of cancer cells from blood by differential filtration. In preparation. PAPANICOLAOU,G. N. Atlas of Exfoliative Cytology. Cambridge, Mass., 1954. Harvard University Press. MCGREW, E. A., ROBERTS, S., WATNE, A., JONASSON, O., and COLE, W. H. Circulating blood. In: Supplement II to Atlas of Exfoliative Cytology, chapt. 10. Edited by Papanicolaou, G. K. Cambridge, Mass., 1960. Harvard University Press (for the Commonwealth Fund). McGRE~, E. A., ROMSDAHL,M. M., and VALAITIS, J. Differentiation of hematopoietic elements from tumor cells in blood. Acta cytol., 6: 551, 1961. VALAITIS, J., MCGREW, E. A., MCGRATH, R., and ROBERTS, S. Capillary cell clusters in the peripheral blood of postoperative cancer and noncancer patients. In preparation. GOLDBLATT, S. and NADEL, E. M. Cancer cells in the circulating blood: a critical review. Acta rytol., 9: 6, 1965. NADEI., E. M. et al. A cautionary note to those

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et al. prognostic value of tumor cells in Arch. Szhrg., 18: 190, 1961. 14. ROBERTS, S., JONASSON, O., LONG, L., R., MCGREW, E., and COLE, W. significance of cancer cells in the blood. Ann. Surg., 154: 362, 1961.

American

the blood. MCGRATH, H. Clinical circulating

Journal

of Surgery