Nitrosamine-induced transplantable pancreatic ductal adenocarcinoma in inbred Syrian hamsters.

A pancreatic ductal carcinoma, designated CBP, was established as a transplantable tumor line in the CB/SsLak inbred strain of Syrian golden hamsters. The tumor, a primary one induced by chronic administration of the nitrosamine N-nitro-bis(2-hydroxyproyl) amine, is a well-differentiated adenocarcinoma that can be propagated by transplantation in syngeneic hamsters. It grows poorly in other hamster strains. The CBP tumor develops in a predictable quantitative manner and metastasizes to regional lymph nodes. Excision of primary transplanted tumor nodules leads to immunity against subsequent secondary tumor challenges. The CBP tumor appears to be a suitable model for biologic and immunologic studies of pancreatic carcinoma in the syrian hamster.

Induction of delayed hypersensitivity reactions in cancer patients by cholesterol-hemisuccinate-treated autologous tumor cells.

Cholesterol-hemisuccinate (CHS) incorporated into tumor cells increases membrane lipid microviscosity and confers enhanced immunogenicity, which can be manifested by delayed hypersensitivity skin reactions. Skin testing was performed in 30 patients with various advanced malignant tumors. Patients were given intradermal injections of 10(6) autologous, irradiated, CHS-treated tumor cells. Control injections consisted of untreated irradiated tumor cells, CHS-treated autologous normal peripheral lymphocytes, or CHS-treated autologous normal tissues. For all patients tested, strongly positive skin reactions were observed when CHS-treated tumor cells were used. Untreated irradiated cells gave negative or very weakly positive reactions. In all cases, normal CHS-treated cells did not elicit any observable skin reactions. CHS-treated cells may have unmasked tumor-associated antigens to which patients may elicit immunologic responses.

Gastrointestinal carcinoma-associated antigen defined by a murine monoclonal antibody.

A murine monoclonal antibody, CHIP, has been prepared against a human pancreatic carcinoma cell line, SHAW. With the use of the avidin-biotin immunoperoxidase technique, the CHIP antibody detected an antigen found in 11 of 20 fixed tissue sections of tumors obtained from patients with pancreatic carcinoma. The antibody also detected the antigen in 25 of 26 colon carcinoma specimens, 4 of 6 gastric carcinoma specimens, and 1 of 2 esophageal adenocarcinoma specimens. The antigen was also found in normal proximal jejunum and colon and in small amounts in pancreatic islets and parathyroid. There was no reactivity with normal pancreatic ductal or acinar cells or with mesenchymal tissues.

Monoclonal antibodies against human pancreatic adenocarcinoma: distribution of DU-PAN-2 antigen on glandular epithelia and adenocarcinomas.

This report describes the distribution of antigen DU-PAN-2, defined by a monoclonal antibody raised against human pancreatic carcinoma cells, on a variety of tumors and nonneoplastic tissues. With the use of an immunoperoxidase technique, the antigen was detected on 16 of 16 pancreatic carcinomas, on 5 of 5 gallbladder or bile duct carcinomas, on the great majority of stomach adenocarcinomas, and, less commonly, on adenocarcinomas of other primary sites. Substantial intratumor heterogeneity of antigen expression was noted. DU-PAN-2 antigen was present on many types of normal glandular epithelial cells but often was more weakly expressed than on the corresponding tumors. The immunomorphology of staining, coupled with biochemical information known about the antigen, supports the notion that the DU-PAN-2 antigen is a mucin-like substance. Its relative restriction of expression on different types of glandular epithelium suggests that DU-PAN-2 antibody might be a useful reagent for helping to determine the site of origin of adenocarcinomas.

212Bismuth linked to an antipancreatic carcinoma antibody: model for alpha-particle-emitter radioimmunotherapy.

For comparison of cytotoxicity from alpha-particle irradiation with that from conventional x-irradiation, 212Bi, an alpha-emitting radionuclide, was attached to a monoclonal antibody that recognizes a cell surface antigen on human pancreatic carcinoma cells. For a given level of survival, the 212Bi-antibody complex was found to be approximately 20 times more efficient in cell killing than x-irradiation and 5 times more cytotoxic when compared with the cytotoxicity of an antigen-negative cell line or an isotype-matched control antibody. High linear energy transfer radioimmunotherapy using alpha emitters linked to monoclonal antibodies may be useful in vivo and in vitro for selectively killing target cell populations, especially those resistant to other forms of treatment.

