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Lung cancer is the leading cause of cancer mortality for women and men in the United States. Given this, there is a need to find new targets to combat lung cancer. This study explores the E1-like ubiquitin-activating enzyme (UBE1L) that associates with the interferon-stimulated gene 15 (ISG15), which complexes with cyclin D1 and other specific proteins. The ubiquitin protease UBP43 removes ISG15 from conjugated proteins. The UBE1L-ISG15-UBP43 pathway was previously proposed to inhibit lung carcinogenesis by repressing cyclin D1 expression. Our prior work is extended here by reporting that UBP43 is enzymatically active in cells. Also, gain of UBP43 expression specifically stabilizes cyclin D1 while UBP43 knock-down destabilizes cyclin D1, but not cyclin E or other D-type cyclins. This occurs by regulating ISG15 complexes with cyclin D1. UBP43 effects on cyclin D1 were not antagonized by cycloheximide treatment. Whether the deconjugase UBP43 was a lung cancer target was independently addressed through engineered gain and loss of UBP43 expression in lung cancer cells. UBP43 knock-down triggered apoptosis in these cells. In contrast, UBP43 over-expression promoted lung cancer cell growth by inhibiting apoptosis. That cyclin D1 plays a key role in conferring these effects was shown by engineered loss of UBP43 along with forced cyclin D1 expression. Cyclin D1 antagonized effects of loss of UBP43. Engineered UBP43 knock-down in lung cancer cells significantly increased apoptosis (P < 0.05), reduced growth (P < 0.05) and inhibited lung cancer formation (P < 0.05) in FVB mice injected via tail veins with syngeneic lung cancer cells. In marked contrast, forced UBP43 over-expression in lung cancer cells antagonized the effects of interferon, cisplatin and all-trans-retinoic acid, indicating that UBP43 can regulate response to anti-neoplastic agents. To ascertain the clinical impact of this pathway, a paired normal-malignant human lung tissue array from 74 cases was examined for immunohistochemical expression profiles of UBP43 and cyclin D1 proteins. Notably, UBP43 was significantly increased in the malignant as compared to the adjacent normal lung tissues (P = 0.04 for adenocarcinoma and P = 0.02 for squamous cell carcinoma). Intriguingly, a direct relationship was found between UBP43 and cyclin D1, validating clinical relevance. Thus, UBP43 knock-down appears to exert its anti-neoplastic effects by destabilizing cyclin D1. Taken together, these findings establish that the deconjugase UBP43 is a tractable target for lung cancer therapy and prevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1655. doi:10.1158/1538-7445.AM2011-1655

Lung cancer is the most common cause of cancer mortality in the United States. New treatments for lung cancer are therefore needed. We previously engineered murine transgenic cyclin E models that recapitulated frequent features of lung cancers in patients. These included chromosome instability, hedgehog pathway and cyclin D1 activation as well as single or multiple pre-malignant and malignant (adenocarcinoma) lung lesions. Metastases were also observed. Cell lines (ED-1 and ED-2) were derived from these murine lung cancers. After tail vein injection of ED-1 or ED-2 cells into syngeneic FVB mice, neoplastic lung lesions form within 1-2 weeks. These models are useful tools to identify promising anti-neoplastic agents. Among a panel of drugs that caused growth inhibition of lung cancer cells and repression of cyclin D1 protein, the histone deacetylase inhibitor (Vorinostat) was found as most potent. Vorinostat treatment caused dose- (1-5μM) and time-dependent (2 and 4 days) growth inhibition and apoptosis induction in murine lung cancer cells. Effects were reversed by washouts of Vorinostat. Similar reversible Vorinostat effects on growth inhibition and apoptosis were observed in human lung cancer cell lines (HOP62, H522 and H23). Vorinostat treatments repressed cyclin D1 and cyclin E proteins, but increased p27 expression in ED-1 cells. Notably, Vorinostat treatments of cyclin E transgenic mice also substantially repressed cyclin D1 expression in pre-malignant and malignant lung tissues. Vorinostat treatment also significantly reduced lung tumor formation (p = 0.047) in FVB mice harboring transplanted syngeneic murine lung cancer cells. To translate studies into the clinic, an Institutional Review Board-approved proof of principle Vorinostat trial was launched for early stage non-small cell lung cancer cases undergoing surgical resection. Patients receive Vorinostat treatments pre-operatively. Post- versus pre-treatment biopsies will be scored for changes in immunohistochemical profiles of cyclin D1, cyclin E, p27, and ki-67. Trial accrual ends January 2011. Results will then be analyzed with findings reported at this meeting. Taken together, Vorinostat treatments repressed lung cancer cell growth by augmenting apoptosis and reducing cyclin D1 expression. Substantial anti-neoplastic effects of Vorinostat were seen in murine transgenic and transplantable lung cancer models. These studies underscore the value of clinically-relevant murine lung cancer models for guiding clinical trials in lung cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 342. doi:10.1158/1538-7445.AM2011-342

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.

