Neoadjuvant therapy and surgical resection for locally advanced non-small cell lung cancer.

During the past 15 years, treatment of stage IIIA (N2) non-small cell lung cancer has evolved considerably because of improvements in patients selection, staging, and combined modality therapy. Results of several clinical trials suggest that induction chemotherapy or chemoradiation and surgical resection is superior to surgery alone. However, the optimal induction regimen has not been defined. An intergroup trial is also underway to determine whether chemoradiation and surgical resection leads to better survival than chemotherapy and radiation alone. Future studies will assess ways to combine radiation and novel chemotherapeutic agents, and will identify molecular abnormalities that predict response to induction therapy.

Immune response against large tumors eradicated by treatment with cyclophosphamide and IL-12.

Previous studies have demonstrated eradication of small (4-8 mm) established murine MCA207 sarcomas by treatment with systemic IL-12. Analysis of the mechanism has revealed a cellular and molecular immune response at the tumor typical of a Th1 cell-mediated, macrophage-effected, delayed-type hypersensitivity (DTH) response. In the current study we investigate the immune response against long term established, large MCA207 tumors induced by combined treatment with IL-12 and cyclophosphamide (Cy), an agent known to potentiate the DTH response. Our results demonstrate that s.c. large MCA207 tumors (15-20 mm) that are refractory to treatment by either IL-12 or Cy alone can be completely eradicated by the combination of Cy and IL-12. IL-12 is apparently the only cytokine capable of mediating tumor eradication, and the effect is dependent on IFN-gamma. The contribution of Cy is probably due to immunopotentiation of DTH rather than to direct cytotoxicity to the tumor. The regression of these large tumors takes >4 wk and, in many cases, is self-sustained, in that little or no additional IL-12 is needed beyond the initial week of administration. Analysis of the cellular and molecular events at the tumor site suggests that the mechanism is a Th1-mediated antitumor immune response.

IL-12 induces T helper 1-directed antitumor response.

Although IL-12 possesses the most potent single-cytokine antitumor efficacy, the mechanism by which IL-12 exerts its antitumor activities remains unclear. Using a complete tumor regression model induced by IL-12 treatment, we demonstrate that the antitumor response induced by IL-12 is mediated by a Th1 cell-directed process, with the macrophage as the effector cell and nitric oxide produced by the activated macrophage as the effector molecule. The induction of the Th1 response by IL-12 depends on the existence of a host T cell response to the tumor before IL-12 administration. IL-12 treatment causes the complete regression of 10-day established s.c. tumors (4-8 mm). Associated with the induction of tumor necrosis, activated macrophages expressing high levels of inducible nitric oxide synthase were found surrounding the tumor. The importance of nitric oxide as the effector molecule was further confirmed by the delay and loss of tumor regression in the presence of a nitric oxide synthase inhibitor in vivo. Examination of tumor-associated T cells indicates that IL-12 induces production of the Th1 cytokine IFN-gamma and suppresses production of IL-2, IL-4, and IL-10 at the tumor site, where these are found to be the predominant cytokines produced by tumor-associated T cells before IL-12 treatment. These findings demonstrate that IL-12 plays an essential role in the induction of an effective Th1 type of cell-mediated immune response against established tumors.

Induction of antigen-presenting capacity in tumor cells upon infection with non-replicating recombinant vaccinia virus encoding murine MHC class II and costimulatory molecules.

The possibility of inducing antigen-presenting capacity in cells normally lacking such capacity, currently represents a major goal in vaccine research. To address this issue we attempted to generate 'artificial' APC able to stimulate CD4+ T cell responses when tumor cells were infected with a single, recombinant, vaccinia virus (rVV) containing the two genes encoding murine MHC class II I-Ak and a third gene encoding the murine B7-1 (mB7-1) costimulatory molecule. To minimize the cytopathic effect and to improve safety, in view of possible in vivo applications, we made this rVV replication incompetent by Psoralen and long wave UV treatment. Tumor cells infected with rVV encoding I-Ak alone, pulsed with hen egg white lysozyme peptide (HEL46-61), induced IL-2 secretion by an antigen-specific T hybridoma. Tumor cells infected with the rVV encoding mB7-1 provided costimulation for activating resting CD4+ T cells in the presence of ConA. Tumor cells infected with the rVV encoding I-Ak and mB7-1, and pulsed with chicken ovotransferrin peptide (conalbumin133-145), induced a significantly higher response in a specific Th2 cell clone (D10.G4.1) as compared to cells infected with rVV encoding I-Ak molecules only. Thus, this replication incompetent rVV represents a safe, multiple gene, vector system able to confer in one single infection step effective APC capacity to non-professional APCs.

