Leukocyte migration inhibition in patients with Ewing's sarcoma by 3-M potassium chloride extracts of fresh and tissue-cultured Ewing's sarcomas.

Leukocyte migration inhibition (LMI) assays were performed to detect cell-mediated immune reactions against tumor-associated antigens (TAA) of Ewing's sarcoma. With the use of crude antigen preparations obtained by 3M KCl extractions of fresh Ewing's sarcoma or of tissue culture cells derived from a pleural effusion of a Ewing's sarcoma patient, assays were performed with leukocytes from these patients, patients with other cancers, and normal donors. The results demonstrated approximately 60% or greater positive LMI reactivity in Ewing's sarcoma patients, as compared to less than 10% reactivity of normal donors, with the use of extracts of either fresh or tissue-cultured Ewing's sarcoma cells. A lower proportion of positive reactivity was observed in patients with breast and lung cancer. Further specificity tests indicated that a smaller proportion of patients with Ewing's sarcoma had LMI reactivity with KCl extracts of tissue-cultured cells derived from breast cancer of fresh lung cancer cells than did patients with the homologous disease. The results indicate that many patients with Ewing's sarcoma have cell-mediated immunity toward TAA on Ewing's sarcomas. Inasmuch as all the LMI assay were performed with allogeneic extracts, the data also suggested that different Ewing's sarcomas possess common antigens and that some breast and lung cancers may share some TAA with Ewing's sarcoma.

Cell culture of human colon adenomas and carcinomas.

Cell lines were established from colon adenomas, including tubular and villous polyps, primary adenocarcinomas, and metastases arising in patients with colon adenocarcinomas. The protocol for cultivating these diverse tissues includes primary cultivation of tissue explants on a type I collagen gel followed by nonenzymatic subculture of the epithelial outgrowth. All early passages were accomplished using low subculture ratios. Cultured cells elaborate morphological structures which are similar to features present in the tissues from which they were cultivated. Specifically, all structural features of colon epithelial cells were identified, including junction formation, prominent microvilli, and mucin secretion, in several cell lines. Five cell lines cultured from colonic neoplasms at different stages of cancer progression were selected for detailed characterization. Cells grown from two tubular polyps had normal human karyotypes. Cells from a villous polyp and all adenocarcinomas were aneuploid with stable marker chromosomes. The established cell lines exhibit distinct phenotypes based on growth characteristics in vitro and in athymic mice; and it is suggested that these cell lines represent useful models for studying the evolution of colon cancer from a benign to an aggressive cell type.

Establishment and characterization of human colorectal cancer cell lines.

A protocol for establishment of human colorectal carcinoma cell lines from fresh surgically removed tissues is described. Twelve human colorectal carcinoma cell lines were established from 6 of 18 primary cancers and four of four metastases. Cell lines from concurrent primary tumors and metastases were established from two individual patients. Two primary cancers gave rise to multiple cell lines with differing biological characteristics. Factors contributing to our success appear to be differential selection on the basis of substrate adherence and the timing of passage. The protocol avoids the use of feeder layers or passage through athymic mice. The established cell lines exhibit a range of karyotypes, morphologies, and growth characteristics.

Phase I trial of hepatic artery infusion of 5-iodo-2'-deoxyuridine and 5-fluorouracil in patients with advanced hepatic malignancy: biochemically based combination chemotherapy.

Eighteen patients with hepatic metastases primarily from colorectal carcinoma were treated on a phase I protocol employing hepatic artery infusion (HAI) of 5-fluorouracil (FUra) and 5-iodo-2'-deoxyuridine (IdUrd) via implantable infusion pump. Patients received a 14-day continuous HAI of 300 mg/day FUra. During days 8-14 of therapy, patients received IdUrd as a separate 3-h HAI daily x 7. Treatment cycles were repeated every 28 days. IdUrd was escalated from 0.1 to 2.86 mg/kg/day x 7. Myelosuppression and stomatitis were mild and not dose limiting. Hepatotoxicity was dose limiting and similar to that reported for 5-fluoro-2'deoxyuridine alone administered as a 14-day infusion every month. One patient developed a clinical picture consistent with sclerosing cholangitis and another had biopsy-proven cholestasis and triaditis. Catheter complications occurred in 7 of 18 patients. Plasma concentrations of FUra during the 7-day continuous HAI of FUra alone were consistently either undetectable or very low (less than or equal to 0.1 microM). At level 3 (1.0 mg/kg/day IdUrd) and beyond, measurable plasma concentrations of FUra, iodouracil, and IdUrd were found at the end of the daily 3-h infusion of IdUrd. The maximum tolerated dose of IdUrd as administered in this trial is 2.2 mg/kg/day x 7 and the recommended starting dose for further clinical investigation is 1.7 mg/kg/day x 7.