Spontaneous lymphosarcoma arising in a nude mouse: characterization in vivo and in vitro.

A lymphosarcoma spontaneously arising in a nude mouse and a continuous cell line (NML-1) derived from it are described and compared. The primary tumor and a transplantable tumor line from it were composed of lymphoid cells, with no C-type viral particles seen by electron microscopy. The culture line was composed of cells with morphologic and functional properties of macrophages; budding C-type particles were abundant. The cells in the tumors produced in nude mice by injection of the NML-1 cells also resembled macrophages morphologically rather than lymphocytes; however, by electron microscopy, no C-type particles were seen. The findings suggest some type of in vivo suppression of complete expression of the virus.

Maintenance of ectopic chorionic gonadotropin and alpha subunit secretion by a human lung cancer cell line (ChaGo) transplanted into nude mice.

ChaGo cells, derived from a human primary carcinoma of the lung, were successfully transplanted into nude mice without any change in morphologic characteristics over four generations and with continued ectopic secretion of human chorionic gonadotropin (HCG) and HCG alpha subunit (HCG-alpha). The concentration of free HCG-alpha was 1,100-fold higher than that of complete HCG in the original ChaGo culture medium but only 35-fold higher in nude mouse plasma, possibly due to slower metabolic clearance of complete HCG. Tumor weights correlated with plasma HCG-alpha but not with HCG. Tumor-bearing mice had significantly heavier uteri than did control mice.

Active immunization of hamsters against pancreatic carcinoma with lipid-treated cells or their shed antigens.

Increasing membrane lipid microviscosity of cells by treatment with cholesterol esters such as cholesterol hemisuccinate (CHS) enhances immunogenicity, probably by exposure of cryptic membrane antigens. Transplantable pancreatic carcinoma lines CBP and LSP-1 grown in inbred hamsters were tested for immunogenicity after CHS treatment. Tumor cells were incubated in CHS to rigidify cell membranes, and they were irradiated and injected i.p. into syngeneic hamsters. Incubation media after CHS treatment, considered to contain shed antigens due to hyperrigidification, were also used for immunization. Two identical immunizations using 10(7) cells or incubation media were performed 14 days apart. In control experiments, non-CHS-treated, irradiated cells were injected. Immunizations were performed using both syngeneic and allogeneic cells and supernatants for both the CBP and LSP-1 systems for specificity experiments. The degree of immunization in the treated hamsters was assessed by the response to a subsequent s.c. challenge with viable tumor cells given 7 days following the last immunization. For the CBP pancreatic cancer line, CHS treatment increased tumor immunogenicity significantly, as demonstrated by diminished tumor growth rate and by increased duration of survival after challenge. With the LSP-1 pancreatic tumor, immunization with CHS-treated cells showed no enhancement of immunogenicity. However, immunization with supernatant of CHS-treated cells resulted in a significant delay of tumor growth and increased survival, suggesting immunization by shed antigens in the CHS incubation medium. Use of CHS-treated cells or shed antigeneic material could be of potential value as a method of active immunotherapy.

Evaluation of the utility of a radioimmunoassay for serum CA 19-9 levels in patients before and after treatment of carcinoma of the pancreas.

By radioimmunoassay we determined circulating levels of a tumor-associated antigen, CA 19-9, in 47 patients with pancreatic adenocarcinoma, to learn if serial testing was useful in predicting prognosis or in detecting disease progression. Before treatment, 42 (89%) had an abnormal serum level, and 45 (96%) had an abnormal level at some time during the disease course. A pretreatment value of less than 1,000 U/mL (normal, less than or equal to 37 U/mL) was found in 38 patients; 20 (53%) had resectable disease. One of nine patients (11%) with a pretreatment value greater than 1,000 U/mL had resectable disease (P2 = .05). Among 14 patients who underwent pancreatectomy and were studied serially, the CA 19-9 level normalized in eight; seven (88%) survived greater than or equal to 18 months. Six patients whose levels did not normalize after pancreatectomy all died in less than 12 months (P2 less than .005). Greatly elevated levels occurred in 11 patients after pancreatectomy 1 to 7 months before clinically apparent recurrence. The other three patients without significant elevations remain clinically free of disease. The data suggest that serial determination of serum CA 19-9 levels are useful as a prognostic indicator and in detecting disease recurrence following pancreatectomy. Concurrent determinations of carcinoembryonic antigen (CEA) levels showed abnormal preoperative values in 28 of 46 patients tested (61%). Concurrent serial postoperative determinations of CEA were available in ten patients. Whereas CA 19-9 values clearly indicated eight recurrences, CEA was helpful in only four. In this small group of patients, CA 19-9 was a better predictor of recurrence.

