Flow cytometric detection of tumor metastases during urologic surgery

PRELIM][NA~'.~ C O M M U N I C A T I O N

FLOW CYTOMETRIC DETECTION OF TUMOt/ METASTASES DURING UROLOGIC SURGERY NEIL JAY NUSBAUM, M.D. ROBERT R. KESTER, M.D. UNNI M. M. MOOPPAN, M.D. HONG KIM, M.D. From the Lee Streich Flow Cytometry Laboratory, and the Departments of Medicine (Hematology/Oncology) and Urolog% Brookdale Hospital Medical Center, and the Health Science Center, Brooklyn, New York

t~ cytometric studies were performed on peripheral blood obtained before,, 7uent to urologic surgery. The results suggest that 7 of 1Z patients had a brief ~,ical manipulation when they had cells from the primary surgical site in the ~Soncentration of such cells was generally < lOe/mL even during t,he intraoperafindings suggest that perioperative tumor seeding is a real concern, a:nd that flow used to study maneuvers to reduce its incidence.

nalignaneies, local control is y, but the patient ultimately etastases. General surgical 2ated attempts to refine the to try to prevent intraoperaspread. Some measures ta.dominal operation for organ early ligation of the tumor's bviously not feasible as part ,h as transurethral resection )r. The long-term managewith a bladder tumor often ystoseopic examinations and rying a theoretical risk of 3ruination. has the capability to screen Lheir light-scattering charae?vides information on their y), as well as their fluores',n labeled with fluorescent or with a fluorophore-antirothelial tumor cells, unlike blood cells, should react eratin antibody. It was hy~w cytometry could screen ~ripheral blood cells to iden-

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tify and sort out a minor subl:,opulakion of circulating tumor cells. In this; pilot .study, evidence for such cells was detect~ed in peripheral blood samples drawn intraoperatively. Material and Methods Under a protocol approved by the Brookdale Hospital Medical Center's Institutional Review Board, and with informed[ consent, blood samples were obtained from patients prior to urologic surgery, during the procedure, and at the time they had postoperative bloodwork. Samples were obtained from patients who were thought, by one of us (R.R.K.), t:o be clinically at risk for perioperative seeding, e.g., patients with bulky lesions. The choice o:F urologic procedure was made by the attending urologist. The study was thus intended to look at the natural history of surgical intervention, not to modify standard surgical procedure. The buffy coat from peripheral blood samples was treated with red ceiL1lysing solution, fixed and stained for analysis on a Faestar (Beeton-Dickinson, Mountain View, CA) flow tyrometer, according to the manu:Facturer's instruction. Tumor cells were identified primarily by a

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TABLE I.

Case No. 1 2 3 4 5 6 7 8 9 10 11

Intraoperative blood samples in i1 patients

Specimen Papillary urothelial carcinoma, grade 1-2 Prostatic hyperplasia Bladder neck tumor with chronic inflammation and calcification. Hyperplastie prostatic tissue and bladder neck tissue with chronic inflammation Right kidney specimen: renal cell carcinoma (no invasion of pelvis, negative margins) Prostatic tissue with poorly differentiated adenoeareinoma Multiple bladder biopsies: papillary urothelial carcinoma grade II; dysplasia; in situ urothelial carcinoma Extensive poorly differentiated adenoearcinoma (prostate) Hyperplasia (prostate) Multiple (prostatic) foei of adenoeareinoma, well to moderately differentiated; chronic inflammation Extensive (prostatic) squamous metaplasia with foei of atypical cells; acute and chronic inflammation Papillary urothelial carcinoma, grade 3, with foei of superfieial invasion; prostatic tissue with glandular hyperplasia

two-step immunofluoreseenee proeedure for cytokeratin with mouse anti-eytokeratin (BeetonDiekinson 7650) and goat anti-mouse FITC (Beeton-Diekinson 9031), as well as by light side scatter and forward scatter criteria. DNA content was evaluated by staining with propidium iodide. In some eases, samples of the reseeted surgical speeimen were disrupted by serial needle passages and then analyzed and compared with cells seen in circulation in the peripheral blood. Comparisons were based on flow eytometry histograms and/or light mieroseopic morphology of the sorted cells. Gating criteria were established using the preoperative sample as a negative control. It was assumed that tumor cells would be rare in the preoperative sample, so that a sample of 104 nucleated cells would consist of white cells (and perhaps a few nucleated red cells) but not tumor cells. The maximum green (FITC) fluorescence on this preoperative sample was then used to set the lower limit of the green gate to screen the intraoperative specimen for circulating tumor cells. Results The technique of flow eytometrie analysis was developed (by N.J.N.) by study of an initial series of specimens, following which 11 patients' intraoperative specimens were studied in detail (Table I). The intraoperative peripheral blood samples for 7 of these 11 patients yielded 152

