Intraperitoneal lymphokine-activated killer-cell and interleukin-2 therapy for malignancies limited to the peritoneal cavity.

Intraperitoneal Lymphokine-Activated Killer-Cell and Interleukin-2 Therapy for Malignancies Limited to the Peritoneal Cavity By Ronald G. Steis, Walter J. Urba, Louis A. VanderMolen, Michael A. Bookman, John W. Smith II, Jeffrey W. Clark, Robin L. Miller, Edward D. Crum, Suzanne K. Beckner, John E. McKnight, Robert F. Ozols, Henry C. Stevenson, Robert C. Young, and Dan L. Longo Autologous lymphokine-activated killer (LAK) cells and recombinant human interleukin-2 (rlL-2) were administered intraperitoneally (IP) to 24 patients with malignancies limited to the peritoneal space. Ten patients had ovarian cancer, 12 had colorectal cancer, and one patient each had endometrial carcinoma and primary small-bowel adenocarcinoma. All ovarian cancer patients, three of twelve colorectal cancer patients, and one patient with endometrial carcinoma had received prior therapy. Patients received IL-2 100,000 U/kg every 8 hours intravenously (IV) for 3 days, and 2 days later underwent daily leukapheresis for 5 days. LAK cells were generated in vitro by incubating the peripheral blood mononuclear cells in IL-2 for 7 days and were then administered IP daily for 5 days through a Tenckhoff catheter (Davol, Inc, Cranston, RI) together with IL-2 25,000 U/kg IP every 8 hours. All but one patient completed at least one cycle of therapy. Toxic side effects included minor to moderate hypotension, fever, chills, rash, nausea, vomiting, abdominal pain

and distension, diarrhea, oliguria, fluid retention, thrombocytopenia, and minor elevations of liver function tests; all of these rapidly improved after discontinuation of IL-2. One patient had a grand mal seizure, and one suffered a colonic perforation; these were felt to be treatment-related. IP fibrosis developed in 14 patients and limited repeated cyclic administration of this therapy in five patients. Two of 10 (20%) ovarian cancer patients and five of 12 (42%) colorectal cancer patients had laparoscopy- or laparotomy-documented partial responses. We conclude that LAK cells and rlL-2 can be administered IP to cancer patients, resulting in moderate to severe short-term toxicity and modest therapeutic efficacy. Further investigation of this form of adoptive immunotherapy modified to address the problem of IP fibrosis and with lower IP IL-2 doses is justified by these initial results. J Clin Oncol 8:1618-1629, 1990. This is a US government work. There are no restrictionson its use.

killer

phenotype of activated natural-killer cells.2 6 The generation of LAK activity from peripheral blood mononuclear cells is entirely dependent on IL-2; other lymphokines appear to be unable to activate these cells to lyse natural-killer cell-resistant

THE

LYMPHOKINE-activated

(LAK)-cell phenomenon was first described by Grimm et al' as the ability of peripheral blood mononuclear cells, following incubation with interleukin-2 (IL-2), to lyse fresh autologous tumor cells. The majority of cancer patients and

targets.7

responsible for LAK activity, but most studies suggest that the majority of this activity resides in cells with the buoyant density and cell-surface

The potential clinical use of LAK cells in cancer patients was first suggested by a series of studies in mouse tumor model systems.'-o Subsequent trials in humans using intravenously (IV) administered LAK cells and IL-2 demonstrated tumor responses among highly selected groups of

From the Medicine Branch, Clinical Oncology Program, National Cancer Institute, National Institutes of Health, Bethesda;Biological Response Modifiers Program,Division of Cancer Treatment; Clinical Immunology Services Program Resources, Inc, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick,MD. Submitted June 5, 1989; accepted April 16, 1990. Sponsored in part by the National Cancer Institute, Department of Health and Human Services, under contract N01-CO-23910 with Program Resources, Inc. The contents of this publication do not necessarily reflect the views or

policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizationsimply endorsement by the United States Government. Address reprint requests to RonaldG. Steis, MD, Biological Response Modifiers Program,National CancerInstitute, Frederick MemorialHospitalCancer Treatment Center,501 WSeventh St, Suite no. 3 Frederick,MD 21701. This is a US government work. There are no restrictions on its use. 0732-183X/90/0810-0006$0.00/0

all normal volunteers evaluated had circulating LAK precursor cells in the peripheral blood.

There is probably not a single effector-cell type

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Journalof ClinicalOncology, Vol 8, No 10 (October), 1990: pp 1618-1629

Downloaded from ascopubs.org by 80.82.78.170 on January 9, 2017 from 080.082.078.170 Copyright © 2017 American Society of Clinical Oncology. All rights reserved.