Preliminary results of a randomized study of adjuvant radiation therapy in resectable adult retroperitoneal soft tissue sarcomas.

Between January 1980 and September 1985, 35 adult patients with resectable retroperitoneal soft tissue sarcomas were entered on a randomized trial comparing two forms of adjuvant radiation therapy. Fifteen patients received the experimental therapy consisting of intraoperative radiotherapy (IORT) to 20 Gy using high-energy electrons followed by low-dose (35 to 40 Gy) postoperative external beam irradiation. Twenty patients received standard therapy consisting of high-dose (50 to 55 Gy) postoperative external beam irradiation. With a minimum follow-up of 15 months, there is no significant difference in the actuarial disease-free survival (DFS) and overall survival (OS) comparing the two groups (median DFS, 34 months; median OS, 38 months). At 5 years follow-up, approximately 40% of patients are alive and 20% of patients remain disease-free. Although there is a trend towards an improvement in in-field local control in the experimental arm, the predominant pattern of failure in both groups was locoregional within the retroperitoneum and/or peritoneal cavity. Acute and late radiation enteritis were significantly reduced in the experimental group. However, four experimental patients developed late (greater than 6 months following treatment) peripheral neuropathy believed related to the use of IORT; all four recovered. We conclude that there is no difference in the therapeutic effectiveness of the combination of IORT and low-dose external beam radiation compared with conventional high-dose radiation as adjuvant treatment in retroperitoneal sarcomas, although the former appears to be less toxic. Newer combined modality treatment strategies are discussed to improve the prognosis in these patients.

Adjuvant chemotherapy for patients with high-grade soft-tissue sarcomas of the extremity.

We have previously reported the results of a randomized trial that demonstrated the survival benefit of adjuvant chemotherapy in the treatment of patients with high-grade extremity sarcomas compared with no chemotherapy. This regimen included doxorubicin, cyclophosphamide, and methotrexate. This report updates and extends our experience. The median follow-up of this trial is now 7.1 years and reveals a 5-year disease-free survival of 75% and 54% for chemotherapy and no chemotherapy groups, respectively (two-sided P [P2] = .037). The 5-year overall survival for patients in this trial was 83% and 60% for the chemotherapy and no chemotherapy groups, respectively, with a trend towards improved survival in the chemotherapy arm (P2 = .124). Because of doxorubicin-induced cardiomyopathy we performed a subsequent randomized trial comparing this high-dose regimen to reduced cumulative doses of doxorubicin and cyclophosphamide without methotrexate. Eighty-eight patients were entered into this trial which has a median follow-up of 4.4 years. The 5-year disease-free and overall survival for patients treated with the reduced doses of chemotherapy was 72% and 75%, respectively, and was not significantly different from the high-dose regimen. No patients developed congestive heart failure on this study. We conclude that adjuvant chemotherapy improves disease-free survival in patients with extremity soft-tissue sarcomas. The overall survival advantage in patients receiving adjuvant chemotherapy in our initial randomized high-dose chemotherapy trial has diminished though it continues to favor the chemotherapy group. A reduced-dose chemotherapy regimen was found to be comparable to the high-dose regimen.

Randomized prospective study of the benefit of adjuvant radiation therapy in the treatment of soft tissue sarcomas of the extremity.