William G North1, Guohong Gao1, Amy Jensen1, Vincent A Memoli2, Jinlin Du11Departments of Physiology and 2Pathology, Dartmouth Medical School, Lebanon, NH, USAAbstract: We previously showed that functional N-methyl-D-aspartate (NMDA) receptors are expressed by human neuroblastoma cells. In this study we demonstrate functional NMDAR1 and NMDAR2 receptors are expressed by small-cell lung cancer (SCLC) classical cell lines NCI H146, NCI H345, and DMS 53, by variant cell line NCI H82, and by most SCLC tumors, and that these receptors are important for the growth of human SCLC tumor xenografts in mice. Reverse transcription-polymerase chain reaction demonstrated mRNA for both receptors, with sequences identical to those for human mRNAs, are expressed in all four cell lines, and these generated proteins of the expected sizes 120 and 170 kDa. Cell viability tests showed cell growth was significantly (P < 0.0001) impaired by NMDAR1 antagonists MK-801 and memantine. Ifenprodil and Ro25-6981, NMDAR2B antagonists at the polyamine site, also significantly (P < 0.001) inhibited the growth/survival of these cells. Alternatively, the glycine-binding antagonist, L701, 324, increased viability to 140% and 120% in NCI H345 and NCI H82 cells after 48 hours of incubation. Immunohistochemistry of SCLC tumors with our polyclonal antibodies gave specific positive staining for the NMDAR1 receptor in 8 of 10 tissues examined. Small amounts of these same antibodies significantly reduced the growth of NCI-H345 cells up to 25% (P < 0.001). When NCI H345 cells were grown as tumor xenografts in mice, the growth of these tumors was reduced by 60% (P < 0.001) by treatments with MK-801 over five days. All of these data point to active NMDAR receptors possibly having an important influence on SCLC growth and survival.Keywords: NMDA, receptors, small-cell lung cancer, inhibition, treatment

MicroRNAs (miRNAs) encode small RNAs that regulate gene expression. The expression profiles of miRNAs have proven useful to improve classification, diagnostic and prognostic information for human cancers. We sought to uncover miRNAs preferentially over-expressed in lung cancers versus adjacent normal lung tissues. We comprehensively interrogated miRNA array expression profiles conducted on lung cancers versus adjacent normal lung tissues isolated from murine transgenic cyclin E models that were engineered. Notably, miR-136, miR-376a and miR-31 were each significantly over-expressed in murine lung cancers as compared to adjacent normal lung tissues. Real-time RT-PCR and in situ hybridization assays independently confirmed these miRNA expression profiles in paired normal-malignant lung tissues from transgenic mice as well as from a well characterized panel of paired human normal-malignant lung tissues. Knock-down of miR-31, but not other highlighted miRNAs, using a synthetic antagonist significantly repressed lung cancer cell growth and in vivo tumorigenicity. Bioinformatically predicted miR-31 target genes were identified and studied. These included two tumor suppressors: large tumor suppressor 2 (LATS2) and PP2A regulatory subunit B alpha isoform (PPP2R2A) that were each substantially increased by miR-31 knock-down and repressed by miR-31 over-expression. Engineered repression of either LATS2 or PPP2R2A significantly antagonized miR-31-mediated growth inhibition. Luciferase binding assays confirmed that miR-31 directly suppressed LATS2 and PPP2R2A through binding to their respective 3′-untranslated regions (UTRs). Interestingly, miR-31 was inversely expressed relative to these identified target genes in both murine and human lung cancers. Clinical relevance was uncovered using miR-31 in situ hybridization and cyclin E immunohistochemistry assays performed on tissue arrays from the population-based New Hampshire Lung Cancer Registry. As expected, cyclin E expression was significantly associated with miR-31 levels. Real-time RT-PCR assays validated LATS2 and PPP2R2A repression in lung cancers versus adjacent normal lung tissues. Thus, these findings implicate miR-31 as an oncogenic miRNA (oncomir) that exerts its effects by repressing specific tumor suppressors. Taken together, miR-31 is proposed as a novel molecular target for lung cancer therapy and chemoprevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5698.