Nonhematologie C e ~ Isolated by Flow Cytometr~ Yes (Fig. ~-) No ~ii:i Yes

ii

No No No Yes (Fig. 1C, D) Yes (Fig. 1E, F) ~: Yes

eolls nonhomatolo c morpholo Fig 1iii!ii some cases, morphology and/or green fluo eence intensity was confirmed to be simila~ii that seen in cells of the tissue obtained ati.i operation. The general scheme of analysis was modif~ slightly for the characteristics of each patie~ tissue in order to optimize the sort. A forw~ scatter threshold typically was used to exc!g2~ small bits of debris, sometimes in conjuneti@ with a red (propidium iodide) gate to sei:~ DNA-containing cells. The most ~mportant~ criterion, however, was to select for cells very strong green (cytokeratin) fluoreseen ea~~~ It should be emphasized that the c y t o k e r ~ positive cells were relatively rare even in t h~~ 'i traoperative specimens. Many of the a n a ~ required an hour or more of sorting t o ~ several dozens of cytokeratin-positive e v e ~ These events were not found in all thej!~i~ • • traoperatlve specimens, and even when f(li were typically 1 cytokeratin-positive c e l i ~ 105-106 white blood cells in the p e r i p ~ blood. This yields an estimate of somewha~4~ der 102 cytokeratin-positive cells per m i l ~ in the peripheral blood during surgical m a ~ lation of bulky urologic lesions• A l t h o u g ~ concentration is low, it corresponds to I~,~.~, more eytokeratin-positive cells in the c i r ~ tion intraoperatively. ~a~,. The next routine sampling point was N postoperative day 1 sample. We were unabN detect circulating cytokeratin-positive :,~!~,

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(A) Papillary urothelial carcinoma (grade 1-2). (B) Prostatic hyperplasia. (C) and (D) Multiple ~ i C ) Joci of adenocarcinoma, well to moderately diJ]erentiated; chronic inflammation. (E) and (F) li ~ e (prostatic) squamous metaplasia with Joci oJ atypical cells; acute and chronic inflammation. /

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with our technique by postoperative day 1, consistent with the hypothesis that cells released into the bloodstream with urologic manipulation would tend to be cleared rapidly. An attempt was also made to look at the DNA histogram of the eytokeratin-positive cells in the intraoperative specimens. This technique demonstrated unacceptably high coefficients of variation. These may reflect both cell fragments (with low DNA content) and cell d u m p s (with high DNA content). DNA histogram analysis might potentially be more successful if combined with vital staining (to eliminate fragments of dead cells) and enzymatic or other additional procedures to separate cells that have adhered to one another. Comment Flow cytometry was used in this pilot study as a technique to look for tumor cells in the peripheral blood, and evidence suggesting their presence was found i n many intraoperative specimens. The population of tumor cells would undoubtedly have been higher in the venous blood draining from the tumor site itself, 2 prior to the filtering action of the pulmonary capillary bed. 3'4 Even if tumor cells do gain access to the systemic circulation, only a small minority of them are likely to produce clinically evident metastatic lesions. 5-v This study demonstrates that in intraoperative blood samples one can often identify cells that by flow cytometric criteria are not of hematologic origin. Some of these cells may represent surrounding normal tissue, tumor stroma, or dead or quiescent tumor cells. A fraction, however, is likely to possess metastatic potential. In our assay system, we attempted to use two-color analysis to look at DNA content (propidium iodide-red); a more sophisticated analysis could be performed using a third color to monitor incorporation of bromodeoxyuridine in the freshly harvested blood sample, to detect tumor cells in the blood that were actively synthesizing DNA. Such cells would be likely candidates to have high metastatic potential, s It is of particular interest that the present techniques did not detect tumor cells in the circulation one-clay postoperatively. The data support the idea that there is a much greater seeding of the blood by tumor cells immediately during a procedure than there is even a short while later. It will require clinical follow-up to determine if a high frequency of periopera-