PURPOSE: This randomized, prospective study assesses the impact of postoperative external-beam radiation therapy on local recurrence (LR), overall survival (OS), and quality of life after limb-sparing resection of extremity sarcomas. PATIENTS AND METHODS: Patients with extremity tumors and a limb-sparing surgical option were randomized to receive or not receive postoperative adjuvant external-beam radiotherapy. Patients with high-grade sarcomas received postoperative adjuvant chemotherapy whereas patients with low-grade sarcomas or locally aggressive nonmalignant tumors were randomized after surgery alone. RESULTS: Ninety-one patients with high-grade lesions were randomized; 47 to receive radiotherapy (XRT) and 44 to not receive XRT. With a median follow-up of 9.6 years, a highly significant decrease (P2 = .0028) in the probability of LR was seen with radiation, but no difference in OS was shown. Of 50 patients with low-grade lesions (24 randomized to resection alone and 26 to resection and postoperative XRT), there was also a lower probability of LR (P2 = .016) in patients receiving XRT, again, without a difference in OS. A concurrent quality-of-life study showed that extremity radiotherapy resulted in significantly worse limb strength, edema, and range of motion, but these deficits were often transient and had few measurable effects on activities of daily life or global quality of life. CONCLUSION: This study indicates that although postoperative external-beam radiotherapy is highly effective in preventing LRs, selected patients with extremity soft tissue sarcoma who have a low risk of LR may not require adjuvant XRT after limb-sparing surgery (LSS).

Patterns of disease recurrence following definitive therapy of adenocarcinoma of the pancreas using surgery and adjuvant radiotherapy:correlations of a clinical trial.

PURPOSE: Between 1980 and 1984, 26 patients with resectable adenocarcinoma of the pancreatic head were enrolled in a National Cancer Institute protocol evaluating intraoperative radiotherapy vs. standard therapy. METHODS AND MATERIALS: After complete excision of their lesions, patients were observed (Stage I), or randomized to intraoperative radiotherapy vs. external beam radiotherapy (Stages II-IV). The intraoperative dose was 20 Gy in a single fraction using 9-20 MeV electrons. The external beam radiotherapy schema involved daily 150-180 cGy fractions to 45-55 Gy in 5-6 weeks. Chemotherapy was not used for primary disease but was administered off protocol for recurrent disease. Median potential followup for the trial was > 9 years, with a median patient survival of 18 months. Perioperative mortality was 27% (7 patients). Of the remaining 19 patients, one remains alive and without evidence of disease 9 years post-therapy. Twelve patients underwent autopsy and 2 required antemortem laparotomy; histopathologic evidence of disease recurrence was analyzed. RESULTS: Of 15 patients evaluable for intra-abdominal control, 7 (47%) suffered local recurrences and 7 (47%) failed regionally, with 5 patients (35%) failing in both areas. Five patients (35%) developed peritoneal seeding. Of 13 patients evaluable for systemic disease, 8 (62%) suffered distant failure. There were no differences in outcome between intraoperative or external beam radiotherapy or observation in this subset of patients. CONCLUSION: This report is among the most rigorous descriptions of patterns of failure after resection of pancreatic cancer. It appears clear that advances in local control of this disease are unlikely to translate into increased survival in the absence of improved systemic therapy.

Surgery with intraoperative radiotherapy for sarcomas of the pelvic girdle: a pilot experience.

Sarcomas of the pelvic girdle represent difficult treatment problems. Many are not treatable for cure, and among the patients who are technically resectable, there is high risk for local tumor recurrence and distant spread. Intraoperative radiotherapy (IORT) has been used in conjunction with surgical resection in five patients with extensive sarcomas of the pelvic girdle. Patients underwent a hemipelvectomy and IORT (dose 20-30 Gy) to the sacral resection margin and surrounding soft tissues. Three patients developed pulmonary metastases within 3 months and eventually died from metastatic disease (8-38 months). Two patients have remained disease-free (43 and 53 months). Four patients (80%) have remained locally free of tumor with follow-ups of 8-53 months. The only treatment complication was late osteonecrosis of the coccyx which appeared 7 months after treatment. By contrast, six historical control patients with sarcomas of the pelvic girdle treated with resection alone showed a local control rate of only 27% over a 40-month follow-up. On the basis of this preliminary experience, it appears that IORT may substantially help to control local disease in patients with grossly resectable sarcomas of the bony pelvis.