Recent progress in elucidating the complex and heterogeneous interactions between malignancy and coagulation or fibrinolysis reactions in humans has clarified the pathogenesis of disseminated intravascular coagulation that occurs with malignancy and has revealed evidence for two distinct pathways of growth regulation based on production by tumor cells of initiators of thrombin formation versus plasminogen activators. We have proposed a preliminary classification of tumors (see Table 2) based on these interactions. Type I tumors are those in which the tumor cells are associated with an intact coagulation pathway that leads to thrombin formation at the tumor periphery but in which the tumor cells lack u-PA. Examples of tumors in this category include SCCL, malignant melanoma, and renal cell carcinoma. Type II tumors are those in which the tumor cells express u-PA but lack an associated coagulation pathway leading to thrombin formation. Examples of type II tumors include prostate cancer, colon cancer, breast cancer, and N-SCLC. Type III tumors are those that express neither of these pathways, or exhibit some other pattern of interaction. Obviously, this formulation must be regarded as hypothetical. However, this concept fits with the limited data available to date from clinical trials. More importantly, this hypothesis can be tested further by means of intervention aimed at interrupting pathways relevant to specific tumor types. Characterization of additional tumor types by the methods described should permit amplification of this classification of tumors and other patterns of interaction may be defined. Exploration of the coagulation-cancer interaction holds considerable promise for gaining new understanding of both the coagulation mechanism and tumor biology. Most intriguing is the prospect that imaginative approaches to cancer treatment may be devised that are not only relatively nontoxic and low cost, but also effective.

A case of adrenal myelolipoma is reported in a patient in whomsymptoms of hematuria and flank pain developed after the patient had sustained blunt trauma. Adrenal myelolipomas are usually found incidentally at autopsy. However, the symptomatic presentation of this patient was probably caused by traumatic intratumor hemorrhage. The etiology and pathogenesis of these tumors are discussed in relation to their occasional symptomatic presentation and to their differential diagnosis.

A135 MicroRNAs (miRNAs) are noncoding small RNAs that regulate gene expression. Expression profiles of miRNAs are useful to improve classification, diagnosis, and prognostic information of specific human malignancies, including lung cancer. We sought to uncover miRNAs preferentially repressed or over-expressed in pre-malignant and malignant lung lesions in recently described transgenic cyclin E mice. These transgenic mice expressed in the lung under control of the human surfactant C promoter wild-type or proteasome degradation-resistant cyclin E species. These mice developed pulmonary dysplasia and adenocarcinoma, recapitulating pre-malignant and malignant lung lesions frequently found in lung cancer patients. Comprehensive miRNA microarray analyses were conducted using independently harvested normal and malignant lung tissues from these transgenic mice. A cluster of miRNAs was preferentially repressed in transgenic lung cancers versus normal lung tissues including: miR-34c, miR-145, miR142-5p, and other miRNAs previously associated with lung carcinogenesis. Expression profiles were independently validated by semi-quantitative and real-time polymerase chain reaction assays. In transgenic mice, single cell expression profiles were studied in pre-malignant and malignant lung lesions by in situ hybridization assays. Concordant results were obtained after analyses of paired normal-malignant human lung tissues representing each histopathologic subtype of non-small cell lung cancer. To address functional roles of repressed miRNAs, novel lung cancer cell lines were derived from murine transgenic wild-type (ED-1 cells) or proteasome-degradation resistant (ED-2 cells) cyclin E expressing lung cancers. ED-1 and ED-2 cells each caused lung adenocarcinomas to form after tail-vein injections into syngeneic FVB mice. Engineered over-expression of each basally repressed miRNA in ED-1 as well as in ED-2 cells markedly (P < 0.001) repressed cell growth. Anti-miR co-transfections antagonized these effects. A mechanism for this growth suppression was found by showing cyclin E, a predicted miR-34c bioinformatic target, was significantly repressed in miR-34c transfectants. Other miRNAs were selected for knock-down in ED-1 and ED-2 cells by virtue of their high basal expression in these cells as well as in murine transgenic lung cancers and human lung cancers versus normal lung tissues. Knock-down of candidate oncogenic miRNAs repressed lung cancer cell growth, which was antagonized by over-expression of the same miRNA. Experiments are underway in miRNA transfected ED-1 cells to assess in vivo tumorigenicity in FVB mice. Taken together, these studies uncovered candidate tumor suppressive and oncogenic miRNAs. We propose these miRNAs are molecular pharmacologic targets for lung cancer therapy and chemoprevention. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A135.