Glutathione S-transferases in human prostate.

A number of human prostatic tissue biopsies have been analyzed for glutathione S-transferase activity, using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. Samples from nine patients (age range 61-90) with benign prostatic hypertrophy who had received no prior chemotherapy had a mean glutathione S-transferase activity of 137 +/- 44 nmol/min per mg with a range of 97-237. A qualitative comparison of the glutathione S-transferase of normal prostate and benign prostatic hypertrophy samples was carried out. Approximately 260-fold purification was achieved using glutathione-Sepharose affinity chromatography, with glutathione S-transferase accounting for approximately 0.19-0.33% of the total protein. Substrate specificity determinations suggested similar, but not identical, glutathione S-transferase subunits in normal prostate and benign prostatic hypertrophy. One- and two-dimensional electrophoresis (isoelectric focusing and 12.5% SDS-polyacrylamide gel electrophoresis) identified at least seven stained polypeptides in the purified glutathione S-transferase preparations. These ranged in Mr from approximately 24,000 to 28,500 and in pI from near neutral to basic. Western blot analysis using polyclonal antibodies raised against rat liver glutathione S-transferase suggested crossreactivity with five of the human isoenzymes in both normal prostate and benign prostatic hypertrophy. One of the glutathione S-transferases, present in both normal prostate and benign prostatic hypertrophy, had an Mr of approx. 24,000 and a near-neutral pI and crossreacted immunologically with a polyclonal antibody raised against human placental glutathione S-transferase (Yf, subunit 7 or pi). These data suggest that four glutathione S-transferases are expressed in human prostate, with subunits from each of the major classes alpha, mu and pi. These are characterized as Ya, Yb, Yb' and Yf (analogous alternative nomenclature subunits 1, 3, 4 and 7).

Phase II trial of preoperative radiation therapy and chemotherapy for patients with localized, resectable adenocarcinoma of the pancreas: an Eastern Cooperative Oncology Group Study.

PURPOSE: A prospective, multiinstitutional trial was initiated in 1991 to examine the tolerance to and efficacy of a program of preoperative chemoradiotherapy (CTRT) and surgical resection for patients with localized adenocarcinoma of the pancreas. PATIENTS AND METHODS: Fifty-three patients were assessable for analysis, with a median follow-up of 52 months for survivors. Radiation therapy (RT) totaling 5,040 cGy in 180 cGy fractions with mitomycin 10 mg/m2 day 2 and fluorouracil (5-FU) 1,000 mg/m2/d continuous infusion days 2 through 5 and 29 through 32 were given as preoperative adjuvant therapy. Twelve patients did not proceed to surgery (one death, one toxicity, three local progression, six distant metastases, one intercurrent illness), whereas 41 patients underwent surgery. Of these, 17 patients did not have resection (11, hepatic and/or peritoneal metastases and six local extension that precluded resection). Twenty-four patients had tumor resection (19 Whipple, four total pancreatectomy, one distal pancreatectomy). RESULTS: Treatment toxicity was primarily hematologic, although a comparable number suffered biliary tract complications, either from obstruction or cholangitis as a result of an occluded stent or the primary tumor. There was one postoperative death. Median survival for the entire group and for the 24 patients with resection was 9.7 and 15.7 months. This survival rate reflected the advanced state of most resected cancers (positive peritoneal cytology, three patients; margins within 2 mm, 13 patients; involved lymph nodes, four patients; and need for superior mesenteric vein (SMV) resection, four patients). Tumor progression was most frequent at metastatic sites. CONCLUSION: This preoperative CTRT protocol was feasible and safe in a cooperative group setting. Entry of patients with advanced tumors probably accounted for the suboptimal resectability and survival results.

