Frequent requirement of hedgehog signaling in non-small cell lung carcinoma.

Although it had previously been suggested that the hedgehog (HH) pathway might be activated in some lung tumors, the dependence of non-small cell lung carcinomas (NSCLC) for HH activity had not been comprehensively studied. During a screen of a panel of 60 human tumor cell lines with an HH antagonist, we observed that the proliferation of a subset of NSCLC cell lines was inhibited. These NSCLC cell lines express HH, as well as key HH target genes, consistent with them being activated through an autocrine mechanism. Interestingly, we also identified a number of NSCLC cell lines that express high levels of the downstream transcription factor GLI1 and harbor enhanced levels of HH activity, but appear insensitive to known HH antagonists. We hypothesized that the high levels of GLI1 in these cells would function downstream of the HH antagonist target, allowing them to bypass the antagonist-mediated block in proliferation. Consistent with this hypothesis, when the levels of GLI1 are knocked down in such cells, they become sensitive to these inhibitors. We go on to show that a large percentage of primary NSCLC samples express GLI1, consistent with constitutive activation of the HH pathway in these samples. Taken together, these results establish the involvement of the HH signaling pathway in a subset of NSCLCs.

Bispecific Antibody MDX-210 for Treatment of Advanced Ovarian and Breast Cancer.

A large number of monoclonal antibodies (MAbs) to various tumor cell lines have been developed (1). However, MAbs have thus far had limited therapeutic impact in oncology, probably in part because many murine MAbs do not effectively recruit immune effector mechanisms, such as complement fixation and antibody-dependent cell-mediated cytotoxicity (ADCC) in humans. Additionally, although humanized MAbs are being developed, when used therapeutically their immunological effectiveness may be limited by high concentrations of nonspecific immunoglobulin (Ig) in patient serum. These nonspecific Ig will compete with conventional MAbs for binding to Type I Fc receptors (FcγRI) on immune effector cells, and may therefore limit conventional MAbs ability to recruit an immune response. Recently, however, clinical efficacy of a humanized MAb directed against HER-2/neu in patients with advanced breast cancer has been demonstrated (2-4). Preclinical data suggests that mechanistically this activity may be as a consequence of modulation of important biologic properties of the HER-2/neu receptor itself, as opposed to through an immunologic mechanism of tumor cell destruction.

The "Spot 14" gene resides on the telomeric end of the 11q13 amplicon and is expressed in lipogenic breast cancers: implications for control of tumor metabolism.

Enhanced long chain fatty acid synthesis may occur in breast cancer, where it is necessary for tumor growth and predicts a poor prognosis. "Spot 14" (S14) is a carbohydrate- and thyroid hormone-inducible nuclear protein specific to liver, adipose, and lactating mammary tissues that functions to activate genes encoding the enzymes of fatty acid synthesis. Amplification of chromosome region 11q13, where the S14 gene (THRSP) resides, also predicts a poor prognosis in breast tumors. We localized the S14 gene between markers D11S906 and D11S937, at the telomeric end of the amplified region at 11q13, and found that it was amplified and expressed in breast cancer-derived cell lines. Moreover, concordant expression of S14 and a key lipogenic enzyme (acetyl-CoA carboxylase) in a panel of primary breast cancer specimens strongly supported a role for S14 as a determinant of tumor lipid metabolism. S14 expression provides a pathophysiological link between two prognostic indicators in breast cancer: enhanced lipogenesis and 11q13 amplification.

Evaluation of p53 mutation in pancreatic acinar cell carcinomas of humans and transgenic mice.

Mutation of the p53 tumor suppressor gene is found in a large number of exocrine pancreatic tumors. The majority of these tumors is of the ductal cell phenotype. We examined 12 human acinar cell carcinomas and 42 transgenic mouse carcinomas (including 36 acinar cell tumors, four islet cell tumors, and two liver metastases of primary acinar cell tumors) for evidence of p53 mutation. Immunohistochemistry was used to identify p53 protein in tumor sections. To evaluate p53 exons 5-8, heteroduplex analysis was used on formalin-fixed, paraffin-embedded human tumor DNA, and single-strand conformation polymorphism analysis was used on frozen mouse tumor DNA. No molecular evidence of p53 mutation was found in any of the tumor DNAs and immunohistochemical data were regarded as negative. This study provides evidence that acinar cell carcinogenesis in both humans and transgenic mice is independent of p53 mutation.

Fibrillin-1 in human cartilage: developmental expression and formation of special banded fibers.

