Pulmonary hilar stereotactic body radiation therapy in the rat.

Stereotactic Body Radiation therapy (SBRT) is an emerging modality of treatment for early stage non-small cell lung carcinoma. Concerns have arisen related to increased toxicities for medial tumors. We have developed a model of high dose, hypofractionated radiotherapy to the pulmonary hilum using the Leksell Gamma-Knife. Sprague-Dawley rats received hypofractionated SBRT to the unilateral lung hilum using a custom immobilization device on the Gamma Knife. Each animal was individually scanned, treatment planned, and treated with either two 4 mm or one 8 mm collimated shots at escalating doses of 20, 40, and 80 Gy to the 50% isodose volume, encompassing the right mainstem bronchus. All animals were carefully followed post-treatment and imaged by plain film and CT. In addition, histopathological analysis of all rats was performed at selected time points. Animals treated with 4 mm collimated shots demonstrated no appreciable changes on plain films or sequential, follow-up CT scans, or histopathologically. Animals irradiated with the 8 mm collimator were less active, gained weight at a reduced rate, and demonstrated histopathological changes in 7/34 animals six months post-irradiation. Cellular atypia and interstitial pneumonitis were found, three of the seven of the animals showed clear bronchial damage and two showed vascular damage. Significant volume and time effects were found. Utilizing a novel Gamma Knife based animal model to study SBRT toxicity, it was found that the bronchus will tolerate small volumes of very high dose radiotherapy. It was postulated that radiation of the surrounding support stroma and normal tissue are important in the etiology of bronchial or hilar damage.

Superior vena cava obstruction due to prostate carcinoma.

Superior vena cava obstruction (SVCO) is considered an oncologic emergency commonly associated with lung carcinoma. The case presented here is that of a 48-year-old man presenting with SVCO, which was diagnosed as metastatic prostate carcinoma localized to the chest. He was treated with goserelin and aggressive radiotherapy with a drop in his prostate-specific antigen levels and symptomatic relief that lasted approximately 12 months. SVCO recurred locally in the chest and the patient died 24 months after diagnosis. This represents a rare presentation of prostate carcinoma and underlines the necessity for tissue diagnosis before local radiotherapy.

Treatment resistant small cell carcinoma of the cervix.

BACKGROUND: Small cell undifferentiated carcinoma of the cervix is an uncommon malignancy with a poor prognosis. Treatment of localized disease has an approximate 40% 5-year survival with multimodality therapies. CASE REPORT: We describe the case of a 24-year-old woman with small cell carcinoma of the cervix that recurred locally despite intensive chemotherapy and radiotherapy. Hysterectomy was performed and the patient is now 18 months disease free. Following treatment, the pathological appearance of the tumor had changed from a typical small cell neuroendocrine malignancy to a more intermediate neuroendocrine cell type. CONCLUSION: Small cell carcinoma of the cervix is a rare aggressive malignancy that may require cytostatic multimodality therapy including chemotherapy, radiotherapy and surgery, even in early stage disease.

Optimal photon energies for IUdR K-edge radiosensitization with filtered x-ray and radioisotope sources.