Threshold dose for peripheral neuropathy following intraoperative radiotherapy (IORT) in a large animal model.

Radiation injury to peripheral nerve is a dose-limiting toxicity in the clinical application of intraoperative radiotherapy, particularly for pelvic and retroperitoneal tumors. Intraoperative radiotherapy-related peripheral neuropathy in humans receiving doses of 20-25 Gy is manifested as a mixed motor-sensory deficit beginning 6-9 months following treatment. In a previous experimental study of intraoperative radiotherapy-related neuropathy of the lumbro-sacral plexus, an approximate inverse linear relationship was reported between the intraoperative dose (20-75 Gy range) and the time to onset of hind limb paresis (1-12 mos following intraoperative radiotherapy). The principal histological lesion in irradiated nerve was loss of large nerve fibers and perineural fibrosis without significant vascular injury. Similar histological changes in irradiated nerves were found in humans. To assess peripheral nerve injury to lower doses of intraoperative radiotherapy in this same large animal model, groups of four adult American Foxhounds (wt 20-25 kg) received doses of 10, 15, or 20 Gy to the right lumbro-sacral plexus and sciatic nerve using 9 MeV electrons. The left lumbro-sacral plexus and sciatic nerve were excluded from the intraoperative field to allow each animal to serve as its own control. Following treatment, a complete neurological exam, electromyogram, and nerve conduction studies were performed monthly for 1 year. Monthly neurological exams were performed in years 2 and 3 whereas electromyogram and nerve conduction studies were performed every 3 months during this follow-up period. With follow-up of greater than or equal to 42 months, no dog receiving 10 or 15 Gy IORT shows any clinical or laboratory evidence of peripheral nerve injury. However, all four dogs receiving 20 Gy developed right hind limb paresis at 8, 9, 9, and 12 mos following intraoperative radiotherapy. These experimental data suggest that intraoperative doses of less than 20 Gy may not result in clinically significant peripheral nerve injury with follow-up of 3.5 years. Longer (5 yrs) follow-up with planned sacrifice of the remaining dogs is scheduled to assess any late peripheral nerve damage.

Television system for verification and documentation of treatment fields during intraoperative radiation therapy.

Intraoperative radiation therapy (IORT) involves direct treatment of tumors or tumor beds with large single doses of radiation. The verification of the area to be treated before irradiation and the documentation of the treated area are critical for IORT, just as for other types of radiation therapy. A television system which allows the target area to be directly imaged immediately before irradiation has been developed. Verification and documentation of treatment fields has made the IORT television system indispensable.

Long term tolerance of thoracic organs to intraoperative radiotherapy.

The tolerance of mediastinal structures to intraoperative radiotherapy (IORT) was investigated in 3 separate animals trials using 49 adult foxhounds and one limited Phase I trial in 4 patients with Stage II or III non-small cell lung cancer (NSCLC). The 1- to 2-year results of these trials have been previously reported with significant toxicity found at dose levels over 20 Gy. We now report the results of five dogs reserved for long term studies and one Stage II NSCLC patient alive at 5 years. Two dogs received 20 Gy IORT and one received 30 Gy IORT to the esophagus, all three to a single 6 cm field with 9 MeV electrons. One control dog underwent surgery without irradiation. One dog received 20 Gy IORT to a single 5 cm mediastinal field with 13 MeV electrons following left pneumonectomy. At 5 years, all five dogs reserved for a long term evaluation were alive and evaluable with minimal endoscopic and radiographic abnormalities. The one patient alive at 5 years for evaluation received 25 Gy IORT to two matched 6 cm fields with 13 MeV electrons. She has stable dyspnea on exertion and there is no evidence of cancer by endoscopy. We conclude, based on these limited data, that IORT in the mediastinum may be safe at dose levels that do not exceed 20 Gy, and further careful evaluation at these lower treatment doses is warranted to determine efficacy.

Tolerance of the canine bladder to intraoperative radiation therapy: an experimental study.