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IP LAK-CELLS AND IL-2 FOR ADVANCED CANCER patients with melanoma, renal cell carcinoma, and colon cancer." This therapy is toxic and requires considerable clinical and laboratory support. Nonetheless, the results of the early trials demonstrated that adoptive immunotherapy can be successfully applied to humans with cancer. Treatment of malignancies limited to the peritoneal space by the local application of LAK-cell and IL-2 therapy is attractive for several reasons. First, since trafficking of in vitro activated lymphocytes may be abnormal, delivery of the LAK cells to the sites of disease might be more readily accomplished following intraperitoneal (IP) therapy than during systemic therapy. Second, the pharmacokinetics of a large molecule such as IL-2 administered IP should favor high local concentrations and low levels systemically. Indeed, one study12 has demonstrated prolonged retention of IL-2 in the peritoneal cavity and low IL-2 levels in the peripheral blood following IP administration of IL-2; maintenance of effective local LAK-cell activation and decreased systemic toxic effects might therefore be possible. Third, LAK activity and other immunologic parameters could be measured directly at the site of the tumor to allow an assessment of those factors that might be important in the therapeutic and toxic effects of this therapy. Finally, laboratory studies using human ovarian carcinoma cells and a variety of human effector cells in a nude mouse model demonstrated significant prolongation of survival following LAK-cell and IL-2 therapy.13 This report describes the therapeutic and toxic effects of our initial attempt to treat patients with tumors limited to the peritoneal space using this approach. Laboratory studies of the immunologic perturbations induced by this therapy are presented elsewhere.14 PATIENTS AND METHODS Patients Eligibility was restricted to patients with a performance status of _ 80% (Karnofsky scale), no history of cardiovascular disease, normal renal (creatinine < 2 mg/dL), hepatic (bilirubin < 2 mg/dL), and pulmonary (single breath carbon monoxide diffusing capacity > 60% predicted) function, a 3 lymphocyte count greater than 1,000/mm , WBC count 3 greater than 4,000/mm , and platelet count greater than 3 100,000/mm . Estimated life expectancy without treatment had to be greater than 3 months. Patients had to have a histologically documented malignancy limited to the peritoneal space as assessed by physical examination, posteroante-

rior and lateral chest radiographs, and computed tomographic (CT) scans of the abdomen and pelvis. Patients with retroperitoneal lymphadenopathy or parenchymal liver metastases were excluded. Tumor had to be measurable or assessable by abdominal or pelvic CT scans or by peritoneoscopic examination. An interval of 28 days was required from completion of prior chemotherapy, radiotherapy, or biological response modifier therapy until the beginning of IL-2 and LAK-cell therapy (42 days if patients received nitrosoureas). Patients with ovarian cancer had to have either failed induction therapy with a combination chemotherapy regimen that included cisplatin or relapsed from a prior complete remission. Colon cancer, small-bowel cancer, and endometrial cancer patients need not have been previously treated, other than with surgery, prior to study entry. After initial evaluation, patients underwent placement of a Tenckhoff catheter (Davol, Inc, Cranston, RI). Patients then underwent "priming" with recombinant human IL-2 (rIL-2; Cetus Corp, Emeryville, CA) 100,000 U (Cetus)/kg IV every 8 hours for 3 consecutive days. In a separate Biological Response Modifiers Program (BRMP) trial of systemically administered LAK cells and IL-2, we observed significant increases in LAK-cell precursor numbers following this abbreviated IL-2 priming regimen (unpublished observations). At the time the current study was initiated, an optimal IL-2 priming regimen had not yet been determined and we opted to use this abbreviated course. Fifty-six hours after completion of IL-2 priming, patients began daily leukapheresis for 5 consecutive days. Peripheral blood mononuclear cells so obtained were separated by centrifugation over a cushion of Ficoll-Hypaque (Pharmacia, Piscataway, NJ), washed and incubated for 7 days with IL-2 at 1,000 U (Cetus)/mL in RPMI-1640 supplemented with 2% human AB serum, glutamine, penicillin, streptomycin, and gentamicin. Penicillin was omitted for patients with a history of penicillin allergy. After 7 days of incubation in IL-2, cells were washed and suspended in 250 mL saline with 5% human serum albumin containing 100,000 U of IL-2 and transported to the bedside for infusion. A mean of 1.03 x 10i' ± 4.5 x 108 cells was obtained by leukapheresis and a mean of 57.7% ± 2.2% of these cells was harvested after 7 days of culture and administered to the patients. Seven-day LAK cells were used in this study because, in our hands, LAK activity of peripheral blood mononuclear cells cultured in IL-2 increased for up to 10 15 days, at which time lytic activity plateaued. Because animal models have shown that the in vivo antitumor activity of LAK plus IL-2 treatments is proportional to the number of cells 8 adoptively transferred, we opted for the 7-day LAK cells rather than the more conventional 3- or 4-day cells. Patients were given approximately 1,800 mL of Inpersol (Abbott Laboratories, North Chicago, IL) (2.5% dextrose) IP and 25,000 U/kg of IL-2 IP just before LAK cell infusions. A test dose of 50 mL of LAK cells was infused and patients were observed for 5 to 10 minutes before the remainder were administered. LAK cells were infused once daily for 5 days and IL-2 (25,000 U (Cetus)/kg) was given IP every 8 hours during the same 5 days. This dose of IL-2 is approximately 75% lower than that used in previous trials of systemically administered LAK and IL-2. In an average-sized patient, this dose would result in peak IP IL-2 levels of 875 U/mL, sufficient to maintain LAK activity locally." After 2 addi-


1620

STEIS ET AL

I4

Systemic Priming 5

IL-2 (1 x 10 Ulkgl8 hr)

0

1

2

3

4

5

6

7

8

· Intraperitoneal Therapy LAK and IL-2 (2.5 x 104 Ulkgl8 hr)

K, i

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

ttttt

t ttt Leukapheresis I J LAK Generation IL-2 (1000 Ulmi) x 7 d)

Fig 1. Treatment schema for patients with peritoneal carcinomatosis receiving IP LAK cells

and IL-2. tional days without therapy, patients underwent an additional cycle of leukapheresis, LAK generation, and LAK-cell and IL-2 infusions. There was no separate IL-2 priming for this part of the study. The treatment schema used in this study is outlined in Fig 1. Routine microbial cultures of LAK cell and peritoneal fluid samples were performed daily. Patients were treated in a standard medical ward but, when necessary, were monitored more closely with an ECG monitor and an automatic blood pressure cuff to facilitate frequent vital sign assessments. Patients were given indomethacin (50 mg orally every 8 hours) and acetaminophen (650 mg orally every 4 hours) in an attempt to prevent or decrease the fever and chills associated with LAK-cell and IL-2 therapy. Patients also routinely received ranitidine, antacids, and meperidine for shaking chills. No other drugs were administered routinely. Toxicity was graded according to the scale developed by the Cancer Therapy Evaluation Program, National Cancer Institute (NCI), as modified by the BRMP, NCI. The study was approved by the Institutional Review Boards of both the Clinical Oncology Program, Division of Cancer Treatment, NCI and the Frederick Cancer Research and Development Center. Written informed consent was obtained from all patients.