The purpose of this work is to determine the most physically effective radiation energy for K-edge absorption of x- or gamma-rays by iododeoxyuridine (IUdR) on Chinese hamster ovary (CHO) cells. Brachytherapy sources (Sm-145, I-125, Yb-169 and Am-241) and x-ray beams (30 kVp, 100 kVp and 100 kVp with gold, gadolinium, lead or tungsten filtration) were investigated for their preferential absorption qualities by IUdR sensitized DNA. The 30 kVp, 100 kVp and 100 kVp with tungsten filtration were then used to irradiate CHO cells, with or without IUdR incorporation (i.e. 10(-5) M of IUdR for 3 days). Radiation absorption calculations were performed to determine the increase in energy absorption in DNA with and without IUdR incorporated. In order to measure the in vitro biological effects of K-edge absorption, cell survival experiments were performed. The radiation physics calculations yielded an iodine dose enhancement ratio (DER) of 1.4+/-0.15. 1.8+/-0.15 and 2.7+/-0.15 for the 30 kVp, 100 kVp and tungsten filtered 100 kVp respectively, for 18% IUdR replacement of thymidine in DNA. The corresponding cell sensitization enhancement ratios (SER), determined from the cell survival assay, were determined to be 1.24+/-0.2, 1.8+/-0.2 and 2.3+/-0.3 for the 30 kVp, 100 kVp and tungsten filtered 100 kVp respectively, for cells with 18+/-2% IUdR incorporation. These SER values are in reasonable agreement with the DER values of 1.4, 1.8 and 2.7. From these radiation calculations and radiobiology experiments we confirm that using x-radiation energies above the K-edge of iodine (33.2 keV) can have a significant effect on cell survival. This effect is due mainly to the increase in the local dose to the DNA for IUdR-sensitized cells compared with the normal DNA which lacks the iodine contrast agent. Our results support the clinical application of IUdR and low-energy brachytherapy, perhaps using new technologies such as the x-ray needle or new isotopes such as Yb-169.

Molecular evidence for the expression of Schwann cell markers in human neuroblastoma.

Human neuroblastoma (NB) cell lines have been suggested to represent a model of neural crest differentiation. The expression of several Schwann-cell-associated antigens was examined by flow cytometry and Northern blot analysis. Variable reactivity of the human NB cell lines was found in both the level and pattern of reactivity. Retinoic acid treatment of cell line SMS-KAN resulted in a neuron-like morphological differentiation and a decrease in several of the glial markers under study. Similarly, Northern blot analysis illustrated myelin-associated glycoprotein expression, and decreased expression of this message with retinoic acid treatment was consistent with the neuron-like morphological changes. Overall, human NB in vitro was found to be multipotential, but we have shown that it is capable of expressing several Schwann cell markers which are modulated during induced differentiation.

Induced morphological changes in human small cell lung carcinoma cells.

Several theories suggest that lung carcinomas are not totally separate entities, but are derived from a common precursor, probably of endodermal origin. The histological classification of lung cancers is complex, with much overlap between groups broadly designated as small cell (SCLC), squamous cell, adenocarcinoma and all others simply termed non-small cell. It is shown here that in vitro exposure of classic, non-adherent SCLC lines to 10 microM 5' bromodeoxyuridine (BrdU) results in a rapid cell-line dependent change to a morphology consistent with an adherent, non-small cell phenotype. Accompanying this morphological shift is a decreased expression of the amplified N-myc protooncogene. These induced changes underline the morphological relatedness of lung carcinoma cell lines.

Human melanoma-associated antigen expression on human neuroblastoma cells: effects of differentiation inducers.

We have described two human melanoma-associated antigens (HMAA), recognized by the murine monoclonal antibodies LS62 and LS109. LS62 recognizes the neuroglandular antigen (NGA), which is overexpressed in neoplastic melanocytes as well as in several tissues of neuroectodermal origin. These antibodies were used to screen six neuroblastoma cell lines and one neuroepithelioma cell line. A melanoma cell line, G361, known to express the two antigens, was used as the positive control. Variable expression of the two antigens was detected in neuroblastoma cells. The surface expression of NGA and of the LS109 antigen was modulated in parallel with the morphological differentiation induced by retinoic acid, 5-bromodeoxyuridine, or cyclic AMP analog/activators. The modulation of the expression of the two HMAA was detected in G361 melanoma cells and in one of the neuroblastoma cell lines, SK-N-SH. These results suggest altered expression of both antigens during melanoma and neuroblastoma cell differentiation in culture.

Effects of retinoic acid and bromodeoxyuridine on human melanoma-associated antigen expression in small cell lung carcinoma cells.