The molecular basis for Marfan's syndrome (MS), a heritable disorder of connective tissue, is now known to reside in mutations in FBN1, the gene for fibrillin-1. Classic phenotypic manifestations of MS include several skeletal abnormalities associated primarily with overgrowth of long bones. As a first step towards understanding how mutations in FBN1 result in skeletal abnormalities, the developmental expression of fibrillin-1 (Fib-1) in human skeletal tissues is documented using immunohistochemistry and monoclonal antibodies demonstrated here to be specific for Fib-1. At around 10-11 weeks of fetal gestation, Fib-1 is limited in tissue distribution to the loose connective tissue surrounding skeletal muscle and tendon in developing limbs. By 16 weeks, Fib-1 is widely expressed in developing limbs and digits, especially in the perichondrium, but it is apparently absent within cartilage matrix. Fib-1 appears as a loose meshwork of fibers within cartilage matrix by 20 weeks of fetal gestation. Until early adolescence, Fib-1 forms loose bundles of microfibrils within cartilage. However, by late adolescence, broad banded fibers composed of Fib-1 are found accumulated pericellularly within cartilage. Because these fibers can be extracted from cartilage using dissociative conditions, we postulate that they are laterally packed and crosslinked microfibrils. On the basis of these findings, we suggest that the growth-regulating function of Fib-1 may reside persistently within the perichondrium. In addition, the accumulation of special laterally crosslinked Fib-1 microfibrils around chondrocytes during late adolescence suggests that growth-regulating activities may also be performed by Fib-1 at these sites.

CD3+ CD8+ CTL activity within the human female reproductive tract: influence of stage of the menstrual cycle and menopause.

The human female reproductive tract (RT) has been analyzed by others with respect to NK cell cytolytic activity, but not CD3+ T cell (CTL) cytolytic activity. Here, we describe the cytolytic capacity of mucosal CD3+ T cells both longitudinally within the RT (Fallopian tube, uterine endometrium, endocervix, ectocervix, and vaginal mucosa) and temporally throughout the menstrual cycle, using a redirected lysis assay system. Cytolysis by CD3+ CD8+ T cells is found throughout the RT and appears to be hormonally regulated, since in the uterine endometrium, the capacity for CD3+ T cell cytolytic activity is present during the proliferative phase of the menstrual cycle and absent during the subsequent secretory (postovulatory) phase. In contrast, in postmenopausal women the entire RT, including the uterus, retains the capacity for strong CD3+ T cell cytolytic activity. These findings suggest that the high levels of estradiol and progesterone present during days 14 to 28 of the menstrual cycle down-regulate CTL activity in the uterus. As a consequence, the absence of this activity may allow implantation of a semiallogeneic embryo that would otherwise be rejected. Further, these studies indicate that CTL activity is regulated differentially in different regions of the RT, persisting in the cervix and vagina throughout the menstrual cycle.

Well-differentiated pulmonary neuroendocrine carcinoma metastatic to the endometrium: a case report.

The female genital tract is an infrequent site of metastasis, particularly for extragenital primaries. The ovary and vagina are the sites within the female genital tract that are the most frequently affected. The uterine corpus, especially the endometrium, is a distinctly unusual site of involvement. Primary lung cancer is the source of metastatic tumor to the female genital tract in less than 5% of patients. In the reported instances of endometrial involvement by a primary lung cancer, adenocarcinoma has been the reported subtype. Here, we report a case of well-differentiated neuroendocrine carcinoma of the lung metastatic to the endometrium in a 68-year-old woman with postmenopausal bleeding. Immunohistochemical studies were performed to confirm the neuroendocrine nature of the neoplasm.

The cytokine microenvironment of human colon carcinoma. Lymphocyte expression of tumor necrosis factor-alpha and interleukin-4 predicts improved survival.