Aerosolized gamma-interferon and lipopolysaccharide enhances cytotoxicity of murine pulmonary alveolar macrophages.

In vivo stimulation of pulmonary alveolar macrophages (PAMs) may enhance tumor cell cytotoxicity. A model using aerosolized gamma-interferon (gamma-IFN) and lipopolysaccharide (LPS) was developed to induce enhanced PAM activation in vivo in C57BL/6 mice. Mice received four doses of aerosol (2 doses/day) consisting of gamma-IFN (10(4) microU/mouse) and LPS (100 micrograms/mouse). Other groups received either gamma-IFN alone, LPS alone, or saline (control). Cells were harvested by bronchoalveolar lavage. Macrophage cell count demonstrated an increase in macrophage recruitment in the gamma-IFN and LPS group. PAMs were evaluated for in vitro cytotoxicity against B16-F10 melanoma cells. Treatment groups demonstrated enhanced cytotoxicity over controls, and the combination (gamma-IFN plus LPS) was significantly better in cell killing than either treatment modality alone (p less than or equal to 0.02). Activated PAMs selectively killed tumor cells, but did not kill the 3T3 fibroblast cell line. Peritoneal macrophages from mice treated by inhalational gamma-IFN + LPS were enhanced (indicating a systemic effect), but not to the same extent as PAMs. These studies suggest that inhalation of gamma-IFN + LPS can selectively enhance in vivo cytotoxicity of murine PAMs. This may potentially be applicable to human tumor management.

Quantitation of local cerebral blood flow and partition coefficient without arterial sampling: theory and validation.

A new technique that requires neither arterial blood sampling nor prior knowledge of the indicator's tissue-blood partition coefficient has been developed for quantitation of local CBF. This technique arises from an existing method that uses the inert, freely diffusible gaseous tracer [18F]methyl fluoride (CH3(18)F) and positron computed tomography. The shape of the arterial blood curve is derived from continuous sampling of expired air. The concentration of CH3(18)F in the arterial blood is assumed to be proportional to the expired gas curve interpolated between end-tidal values. The absolute scale of the blood curve is determined by fitting a series of venous blood samples to a multicompartment model. Four validation studies were performed to compare values derived using the venous scaled expired breath input function with those derived using direct arterial samples. The proposed method gave higher flow values than the standard arterial sampling method by an average of 4.4%. These validation studies and data from both normal and patient scans suggest that the method provides the quantitation necessary for interstudy comparisons yet avoids the trauma of an arterial puncture.

Does omentectomy prevent malignant small bowel obstruction?

Because the omentum collects and disseminates cancer cells, omentectomy is an integral part of ovarian cancer surgery. We postulate that the omentum serves a similar function in colon cancer and may contribute to post-operative malignant small bowel obstruction (S.B.O.) and that routine omentectomy during colectomy would reduce the incidence of S.B.O. Fischer 344 rats and a transplantable carcinogen-induced rat colon cancer were used to test: (1) whether the omentum is a unique site of intra-abdominal colon tumor implantation which contributes to S.B.O.; and (2) whether omentectomy at the time of tumor implantation would reduce the incidence of S.B.O. Statistical analysis confirmed that animals undergoing omentectomy had a significantly lower incidence of omental tumors and malignant S.B.O. (26 per cent and 16 per cent respectively) when compared with sham operated animals (75 per cent and 85 per cent respectively, P less than 0.001). These data suggest that the omentum is a source of bowel obstruction from implantation and growth of tumour cells in the rat model. Although this could be tested in other animal systems, the addition of routine omentectomy to colectomy is simple, not time-consuming, and may reduce postoperative morbidity.

Brief communication: technical modifications of the human agarose microdroplet leukocyte migration inhibition assay.

Direct leukocyte migration inhibition assays using the capillary tube technique can be used to demonstrate cell-mediated immunity in vitro. Unfortunately, the cumbersome nature of this technique makes it time consuming and difficult to perform. Similar results have been obtained using the direct agarose microdroplet leukocyte migration inhibition assay. In this paper, modifications of the agarose technique are outlined which insure standardization of droplets and ease of performance of the assay. Additionally a technique is described to reduce the time required for calculation of results.