Evaluation of somatostatin-receptor scintigraphy for detecting neuroendocrine tumors.

BACKGROUND: Somatostatin-receptor scintigraphy (SRS) has gained attention as an imaging modality for neuroendocrine tumors (NETs). The purpose of this study was to present one of the first American series evaluating the ability of SRS to detect local and distant disease caused by NETs. METHODS: Medical records were reviewed from 35 patients who underwent a total of 38 studies using 111In-pentetreotide between 1993 and 1995. Twenty-two patients had islet cell tumors, seven had carcinoid tumors, and six had other NETs. RESULTS: The overall sensitivity of SRS was 74% for detecting local disease (primary tumor +/- regional lymph node metastases) in all NETs, excluding insulinoma, 75% in gastrinoma, 0% in insulinoma, 78% in other islet cell tumors, and 50% in carcinoids. For detecting distant disease, the overall sensitivity of SRS was 67% for all NETs, excluding insulinoma, 100% for gastrinoma, 50% for other islet cell tumors, and 80% for carcinoids. Specificity and positive predictive value were 100% for all tumors. Negative predictive value ranged from 33% to 100%. CONCLUSIONS: A positive SRS study strongly predicts the presence of tumor (100% positive predictive value in our study). However, unlike the European reports of very high sensitivity (80% to 88%), we found that SRS had a lower sensitivity (67% for all NETs excluding insulinoma and 71% for noninsulinoma gastroenteropancreatic NETs). Thus negative SRS in patients with NETs must be viewed cautiously, because the false-negative rate is high, and this limits the use of this method in the most difficult patients.

Cytokine production and antitumor effect of a nonreplicating, noncytopathic recombinant vaccinia virus expressing interleukin-12.

BACKGROUND: Cancer gene therapy with interleukin-12 (IL-12) has generated much interest because of the potent antitumor effects of this cytokine. The purpose of this study was to construct a nonreplicating, noncytopathic recombinant vaccinia virus (irecVV) encoding murine IL-12 (ivKT0327mIL-12) and to assess its biologic activity and antitumor effects. METHODS: ivKT0327mIL-12 was constructed by inserting the genes encoding mIL-12 into the VV genome by homologous recombination. Psoralen and long-wave ultraviolet light were used to render the virus incapable of replication or cell lysis. Cytokine production was assessed by infecting tumor cell lines and measuring mIL-12 in the culture supernatants by using a bioassay. C57BL/6 mice were injected subcutaneously in a randomized, blinded fashion with 5 x 10(5) MCA 101 fibrosarcoma cells or 5 x 10(5) PAN 02 pancreatic tumor cells infected in vitro with either ivKT0327mIL-12 or ivKT033, a control irecVV containing no cytokine genes. Emergence of tumor and tumor size were measured. RESULTS: Tumor cells infected with ivKT0327mIL-12 produced large amounts of biologically active mIL-12 (up to 793 ng/10(6) cells/24 hr) but continued to proliferate in vitro with no cytopathic effect. Mice injected with MCA 101 infected with ivKT0327mIL-12 had significantly delayed tumor emergence (p < 0.03) and decreased tumor size (p < 0.003) compared with mice injected with ivKT033-infected MCA 101. Tumor growth was also significantly smaller in a similar experiment with PAN 02 (p < 0.01). CONCLUSIONS: Tumor cells infected with irecVV encoding mIL-12 express high levels of biologically active mIL-12 in vitro and exhibit delayed tumor establishment, significant tumor growth inhibition, or both, in vivo.

In vivo murine tumor gene delivery and expression by systemic recombinant vaccinia virus encoding interleukin-1beta.