The dispersed neuroendocrine system includes cells with different embryological derivations, sharing a common neuroendocrine (NE) program, as indicated by the expression of NE markers, some of which are shared antigenic determinants. We report here that the small cell lung carcinoma cells NCI-H69 express the two human melanoma-associated antigens (HMAA) NGA/LS62 an LS109. Incubation of NCI-H69 cells with maturational inducers, such as retinoic acid and bromodeoxyuridine (BrdU), upregulated the expression of both HMAA. Exposure to BrdU for 4 weeks induced the appearance of a different phenotype in subpopulations of NCI-H69 cells, which became epithelioid, substrate-adherent, grew in monolayer and continued to express NE-associated antigens in variable amount. The shift in phenotype was not reversible after BrdU withdrawal and was maintained for at least 6 months in continuous culture. The substrate adhesion of NCI-H69 cells was paralleled by a change in NGA glycosylation pattern, thus suggesting a possible functional role for NGA in cell substrate adhesion/recognition.

Melanocytic differentiation of human neuroblastoma: expression of a human melanosome-associated antigen.

Five human neuroblastoma cell lines were examined for expression of a human melanosome-associated antigen (HMSA). Only cell line SK-N-SH reacted with a monoclonal antibody, HMSA-2, shown to recognize melanosomal glycoproteins. To further characterize the melanocytic lineages of SK-N-SH, three morphologically distinct clones designated SK-N-SH-N (neuroblast type), SK-N-SH-F (fibroblast type), and SK-N-SH-EP (epithelial type) were established by colony formation cloning. By fluorescence-activated cell sorter analysis and tyrosinase assay, we found that only SK-N-SH-EP and SK-N-SH-F reacted with HMSA-2 and had tyrosinase activity. These results suggest that epithelial-type and fibroblast-type cells appear to possess the melanocytic potential, but not neuroblast-type cells. Furthermore, SK-N-SH-EP was found to spontaneously convert to neuroblast-type or fibroblast-type cells, whereas SK-N-SH-N and SK-N-SH-F clones have remained morphologically stable. Our results suggest that at least one neuroblastoma cell line, SK-N-SH, may be an excellent model for investigating clonal maturation and the melanocytic differentiation of neuroblastoma.

Alterations in plasminogen activator and inhibitor activity during the differentiation of a human neuroblastoma cell line, SMS-KAN.

The fibrinolytic enzyme profile of SMS-KAN human neuroblastoma cells was found to vary dramatically during the differentiation process. Five maturational agents--retinoic acid, dibutyryl cAMP, 5-bromodeoxyuridine, sodium butyrate and phorbol myristate acetate were tested for their effects on cellular morphology, DNA synthesis, plasminogen activator (PA) and PA inhibitor (PAI) activity. SMS-KAN cells secrete urokinase (UK) and tissue PA (tPA) as well as a possibly unique PAI. Treatment of cells with 1 microM RA resulted in an inhibition of proliferation, extension of neurite-like processes indicative of differentiation, as well as a switch from secretion of UK to tPA and a reduction in PAI secretion. Other agents which caused neural process formation and decreased cell proliferation also induced alterations in PA/PAI while agents which had no detectable effect on cell growth induced little change in the fibrinolytic enzyme profile.

Effect of retinoic acid on human neuroblastoma: correlation between morphological differentiation and changes in plasminogen activator and inhibitor activity.

The relationship between plasminogen activator (PA)/plasminogen activator inhibitor (PAI) activity and morphological differentiation was investigated in human neuroblastoma (NB) cells treated with retinoic acid (RA). Conditioned medium from nine NB cell lines and one closely related neuroepithelioma line was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. All NB cell lines were shown to secrete urokinase (UK)-type PA (mol. wt., 52 kDa), and all except two produced tissue PA (mol. wt., 65 kDa). Identification of the PAs was made based on molecular weight and sensitivity to inhibition by anti-UK and anti-tPA antibodies. Several cell lines expressed PA inhibitory molecules; two molecular-weight forms were observed (35 and 40 kDa) in different cell lines. Complex formation with [125]I-labelled proteases revealed specific binding with UK and trypsin but not thrombin, plasmin, or kallikrein. After treatment for 6 days with 1 microM RA, six of the cell lines exhibited an increase in cell-associated and/or secreted tPA activity, corresponding to morphological differentiation of the cells as manifested by extensive neurite outgrowth. A decrease in UK and UK-complex secretion was observed in several of these cell lines. Three cell lines exhibiting no detectable morphological alterations with RA treatment also showed no dramatic changes in PA/PAI activity. These results suggest that morphological differentiation of NB cells may be associated with alterations in the regulation of PA activity.