A lysosomal study of cold-induced hyperthyroidism in the rat: comparison to human hyperthyroid tissue.

Prolonged exposure to cold in the adult rat is considered an acceptable model to study experimental hyperthyroidism. In our experiments adult rats were exposed to cold (1 degree to 4 degrees C) for various periods of time, after which their thyroid tissue and plasma were assayed for lysosomal enzyme activity. In additional animal experiments we examined the cold-exposed rats during propranolol treatment. After 2 weeks of exposure to cold, rat thyroid glands were hypertrophied and hypervascular. There was a significant increase in all three lysosomal enzymes measured in the rat thyroid tissue compared to age-matched controls. The percent of enzyme activity found in the various subcellular fractions did not change. The plasma triiodothyronine concentration increased slightly in the cold-exposed rats, whereas there was no change in thyroxine or lysosomal activity. Propranolol had no effect. In separate experiments human thyroid tissue from patients with diffuse toxic goiter or histologically normal thyroid tissue was examined. Similar to cold-exposed rat thyroid tissue, diffuse toxic goitrous tissue showed a twofold to threefold increase in lysosomal enzyme activity compared to control tissue. These data suggest that increased thyroidal lysosomal enzyme synthesis is one of the factors operative in the increased secretion of active thyroid hormones in Graves' disease.

Do operations facilitate tumor growth? An experimental model in rats.

Enhancement of tumor growth by operation is a concern often expressed by surgeons and patients anticipating cancer surgery. Two series of experiments were performed in which Fischer 344 rats and a carcinogen-induced transplantable rat colon cancer were used to test whether anesthesia and operation facilitate tumor implantation and growth. In the first experiments two groups of rats were given intraperitoneal tumor cells. One group underwent sham laparotomy; the second did not undergo surgery. In the second set of experiments rats were injected subcutaneously with tumor cells and then divided into four groups. The first group did not undergo laparotomy. The second underwent laparotomy on day 1, the third on day 15, and the fourth on days 15 and 29 after tumor implantation. Animals were followed for the incidence and growth rate of tumors that developed. The initial experiments demonstrated that 89% of the operated versus 49% of the nonoperated animals developed a tumor (p less than 0.001). The second experiment demonstrated that: animals undergoing multiple operations have a higher incidence of subcutaneous tumor nodules than nonoperated animals (p less than 0.05); animals undergoing multiple operations have a higher incidence of subcutaneous tumor nodules than animals undergoing a single operation (p less than 0.05); animals undergoing multiple operations had larger size tumor masses than the nonoperated animals (p less than 0.05) and than animals undergoing only one operation (p less than 0.04). This study supports the hypothesis that multiple operations and anesthesia may enhance tumor implantation and growth of metastases. This should be considered when designing therapy for patients with cancer.

Neoadjuvant chemotherapy, radical resection with intraoperative radiation therapy (IORT): improved treatment for gastric adenocarcinoma.

BACKGROUND: Adenocarcinoma of the stomach and gastroesophageal junction results in substantial morbidity, locoregional recurrence, and death. Surgical procedures, even with adjuvant therapy, have not significantly improved survival. This study evaluated the toxicity, response rate, locoregional control, and survival of patients with locally advanced gastric cancer that was treated with neoadjuvant multimodality therapy. METHODS: Patients with stage IIIA or early stage IV gastric adenocarcinoma received neoadjuvant 5-fluorouracil, Leucovorin, Adriamycin, and Cisplatin and underwent gastrectomy or esophagogastrectomy with intraoperative radiotherapy (IORT; 1000 cGY) to the gastric bed and postoperative radiation therapy. RESULTS: Nine of 15 patients (60%) with transmural extension and/or nodal metastases received IORT. There were 2 pathologically complete responses at the primary site. Eleven of 15 patients (73%) had tumor in perigastric lymph nodes; however, 9 of 15 patients (60%) had mucin-filled nodes without tumor cells. Neoadjuvant treatment did not increase operative morbidity rates. Ten of 15 patients (67%) remain free of disease (median, 27 months; range, 6-60 months). Five patients died 13 to 41 months (median, 17 months) after diagnosis. CONCLUSIONS: Neoadjuvant multimodality therapy with neoadjuvant 5-fluorouracil, Leucovorin, Adriamycin, and Cisplatin, radical resection with IORT, and postoperative radiation therapy is safe, can downstage tumors, provides improved locoregional control, and appears to cause significant tumor regression that may result in long-term survival or cure.

