Parenteral Nutrition Does Not Stimulate Tumor Proliferation in Malnourished Gastric Cancer Patients

By Pacelli, Fabio Bossola, Maurizio; Teodori, Laura; Trinca, Maria Luisa; Tortorelli, Antonio; Rosa, Fausto; Doglietto, Giovan Battista

ABSTRACT. Background: The present study evaluated the effects of preoperative parenteral nutrition (PN) on tumor cell proliferation in malnourished gastric cancer patients. Methods: Twenty malnourished patients affected by gastric cancer were randomized to receive the standard hospital oral diet (control group) or the standard hospital oral diet plus PN (PN group; 0.2 g/kg/d of nitrogen and 30 nonprotein kcal/kg/d). Samples of tumor tissue and surrounding health mucosa were taken by endoscopic biopsies and from the operative specimen, immediately after resection. Tissues were sent for histologic examination and prepared for flow cytometry and for the measurement of the uptake of bromodeoxyuridine (BduR) by cells after in vitro exposure to the agent. The BduR uptake is largely used to assess the proportion of cells actively synthesizing DNA and is one of the principle methods used to measure tumor proliferation. Results: In the PN group, the mean percentage of tumor cells incorporating BduR was 2.51% +- 1.7% in the endoscopic samples and 1.52% +- 0.8% in the operative specimens (p = .2). In the normal mucosa, the mean percentage of cells incorporating BduR was 2.24% +- 1.8% and 1.13% +- 1.1%, respectively (p = .1). In the control group, the percentage of cells incorporating BduR in the normal mucosa was 1.26% +- 1.1% at endoscopy and 0.41% +- 0.3% at surgery (p = .2), whereas the percentage of cells incorporating BduR in the tumor tissue was 1.41% +- 1.2% at endoscopy and 0.48% +- 0.6% at surgery (p = .2). The percentage of S-phase cells documented by flow cytometry in the PN group was: in the tumor, 6.6% +- 2.9% in the endoscopic samples and 5.7% +- 2.5% in the operative specimens (p = .6); in the normal mucosa, 5.8% +- 2.5% at endoscopy and 5.4% +- 0.9% at surgery (p = .7). In the control group, the percentage of proliferating cells measured by flow cytometry was 4.9% +- 3.2% in the normal mucosa taken by endoscopic biopsy and 5.3% +- 1.4% in the normal mucosa taken from the operative specimens (p = .8), whereas it was 11.4% +- 7.2% in the tumor taken with endoscopic biopsy and 9.7% +- 4% in the tumor tissue taken from the surgical specimens (p = .7). Conclusions: The present study suggests that PN does not stimulate tumor proliferation in malnourished patients affected by gastric cancer. (Journal of Parenteral and Enteral Nutrition 31:451- 455, 2007) Patenterai nutrition (PN) is widely used in malnourished cancer patients who are candidates for major abdominal surgery. Numerous prospective, randomized trials have demonstrated that it effectively reduces postoperative complications.1’2 However, major concern about the use of PN in cancer patients still exists because nutrients administered to prevent or correct malnutrition in cancer patients might, at least theoretically, stimulate tumor proliferation. Indeed, data in tumor-bearing animals would indicate that PN can lead to an increase in tumor volume, weight, mitotic activity, and number of metastases.3″8 Whether this also applies to humans is still a matter of discussion8″18 because studies performed in cancer patients are few and have led to conflicting results. Indeed, apart from anecdotal reports,19″21 fewer than 10 studies have been conducted aimed at determining the impact of PN10,11,13,14,16,18 or enterai nutrition.17