Recognition of an in vivo immune response to human neuroblastoma modulation of antigen expression by retinoic acid.

Neuroblastoma is one of the most common solid tumors of childhood and is notable for its ability to spontaneously regress and, in some instances, to differentiate to less malignant ganglioneuromas. Since immune mechanisms may account for these phenomena, identification of in vivo immune responses to tumor cell surface antigens may be important to the progression of the disease. As determined by analysis on the fluorescence-activated cell sorter, sera from 10 of 18 neuroblastomas patients were found to contain antibodies to a cell surface antigen present on subpopulations of cells from human neuroblastoma cell lines maintained in vitro. Eight human neuroblastoma cell lines were examined and found to vary in reactivity with sera. Induction of differentiation of cell lines with retinoic acid (RA) in vitro resulted in most cell lines bearing higher percentages of positive cells but with a decreased mean cell fluorescence. Preliminary Western blot analysis of lysates of the human cell lines NMB/N7, SMS-KAN, and SK-N-MC showed two principal antigen bands on reducing gels. Comparison of sera from different individuals on lysates of cell lines showed reactivity principally with bands of 105-110 kD and 65-70 kD and an additional minor band of slightly lower molecular weight with the higher titer sera. The ability of different sera to recognize a common antigen pattern suggests that this represents an immunodominant cell surface antigen. Examination of reactivity of other cell lines in this system showed that positive sera reacted with all neuroblastoma lines examined, one neuroepithelioma (SK-N-MC), two melanoma lines (MeWo, G361), and one adrenal-derived adenocarcinoma (SW-13).

Expression of markers shared between human natural killer cells and neuroblastoma lines.

Neuroblastoma is a tumor of neuroectodermal origin arising most commonly from the adrenal medulla. We have examined the ability of several monoclonal antibodies which recognize markers predominantly expressed on human natural killer (NK) cells to react with neuroblastoma cell lines in vivo derived sections of tumor. HNK-1 (Leu 7) is a monoclonal IgM antibody which recognizes a carbohydrate epitope on NK cells and a wide range of tumor cell types. We have shown that HNK-1 recognizes the human neuroblastoma lines SMS-KCNR, SMS-KAN, NMB/N7, and IMR/5. Expression of this antigen on cell lines can be slightly increased by retinoic acid-induced differentiation of the cells. N901 (NKH1), a monoclonal antibody raised against interleukin 2-dependent human NK cell lines also recognizes all human neuroblastoma cell lines examined. This expression is independent of differentiation induction and levels remain unaltered following retinoic acid treatment of the cell lines. Lastly, with monoclonal antibody 49H.8, it has been found that reactivity of the lines is weak until induction of differentiation, after which highly significant increases of reactivity are seen. 49H.8 recognizes several cryptic carbohydrate antigens with varying affinities, shown to identify mouse and rat NK cells. In contrast to other NK markers, human neuroblastoma cell lines did not express significant reactivity with B73.1, Leu 11b, or Leu 18. Immunohistochemical staining of sections of human neuroblastoma tumors correlated with the in vitro findings; however, staining with N901 and 49H.8 was only seen on frozen sections, not paraffin-embedded. The significance of shared NK cell-neuroblastoma/neuron antigens is currently under investigation.

Adhesion properties of a neuronal epitope recognized by the monoclonal antibody HNK-1.

A carbohydrate epitope on adhesion proteins of the developing nervous system, and on myelin-associated glycoprotein, is recognized by the monoclonal antibody HNK-1. The HNK-1 epitope bearing proteins and the monoclonal antibody alter, in a dose-dependent manner, the interaction between neurons and neurite-promoting substrate-attached materials released from cultured neural cells.

The characterization and cellular distribution of a family of antigens related to myelin associated glycoprotein in the developing nervous system.