BACKGROUND: The functional significance of cytokines expressed in situ by tumor cells and tumor infiltrating lymphocytes (TIL) in human colon carcinomas is largely unknown. METHODS: We assessed TIL expression of interleukin-2 (IL-2), IL-4, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), granulocyte-macrophage colony stimulating factor (GM-CSF), IL-10, and transforming growth factor-beta (TGF-beta), and tumor cell expression of IL-10 and TGF-beta in situ in 49 primary colon carcinomas and 20 metastases using immunohistochemistry. RESULTS: The percentage of primary colon carcinoma samples in which > 20% of TIL expressed each cytokine was as follows: IL-4: 47%; TNF-alpha: 22%; TGF-beta: 10%; IFN-gamma: 6%; IL-2:2%; IL-10: 0%; and GM-CSF: 0%. Lymphocytes more commonly infiltrated colon carcinoma primaries than metastases, and TIL expression of IL-4 and TNF-alpha was more common in primary than metastastatic carcinomas. Expression of TNF-alpha by even a small proportion (> or = 3%) of the TIL in a colon carcinoma specimen was associated with better overall survival (P = 0.01) when compared with patients with little or no TIL TNF-alpha expression (5-year survival 82% vs. 47%). Expression of IL-4 by > or = 20% of colon carcinoma TIL was also associated with improved survival (P = 0.01; 5-year survival 87% vs. 50%). The expression of IL-10 or TGF-beta by colon carcinoma TIL or colon tumor cells themselves was not associated with impaired survival. Benign epithelial cells stained positively for IL-10 and TGF-beta more frequently than tumor cells (P < 0.001). CONCLUSIONS: There are differences between the immune microenvironment of primary tumors and metastases. Although IL-10 is expressed by colon carcinoma cells and TIL, it is unlikely that it plays an important immunosuppressive role. TNF-alpha and IL-4 are commonly expressed by colon carcinoma TIL and both are associated with improved survival.

In situ cytokine production by breast cancer tumor-infiltrating lymphocytes.

BACKGROUND: Human breast cancers progressively grow despite the presence of extensive lymphocytic infiltration and specific antitumor immune recognition, thereby calling into question the competency of breast tumor-infiltrating lymphocytes (TIL). The function of breast TILs in vivo and their possible role in the suppression of an antitumor immune response are largely unknown. METHODS: The cytokines produced in situ by lymphocytes in 89 breast carcinomas and 14 benign breast lesions were assessed using immunohistochemistry. RESULTS: The majority of tumor and benign breast samples contained T-cell infiltrates, which were disclosed using an anti-CD3 antibody stain. The percentage of tumor samples in which > or =3% of the lymphocytes were producing cytokines was as follows: interleukin (IL)-2 45%, IL-4 36%, tumor necrosis factor-alpha (TNF-alpha) 28%, transforming growth factor-beta 1 (TGF-beta 1) 20%, IL-10 11%, interferon-gamma (IFN-gamma) 4%, and granulocyte-macrophage colony-stimulating factor (GM-CSF) 3%. Production of IL-2, IL-4, and TGF-beta 1 by TILs in breast cancers exceeded that detected in benign breast lesions (p < 0.005). Significantly more tumor samples contained lymphocytes producing IL-2, IL-4, TGF-beta 1, and TNF-alpha than IFN-gamma and GM-CSF (p < 0.002 for each comparison). One or more of the potentially immunoinhibitory cytokines-IL-4, IL-10, or TGF-beta 1-were produced by lymphocytes in 44% of the specimens. No significant associations were seen between lymphocyte production of a particular cytokine and disease-free survival (median follow-up 43 months). CONCLUSIONS: Immunohistochemical techniques can be used to detect cytokine secretion by TILs in preserved tissue. The relative lack of secretion of IFN-gamma and GM-CSF, rather than a deficiency of IL-2, may explain why the antitumor immune response to breast cancer is impaired.

Cellular localization of enzymatically active thrombin in intact human tissues by hirudin binding.

Cellular sites of coagulation activation within complex, intact tissues have been studied by immunohistochemical techniques. Hirudin, a specific and high affinity inhibitor of the active site of thrombin, together with antibody to hirudin were applied to sections of AMeX-fixed specimens of normal lung, kidney, placenta, freshly incised skin and unperturbed skin obtained at fresh autopsy; to rheumatoid synovial tissue; and to malignant tissue from a variety of tumor types. Staining for thrombin was observed selectively on pulmonary alveolar, rheumatoid synovial, and placental macrophages that express an intact extrinsic coagulation pathway. Staining was also observed restricted to the endothelium of capillaries in freshly incised skin but not in either unperturbed skin or in aged incisions. Staining of tumor cell bodies was observed in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma tissues that we found previously to show tumor cell-associated procoagulant activity. This staining occurred commonly on cells within the tumor mass that were distant from stromal fibrinogen/fibrin. By contrast, tumor-associated macrophage but not tumor cell staining was seen in adenocarcinoma and squamous cell carcinoma of the lung, and little or no staining was seen colon cancer tissue. Negative controls in which either the hirudin probe or its antibody were omitted failed to show staining. These results are in accord with previous findings and suggest that such techniques may be useful for studying the cellular sites of thrombin generation in intact tissues. We postulate that administration of potent and specific thrombin antagonists, such as hirudin, to patients with relevant tumor types might be followed by homing of hirudin to tumor cells in vivo so that effects of local thrombin generation on malignant progression can be determined.