DNA flow cytometry is a quantitative measure of DNA content and proliferative activity (S-phase fraction), representing a reliable approach to the study of tumor proliferation and cell cycle kinetics.19″21 In 1983, Dolbeare et al22 reported the development of a technique studying cell cycle kinetics that allows the simultaneous measurement of total cellular DNA content and the proportion of cells actively synthesizing DNA, as evidenced by their ability to incorporate bromodeoxyuridine (BduR), a synthetic analog of thymidine. Uptake of BduR occurs in viable cells after in vivo or in vitro exposure to the agent. secondary labeling of cells with a fluorescent monoclonal antibody against BduR allows their rapid detection by chemical and fluorescent techniques. Its ease of administration and lack of radioactivity or toxicity at diagnostic doses have resulted in its increasing popularity among investigators who are interested in examining phase fractions in vivo for prognostic and therapeutic proposals.23″25 The accuracy of this method has been well documented after in vivo and in vitro administration and compares favorably with established alternative methods used in the study of cell cycle kinetics.26

The present study was aimed at evaluating the effect of preoperative PN on tumor cell proliferation in malnourished gastric cancer patients. Tumor cell kinetics was measured by flow cytometry and BduR incorporation in vitro.

MATERIALS AND METHODS

The protocol was approved by the Ethical Committee of the Universita Cattolica del Sacro Cuore of Rome, Italy. Informed consent was obtained from the patients before entering the study.

Patients with a clinical or pathologic diagnosis of cancer of the stomach were evaluated for possible inclusion in the study. Eligibility criterion was previously untreated localized neoplastic lesion amenable to surgical resection. Sufficient size and accessibility of the tumor, evidence of malnutrition (weight loss >10% with respect to usual body weight), acute and chronic renal failure (serum creatinine >1.3 mg/dL), liver failure, cardiac (New York Heart Association class >2) or pulmonary (arterial PaO^sub 2^ <69 mm Hg) dysfunction, HIV (human immunodeficiency virus) infection, sepsis, autoimmune disorders, metabolic acidosis, radiotherapy, or chemotherapy during the prior 12 months represented exclusion criteria.

Patients considered eligible underwent nutrition assessment (measurement of triceps skinfold thickness, midarm muscle circumference, serum prealbumin and transferin levels, immune incompetence measured by delayed cutaneous hypersensitivity PDCH], measurement of total number of lymphocytes). All patients underwent esophagogastroscopy. Endoscopy was performed after an overnight fast, and biopsies were taken from apparently viable tumor tissue and surrounding apparently health mucosa. Multiple site sampling was carried out in different parts of the tumor mass and of the surrounding mucosa. Tissue samples were sent for routine histologie examination and prepared for measurement of BduR uptake in viable cells after in vitro exposure to the agent and flow cytometry.

Thereafter, patients were randomized to receive the standard hospital oral diet (control group) or the standard hospital oral diet plus PN (PN group). Randomization was performed by using a central computerized system. The PN formula contained 0.2 g/kg/d of nitrogen and 30 nonprotein kcal/kg/d. The PN was given as a balanced mixture of o-glucose, lipids (20% Intralipid; Fresenius Kabi, Verona, Italy), and amino acids (Freamine III; Baxter, Italy). Electrolytes, vitamins (Cernevit; Clintec Baxter, Italy), and trace elements were administered according to current recommendations. Fat represented 40% of the nonprotein calories (Table I). The mixture was prepared in the hospital pharmacy under sterile conditions and was delivered in 3-L infusion bags. Vitamins were infused through a separate line. The daily infusion was given from 8:00 AM until 8:00 AM the following day. Patients were allowed to move around freely in the hospital ward until the day of operation.

TABLE I

Parenteral nutrition prescription

In all patients, partial or a total gastrectomy was performed. Immediately after resection, the operative specimen was opened and multiple tissue samples from the tumor and the surrounding mucosa were taken. Tissues were taken as far as possible from the location of the first mucosa. Multiple site sampling was carried in different parts of the tumor mass and of the surrounding mucosa. Tissues were sent for measurement of uptake of BduR in viable cells after in vitro exposure to the agent and for flow cytometry. Tumors Were staged according to the classification of the American Joint Commission on cancer staging of gastric cancer.27