PURPOSE: This study investigates the feasibility of systemic gene delivery in a tumor-bearing host using a vaccinia virus-based in vivo gene delivery and expression system. METHODS: A recombinant vaccinia virus encoding human interleukin-1beta (hIL-1beta) was constructed with a strong synthetic vaccinia virus late promoter driving hIL-1beta gene expression. C57BL/6 mice bearing established subcutaneous pancreatic tumors were injected intravenously in a blinded, randomized fashion with different doses of either the recombinant vaccinia virus(vMJ601hIL-Ibeta), control vaccinia (wild-type or TK-deficient), or saline. Toxicity was assessed, serial tumor sizes were measured, and viral titers and the amount of hIL-1beta in tumor, liver, and spleen were determined. RESULTS: High viral titers (10(6) PFU/g) were detected in tumors for all three viruses on postinjection day 3, and tumor viral titers persisted at high levels until day 9. In contrast, viral titers were initially 104-fold lower in nontumor tissues and decreased to undetectable levels by day 9. vMJ60hIL-1beta was rapidly cleared from liver and spleen by day 3 (titer levels < 100 PFU/g), while tumor titer levels persisted at 8.5 x 10(6) PFU/g. hIL-1beta was measurable in three of three tumors from vMJ601hIL-1beta treated mice on postinjection day 3, one of three on day 6, and one of three on day 9; no hIL-1beta was detected in any other tumors or normal tissues. Wild-type vaccinia had no antitumor effects. Treatment with two different doses of vMJ601hIL-1beta resulted in a consistent and significant decrease in tumor size in repeatable experiments as compared to controls. Histologic analysis revealed tumor cell necrosis with a surrounding neutrophil infiltrate in the vMJ601hIL-1beta treated tumor. CONCLUSION: These data show that recombinant vaccinia virus encoding hIL-1beta given intravenously preferentially localizes and amplifies in tumor tissue, is rapidly cleared from liver and spleen, produces measurable hIL-1beta in tumor but not normal tissues, and inhibits growth of established solid tumors in mice. Recombinant vaccinia virus encoding therapeutic genes may be a practical, efficient vehicle for direct in vivo gene transfer and expression in the treatment of cancer.

Prevention of murine breast cancer by vaccination with tumor cells modified by cytokine-producing recombinant vaccinia viruses.

BACKGROUND: Cancer gene therapy expressing specific levels of multiple genes has not been adequately tested. This study investigates the antitumor effects of recombinant vaccinia viruses (recVVs) that express predictable levels of single and multiple cytokines in a passive immunization murine breast cancer (C3HBA) model. METHODS: Seventeen recVVs encoding different cytokine combinations under weak and strong VV promoters were constructed. Cytokine production was measured in vitro by enzyme-linked immunosorbent assay. Mice were immunized with infected C3HBA cells and rechallenged 10 days later with 10(6) parental tumor cells. Controls were treated with saline or recVVs not expressing cytokines. Tumors were measured for 24 days. Data were analyzed using Fisher's exact test and the Breslow-Gehan-Wilcoxon test. RESULTS: recVVs encoding multiple cytokines induced secretion of each cytokine at predictable levels corresponding to VV promoter strength. Ten of 10 controls developed tumors by day 11 after rechallenge. recVVs producing large but not small amounts of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) alone resulted in complete protection in all immunized mice (p = 0.0003) for 24 days and disease-free survival (DFS) was significantly prolonged versus controls (p < 0.001). GM-CSF under a weak promoter was also effective in combination with human interleukin-1 beta (hIL-1 beta; p = 0.0220; DFS, p = 0.031) or interferon-gamma (IFN-gamma; p = 0.0037; DFS, p = 0.003). Effectiveness of hIL-1 beta and IFN-gamma vaccines depended on cytokine combinations and not the amount of cytokine produced. IL-2 or tumor necrosis factor-alpha vaccines had no significant antitumor effect. CONCLUSIONS: (a) recVVs can simultaneously express controllable levels of two cytokines; (b) tumor cells modified by recVVs generate systemic antitumor immunity; and (c) strength of immunity appears to be related to the amounts and specific cytokine(s) produced.

Large cystic/solid thyroid nodules: a potential false-negative fine-needle aspiration.