Assessment of Tumor Response Tumor responses were assessed by repeat peritoneoscopy with appropriate biopsies and cytologic examination of peritoneal fluid 1 month after completion of one full cycle of

therapy. If no tumor was found at peritoneoscopy, or if peritoneoscopy could not be accomplished because of technical problems, a laparotomy was performed. If additional treatment cycles were administered, treatment was initiated as soon as abdominal incisions had healed sufficiently, generally less than 3 weeks from reevaluation. For patients having no macroscopic tumor at restaging peritoneoscopy or laparotomy, blind biopsies of peritoneum at sites of previous disease, at accessible areas throughout the pelvis, abdomen, under the diaphragms, and over the liver were performed. Serum samples for determination of tumor markers carcinoembryonic antigen (CEA) and CA-125 were obtained serially. Standard criteria for assessment of tumor responses were used. However, for tumors diffusely involving the peritoneal space, such assessments are impractical and difficult to reproduce. For these reasons, descriptions of the findings at peritoneoscopy and/or laparotomy are included for responding patients. RESULTS Patient Data

Twenty-four patients were treated. Clinical and response data for the patients with ovarian cancer and colon cancer are given in Tables 1 and 2, respectively. All 10 patients with ovarian cancer had been treated previously with combina-

Table 1. Clinical Characteristics of Patients with Ovarian Cancer Patient No.

Age (years)

Prior Therapy

1 2 3 4 5 6 7 8 9 10

48 37 57 39 42 38 57 41 73 55

CHEX-UP, IP 5-FU, CP, CBDCA AP, XRT, megestrol CP, CBDCA CHAD CP CAP, CP, CBDCA, TCNP, IP oclarubicin CP CP,XRT CAP CAP

Largest Tumor > 2 cm No No Yes No No No No No No No

Response NR PR NR PR NR NR NR NR NR NR

Abbreviations: C, cyclophosphomide; H or HEX, hexamethylmelamine; U or 5-FU, fluorouracil; IP, intraperitoneal; P or D, cisplatin; A, doxorubicin; XRT, whole abdominal radiation therapy; CBDCA, carboplatin; TCNP, tricyclic nucleoside phosphate; NR, no response; PR, partial response.


1621

IP LAK-CELLS AND IL-2 FOR ADVANCED CANCER Table 2. Clinical Characteristics of Patients with Colon Cancer Patient No.

Age/Sex

Prior Therapy

Largest Tumor -2 cm

11 12 13 14 15 16 17 18 19 20 21 22

57/M 53/F 38/M 46/M 53/M 64/M 33/F 65/M 41/F 27/M 56/M 42/M

IP 5-FU None IP activated monocytes None IV 5-FU None None None None None None None

Yes No Yes Yes Yes Yes Yes Yes Yes No Yes No

Response PR NR NR PR NR PR NR NR PR PR NA NR

Abbreviations: IP, intraperitoneal; 5-FU, fluorouracil; PR, partial response; NR, no response; NA, nonassessable.

tion chemotherapy. Eight patients (patients no. 1, 3, and 5 to 10) had persistent disease following combination chemotherapy, and two (patients no. 2 and 4) had relapsed disease following prior complete responses to chemotherapy or radiation therapy. Of 12 patients with colon cancer, two had failed fluorouracil, one had failed experimental therapy with interferon gamma-activated autologous monocytes, and nine were previously untreated. One patient with endometrial carcinoma had not responded to medroxyprogesterone acetate, and the one patient with small-bowel adenocarcinoma had not received any treatment after the initial surgery. Response Analysis To be assessable for response, patients must have completed at least one cycle of IL-2 priming and 5 days of IP LAK-cell and IL-2 infusions. Patient no. 21 developed a colonic perforation at a site distant from sites of tumor involvement shortly after beginning IL-2 priming and was ultimately removed from study; he is the only patient nonassessable for response. If improvement in clinical status occurred after one cycle, additional treatment was offered for as long as continuing benefit was observed. Most patients on this study did not have any standard treatment options and further treatment was offered even to patients with stable disease in whom a subjective improvement had occurred. However, only those patients meeting objective criteria for tumor response were labeled as responders. In total, 12 patients received only one treatment cycle, four patients received two cycles, five patients received three cycles, and one each of

the remaining patients received four or five cycles of therapy. Tumor responses are shown in Tables 1 and 2. Overall, seven of 23 patients (30%) showed a partial response to therapy. These responses were noted in two of 10 (20%) patients with ovarian cancer and five of 12 (42%) with colon cancer. Neither the patient with endometrial nor smallbowel carcinoma responded. Responses were not limited to patients with tumors less than 2 cm in maximum diameter; four of 10 (40%) patients with tumors larger than 2 cm in diameter responded compared with three of 13 (23%) patients with smaller tumors. Responses were assessed by peritoneoscopy (four patients) and/or laparotomy (four patients). Changes in CA-125 levels correlated with other assessments of tumor response in patients with ovarian cancer; levels fell in patients who had surgically documented responses, and were either stable or increased in nonresponders. Four of five responding colon cancer patients had low normal baseline CEA levels that remained low during therapy. The fifth patient had elevated baseline CEA levels that fell during treatment. Four of the seven nonresponding colon cancer patients had elevated CEA levels before therapy that fell progressively during treatment. As these patients had clearly not responded to treatment, serum CEA levels do not appear to be suitable markers for response assessment in this setting. Since the majority of patients had diffuse intraabdominal disease rather than easily measurable tumor masses, pretreatment and posttreatment tumor evaluations of responding patients are presented here in detail. Patient no. 2 had