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tive seeding is a risk facto: ease. 0 Although the present , single antibody preparatJ keratin, it is possible that could be obtained by empk cytokeratins. 1°,11 One cot such techniques to look at expression~2 or other anti~ terest ~3 on the circulating tion, the specificity of det keratin-positive cells could of multicolor analysis, if color detector were availa[ a hematopoietic marker. ~[ to exclude from the sorting topoietic cells that had n, the anti-cytokeratin anti technique has been report one lymphoblast per 10 mononuclear cells, TM and , efficient at making the needed to differentiate a peripheral blood cells. It is noteworthy in part: the cases had circulating cells but a benign pathok primary lesion. The most tion is that urologic ma transurethral prostatector sient seeding of the blo¢ whether benign or malig mary site. The urothelial c cance in the circulation a with markers for maligna normal immunologic marl meter flow eytometry sho~ ing what fractions of the cells are presumptively m~ Finally, the present stud for trials of perioperative to decrease the risk of mei motherapeutic maneuvers have recently shown son with bulk disease. 15 It successful in dealing with number of tumor cells th~ tion intraoperatively, whel exposed to the blood level~ would be of interest tc whether or not ehemott diately preoperatively eau percentage of bromodeo: tion among tumor cells fc traoperatively. The u l

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~vhether or not such perioperative maneuvers ~;~rease the rate of death from distant metastases. CHC 134 Brookdale Hospital Medical Center 1275 Linden Boulevard Brooklyn, New York 11212

(DR. NUSBAUM) F u n d i n g was provided in part by kemia F o u n d a t i o n a n d the Louis adation, a n d to Thomas A b r a h a m

References to EG, and Haskell CM: Oncology, in es of Surgery, New York, McGraw-Hill 984, pp 338-339. /P, and Weiss L: Hematogenous disman renal adenocareinomas, Br J Can,n between circulating tumor ceils and r J Cancer 48:665 (1983). ieron A, and Holdand M: Fate of tumor :ride of heart in BALB/e mice: role of ~0:657 (1988).

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5. Roberts LL, et aI: Prognostic significance of cancer cells in the circulating blood: a ten-year evaluation, Am J Surg 113:757

(1967). 6. Murphy P, et al: Mechanisms of organ selective tumor growth by bloodborne cancer cells, Br J Cancer 57:19 (1988). 7. Onoda JM, et ah Separation of high and low metastatic potential subpopulations from tumors by centrifugal elutriation, Proc Soc Exp Biol Med 187:250 (1988). 8. Nemoto R, et ah S-phase fraction of human bladder tumor measured in situ with bromodeoxyuridine labeling, J Urol 139: 286 (1988). 9. Sandier HM, and Hanks GE: Analysis of the possibility that transurethral resection promotes metastasis in prostate cancer, Cancer 62:2622 (1988). 10. Brauer MK, Peehl DM, Stamey TA, and Bostwick DG: Keratin immunoreaetivity in the benign and neoplastic human prostate, Cancer Res 45:3663 (1985). 11. Moll R, et ah Cytokeratins in normal and malignant transitional epithelium, Am J Pathol 132:123 (1988). 12. Pauwels RPE, et ah Blood group isoantigen deletion and chromosomal abnormalities in bladder cancer, J Urol 140:959

(1988). 13. Ornotoft TF, Petersen SE, and Wolf H: Dual-parameter flow eytometry of transitional cell carcinomas: quantitation of DNA content and binding of carbohydrate ligands in cellular subpopulations, Cancer 61:963 (1988). I4. Ryan DH, et al: Improved detection of rare CALLA-positive cells in peripheral blood using multiparameter flow cytometry, J Immunol Methods 74:115 (1984). I5. Seher HI, et al: Neoadjuvant M-VAC (methotrexate vinblastine, doxorubiein and eisplatin) effeet on the primary bladder lesion, J Urol 139:470 (1988).

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