BACKGROUND: False-negative fine-needle aspiration (FNA) biopsy results of thyroid nodules are of particular concern because they imply missed malignant lesions. The purpose of this study was to identify characteristics of thyroid nodules that may lead to false-negative cytologic interpretation after FNA. METHODS: We reviewed 90 consecutive patients who underwent preoperative FNA of thyroid nodules followed by thyroid surgery during a period of 27 months, including their clinical data, type and size of thyroid nodule, FNA cytology results, and final pathology report of the surgical specimen. RESULTS: Thyroid nodules that had the highest probability of malignancy were those that were large (3 cm or larger), cystic/solid, or large and cystic/solid. The overall false-negative rate for preoperative FNA was 11%. Large, cystic/solid, and thyroid nodules with both characteristics had false-negative rates of 17%, 25%, and 30%, respectively, compared with 0%, 9%, and 17% for small (less than 3 cm), solid, and solid nodules 3 cm or larger, respectively. CONCLUSIONS: Because of the high prevalence of malignancy in thyroid nodules that are large (3 cm or larger), cystic/solid, or large and cystic/solid and the high false-negative rate of FNA in diagnosing these lesions, thyroid lobectomy for diagnosis should be strongly considered in these patients even when FNA cytologic finding is interpreted as benign.

High cytokine production and effective antitumor activity of a recombinant vaccinia virus encoding murine interleukin 12.

We have constructed a recombinant vaccinia virus (recVV), vKT0334 mIL-12, containing the genes encoding the p35 and p40 subunits of murine interleukin-12 (mIL-12). In vitro experiments demonstrated that vKT0334 mIL-12 efficiently infected a variety of murine and human tumor cell lines and produced very high amounts (1.5 micrograms/10(6) cells/24 h) of biologically active mIL-12. Mice injected s.c. with 10(6) MCA 105 sarcoma cells, followed by injection at the same site with saline or a control recVV, vKT033, containing no mIL-12 genes, all developed progressively growing tumor, whereas 60% of animals injected with vKT0334 mIL-12 remained tumor free (P < 0.0005). Furthermore, tumor growth was significantly reduced in the remaining mice treated with vKT0334 mIL-12 that did develop tumor compared with mice treated with vKT033 (P < 0.03) or saline (P < 0.0001). We conclude that recVV expressing high levels of mIL-12 offers an effective in vivo method of cytokine gene delivery and expression in tumors with subsequent antitumor effect.

In vivo gene therapy of a murine pancreas tumor with recombinant vaccinia virus encoding human interleukin-1 beta.

BACKGROUND: Recombinant vaccinia virus (VV) encoding human interleukin-1 beta (vMJ601hIL-1 beta) given intravenously persists in tumor tissue and expresses hIL-1 beta for at least 9 days after treatment and is associated with significant retardation of tumor growth. To document the significance of this approach and to further elucidate the mechanism, this study compares the antitumor effect of vMJ601hIL-1 beta administered either intravenously or intratumorally and intravenous recombinant hIL-1 beta protein. METHODS: C57BL/6 mice with established subcutaneous pancreatic tumors were randomized to treatment with intravenous or intratumoral vMJ601hIL-1 beta, wild-type VV, saline solution, or intravenous recombinant hIL-1 beta protein in a blinded fashion. Toxicity and tumor size were measured. Data were analyzed with the Kruskal-Wallis and Wilcoxon tests. RESULTS: Treatment with intratumoral vMJ601hIL-1 beta repeatedly resulted in significant reduction in tumor size as compared with saline treated controls (p < 0.001). Tumor growth inhibition was consistently similar after intravenous or intratumoral vMJ601hIL-1 beta administration (p > 0.52). Wild-type VV given intratumorally or intravenously had no antitumor effect versus saline controls (p > 0.30). No significant toxicity or deaths resulted from vMJ601hIL-1 beta treatment. Recombinant hIL-1 beta protein administered intravenously caused severe toxicity (median lethal dose approximately 100 micrograms/kg), and no significant antitumor effect was observed at sublethal doses versus saline controls (p = 0.19). CONCLUSIONS: Direct, in vivo hIL-1 beta gene delivery and expression by recombinant VV given intravenously or intratumorally results in significant tumor growth inhibition, which appears to be a consequence of local, intratumoral vaccinia infection and production of hIL-1 beta.

Construction and expression in tumor cells of a recombinant vaccinia virus encoding human interleukin-1 beta.