Prevention of rat colon cancer metastases by perioperative immunostimulation.

Major intra-abdominal operations result in profound immunodepression. In addition, manipulation of malignant tumors may release tumor cells into the systemic and portal circulations. The additive effects of immunodepression and tumor cell release may enhance the metastatic potential of tumors. Perioperative correction of immune depression by levamisole can restore lymphocyte proliferation levels in rats. We have developed a model in which rat colon carcinoma cells transplanted into the portal venous system consistently induce hepatic metastases by 4 weeks and death within 9 weeks. Rats pretreated with levamisole (4 mg/kg administered intraperitoneally) the day before and the day of tumor implantation developed fewer metastases (41% of animals treated with levamisole compared with 6% of animals not treated with levamisole had less than or equal to two metastases per liver). Twenty percent of the rats treated with levamisole developed no hepatic metastases. Comparison of median liver weights between the group treated with levamisole and the nontreated, tumor-bearing group was highly significant (p less than 0.005). We conclude that the perioperative period is critical for the implantation and growth of metastases and that perioperative immunostimulation may be a factor in decreasing the incidence of metastases. This model may have relevance to the adjuvant treatment of human colon cancer.

Lymphokine-activated killer cell suppressor factor in malignant effusions.

We examined the possibility that tumor-released products inhibit lymphokine-activated killer cell activation. Lymphokine-activated killer cells from human peripheral blood lymphocytes were activated with recombinant interleukin 2 for 4 days in the presence of malignant effusions or conditioned media from cultured cell lines (10% vol/vol). Eight of 10 malignant effusions/media suppressed the induction of lymphokine-activated killer cell cytotoxicity, as measured in a 4-hour sodium chromate release assay. Seven of 10 effusions/media inhibited lymphokine-activated killer cell proliferation. Suppression was both dose and time dependent. A representative suppressive effusion was fractionated by agarose gel chromatography, treated with detergents disruptive of ionic bonds and lipids, and refractionated using polyacrylamide gel chromatography. Seven suppressive fractions ranging in molecular weight from 1 x 10(5) to 3 x 10(5) d were isolated. It is speculated that this suppressor factor may represent a large multimeric structure with ionic-bonded individual suppressive components.

Avoidance of artifacts on computerized tomograms by selection of appropriate surgical clips.

Surgical clips (metallic or plastic) are frequently used for hemostasis and tumor marking. This study evaluated the radiographic and computerized tomographic appearance of different clips and their relative interference with computerized tomographic scans. Metallic clips (stainless steel, tantalum, and titanium) can all be seen on plain radiographs. Tantalum clips caused extensive distortion on computerized tomographic scans which would interfere with scan interpretation. Both stainless steel and titanium clips resulted in much less artifact and interference on computerized tomographic scans. Recent studies have suggested that there may be some risk of torsion of stainless steel clips in nuclear magnetic resonance scanners resulting in tissue damage. Absorbable plastic clips cannot be seen on plain film but are visualized on computerized tomographic scans and do not appear to cause scan artifact. Overall, we recommend the use of either titanium hemostatic clips when tumor marking on plain film is required or plastic clips when tumor marking is less important.

Percutaneous transhepatic cholangiography, drainage, and biopsy in patients with malignant biliary obstruction. An alternative to surgery.

We retrospectively reviewed our experience with 100 patients with malignant biliary obstruction who underwent percutaneous transhepatic cholangiography alone or in combination with percutaneous drainage. On the basis of this study, we found percutaneous transhepatic cholangiography to be a safe and effective procedure for demonstrating the site, nature, and extent of obstructive lesions. Percutaneous cholangiography was successful in 98 percent of patients, and only one patient (4.1 percent) had a significant complication. Likewise, percutaneous drainage has been highly successful in providing palliative biliary drainage in patients with unresectable malignancies. Percutaneous drainage was successful in 74 of 76 patients (97.4 percent). Five of these patients (6.6 percent) had significant complications, including one death (1.3 percent). The mean survival period of patients with carcinoma of the bile ducts was 29 months, whereas for those with carcinoma of the pancreas it was 3.4 months. Thus, mortality and complication rates were lower, and patient survival rates were similar or improved when compared with those of patients palliated by surgical bypass. Percutaneous drainage thus provides a satisfactory alternative to surgery. Biopsy performed in conjunction with these procedures can often provide a definitive diagnosis. Final tissue diagnoses were made in 20 of 23 patients (87 percent) by transcatheter or percutaneous biopsy.