1622

STEIS ET AL

ovarian carcinoma with twenty 1 to 5 mm nodules and a 1 cm nodule in the midabdomen at the time of her initial staging peritoneoscopy. Cytologic examination of peritoneal fluid at that time showed malignant cells. The CA-125 level was 66. After completion of one cycle of therapy a repeat peritoneoscopy showed a number of adhesions, but no gross tumor was seen, and cytologic examination of peritoneal fluid failed to show any malignant cells. The CA-125 level had fallen to 9.1. At subsequent laparotomy, three nodules, all less than 1 cm in diameter, were found along the posterior surface of the lesser curvature of the stomach, an area not visualized during the initial staging peritoneoscopy. A biopsy showed ovarian carcinoma. No other sites of disease were found. Treatment was continued for two additional cycles. Dense adhesions then prevented delivery of further therapy. The patient ultimately developed progressive disease documented by laparotomy 6 months after initial documentation of tumor response. Patient no. 4 had ovarian carcinoma with twenty to twenty-five 1 to 3 mm nodules on the right hemidiaphragm and surface of the liver at baseline staging peritoneoscopy. A biopsy showed ovarian carcinoma. Cytologic examination of peritoneal fluid showed malignant cells. The CA-125 level was 102. After two cycles of LAK-cell and IL-2 therapy, no macroscopic tumor was seen during repeat peritoneoscopy but one of 19 blind biopsies showed malignant cells. Cytologic examination of peritoneal fluid did not show malignant cells, and the CA-125 level had fallen to 15. Treatment was continued, but by the fourth cycle, extensive peritoneal fibrosis had developed and limited the IP distribution of LAK cells and IL-2. The patient was found to have disease progression at laparoscopy 4 months after the first restaging peritoneoscopy. Patient no. 11 had diffuse peritoneal seeding and malignant cells in the peritoneal fluid at the time of his baseline peritoneoscopy. Baseline CEA level was normal. Repeat peritoneoscopy after completion of one cycle of LAK and IL-2 therapy was not possible because of the development of adhesions. Visual inspection at laparotomy showed a 50% to 75% decrease in the amount of tumor in the abdomen. Cytologic examination of peritoneal fluid did not show

tumor cells. Three additional treatment cycles were administered, but progressive fibrosis of the peritoneal cavity prevented delivery of further therapy. Disease progression was observed at laparotomy 7 months after initial documentation of response to LAK and IL-2 therapy. Patient no. 14 had colon cancer with multiple omental and mesenteric tumor implants at his initial laparotomy. The baseline CEA level was normal. After completion of one cycle of therapy, he developed a small bowel perforation secondary to the Tenckhoff catheter, and at laparotomy, no gross tumor was found. One of 13 blind biopsies did, however, show tumor. Two additional cycles of therapy were given but treatment was ultimately stopped because of progressive peritoneal fibrosis and catheter malfunction. The patient ultimately developed progressive disease at laparotomy 10 months after initial documentation of disease response. Patient no. 16 had metastatic colon carcinoma with multiple biopsy-proven peritoneal tumor nodules, a large tumor mass in the omentum, and "suspicious" cells in the peritoneal fluid during the baseline peritoneoscopy. The baseline CEA levels were normal. At restaging peritoneoscopy after three cycles of IP LAK and IL-2, no gross disease was present but one of nine blind biopsies showed tumor. Cytologic examination of peritoneal fluid was unremarkable. Because of fungal peritonitis, further IP LAK-cell and IL-2 therapy was delayed, and tumor progression, assessed by repeat peritoneoscopy, occurred 6 months later. Patient no. 19 had colon cancer with a 2 x 3 cm nodule on the left hemidiaphragm and malignant ascites at the time of her baseline evaluation. The CEA level was 93. A laparotomy was performed when she developed severe abdominal pain of unknown etiology after her third cycle of IP LAK-cell and IL-2 therapy. A 1 x 1 cm nodule was present on the diaphragm but was not biopsied. Three of three blind biopsies were negative, and cytologic examination of peritoneal fluid showed no malignant cells. The CEA level at this time was 1.1. This patient is considered a partial responder because biopsy of the one suspicious nodule in her peritoneal cavity at the time of her laparotomy was not performed for technical reasons. She, unfortunately, has been


1623

IP LAK-CELLS AND IL-2 FOR ADVANCED CANCER

lost to follow-up, but was alive and without disease progression 2 months after laparotomy. Finally, patient no. 20 was found to have extensive metastatic colon cancer at laparotomy with numerous tumor nodules less than 1 cm involving pelvic peritoneum, small-bowel mesentery, and omentum. After omentectomy and removal of the primary tumor in the left colon, he was referred to the BRMP where peritoneoscopy showed no gross disease, but malignant cells were found in washings, and disease was present in two of nine blind biopsies. Baseline CEA levels were normal. At repeat peritoneoscopy after three cycles of therapy, atypical cells not diagnostic of malignancy were present in washings of the peritoneal cavity, no gross disease was seen, and none of 15 blind biopsies showed malignant cells. However, at subsequent laparotomy, several 2 to 3 mm peritoneal nodules containing malignant disease were found. Response duration cannot be determined as the patient refused further treatments. Responding patients received a median of two treatment cycles (range, one to three) before tumor response was documented. Nonresponders received a median of one treatment cycle. Only one responding patient (patient no. 4) had disease progression on therapy, probably because of inadequate IP distribution of the LAK cells and IL-2. Catheter malfunction due to IP fibrosis prevented further therapy in four responding patients, fungal peritonitis delayed further therapy in one patient, and one responding patient each was lost to follow-up or refused further