Studies of possible mechanisms for the effect of urokinase therapy in small cell carcinoma of the lung.

Urokinase-type plasminogen activator has been administered by other investigators to patients with small cell carcinoma of the lung (SCCL) in an attempt to induce lysis of fibrin that is known to exist in the connective tissue stroma of this tumour type and that may support tumour growth. To study the fate of infused urokinase in this disease, a biopsy of a scalp metastasis was obtained from a patient with SCCL (entered on a phase I clinical trial of urokinase plus combination chemotherapy) immediately following urokinase infusion during the fourth course of therapy a time when this tumour mass had decreased to approximately 25% of its original size. Immunohistochemical procedures revealed abundant stromal fibrin in accord with previous observations from this laboratory. By contrast, urokinase, that is not a feature of small cell tumour cells, was present on the tumour cells in this specimen. Urokinase infusion was associated with a rapid increase in the amount of this enzyme associated with isolated peripheral blood monocytes. These results are consistent with uptake of infused urokinase onto monocytes and possibly tumour cells. It is postulated that substantial tumour fibrinolysis may not accompany such therapy and that urokinase, or its amino terminal fragment that bears the growth factor domain of this molecule, may bind to and alter the growth of the tumour cells.

Factors regulating the production of vasopressin-associated human neurophysin by small-cell carcinoma of the lung: evaluation by computer-enhanced quantitative immunocytochemistry.

Expression of the vasopressin gene appears to be a property common to all small-cell lung tumours. For some cultures of small-cell lung carcinoma (SCCL), Northern and Western Blot analyses have revealed that expression of this gene and its protein products are regulated by cAMP and glucocorticoids. In this study, these evaluations have been extended by examining the production of vasopressin-associated human neurophysin (VP-HNP) by computer-enhanced quantitative immunocytochemistry in a classical cell-line (H69) of SCCL, and defining the amount of protein in cells by area of positive staining above an arbitrarily set threshold. Intracellular cAMP was raised by incubating cells with either 8,Br-cAMP (0.5 mM) and IBMX (0.5 mM), or with forskolin (25 microM) and IBMX (0.5 mM). Both of these treatments caused a significant increase in the amount of positive VP-HNP immunoreactivity in the cells, an increase that was further enhanced by simultaneous administration of dexamethasone (0.1 microM). Addition of dexamethasone alone, however, caused a significant decrease in VP-HNP levels. Results confirm earlier findings from Western Blot analysis revealing the influence these agents have on production of vasopressin gene-related proteins by H69 cells, and indicate that computer-enhanced quantitative immunocytochemistry can be effectively used to provide a suitable index of this production.

Sex hormone receptors in non-small-cell lung cancer in human beings.

To investigate whether sex hormone receptors exist in the resected non-small-cell lung cancer in human beings and to determine a link between the pulmonary carcinogenesis and the sex receptor status of the lung cancer tissue, we reviewed the case histories of 64 patients who underwent resectional therapy for non-small-cell lung cancer between 1988 and 1990 (38 men and 26 women, mean age 65 years). Mouse monoclonal immunoglobulin G antibodies were used for immunohistochemical detection of estrogen receptors and progesterone receptors in the acetone-fixed specimen. The control group consisted of normal lung tissue from the patients with and without bronchogenic carcinoma and breast cancer tissue from the patients with estrogen and progesterone receptor immunoreactivity. No evidence of estrogen and progesterone receptor immunoreactivity was present in the normal lung tissue. All but two patients had immunoreactivity (97%) for estrogen receptors in the lung cancer tissue (p < 0.001). The differences for sex and for histologic subtypes were not statistically significant. Observed actuarial survival at 3 years was 83% for all patients with estrogen receptor immunoreactivity: 94% for women and 75% for men (p < 0.05). We found no correlation between the hormone receptor status and the type, clinical features, or prognosis of the non-small-cell lung cancer. We conclude that an abundance of estrogen receptors is hosted only in cancerous tissue, not in normal pulmonary tissue. Improved identification and definition of estrogen receptors in the nontarget lung cancer tissue offer a possibility of antiestrogen therapy for patients with advanced bronchogenic carcinoma.

