Insulin-like growth factor-I can mediate autocrine proliferation of human small cell lung cancer cell lines in vitro.

The effect of insulin-like growth factor I (IGF-I) on growth of small cell lung cancer (SCLC) cell lines was studied. Western blot analysis of whole cell lysates of cell lines NCI-H345 and NCI-N417 demonstrated the presence of a 16-kD band consistent with an IGF-I precursor molecule. Scatchard plot analysis of cell line NCI-H345 using 125I-labeled IGF-I demonstrated two high affinity specific binding sites (Kd 1.3 and 4.0 nM with maximal rate (Bmax) 200 and 500 fmol/mg protein, respectively). The exogenous addition of IGF-I, IGF-II, or insulin resulted in marked proliferation of human SCLC cells as evaluated using an in vitro growth assay. These peptides stimulated the growth of SCLC cell lines NCI-H82, NCI-H209, NCI-H345, and NCI-N417. The concentration of IGF-I producing maximal SCLC cell growth was 10-100-fold less than that of insulin or IGF-II, whereas the maximal growth stimulated by the optimal concentration of these peptides were similar. An MAb that specifically binds to the IGF-I receptor (but not to the insulin receptor) mediates a dose-dependent inhibition of cell growth in basal media as well as IGF-I, IGF-II, or insulin-supplemented media. The IGF-I receptor thus appears to be the common pathway for the mitogenic activity by IGF-I, IGF-II, and insulin for human SCLC cell lines. The demonstration of an IGF-I precursor molecule, specific IGF-I receptor binding, IGF-I-mediated growth stimulation, and inhibition of basal cell growth by an MAb to the IGF-I receptor suggests that an IGF-I-like molecule can function in vitro as an autocrine growth factor for human SCLC cell lines.

Growth control of lung cancer by interruption of 5-lipoxygenase-mediated growth factor signaling.

Signal transduction pathways shared by different autocrine growth factors may provide an efficient approach to accomplish clinically significant control of lung cancer growth. In this study, we demonstrate that two autocrine growth factors activate 5-lipoxygenase action of the arachidonic acid metabolic pathway in lung cancer cell lines. Both growth factors increased the production of 5(S)-hydrooxyeicosa-6E,8Z,11Z,14Z-tetraeno ic acid (5-HETE), a major early 5-lipoxygenase metabolic product. Exogenously added 5-HETE stimulated lung cancer cell growth in vitro. Inhibition of 5-lipoxygenase metabolism by selective antagonists resulted in significant growth reduction for a number of lung cancer cell lines. Primary clinical specimens and lung cancer cell lines express the message for the 5-lipoxygenase enzymes responsible for the generation of active metabolites. In vivo evaluation demonstrated that interruption of 5-lipoxygenase signaling resulted in enhanced levels of programmed cell death. These findings demonstrate that 5-lipoxygenase activation is involved with growth factor-mediated growth stimulation for lung cancer cell lines. Pharmacological intervention with lipoxygenase inhibitors may be an important new clinical strategy to regulate growth factor-dependent stages of lung carcinogenesis.

Antigenic cross-reactivity between adenocarcinoma of the breast and fibrocystic disease of the breast.

Peripheral lymphocytes obtained from 12 of 13 patients who had fibrocystic disease (FCD) of the breast were specifically cytotoxic to breast adenocarcinoma cells, as measured in vitro by the microcytotoxicity test. Sera from 6 women with active FCD or metastatic breast cancer could specifically block the cytotoxicity of lymphocytes from either population of patients against cancer cells. This implied extensive antigenic cross-reactivity between benign and malignant hyperplastic disease of the breast. Sera from 4 individuals clinically free of FCD or breast fibroadenoma (FAD) neutralized the blocking activity in the sera of patients with metastatic breast cancer. Patients "cured" of FCD or FAD represented a pool of potential plasma donors for immunotherapy of recurrent breast cancer.

Preclinical evaluation of an anti-autocrine growth factor monoclonal antibody for treatment of patients with small-cell lung cancer.