The antigenic epitope detected on myelin associated glycoprotein (MAG) by the monoclonal antibody HNK-1 (Leu 7) was sensitive to degradation by trifluoromethane-sulfonic acid (TFMS) and is therefore probably carbohydrate in nature. This antigen was found to be widely distributed within the rat and chicken embryonic nervous system and was present on cultured central and peripheral neurons (100%), oligodendrocytes (100%) and astrocytes (70-80%) as detected by double marker immunofluorescence. The antigen could be removed from cultured neurons by trypsinization and its resynthesis was blocked by cycloheximide, suggesting that the carbohydrate epitope detected by HNK-1 was attached to a de novo synthesized protein. Several molecular species were detected on Western blots of detergent extracts from 13-15d rat embryonic brain and neuron-enriched cultures from chick spinal cord and dorsal root ganglia. Protein components with molecular weights in the ranges of 90-100 kd to 280 kd were observed and comprise a family of glycoproteins containing the HNK-1 reactive carbohydrate epitope present on MAG. These glycoproteins could play a role in intercellular interactions within the developing nervous system.

Differential expression of the Leu-7 antigen on human lung tumor cells.

The HNK-1 monoclonal antibody detects an antigen (Leu-7) on a subpopulation of large granular lymphocytes which have natural killer cell function. Recently this antigen has been found on nonhemopoietic tissues. In the present study human lung tumor cells were examined for the presence of Leu-7 antigen using an enzyme-linked immunosorbent assay, immunoperoxidase staining, and fluorescence-activated cell sorter analysis. All small cell lung tumor cells tested were Leu-7 positive. In contrast only two of seven biopsy specimens from small cell lung cancer patients were Leu-7 positive. Several large cell lung tumor lines were Leu-7 positive while an adenocarcinoma and squamous cell carcinoma were negative. These results indicate that expression of Leu-7 antigen on lung tumor cells is heterogeneous both in vitro and in vivo. Small cell lung tumor lines have been reported to undergo histological conversion in vitro accompanied by the loss of a number of biochemical markers. In our study histologically converted cells exhibited much less reactivity with HNK-1 than did the parent cells. These results indicate that the degree of expression of Leu-7 antigen may be under the control of differentiation-related events. Thus monoclonal antibody HNK-1 has been very useful in studying heterogeneity within and among lung tumor cells.

Accelerated regenerative neurite formation by a neuronal surface epitope reactive with the monoclonal antibody, Leu 7.

A family of glycoproteins sharing an epitope with myelin associated glycoprotein as recognized by the monoclonal antibody Leu 7 (HNK-1) has been found to be present on neurons grown in culture from embryonic chicks and rats. Immunofluorescent staining demonstrates that, in vitro, 100% of the neurons from dorsal root ganglia and spinal cord from 7-8 day chick embryos react with Leu 7. Analysis of in vitro regenerative neurite formation by neurons on substrates enriched with Leu 7 showed accelerated regenerative process formation under limiting conditions. These results indicate that the Leu 7 epitope on neurons is appropriate for substrate adhesion and promotes rapid process extension.

The cooperative effect of the satin and beige mutations in the suppression of NK and CTL activities in mice.

The functional activity of natural killer (NK) cells has been found to be modulated by several point mutations associated with coat color. The most commonly studied gene, beige (Bg), has been found to block a postrecognition event in the lytic cycle. Four other coat color mutations in the mouse (satin, leaden, fuzzy, pale ears) were studied for their effect on NK cell function, and only one, satin (Sa), was found to be suppressive. When both the Sa and Bg mutations were present in the same animal, their effects were synergistic in the suppression of NK levels. Normal numbers of NK cells were present in these double mutants, as determined by the frequency of IgG2b binding cells and by antiasialo GM1 staining. The ability of Sa/Bg NK cells to recognize and bind targets suggests that the defect is localized in the postbinding cytolytic pathway. These genes were not specific for NK cells and also suppressed alloimmune cytolytic T lymphocyte function. Since Sa/Bg mice are much more suppressed in NK function than Bg mice, we suggest that this double mutant may be a better model for NK deficiency in vivo.