Germline RET mutations in MEN 2A and FMTC and their detection by simple DNA diagnostic tests.

Multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC) are two closely related cancer syndromes inherited in an autosomal dominant manner. Mutations in the RET proto-oncogene were found in MEN 2A and FMTC families. In this study we report seven different germline mutations in the RET proto-oncogene in five of five MEN 2A and five of six FMTC families. Each of the mutations involves a cysteine residue in the extracellular cysteine-rich domain of the RET receptor tyrosine kinase. We developed simple polymerase chain reaction based diagnostic tests for all seven mutations in these families.

Products of vasopressin gene expression in small-cell carcinoma of the lung.

Small-cell neuroendocrine carcinoma of the lung is known to express products related to the vasopressin gene, although these products have been reported to sometimes differ from those generated by neurones of the hypothalamo-neurohypophyseal system. To further investigate vasopressin gene expression in neuroendocrine carcinomas, we performed immunohistochemistry on 24 histologically classified small-cell carcinomas using antibodies directed against different regions of the vasopressin precursor. All of the tumours examined contained at least two parts of the vasopressin precursor, suggesting that vasopressin might have a biological role in these tumours and indicating a role for these products in tumour diagnosis and treatment. Sixty-seven per cent of the tumours contained immunoreactivity for all major regions of the precursor: vasopressin, vasopressin-associated human neurophysin, the bridging region between the hormone and the neurophysin, and vasopressin-associated human glycopeptide. However, 33% of the tumours examined appeared to express only part of the vasopressin precursor, as evidenced by the absence of immunoreactivity for the neurophysin and/or the glycopeptide. These results support the proposition that both normal and abnormal vasopressin gene expression occurs in small-cell carcinoma of the lung.

Cell lines derived from pancreatic tumors of Tg(Ela-1-SV40E)Bri18 transgenic mice express somatostatin and T antigen.

Continuous cell lines have been isolated from islet cell, small cell anaplastic and acinar cell carcinomas arising in the pancreas of transgenic mice, line Tg(Ela-1-SV40E)Bri18. These mice carry the pseudogene construct composed of elastase-1 promoter linked to the SV40 T antigen. Cells derived from islet cell or small cell anaplastic tumors secreted insulin and somatostatin during the early period of culture. Phenotypic alterations occurred during culture, whereby insulin secretion ceased and cells instead secreted somatostatin, indicating a change from beta-cell to delta-cell phenotype. Acinar cell lines did not secrete amylase or lipase.

Soluble normal and mutated DNA sequences from single-copy genes in human blood.

Healthy individuals have soluble (extracellular) DNA in their blood, and increased amounts are present in cancer patients. Here we report the detection of specific sequences of the cystic fibrosis and K-ras genes in plasma DNA from normal donors by amplification with the polymerase chain reaction. In addition, mutated K-ras sequences are identified by polymerase chain reaction utilizing allele-specific primers in the plasma or serum from three patients with pancreatic carcinoma that contain mutated K-ras genes. The mutations are confirmed by direct sequencing. These results indicate that sequences of single-copy genes can be identified in normal plasma and that the sequences of mutated oncogenes can be detected and identified with allele-specific amplification by polymerase chain reaction in plasma or serum from patients with malignant tumors containing identical mutated genes. Mutated oncogenes in plasma and serum may represent tumor markers that could be useful for diagnosis, determining response to treatment, and predicting prognosis.

Vasopressin and oxytocin production by non-neuroendocrine lung carcinomas: an apparent low incidence of gene expression.

In previous studies we have demonstrated the high incidence of vasopressin gene expression as a characteristic feature of small-cell carcinoma of the lung. In the present study we examined expression of this gene in non-neuroendocrine tumors to determine if vasopressin production is a common feature of all lung tumors. We carried out the immunohistochemical evaluation of 22 non-neuroendocrine tumors (12 adenocarcinomas and 10 squamous-cell carcinomas) with antibodies to vasopressin, to oxytocin, and to their related neurophysins. The antibody preparations directed against vasopressin, oxytocin, or oxytocin-associated human neurophysin did not react with any of the tumors examined. Of two monoclonal antibodies to vasopressin-associated human neurophysin used, one did not react with any of the tumors, while the other stained neoplastic cells in only one adenocarcinoma and one squamous-cell carcinoma. These findings, taken with previous reports, indicate that among lung carcinomas, a high incidence of vasopressin/oxytocin gene expression is confined to neuroendocrine tumors.