We have evaluated an anti-autocrine growth factor monoclonal antibody for potential use in the treatment of patients with small-cell lung cancer. The monoclonal antibody, designated 2A11, binds to the C-terminal region of the autocrine growth factor gastrin-releasing peptide and neutralizes its growth-promoting effects in vitro and in vivo. Equilibrium-binding analysis demonstrated that the peptide binds to the antibody (dissociation constant = 1.5 x 10(-10) at least as avidly as it binds to the tumor peptide receptor. Pharmacokinetic studies in normal BALB/c mice demonstrated an initial clearance half-life (alpha t1/2) of 24.3 +/- 4 hours and a secondary clearance half-life (beta t1/2) of 1039.6 +/- 309 hours, and biodistribution studies revealed a distribution pattern which generally reflected blood flow. Single intravenous infusions of 2A11 (20 mg/20-25-kg dogs) into normal mongrel dogs with surgically created gastric fistulas antagonized the stimulatory effects of exogenously infused gastrin-releasing peptide or bombesin on plasma gastrin release and gastric acid secretion. Toxicology studies in normal dogs (with gastric fistulas) infused with 50 mg 2A11 intravenously three times a week for 4 weeks failed to reveal any adverse behavioral, clinical, or pathological effects. Four of six dogs developed an immune response to 2A11. Anti-idiotypic antibodies elicited in two cases did not mimic the functional effects of the peptide. We conclude that the concept of immunoblockade of an autocrine growth factor appears feasible in vivo.

Relationship of arachidonic acid metabolizing enzyme expression in epithelial cancer cell lines to the growth effect of selective biochemical inhibitors.

Arachidonic acid (AA) metabolizing enzymes are emerging as significant mediators of growth stimulation for epithelial cells. The relative contribution of the various family members of AA metabolizing enzymes to epithelial cancer cell growth is not known. To study this question, we first analyzed a series of epithelial cancer cells to establish the relative frequency of expression for the various enzymes. We analyzed the expression of five AA metabolizing enzymes as well as 5-lipoxygenase activating protein (FLAP) in a panel of human epithelial cancer cell lines (n = 20) using reverse transcription-PCR. From this analysis, we found that cyclooxygenase-1 (COX-1), 5-lipoxygenase (5-LOX), and FLAP were universally expressed in all cancer cell lines tested. For the remaining enzymes, the expression of COX-2, 12-LOX, and 15-LOX varied among cell lines, 60, 35, and 90%, respectively. Although the pattern of expression varied among the different cell types, all of the enzymes were expressed in all major cancer histologies. Using a panel of selective biochemical AA metabolizing enzyme inhibitors, we then evaluated the effect of these agents on cell lines with known expression status for the AA metabolizing enzymes. For the enzymes that were not universally expressed, growth inhibition by selective biochemical inhibitors did not closely correlate with the expression status of specific enzymes (P > 0.05). For the universally expressed enzymes, the LOX inhibitors were more potent growth inhibitors than the COX inhibitors. The frequent expression of the AA metabolizing enzymes suggests that AA metabolism pathway may be modulated in response to xenobiotic exposure during carcinogenesis. Although establishing a priori AA metabolizing enzyme status was not consistently informative about what AA metabolizing enzyme inhibition would be most growth inhibitory, the frequent inhibition of many epithelial cancers by these biochemical inhibitors opens a new avenue for cancer therapy and intervention in carcinogenesis.

[Psi 13,14] bombesin analogues inhibit growth of small cell lung cancerin vitro and in vivo.

Bombesin/gastrin releasing peptide (BN/GRP) functions as an autocrine growth factor in small cell lung cancer (SCLC). Previously, this autocrine growth cycle was disrupted by a monoclonal antibody which binds to the carboxyl terminal of BN and neutralizes the peptide so that it is unable to interact with the BN/GRP receptor. Here a series of BN analogues were synthesized which have a reduced peptide bond near the carboxyl terminal. The analogues inhibited specific binding of 125I-GRP to SCLC cell line NCI-H345 in a dose-dependent manner and the analogue [D-Nal6, Psi13,14, Phe14] BN6-14 was approximately 6-fold more potent than was (Psi13,14, Leu14)BN with a 50% inhibition concentration value of 5 nM. [DNal6, Psi13,14, Phe14]BN6-14 and [Psi13,14, Leu14]BN had no effect on the cytosolic Ca2+ levels but antagonized the increase in cytosolic Ca2+ caused by 10 nM BN. [Psi13,14, Leu14]BN (1 microM) inhibited the growth of SCLC in vitro using a clonogenic assay by approximately 70% Also, injection of [Psi13,14, Leu14]BN (10 micrograms, s.c.) inhibited the growth of SCLC xenografts in nude mice in vivo by approximately 50%. These data suggest that the autocrine growth cycle of BN/GRP in SCLC may also be disrupted by peptide antagonists which bind to the BN receptor.