BduR Incorporation and Antibody Treatment

As previously reported,28 the specimens were minced into small fragments and transferred to a flask containing adequate medium and 30 mmol/L BduR, with an equimolar amount of deoxycytidine. The cells were harvested and washed, and the pellet was resuspended in 70% ethanol and washed with PBS containing EDTA 0.1 N. The pellet was resuspended in 0.5 mL of 3-N HCl for 40 minutes, and 0.1 M Na^sub 2^B^sub 4^O^sub 7^ was then added. The suspension was washed a few times with PBS containing EDTA and 0.5% Tween-20 and filtered with a 100-[mu]m nylon mesh. The cells were treated with the anti-BduR MAb (Partec, Arlesheim, Switzerland) for 1 hour. The MAb was then removed and the cells were washed several times with PBS. After the last centrifugation, the pellet was treated with a goat antimouse IgG (courtesy of Ortho, Milan, Italy), arid the cells were examined immediately with a fluorescence microscope. A minimum of 500 cells was counted for each slide.

Flow Cytometry

DNA/total protein content analysis was previously reported.28’29 Briefly, the specimens were minced with scissors, washed in calcium- and magnesium-free buffer, and treated with 0.05% collagenase and 0.01% hyaluronidase (SERVA, Heidelberg, Germany) for 45 minutes at 37[degrees]C. The cell suspension was then fixed in 70% ethanol and stored at 4[degrees]C. For DNA/protein staining, an aliquot (200,000 cells) of the ethanol-fixed cell suspension was stained with 2 mg/L DAPI (4′,6-diamidino-2-phenylindole) and 10 mg/L sulfo-rhodamine 101 (SR 101; SERVA), and approximately 20,000 cells were measured with a PAS II flow cytometer equipped with a 16-bit minicomputer (Partec, Munster, Germany). TABLE II

Patients’ characteristics

TABLE IV

Percentage of cells in the S-phase as measured by flow cytometry

Statistical Analysis

Data are given as mean +- SD. Student’s t-test for unpaired data was used for the statistical evaluation of the results. A p value < .05 was considered statistically significant.

RESULTS

Patients’ characteristics are shown in the Table II. Patients of the PN group received artificial nutrition for 12 +- 0.5 days, whereas the control patients were operated on 10.5 +- 5 days after endoscopy. During this time, body weight decreased by 0.97 +- 1.27 kg in the control group and increased by 0.8 +- 0.9 kg in the PN group.

All patients were affected by the diffusive form of gastric adenocarcinoma according to the Lauren classification.30

BduR Incorporation

As shown in Table III, the percentage of cells of the normal mucosa incorporating BduR was similar in the PN group (2.24% +- 1.8% in the endoscopic samples and 1.13% +- 1.1% in the operative specimens; p = .12) and in the control group (1.26% +- 1.1% at endoscopy and 0.41% +- 0.31% at surgery; p = .20). Similarly, the mean percentage of tumor cells incorporating BduR was, in the PN group, 2.51% +- 1.7% in the endoscopic samples and 1.52% +- 0.8% in the surgical specimens (p = .13). In the control group, it was 1.41% +- 1.2% at endoscopy and 0.48% +- 0.6% in the surgical specimens (p = .23).

TABLE III

Percentage of cells incorporating bromodeoxyuridine in vitro

Flow Cytometry

As shown in Table IV, in the PN group no statistically significant differences in the percentage of S-phase cells were observed in the tumor samples obtained by endoscopy (6.6% +- 2.9%) and at surgery (5.7% +- 2.5%; p = .6). In the control group, the percentage of S-phase cells was similar in the tumor samples obtained by endoscopy (11.4% +- 7.2%) and in the tumor surgical specimens (9.7% +- 4%; p = .7). Similarly, the percentage of S- phase cells was similar in the normal mucosa obtained by endoscopy and at surgery, both in the PN group (5.8% +- 2.5% and 5.4% +- 0.9%, respectively; p = .7) and in the control group (4.9% +- 3.2% and 5.3% +- 1.4%, respectively; p = .8).