An experimental study of bladder tolerance to intraoperative radiotherapy (IORT) was designed using a large animal model (adult American Foxhounds, weight 25-30 kg) to access acute and late radiation effects. Dogs were subjected to laparotomy where the bladder was mobilized and IORT was delivered using a 5 cm circular cone through a cystotomy incision with 12 MeV electrons. The bladder trigone including both ureteral orifices and the proximal urethra was irradiated in groups of 3 dogs with doses of 0, 20, 25, 30, 35, and 40 Gy. Dogs were followed clinically with repeat urinalysis, intravenous pyelogram (IVP), and cystometrogram at 1 month and then Q6 months for up to 4 years. One dog from each dose group was sacrificed electively at 1 and 2 years, whereas the other dog is being followed clinically for a minimum of 4 years. Complete autopsies were performed with particular attention to genitourinary and pelvic structures. No clinically detectable acute toxicity resulted from IORT to the bladder. Three of 15 IORT dogs (1 each at 25, 35, and 40 Gy) showed obstruction of a ureteral orifice with 2 dogs dying of renal failure secondary to bilateral hydronephrosis within 1-2 years of treatment. The remaining 12 IORT dogs and 3 control dogs have normal repeat IVP's and renal function with up to 4 years of follow-up. Serial cystometry demonstrates no major loss of bladder contractility or volume. At autopsy, histological changes of mucosal thinning and telangiectasia with submucosal fibrosis were confined to the IORT field and appeared dose-related. However, the bladder epithelium remained intact at all doses. The ureterovesical junction in animals receiving 20 Gy showed mild fibrosis of the lamina propria and moderate chronic inflammation. Above 20 Gy, these histological changes at the U-V junction were more pronounced with gross stenosis in 3 animals as predicted by the IVP. We conclude that the bladder trigone will tolerate IORT to 20 Gy without major clinical sequellae. Above 20 Gy, progressive inflammation and fibrosis of the U-V junction resulted in obstructive hydronephrosis in three animals within 1-2 years of IORT. The bladder mucosa remained intact with doses to 40 Gy, although submucosal fibrosis and chronic inflammation were evident and appeared dose-related. However, bladder function as measured by cystometry showed essentially no change with follow-up to 4 years. From this large animal study, IORT for early-stage bladder carcinoma is technically feasible and deserves a careful clinical study.

Response of canine esophagus to intraoperative electron beam radiotherapy.

Tolerance of esophagus to intraoperative radiotherapy (IORT) was investigated in dogs. Thirteen adult foxhounds were subjected to right thoractomy, mobilization of the intrathoracic esophagus, and IORT to a 6 cm full-thickness esophageal segment using 9 MeV electrons at doses of 0, 2,000, or 3,000 cGy. Dogs were followed clinically and were evaluated at regular intervals after treatment with fiberoptic esophagoscopy, barium swallows, and postmortem histologic evaluations. One sham-irradiated control dog showed no abnormalities during follow-up of 24 months. Seven dogs receiving 2,000 cGy IORT showed transient mild dysphagia and mild esophagitis, but no clinically or pathologically significant complications. Five dogs receiving 3,000 cGy demonstrated severe ulcerative esophagitis within 6 weeks of treatment which progressed to chronic ulcerative esophagitis with stricture formation by 9 months following IORT. One 3,000 cGy dog died at 13 months from an esophageal perforation. On the basis of a pilot experience using 13 experimental animals, it was concluded that intact canine esophagus tolerates IORT well to doses of 2,000 cGy, but doses of 3,000 cGy pose serious and potentially lethal risks. The clinical application of IORT to the treatment of human intrathoracic neoplasms requiring esophageal irradiation should be approached with caution, particularly at doses exceeding 2,000 cGy.

Intraoperative radiation therapy at the National Cancer Institute: technical innovations and dosimetry.

The technical complexity of intraoperative radiotherapy (IORT) requires modification of the standard physical and dosimetric methods used in external electron beam therapy. At the National Cancer Institute, a number of technical innovations have been integrated into ongoing clinical studies of IORT. These include: (1) an electron beam applicator system that is significantly different from other IORT systems and includes customized "squircle" applicators; (2) peripheral dose shields; (3) a modified surgical table replacing the standard radiation treatment couch; and (4) routine use of multiple IORT fields that necessitates field matching. The IORT applicator system and related devices and techniques are dosimetrically characterized in detail both for use in the IORT program and in order to illustrate many useful facets of electron dosimetry.