The correct dose: pharmacologically guided end point for anti-growth factor therapy.

Strategies to block the effects of tumor growth factors, such as estrogen, and to recruit other regulatory elements, such as with retinoids, have focused interest on the possibility of successful tumor intervention approaches. Approaches that neutralize the effects of critical molecules that drive tumor promotion are attractive targets for evaluation as new intervention agents. Clinical intervention trials with early stage patients or with subjects from "high risk" populations impose stricter types of constraints than conventional chemotherapy approaches in advanced stage patients. The potential for short-term toxicity has to be considered, as it may affect subject accrual or compliance. The longer expected survival of intervention subjects mandates closer attention to the possibilities of unexpected long-term toxicities with chronic administration of an intervention agent. As part of a Phase I clinical trial evaluating the utility of a monoclonal antibody directed against the autocrine growth factor, gastrin-releasing peptide to block the growth of small cell lung cancer, we developed a mathematical model to predict the requisite amount of antibody to neutralize growth factor effect. This model requires knowledge of the equilibrium concentration of the secreted growth factor, specific receptor, and bioavailability of the antibody in the tumor interstitium. A range of possible target doses of antibody can be developed to address the potential for heterogeneity frequently encountered in such systems, including a range of levels for peptide production and specific receptor expression. This approach could be applied to rationally derive treatment or intervention in which specific information regarding the relevant binding parameters is available. Through refinement of this modeling approach more context-specific dosing of agonist/antagonists could be determined which may decrease side effects associated with the drug administration.

Expression of heterogeneous nuclear ribonucleoprotein A2/B1 in bronchial epithelium of chronic smokers.

The monoclonal antibody 703D4, which binds heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), has been reported to detect lung cancer more than a year earlier than routine chest X-ray or cytomorphology. To explore the biological basis of this detection, we studied the expression of this antigen in the central airways of smokers with evidence of bronchial metaplasia using specimens from a previously reported, randomized retinoid chemoprevention trial. By analyzing 1078 available biopsy specimens from 147 individuals at baseline and 68 individuals who completed the intervention, we frequently detected overexpression of hnRNP A2/B1 in normal and abnormal bronchial epithelium (i.e., in 41% of normal and 37% of squamous metaplasia samples). There was no correlation between hnRNP A2/B1 overexpression and the different histological changes. In cases with hnRNP A2/B1 overexpression, immunoreactivity was homogeneously expressed in all biopsied sites. For the 68 cases with serial biopsies, there was no significant modulation of hnRNP expression by retinoid intervention or smoking status. With lung cancer cell lines, 0.5-4 microM concentrations of 13-cis-retinoic acid reduced hnRNP A2/B1 overexpression by immunocytochemistry. We conclude that hnRNP A2/B1 overexpression is frequently found in central airways of chronic smokers, consistent with the pattern of expression that we reported previously in airways surrounding resected primary lung cancers. Oral 13-cis-retinoic acid at a dose of 1 mg/kg has no demonstrable effects on modulating hnRNP A2/B1 expression in proximal bronchial epithelium.

Phase I and imaging trial of a monoclonal antibody directed against gastrin-releasing peptide in patients with lung cancer.