Immunohistochemical techniques applied to fresh frozen sections of metastatic malignant melanoma tissue revealed abundant fibrinogen (or fibrin I) in perivascular areas throughout the tumor connective tissue stroma. Fibrin was readily detected in a focal distribution in the connective tissue around nodules of viable tumor. Staining for D-dimer of cross-linked fibrin (using an antibody that cross-reacted with fragment D of fibrinogen) coincided with staining for fibrin. Diffuse staining of tumor cell bodies was observed for Factor X, and Factor XIII ("a" subunit) was detected in scattered areas of connective tissue throughout the tumors. Factor VII was not detected, and only rare tumor cells stained for tissue factor. These results support the concept that a tumor cell-associated, thrombin-generating pathway exists in situ in malignant melanoma tissue that includes Factor X but neither tissue factor nor Factor VII. By contrast, tumor cell staining was observed rarely for urokinase and to a variable extent for tissue plasminogen activator.

18054 Background: Transcriptional repression of cyclin D1 occurs during responses to erlotinib (E) both in vitro and in vivo. Cyclin D3 has overlapping function with cyclin D1 but has distinct transcriptional regulation. Methods: The expression of cyclin D3 was compared in E sensitive cell lines (H358, H441) and an E resistant cell line (A549). Cyclins D1, D2, and D3 were independently overexpressed in E sensitive NIH 3T3 cells by plasmid transfection. Biopsy tissues from a proof-of-principal clinical trial of E were assessed for cyclin D3 expression. Results: A549 cells were resistant to E and expressed high levels of cyclin D3 RNA and protein compared to E sensitive cell lines. Overexpression of cyclin D1 and cyclin D3 conferred partial resistance to E in NIH 3T3 cells while cyclin D2 had no significant effect. Comparison of cyclin D3 immunostaining in tumor biopsies from patients before and after treatment with E revealed an increase in the percentage of cyclin D3 expressing cells following treatment with E. Conclusions: Cyclin D3 confers resistance to E in vitro and in vivo. Drugs such as retinoids and rexinoids that target cyclin D3 expression may prove useful for enhancing sensitivity to E. No significant financial relationships to disclose.

15527 Background: ACC is a rare malignancy with a very poor prognosis. Surgery is the only potential curative option. Gefitinib is an oral EGFR inhibitor that may have activity in solid tumors that express EGFR. ACC over expresses EGFR in a high proportion of cases. Methods: From April 2004 through December 2006, the ACC Working Group conducted a phase II trial of Gefitinib as second line, monotherapy in patients with pathologically confirmed unresectable ACC who had progressed on mitotane or chemotherapy. All prior systemic therapy was discontinued 28 days prior to starting gefitinib. Patients were ineligible if: had received prior therapy with any EGFR inhibitor, pregnant or breast feeding, had other co-existing malignancies (other than basal cell carcinoma or cervical cancer in situ), had an ECOG PS > 2, absolute neutrophil counts < 1,500, or platelets < 20,000. Patients were not allowed concomitant use of phenytoin, carbamazepine, rifampicin, barbiturates, or St John’s Wort. Patients took gefitinib 250 mg orally once a day. Each cycle was 21 days with radiological assessment every 6 weeks.Response rate as determined by RECIST criteria was the primary endpoint. Results: 19 patients accrued to the study (18 with measurable disease and 1 without). Pt Characteristics: Female 79% (15/19); Median age 48 (range 26–74); 84% (12 female and 4 male =15/19) of the patients had steroid secreting tumors. Grade 3 toxicity was noted in 2 patients and included, hypertension and lower extremity edema and elevated liver transaminases. No grade 4 toxicities occurred. Of 19 patients evaluable, there were no complete responders, partial responders or patients with stable disease (0% response rate; 95% CI: 0%-18%). Conclusions: Gefitinib demonstrated no activity in patients with unresectable ACC. This study is now closed. This study demonstrated the ability to successfully accrue to a trial of novel agents in rare tumors in a multicenter setting. [Table: see text]