BACKGROUND: Human interleukin-1 beta (hIL-1 beta) injected intratumorally has demonstrated growth inhibition of transplanted subcutaneous tumors in mice, regression of metastatic lesions, resistance to tumor rechallenge, and increased survival. Vaccinia virus (VV) can be genetically engineered to produce cytokines and may be an effective vector for gene therapy of cancer. This study was designed to (a) construct a VV expressing hIL-1 beta, (b) assess tumor cell infection in vitro with this construct, (c) measure hIL-1 beta production, and (d) assess the bioactivity of the secreted cytokine. METHODS: The hIL-1 beta gene was amplified from a plasmid clone using polymerase chain reaction (PCR) and then cloned into a homologous recombination (HR) and expression vector, which was used to insert the hIL-1 beta gene into the VV genome. Selection of the recombinant VV (vMJ601hIL-1 beta) was based on inactivation of viral TK and expression of beta-galactosidase. vMJ601hIL-1 beta infectivity and cytokine production was assessed by infecting tumor cell lines and analyzing culture supernatants for hIL-1 beta. Bioactivity of the hIL-1 beta produced was demonstrated using an IL-1 dependent T helper cell line. RESULTS: The hIL-1 beta gene was successfully cloned into the VV genome by HR, which was confirmed by PCR. vMJ601hIL-1 beta efficiently infected tumor cells, as shown by increased hIL-1 beta secretion (0 to > 500 ng/ml) and morphologic evidence of viral cytopathic effect. vMJ601hIL-1 beta-infected cells secreted large amounts of hIL-1 beta (mean 772 ng/10(6) cells/24 h). The secreted hIL-1 beta was bioactive (mean bioactivity 6.8 x 10(8) U/mg of hIL-1 beta). CONCLUSIONS: (a) hIL-1 beta can be cloned into VV, (b) vMJ601hIL-1 beta retains its infectivity, (c) a large amount of hIL-1 beta is secreted, and (d) the secreted hIL-1 beta is bioactive. Recombinant VV may allow in situ cytokine gene delivery and expression in established tumors.

Management of patients with Zollinger-Ellison syndrome.

Zollinger-Ellison syndrome (ZES) is caused by gastrin-secreting tumors called gastrinomas. Patients commonly present with peptic ulcer disease and may have recurrent, multiple, and atypically located ulcers, e.g. in the jejunum. Alternatively, severe diarrhea may be the only presenting symptom. Patients with multiple endocrine neoplasia Type I (MEN-I) and ZES become symptomatic at an earlier age than patients with sporadic ZES. Patients with ZES have elevated fasting serum gastrin concentrations (> 100 pg/ml) and basal gastric acid hypersecretion (> 15 mEq/h). The secretin stimulation test is the best test to distinguish ZES from other conditions resulting in elevated gastrin levels. Gastric acid hypersecretion can be controlled in virtually all patients with H2-receptor antagonists or omeprazole, thus rendering total gastrectomy unnecessary. Computed tomography (CT), magnetic resonance imaging (MRI), radionuclide octreotide scanning, endoscopic ultrasound, and the selective arterial secretin injection test are the recommended imaging studies for localization of gastrinoma; nevertheless, 50% of gastrinomas are not evident on preoperative imaging studies. All patients with sporadic gastrinoma who do not have unresectable metastatic disease should undergo exploratory laparotomy for potential curative resection. With increased awareness of duodenal tumors, gastrinoma can be found in 80-90% of patients. Surgery may be the most effective treatment for metastatic gastrinoma if most or all of the tumor can be resected. The management of patients with MEN-I and ZES remains controversial. Some clinicians advocate an aggressive surgical approach, whereas others have had little success in rendering patients eugastrinemic.

Endocrine tumors of the pancreas.

Pancreatic endocrine tumors are rare, yet can cause significant morbidity due to excessive secretion of hormones. Octreotide is effective in reducing the plasma concentrations of many of these hormones. The availability of potent H2-receptor antagonists and omeprazole has altered the emphasis in patients with Zollinger-Ellison syndrome away from total gastrectomy and towards resection of the gastrinoma for potential cure. Fifty percent of insulinomas and gastrinomas are not evident on preoperative imaging studies, despite their sophistication. Calcium angiography, endoscopic ultrasonography, isotope-labeled octreotide scanning, and injection of methylene blue during secretin angiography are recent imaging modalities that have shown promise in the localization of these tumors. Intraoperative ultrasound has emerged as the best method for operative detection of insulinomas. Duodenotomy and intraoperative endoscopic transillumination are especially important in the surgical management of Zollinger-Ellison syndrome because 30% to 40% of gastrinomas are located in the duodenum. The management of patients with multiple endocrine neoplasia type 1 and Zollinger-Ellison syndrome continues to be controversial. Some advocate an aggressive surgical approach, whereas others have had little success in rendering patients eugastrinemic.