Small cell lung cancer (SCLC) cells express and secrete bombesin-like peptides (BLP) that can activate specific receptors that stimulate the growth of these cells. A murine monoclonal antibody, 2A11, which binds to the BLP, gastrin-releasing peptide with high affinity, has been reported to decrease the growth of SCLC cells in vitro and in athymic nude mice. A Phase I trial in lung cancer patients was performed using multiple doses of 2A11. Thirteen patients with lung cancer received 12 doses of 2A11 antibody three times a week for 4 weeks at one of four dose levels. Serum samples were obtained prior to initiation and before each dose of 2A11 antibody therapy for measurement of 2A11 antibody levels and determination of serum human anti-mouse antibody levels. A pilot imaging evaluation using 111In conjugated 2A11 monoclonal antibody was also performed in the same patients to aid in the study of pharmacokinetics and biodistribution. No toxic reactions were observed, and none of the patients developed detectable human antimouse antibody; however, no objective antitumor responses were observed. The mean trough serum 2A11 levels in patients increased with increasing dose level: 0.26+/-0.2 microg/ml, 6.7+/-6 microg/ml, 71.5+/-60 microg/ml, 248+/-184 microg/ml for dose levels 1 mg/m2, 10 mg/m2, 100 mg/m2, and 250 mg/m2, respectively. At each dose level, sustained detectable serum levels of the monoclonal antibody were achieved. Tumor uptake was noted in 11 of 12 patients who were injected with 111In conjugated 2A11. Because no dose-limiting clinical toxicity was observed, a mathematical model was used to define the recommended Phase II dose of 250 mg/m2. This trial established that repeated doses of monoclonal antibody 2A11 could be given safely to patients, and sustained levels could be achieved for a 4-week schedule. Further evaluation of the antitumor effects of 2A11 is warranted.

Rational targets for the early detection of lung cancer.

The fact that routinely effective treatments for disseminated lung cancer are not available has prompted the search for effective early detection systems. It is important to identify lung cancer while it is still confined to the bronchial epithelium and is potentially curable with local modalities. We have previously reported on an immunologically based assay to identify antigens expressed on shed bronchial epithelial cells. This assay resulted in a statistically significant correlation of immunostaining with the eventual development of lung cancer 2-4 years prior to routine clinical detection. Attempts to further improve this approach require an understanding of the basis for its success. Based on the work of Hakomori and coworkers, this difucosylated Lewis X structure would be a likely marker of carcinogenic transformation of the bronchial epithelium. In fact, an antibody to this structure was useful for sputum immunocytochemistry analysis for early lung cancer detection. Other carbohydrate structures would also be reasonable markers to evaluate for early detection application, based on the known pattern of expression of these structures in fetal, dysplastic, and neoplastic lung tissue. Another antibody used for sputum immunostaining recognizes a 31-kd protein structure; the antibody is not a known member of a likely class of early detection targets. The reported cases of lung cancer missed by the immunostaining approach included principally adenocarcinoma of the lung, suggesting that the addition of a marker(s) of that type of morphologic differentiation should be considered. Markers to dissect the various forms of lung adenocarcinoma are being characterized and are available for evaluation in early detection applications.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent molecular advances in the approach to early lung cancer detection and intervention.

Lung cancer is a major contributor to overall cancer mortality. Detecting lung cancer while it is still a localized process is a long-cherished goal for improving the outcome of this disease. Recent developments suggest that we are approaching this capability. We next have to think about how to implement a change in our approach to lung cancer management to derive the benefit of better detection capability. This is an area in which our growing understanding of lung cancer biology is providing clues on improving the inhibition of cancer progression.

Antitumor activity of a monoclonal antibody directed against gastrin-releasing peptide in patients with small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) cells express and secrete gastrin-releasing peptide (GRP) which binds to receptors and stimulates growth of these cells. A murine monoclonal antibody, 2A11, which binds GRP with high affinity, decreased growth of SCLC cells in vitro and in athymic nude mice. A phase 1 trial and pharmacokinetic modeling in patients with lung cancer has defined the phase 2 dose of 2A11 but the antitumor activity in patients is unknown. METHODS: Thirteen patients with previously treated SCLC received 2A11 at 250 mg/m2 over 1 h three times per week for 4 weeks. Serum GRP, urine GRP, serum levels of 2A11, and human antimouse antibodies (HAMA) were determined. RESULTS: One of 12 (8%; 95% confidence interval, 0 to 38%) evaluable patients had complete resolution of radiographically detectable tumor lasting 4 months. Four patients (33%) had stable disease. No toxic reactions were observed. The pretreatment serum GRP level of the responding patient was 3.1 fmol/mL and the median of nine nonresponding patients was 7.3 fmol/mL (range, <1.0 to 29.0). The mean trough serum 2A11 level was 49+/-18 microg/mL in the responding patient and 32 to 487 mg/mL (median, 117) in 10 nonresponding patients. HAMA did not increase during 2A11 administration in any patient. CONCLUSIONS: Interruption of the GRP autocrine growth factor loop with 2A11 results in clinical antitumor activity in a minority of patients with previously treated SCLC. Further evaluation of the antitumor effects of 2A11 is warranted to define characteristics associated with response to 2A11.