therapy. Thus, any potential benefit to responding patients of repeated cycles of therapy could not be determined. There was no clear relationship between the number of cycles that could be administered and degree or duration of response. Toxicity The clinical toxicities and laboratory abnormalities that developed during the priming and IP therapy portions of the treatment are shown in Tables 3 and 4, respectively. In general, most toxic effects were similar to those previously reported in patients receiving systemic LAK-cell and IL-2 treatments." Our impression was that the side effects were both less severe and were encountered less frequently during the IP portion of the study than during systemic IL-2 priming. Repeated cycles of therapy were not associated with cumulative or worsening toxicities except for progressive IP fibrosis. Fluid retention, weight gain, progressive accumulation of ascites and edema, fevers, chills, rash, nausea, vomiting, and diarrhea were observed frequently during both systemic and IP treatments; these side effects were difficult to manage and only partially controlled by medical therapy, but they were not dose-limiting in any patient. Dyspnea was more common during IP than systemic treatments and was due to abdominal distention and elevated diaphragms rather than to increased interstitial fluid in the lungs. It was relieved by removal of abdominal fluid. Abdominal pain with rebound abdominal tenderness developed in all patients

Table 3. Clinical Toxicities of Systemic IL-2 Priming and IP LAK- Cell and IL-2 Therapy Priming: Assessable Courses 45

Weight gain Fever Chills Rash Edema Nausea and vomiting Diarrhea Stomatitis Dyspnea Abdominal pain Oliguria Hypotension requiring pressors Confusion

IPTherapy: Assessable Courses 64

Any %

a Grade 3 %

Any %

- Grade 3%

82 67 56 67 76 78 56 4 7 53 24 31 9

4 2 0 0 2 9 2 0 4 16 0 0 2

83 62 28 47 78 83 49 2 23 94 27 9 6

11 0 0 0 3 6 0 0 16 22 0 0 2

NOTE. Numbers represent the percentage of courses during which the indicated toxic effect was observed.


1624

STEIS ET AL Table 4. Laboratory Abnormalities in Patients Receiving Systemic IL-2 Priming and IP LAK-Cell and IL-2 Therapy Priming: Assessable Courses 45

Anemia Granulocytopenia Thrombocytopenia Hyperbilirubinemia Elevated creatinine

IPTherapy: Assessable Courses 64

Any %

- Grade 3 %

Any %

- Grade 3 %

82 18 60 22 7

7 9 13 0 4

97 3 16 2 12

6 2 8 0 2

NOTE. Numbers represent the percentage of courses during which the indicated abnormality was observed.

during IP administration of LAK cells. It was not relieved by paracentesis. Peritoneal fluid studies (see below) suggested that this pain was due to a noninfectious peritonitis. One patient developed very severe abdominal pain after an IP infusion of LAK cells. The pain could not be distinguished clinically from an acute abdominal catastrophe. Laparotomy showed only diffuse erythema and the patient recovered uneventfully. Although this patient achieved a partial response, overall there was no relationship between any toxic effect and tumor response. Abdominal pain was present during approximately one half of the IL-2 priming cycles. In nearly all cases, this was in patients who had received an earlier cycle of IP LAK cells and IL-2 suggesting reactivation of a smoldering peritoneal inflammatory process. Oliguria was observed with equal frequency during the systemic and IP therapy portions of the study but responded to fluid administration, diuretics, or dopamine in most cases. Hypotension was most frequently observed during systemic IL-2 treatments but was corrected with fluids or pressor agents and did not result in discontinuation of therapy in any patient. Four patients had hematemesis that in all cases stopped spontaneously. This was probably related to indomethacin administration and was aggravated by severe thrombocytopenia in one patient. One patient developed IP bleeding after insertion of a Tenckhoff catheter. This resolved spontaneously without intervention. Infections developed in 17 patients and included IV catheter exit-site infections in eight, Tenckhoff catheter exit-site infections in two, bacteremia in five, and peritonitis in four. Urinary tract infections complicated the course of therapy in six patients. One patient developed a necrotizing fasciitis at the exit site of her Tenckhoff catheter and required a prolonged course of antibiotics, excision of necrotic tissue, and subsequent skin grafting. Staphylococcus epidermidis

was the organism most frequently responsible for these infections (seven patients), followed by Staphylococcus aureus (three patients). A variety of different gram-negative organisms was responsible for the remainder. One patient developed fungal peritonitis after receiving LAK cells contaminated with Candida parapsilosis. This resolved after a course of systemic amphotericin B. Although granulocytopenia developed in some patients, most infections developed in the face of adequate neutrophil counts. Treatments with IL-2 can result in suppression of neutrophil Fc receptor expression and chemotaxis, 16 and these functional abnormalities may in part be responsible for the high incidence of infections observed in our patients. Five patients developed atrial arrhythmias including frequent premature atrial contractions in two, regular supraventricular tachycardias in two, and multifocal atrial tachycardia, which evolved into atrial fibrillation in one. All atrial arrhythmias were asymptomatic and resolved uneventfully. The episode of atrial fibrillation converted to normal sinus rhythm with digoxin. Asymptomatic, frequent unifocal premature ventricular contractions occurred in seven patients, and one patient had six beats of ventricular tachycardia that terminated spontaneously. IL-2 and LAK-cell therapy was continued in all patients without incident. Significant (ie, wgrade III) confusion was observed during 2% of the treatment cycles and resolved in all cases without treatment. One patient developed a grand mal seizure that could not be explained on the basis of metabolic or structural causes. One patient developed a colonic perforation during systemic priming with IL-2. The site of perforation was distant from the peritoneal end of the Tenckhoff catheter and was not at a site of involvement of the serosa of the colon with tumor. This rare complication of IL-2 and LAK-cell therapy has been observed and