7136 Background: Bexarotene (Targretin) is a selective retinoid X receptor (RXR) agonist that appears to prolong survival in pts with NSCLC. To determine mechanisms of action, preclinical studies and a short-term, proof-of-principle clinical trial were performed. Methods: BEAS-2B immortalized human bronchial epithelial cells were treated with bexarotene and effects on EGFR and cell cycle regulatory proteins were determined by Western analyses. To evaluate bexarotene tissue pharmacokinetic (PK) and pharmacodynamic effects, 12 pts with pathologically confirmed stage I and II NSCLC were enrolled. Subjects were given 300 mg/m2/day of bexarotene orally for 7 to 9 days prior to surgical resection. Resected tumor tissue was evaluated for biomarker expression and tumor tissue bexarotene concentrations. Plasma bexarotene concentrations were measured at the time of resection. Biomarker changes were assessed immunohistochemically comparing pre- and post-treatment tissues by pathologists blinded to the tissue origin ...

4770 Background: Primary neoplasms of the adrenal cortex are extremely rare malignancies, estimated to represent 0.05–0.2% of all cancers diagnosed in the United States annually. These cancers carr...

3093 Background: Erlotinib (OSI-774, Tarceva™) is an epidermal growth factor receptor (HER1) tyrosine kinase inhibitor (TKI) with clinical activity in lung cancer. Mechanisms engaged during clinical response to erlotinib need to be determined. Methods: Using cultured human bronchial epithelial cells, in vitro effects of erlotinib were investigated. Cells were treated with erlotinib and effects on HER1 and cell cycle regulatory proteins were determined by immunoblot analyses. To evaluate further molecular changes in patient tumor specimens, a phase II pharmacokinetic and pharmacodynamic trial was performed in patients with aerodigestive tract cancers, who were candidates for serial tumor sampling. Five patients were enrolled and 4 patients were evaluable (3 NSCLC, 1 laryngeal SCC). Each received erlotinib at an oral dose of 150mg daily. Detailed plasma pharmacokinetics were performed on days 1 and 8. Tissue and plasma concentrations were determined at the second tissue sampling on day 9. Changes in the des...

Primary neuroendocrine tumors of the larynx appear to be extremely rare. We report a case of well-differentiated neuroendocrine carcinoma arising in the larynx of a 61-year-old white woman. This case was initially diagnosed as a lymph node metastasis in the neck from an unknown primary carcinoma. Extensive workup subsequently revealed that the larynx was the primary site of the tumor. An electron microscopic study of both the primary and metastatic tumors showed numerous neurosecretory-type granules. Immunohistochemical studies revealed positive immunoreactivity against calcitonin, somatostatin, and ACTH. However, the tumor also displayed focal exocrine differentiation and mucosubstance production. The patient underwent a supraglottic laryngectomy and radical neck dissection and was free of tumor 22 months postoperatively.

SummarySystemic activation of the coagulation mechanism is known to exist in patients with colon cancer. The mechanism of such activation was investigated using immunohistochemical techniques applied to fresh frozen sections of resected primary colon cancer specimens. Tumor cells stained for tissue factor, factor V, and urokinase-type plasminogen activator. Perivascular and intercellular areas stained for fibrinogen and the “a” subunit of factor XIII. Staining was minimal or absent for protein C, protein S, plasminogen activator inhibitors 1-3, factor VII, factor X, and fibrin (the antigenic site on the amino-terminal portion of Bβ chain that is exposed following thrombin cleavage of fibrinopeptide B was not detected). The lack of an intact thrombin- generating pathway in situ associated with viable colon cancer cells is consistent with the findings of others that coagulation activation in colon cancer may be triggered by a soluble tumor product that exerts its effect at sites distant from the tumor. These results may explain the absence of clinical responsiveness of colon cancer to antithrombotic drug therapy and may clarify therapeutic strategies for this common tumor.