Moving to the routine management of pre symptomatic lung cancer.

Lung cancer is the world's leading cause of cancer death. Since progress in the treatment of this cancer has been exceedingly slow, the upswing in tobacco consumption in many sectors becomes even more tragic. One area for cautious optimism is the recent pilot reports of improved early lung cancer detection using new spiral CT techniques from institutions in Japan and New York. The prospect of improved early detection in a major cancer raises a number of public health concerns and highlights the importance of critical validation of this proposed new tool. From experience with early detection-based management of other cancers, it is evident that the entire process of detection, case validation, intervention, monitoring and public education needs to be carefully developed. The International Association for the Study of Lung Cancer has worked with the National Cancer Institute over the last decade to nurture interest and expertise in conducting population-based management of early lung cancer. A distillation of this process up to the current time is reviewed in this manuscript.

Peptide amidating activity in human bronchoalveolar lavage fluid.

Monitoring respiratory epithelial biology may reveal individuals with incipient lung cancer. The expression of neuroendocrine (NE) markers in pulmonary epithelium is thought to be central to lung development, repair of injury and may contribute to carcinogenesis. In this study, we evaluate several candidate NE markers to determine the feasibility of prospective analysis of clinical specimens. The potential NE markers include the enzyme L-DOPA decarboxylase (DDC), the neuropeptide gastrin releasing peptide (GRP), and peptidyl-glycine alpha-amidating monooxygenase (PAM), the bifunctional enzyme responsible for the final bioactivation step of many neuropeptides. A comparison of PAM activity and DDC levels in 30 lung cancer cell lines indicated that peptide amidating activity may be an indicator of NE status. Bronchoalveolar lavage (BAL) fluid from subjects at risk of developing second primary lung cancer and from volunteers was obtained. The activity of the first PAM enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), ranged from not detectable to 507 pmol/h/mg protein in 57 specimens. The second PAM enzyme, peptidylamidoglycolate lyase (PAL), ranged from not detectable to 414 pmol/h/mg protein in 56 specimens. Using cluster analysis by the average linkage method, a group of enzyme values with PHM greater than 230 pmol/h/mg protein was determined. Long-term follow-up of these patients for new second primary lung cancers may help to determine the potential predictive value of PAM detected in the BAL fluid.

Antigenic cross reactivity between benign prostatic hyperplasia and adenocarcinoma of prostate.

Tissue cultures were established from biopsy specimens of adenocarcinoma of the prostate (ACP) and benign prostatic hyperplasia (BPH). Generally, peripheral blood lymphocytes from BPH and ACP patients were cytotoxic to both ACP and BPH cells, but not normal fibroblasts nor cells cultured from other types of malignant tissue. Peripheral blood lymphocytes from normal control patients or from patients with other types of cancer were not cytotoxic to ACP- or BPH-derived cells. These findings are consistent with a cross reactive autoimmune response in ACP and BPH patients, directed against a common antigen(s) present on both ACP and BPH cells.

Lung cancer: rational strategies for early detection and intervention.

The current mortality rate for lung cancer, which approaches 90% at two years, is not decreasing despite intensive clinical trials attempting to improve systemic therapy. New drug discoveries and dose intensification approaches have not resulted in more effective anti-tumor control. An alternative approach using sputum immunocytology for early lung cancer detection was recently reported. The proposed basis for this early detection reflects the underlying biology of immunodominant carbohydrate tumor-associated antigens, which are recognized as a class of oncofetal antigens. The process of bronchial epithelial carcinogenesis is associated with an evolution of carbohydrate display which reverses the sequence originally associated with fetal organ development. By elucidating this pattern of fetal carbohydrate display, a precise map of stage of bronchial carcinogenesis may emerge.