1625


reported previously in four of 315 patients receiving systemic treatment.17 Laboratory abnormalities developed in the majority of patients (Table 4). They included anemia, granulocytopenia, thrombocytopenia, hyperbilirubinemia, and elevations in serum creatinine levels. The incidence and severity of these problems were greater during IL-2 priming than during IP therapy. Minor elevations in SGOT, SGPT, and alkaline phosphatase levels were occasionally observed but were limited to less than two times the upper limits of normal. Laboratory abnormalities returned to baseline levels after completion of therapy. All patients required transfusions of RBCs during the course of treatment. The median number of units required was 6 (range, 2 to 18) and the average number of units required per cycle of therapy was 1.4. Although there were five patients who had clinical evidence of bleeding, transfusion requirements for the most part were a reflection of the phlebotomy requirements of the study, unavoidable RBC loss during pheresis, and, possibly, the bone marrow suppressive effects of IL-2. Eight patients, including seven of the 10 patients with ovarian cancer, required platelet transfusions. In one patient with ovarian cancer with persistent thrombocytopenia after treatment with cyclophosphamide, cisplatin, and carboplatin, severe thrombocytopenia refractory to platelet transfusions resulted in persistent gastrointestinal bleeding. Three weeks after therapy, the platelet count rose and bleeding ceased. For those patients requiring platelet transfusions, the median number of units of platelets

required was 14 (range, 6 to 85 units) with an average of 1.8 units per cycle of treatment. Specimens of peritoneal fluid were obtained during 34 of the 64 cycles of IP therapy. The fluid levels of total protein and lactic dehydrogenase (LDH) tended to increase progressively during the course of therapy. The median peak total protein level was 2.6 g/dL (range, 0.7 to 4.8), and the median peak LDH level was 349 U/mL (range, 104 to 1330). Cell counts in the peritoneal fluid tended to increase progressively and peaked at a median of 2,426 cells per cubic millimeter (range, 99 to 15,222). The proportions of different cell types varied from patient to patient but granulocytes and eosinophils predominated in most patients. The development of fibrosis in the peritoneal cavity was a significant and unexpected late complication of this therapy. Fibrosis was the apparent cause of catheter "malfunction" in 14 patients. Fluid could usually be infused into the abdominal cavity of these patients without difficulty but could not be withdrawn. Eleven patients developed cystic structures in the abdominal cavity that were lined by fibrous tissue. These pseudocysts usually enveloped the peritoneal end of the Tenckhoff catheter and when iodinated contrast agents were infused through the Tenckhoff catheter their distribution in the peritoneal cavity was limited by the wall of the pseudocyst (Fig 2). Patients with fibrosis-related catheter malfunction did not have more surgical procedures prior to study entry than those without catheter malfunction. Four patients required catheter replacement for continued administra-

Fig 2. Pseudocyst formation in the abdomen of a patient 1 month after treatment with IP LAK cells and 112. CT scans were obtained (A)before and (B) after infusion of contrast material through the Tenckhoff catheter.


1626

STEIS ET AL

tion of therapy. In five patients, further therapy could not be administered because of the development of extensive IP fibrosis, and in three patients, a posttreatment peritoneoscopy could not be performed because of extensive intraabdominal adhesions. Even in those patients who had no outward signs suggesting IP fibrosis, varying degrees of fibrosis were observed at restaging evaluation. No patient developed bowel obstruction because of adhesions. Four patients suffered bowel perforations during the course of this treatment. Two perforations were due to the Tenckhoff catheter or Tenckhoff catheter placement. One occurred during IL-2 priming, and one developed after the third cycle of therapy, although the site of perforation could not be found at laparotomy, nor did perforation recur postoperatively. DISCUSSION IP administration of autologous LAK cells and IL-2 to patients with malignant disease limited to the peritoneal cavity caused tumor regressions in seven of 23 (30%) assessable patients. Although multiple and significant toxic effects were observed and this therapy is cumbersome and difficult to deliver, the treatments resulted in regressions of refractory cancers. These preliminary results suggest that further exploration of modifications of this type of therapy is warranted. Regional therapy with LAK cells and IL-2 seemed a particularly attractive application of adoptive immunotherapy. Chapman et a1'2 reported prolonged retention of IL-2 in the peritoneal cavity and low serum IL-2 levels following IP administration of between 1 x 105 and 5 x 107 Cetus U/m 2 of IL-2 in small numbers of patients with ovarian cancer. Yasumoto et al' 8 obtained analogous results in patients with lung cancer and malignant pleural effusions following repetitive daily intrapleural administrations of 1,000 U of IL-2. Analysis of IL-2 levels and effector-cell function in our patients' 4 showed very high IP IL-2 levels and maintenance of LAK-cell activity in vivo in ascites for the duration of IL-2 administration. These findings confirm that it is possible to obtain high IL-2 levels at the site of the tumor, significantly lower serum IL-2 levels, and acceptable systemic toxic effects using the intracompartmental approach. Indeed, when direct compari-