Is percutaneous nephrostomy for hydronephrosis appropriate in patients with advanced cancer?

Twenty patients with advanced pelvic malignancy and secondary hydronephrosis underwent percutaneous nephrostomy between July 1982 and October 1986. Improvement in renal function occurred in 17 patients (85 percent), and survival ranged from 4 days to 2 years. Median survival was 13 weeks, and 55 percent of the patients required multiple hospitalizations for urosepsis. In addition, 55 percent required multiple tube changes. Thirty-five percent of the patients never left the hospital and an additional 35 percent spent less than 6 weeks at home before they died. Median survival for eight patients with primary cancers most frequently associated with carcinomatosis was 7 weeks, and 63 percent of these patients died during hospitalization. The factors of limited survival, significant morbidity, in-hospital mortality, and poor quality of life should be considered before recommending percutaneous nephrostomy in patients with advanced cancer.

A pilot study of preoperative chemoradiation for patients with localized adenocarcinoma of the pancreas.

BACKGROUND: We hypothesized that delivering adjuvant radiotherapy (RT) preoperatively with chemotherapy might enhance local control of the cancer and patient tolerance for the intervention. METHODS: Thirty-four patients with localized pancreatic cancer (24 head, 8 head and body, 2 body and tail) were treated during the past 6 years with an intramural protocol consisting of 5-fluorouracil (1,000 mg/m2 on days 2 to 5 and 29 to 32) and mitomycin-C (10 mg/m2 on day 2) given with preoperative external beam RT (median 5,040 cGy). Nine patients did not have surgery: 1 refused, 1 died of cholangitis, and 7 were noted to have distant (5) or unresectable local cancer (2) after RT. Of the 25 patients who underwent celiotomy, 11 had liver (8) or peritoneal (3) metastases and 3 had palliative pancreatectomies (2 with liver metastasectomy and 1 with hepatic artery and portal vein replacement). The remaining 11 patients (44% of the cohort with surgery, 32% of all patients) had potentially curative (PC) resections (5 total pancreatectomy, 5 Whipple, 1 distal pancreatectomy). Median tumor diameter by computed tomographic scan was 3.75 cm (range 3 to 5) for the 11 patients who received PC resections and 4.5 cm (range 3 to 7.5) for all patients. Of the 11 patients with PC resections, 8 had evidence of superior mesenteric, portal or splenic venous involvement and 4 had been deemed unresectable at previous celiotomies. RESULTS: One patient developed respiratory failure and one died postoperatively, yielding a 9% rate of major morbidity and mortality. Median follow-up of the surviving patients with curative resection is 33 months (range 14 to 70). Their median survival from the time of tissue diagnosis is 45 months with a median disease-free survival of 27 months. The product limit estimate of 5-year survival is 40% (95% confidence bounds +29%, -30%). One patient had a microscopically positive resection margin, which was a falsely negative frozen section margin at the pancreatic neck. Two patients had positive regional lymph nodes. Five patients have been diagnosed with recurrent cancer. Only 1 has had a local/regional component to the recurrence. CONCLUSIONS: Preoperative RT and chemotherapy followed by resection is well tolerated and safe for patients with locally advanced pancreatic cancer. This approach provides tumor free resection margins and offers prolonged survival to patients with truly localized pancreatic cancer.

CT-guided needle localization for intraoperative biopsy of the head and neck.

Needle-localized intraoperative biopsy was first described for the nonpalpable breast mass using mammography for needle placement. This technique can be adapted by substituting CT (or MRI) for mammography. It can be a valuable tool in localizing the nonpalpable areas of concern in the head and neck, especially when the location is obscure or the patient has undergone previous radiation and/or surgical therapy.