DISCUSSION

The present study shows that preoperative PN does not interfere with tumor proliferation in malnourished patients affected by gastric cancer. The percentage of cells incorporating BduR did not increase after PN either in the normal mucosa or in the tumor tissue. Indeed, after PN a moderate and not significant decrease of proliferating cells was observed. In addition, the percentage of cells in the S-phase did not increase after PN either in the normal mucosa or in the tumor tissue.

In the present study, we used both DNA flow cytometry and BduR uptake after in vitro exposure to the agent to assess tumor proliferation. DNA flow cytometry is a quantitative measure of DNA content and proliferative activity (S-phase fraction), representing a reliable approach to the study of tumor proliferation and cell cycle kinetics.19″21 BduR uptake after in vitro exposure to the agent is used to assess the proportion of cells actively synthesizing DNA. The accuracy of this method has been well documented after in vivo and in vitro administration and compares favorably with established alternative methods used in the study of cell cycle kinetics.28

The question of whether PN is capable of interfering with tumor cell proliferation is still largely unanswered. In fact, experimental and clinical studies on PN1S effect on tumor proliferation have led to inconsistent and conflicting results.9″18 Moreover, in some reports a miscellaneous cancer group has been included,10 whereas in other studies, well-nourished patients, for whom artificial nutrition has no clinical indication, have been included.9’12’15 In the present study, we included only malnourished patients affected by gastric cancer. Our results are in agreement with those of 2 previous reports in which the effect of PN on tumor cell kinetics was studied by analysis of incorporation of triated thymidine or BduR in tumor cells.12,14

In the present study, a discrepancy was observed between the LI according to the BduR method and the percentage of S-phase cells measured by flow cytometry. An overestimation of the S-phase compartment by flow cytometry can occur. Alternatively, dead, dying, or very-slow-cycling cells might be unable to incorporate BduR but may be able to take up the dye and therefore demonstrate their status as S-phase cells. The occurrence of S-phase quiescent cells is known,31 and their presence in the S portion of the DNA histograms has been reported for in vitro cell populations. A similar discrepancy was observed by Teodori et al28’29 in a study investigating cytokinetics of lung tumors. However, it has been shown that the BduR method based on in vitro incorporation and simple fluorescence microscopy can yield important information concerning the in vivo cell proliferation in human tumors.28,29 In addition, when used in parallel to and in comparison with another approach (DNA content measured by cytometry), it provides important parameters such as magnitude of cell loss, the percent of supposedly quiescent cells, and therefore a more general view on actual in vivo cell proliferation.28,29

Interestingly, cells of the normal gastric mucosa demonstrated BduR incorporation rates similar to those of tumor cells of the same patients. It is common that the constantly self-renewing epithelium of the gastrointestinal tract shows a high rate of DNA synthesis. Similar results have been obtained also in other solid tumors.24,25,33,34

As in the PN group, we did not observe significant differences in the control group in percentage of cells incorporating BduR and of cells in the S-phase measured by flow cytometry between the tissues taken during endoscopy and those taken from the surgical specimen.

In conclusion, the present study shows that PN given preoperatively in malnourished patients affected by gastric cancer does not stimulate tumor proliferation measured by flow cytometry and BduR incorporation in vitro.

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Fabio Pacelli, MD*; Maurizio Bossola, MD*; Laura Teodori, MD[dagger]; Maria Luisa Trinca, MD[dagger]; Antonio Tortorelli, MD*; Fausto Rosa, MD*; and Giovan Battista Doglietto, MD*

From the *Istituto di Clinica Chirurgica, Universita Cattolica del Sacro Cuore, Roma, Italy; and the [dagger] Divisione di Fisica e Scietue Biomediche, ENEA,

CRE, Casaccia, Roma, Italy

Received for publication March 29, 2007.

Accepted for publication June 26, 2007.

Correspondence: Maurizio Bossola, MD, Istituto di Clinica Chirurgica, Universita Cattolica del Sacro Cuore, Largo A. Gemelli, 8-00168 Roma, Italy. Electronic mail may be sent to maubosso@tin.it.

Copyright American Society for Parenteral and Enteral Nutrition Nov/ Dec 2007

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