sons in the same patients are made (Tables 3 and 4), systemic toxicity is less during IP administration of IL-2 than during IV therapy. Of course this comparison is complicated by the fourfold lower IL-2 dose used during IP therapy (25,000 U/kg IP every 8 hours v 100,000 U/kg IV every 8 hours) and the longer duration of IP than IV treatments (5 days v 3 days). It is clear, however, that IP administration of IL-2 at the dosage used in this study results in IP IL-2 levels 50- to 100-fold higher than concurrent serum levels' 4 and that acute systemic toxic effects were less severe than during IV therapy and not doselimiting. Several unexplained toxic effects of this therapy were observed. First was the development of diffuse abdominal pain following IP instillation of LAK cells and IL-2. A previous study using IP IL-2 without LAK cells' 2 did not report significant abdominal pain and rebound abdominal tenderness as side effects. Similarly, pleuritic pain was not reported in patients receiving intrapleural IL-2.' 8 LAK cells themselves, therefore, must be responsible, at least in part, for the abdominal pain. Because LAK cells had never previously been given IP and their toxic effects had not been defined, we elected to give LAK cells IP without IL-2 in the first cycle of therapy in the first three patients entered onto this study. All three rapidly developed abdominal pain with rebound abdominal tenderness that was severe in one case. Peritoneal fluid studies in these patients and in patients receiving both IL-2 and LAK cells showed changes consistent with a noninfectious peritonitis. LAK cells themselves, a product they produce, or products of cells recruited into the peritoneal cavity are thus likely responsible for the pain and abnormal peritoneal fluid findings observed. This toxicity did not limit delivery of therapy and was managed successfully with parenteral analgesics. However, the intraperitoneal fibrosis clearly limited the duration of potentially effective therapy. This side effect was more severe and more common following several cycles of therapy and was not related to the number of abdominal surgical procedures performed prior to study entry. We probably have also underestimated the frequency of this problem because, in general, only responding patients were serially evaluated


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for tumor response with peritoneoscopy or laparotomy at which time fibrosis was readily apparent. Fibrosis resulted in poor catheter function (ie, inability to drain peritoneal fluid) and inability to uniformly treat the entire peritoneal surface. Since only responding patients received multiple treatment cycles, it was in just those patients who would most likely benefit from further treatment that additional therapy could not be given because of IP fibrosis. Thus, the development of fibrosis in our patients represents a serious limitation of this therapy. Stewart et a1' 9 administered LAK cells and IL-2 IP to patients with ovarian cancer and did not observe peritoneal fibrosis. The IL-2 doses used in their study were lower than in our study. Analysis of peritoneal IL-2 levels in our patients1 4 showed very high IL-2 levels that are 50- to 100-fold higher than necessary to maintain LAK-cell activity in vitro. Lower IL-2 doses may be less fibrogenic yet still capable of maintaining LAK-cell activity and antitumor activity in vivo. Other potential causes of fibrosis in our patients should also be considered. Activated lymphocytes, including LAK cells, release transforming growth factor-beta, 20 and this molecule is known to induce synthesis of collagen by fibroblasts.21 Eosinophils were found in the ascites of our patients, and they may release eosinophil major basic protein into the ascites. This protein, found in eosinophil granules, may be responsible for the fibrosis observed in nodular sclerosing Hodgkin's disease 22 and endomyocardial fibrosis in the hypereosinophilic syndrome. 23 IL-1 is mitogenic for fibroblasts and results in increased collagen synthesis. 24 This material is produced by many cells, including activated monocytes, which were found in the ascites of our patients. 14 It has been suggested that IL-1, which can be detected in peritoneal fluid of patients undergoing chronic ambulatory peritoneal dialysis, 25 might be responsible for the IP fibrosis in this clinical setting. The contribution of any or all of these agents to the fibrosis in our own patients is unknown, but levels of these mediators in ascites are being determined in an attempt to understand and ultimately prevent this treatment-related complication. In this study, tumor response was judged by peritoneoscopy and/or laparotomy. Because of the tendency of these types of tumors to diffusely involve the peritoneal cavity, assessments of tu-

mor response may be difficult since a complete examination of the entire abdominal cavity may not be possible. We therefore obtained serial serum CEA and CA-125 levels to aid in tumor response assessments. To our surprise, although CA-125 levels correlated with surgical assessments of tumor response, CEA levels fell in all patients with colon carcinoma regardless of tumor response. Importantly, we saw no example of a surgically documented tumor response with an accompanying increase in serum markers. The discordance between changes in serum CEA levels and surgical assessments of tumor response in some of our colon cancer patients has not been reported previously among patients receiving systemic LAK-cell and IL-2 treatments and suggests that production and/or metabolism of CEA is altered in patients receiving this therapy. Colon carcinoma cell lines express greater amounts of surface-bound CEA following in vitro treatments with interferon (IFN) alfa, 26 and melanoma cell lines show enhanced cellsurface expression and/or shedding of tumorassociated antigens after treatment with IFN alfa or IFN beta.27 Thus, alterations in tumor antigen expression and/or shedding may occur more commonly during treatments with immunomodulatory agents than is currently recognized. Until more information is available on the in vivo effects of biologic agents on serum levels of tumor-associated antigens, assessments of tumor response in these types of studies should continue to include measurements of tumor size. Studies using these therapies that report tumor responses based solely on tumor marker levels should be interpreted with caution. We opted to use both LAK cells and IL-2 in this study because animal tumor model studies with several different tumor types consistently showed better antitumor effects with the combination than with IL-2 alone.8"'0 IL-2 alone, however, was not without antitumor activity in these models and has brought about tumor regressions when used alone in humans. 28 It has been shown that cells capable of developing LAK-cell activity in vitro are present in ascites and pleural fluid of patients with advanced cancers 29 and that cells with LAK-cell activity can be obtained directly from the ascites and pleural fluid of patients receiving regional IL-2 therapy. 12'18 Thus, it is possible that cytolytic effector cells could


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have been recruited into the abdominal cavity and activated in our patients without the need for ex vivo generation and subsequent IP administration of LAK cells. However, although direct comparisons are difficult, it appears that the number of lytic units generated in vivo following intrapleural or IP administration of IL-2 is fairly low in comparison to what was administered to our own patients. If large numbers of LAK cells are important for tumor responses, as suggested by the studies in animals, ex vivo expansion of cell numbers may allow larger numbers of cells to accumulate in the peritoneal cavity than would be possible with cytokines alone. As previously reported, however,14 in the range of cell numbers administered to our patients, we observed no correlation between response and numbers of LAK cells or amount of LAK-cell activity administered. Studies in animal tumor model systems have also demonstrated that when LAK cells are used in adoptive immunotherapy, the greatest antitumor effects were observed with the highest tolerable dose of IL-2. 8-l In our patients, it has been demonstrated that very high peritoneal levels of IL-2 and substantial LAK activity can be maintained in the peritoneal fluid of patients for as long as IL-2 is administered.1 4 Thus, conditions found optimal for tumor eradication in animal model systems, namely, large numbers of LAK cells and high concentrations of IL-2, were achieved in our patients at the site of the tumor. In order to increase the antitumor efficacy of this approach, a method is needed to prevent the development of IP fibrosis and thus allow administration of repeated cycles of therapy. Toward this end, we currently are developing studies that will incorporate collagen synthesis inhibitors,

such an IFN gamma 30 and colchicine,31 and lower IL-2 doses into the regimen described in this report. IP therapy with other biological agents has previously been reported to produce tumor responses. Berek et a132 observed tumor responses in patients with ovarian cancer following IP administration of IFN alfa-2b. However, tumor responses were limited to patients with tumors less than 5 mm in diameter; no responses were seen in patients with larger tumors. Constitutional symptoms, fever, and abdominal pain developed with this treatment, but peritoneal fibrosis was not a significant problem. Berek et a133 administered Corynebacteriumparvum IP to patients with ovarian cancer and also noted tumor responses but, again, only in patients with tumors less than 5 mm in diameter. Toxicities similar to those reported here, including fever, abdominal pain, and hypotension, were observed with this therapy and peritoneal fibrosis, occasionally severe, developed. Six of 21 patients required Tenckhoff catheter replacement because of loculation of dialysis fluid or catheter clogging. It is clear that IP biological therapies of several different types are capable of producing tumor responses but with similar overlapping toxicities. However, the toxicity that appears to limit treatment is peritoneal fibrosis. Studies using this approach should incorporate strategies to prevent or minimize this problem. ACKNOWLEDGMENT The authors acknowledge the support of the nursing staff of Frederick Memorial Hospital, the administrative staff of Program Resources, Inc, and the secretarial help of Joann Ciufolo and Sharon Lewis who assisted in the preparation of the manuscript.

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20. Kasid A, Bell GI, Director EP: Effects of transforming growth factor-# on human lymphokine-activated killer cell precursors. J Immunol 141:690-698, 1988 21. Roberts AB, Sporn MB, Assoian RK, et al: Transforming growth factor type 0: Rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci USA 83:4167-4171, 1986 22. Butterfield JH, Kephart GM, Banks PM, et al: Extracellular deposition of eosinophil granule major basic protein in lymph nodes of patients with Hodgkin's disease. Blood 68:1250-1258, 1986 23. Tai PC, Ackerman SJ, Spry CJ, et al: Deposits of eosinophil granule proteins in cardiac tissues of patients with eosinophilic endomyocardial disease. Lancet 1:643-647, 1987 24. Goldring MB, Birkhead J, Sandell LJ: Interleukin I suppresses expression of cartilage-specific types II and IV collagens and increases types I and III collagens in human chondrocytes. J Clin Invest 82:2026-2037, 1988 25. Krane SM, Goldring MB: Potential role for interleukin-1 in fibrosis associated with chronic ambulatory peritoneal dialysis. Blood Purif 6:173-177, 1988 26. Greiner JW, Hand PH, Noguchi P, et al: Enhanced expression of surface tumor-associated antigens on human breast and colon tumor cells after recombinant human leukocyte a-interferon treatment. Cancer Res 44:3208-3214, 1984 27. Giacomini P, Aguzzi A, Pestka S, et al: Modulation by recombinant DNA leukocyte (a) and fibroblast (fl) interferons of the expression and shedding of HLA- and tumorassociated antigens by human melanoma cells. J Immunol 133:1649-1655, 1984 28. Lotze MT, Chang AE, Seipp CA, et al: High-dose recombinant interleukin-2 in the treatment of patients with disseminated cancer: Responses, treatment-related morbidity, and histologic findings. JAMA 256:3117-3124, 1986 29. Blanchard DK, Kavanagh JJ, Sinkovics JG, et al: Infiltration of interleukin-2-inducible killer cells in ascitic fluid and pleural effusions of advanced cancer patients. Cancer Res 48:6321-6327, 1988 30. Jimenez SA, Freundlich B, Rosenbloom J: Selective inhibition of human diploid fibroblast collagen synthesis by interferons. J Clin Invest 74:1112-1116, 1984 31. Diegelmann RF, Peterkofsky B: Inhibition of collagen secretion from bone and cultured fibroblasts by microtubular disruptive drugs. Proc Natl Acad Sci USA 69:892-896, 1972 32. Berek JS, Hacker NF, Lichtenstein A, et al: Intraperitoneal recombinant a-interferon for "salvage" immunotherapy in stage III epithelial ovarian cancer: A Gynecologic Oncology Group study. Cancer Res 45:4447-4453, 1985 33. Berek JS, Knapp RC, Hacker NF, et al: Intraperitoneal immunotherapy of epithelial ovarian carcinoma with Corynebacteriumparvum. Am J Obstet Gynecol 152:10031010, 1985


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