A thermoplastic polymer, Delrin 150 (polyoxymethylene homopolymer), with creep resistance ten times that of ultrahigh-molecular-weight polyethylene, is used as a material for total joint prostheses. A study was made of the local and systemic host response to this polymer when implanted in three different mammalian species. 316 LC stainless steel was used as a control. The materials were implanted into muscle and bone as solid cylinders. A total of 446 samples were implanted into 74 animals. The duration of implantation ranged from 2 weeks to 2 years. A semi-quantitative evaluation of local tissue reaction was performed. For each implant, 16 histological criteria were graded for severity of host tissue reaction. The liver, spleen, kidneys, pancreas, and lungs from each animal were also studied for evidence of systemic toxicity. The polymer implants exhibited a mild tissue reaction with the same characteristics as the control. Local tumor formation, bone osteolysis, and surrounding muscle necrosis were not seen. No pathological changes compatible with systemic toxicity by Delrin 150 were observed in the study of the organs. Delrin 150 in solid form did not exhibit local or systemic toxicity and is therefore biocompatible by this study. Powder implantation studies should be performed to simulate tissue response to wear particles.

Eighteen head and neck paragangliomas were studied by light microscopy and light microscopic immunohistochemistry by the peroxidase technique for the presence of NSE (neuron-specific enolase), serotonin, and a battery of neuropeptides. Seven of these tumors were also studied by electron microscopy. All 18 cases demonstrated immunostaining for NSE; 10 of the 11 carotid body tumors had immunostaining for multiple hormones. Considering all 18 cases, the most frequently demonstrated hormonal substances were in order: serotonin, leu-enkephalin, gastrin, substance P, vasoactive intestinal polypeptide (VIP), somatostatin, bombesin, calcitonin, and alpha MSH. In several tumors, adjacent-step sections stained for different hormonal substances strongly suggested reactivity for more than one hormone in given tumor cells. By electron microscopy, all 7 cases studied displayed considerable heterogeneity of the neurosecretory granules with respect to size, shape, and electron density. This demonstrated that branchiomeric paragangliomas are capable of producing a spectrum of neuropeptides in addition to their known amine content. The presence of immunoreactive serotonin in most of these neoplasms was confirmed. In addition to these findings, neurofibrils within the substance of carotid body paragangliomas demonstrated immunoreactivity for somatostatin and a gastrinlike neuropeptide. The significance of the neuropeptides in these neoplasms and their possible presence and role in normal and hyperplastic paraganglia remain to be defined.

Through immunohistochemical techniques, blood coagulation factors were identified in situ in fresh frozen sections of small cell carcinoma of the lung. Prothrombin/thrombin, factor VII, factor X, and antithrombin III were present in intercellular spaces and associated with tumor cells. Factor IX, factor XI, prekallikrein, and high molecular weight kininogen were identified as being associated with tumor cells but did not exist in intercellular spaces. Variable connective tissue staining but no tumor-related staining was observed for factor V, factor VIII-related antigen, factor XII, the B subunit of factor XIII, alpha 1-antitrypsin, alpha 2-macroglobulin, or alpha 2-antiplasmin. Neither consecutive tissue nor the tumor manifested platelet Ib and IIbIIIa surface glycoproteins. These divergent staining patterns suggested that the detected clotting factors had not merely diffused from permeabilized blood vessels, but were selectively localized in situ. While conditions may exist within tumor tissue that both retard and promote thrombin generation, we propose that interactions between the observed coagulation factors ultimately lead to local thrombin formation, which is responsible for the conspicuous fibrin deposits already described in small cell carcinoma of the lung. Thrombin formed locally might contribute to progression of this tumor. Inhibition of local thrombin formation by warfarin therapy could explain the beneficial effects of warfarin therapy in treating small cell carcinoma of the lung.

Thrombin-generated cleavage sites of human fibrinogen have been identified adjacent to viable tumor cells in fresh frozen sections of small cell carcinoma of the lung (SCCL) by means of immunohistochemical techniques using mouse monoclonal antibodies to the N-terminal peptides of the fibrinogen alpha and beta chains. These results indicate that thrombin is generated in situ in this tumor type. Based on previous evidence for the existence of an initiator of coagulation together with coagulation factor intermediates associated with viable SCCL tumor cells in situ, and also for the favorable effects of anticoagulant therapy with warfarin in SCCL, we postulate that local tumor cell-induced thrombin formation may contribute to self-regulated progression of SCCL through deposition of fibrin and stimulation of cell proliferation. These results suggest novel treatment strategies for this particular tumor type and justify efforts to identify other tumor types in which similar mechanisms exist.

Twenty-five strictly defined bronchopulmonary carcinoids were studied by light microscopic immunohistochemistry by the peroxidase technique for NSE (neuron-specific enolase), serotonin, and a broad spectrum of neuropeptides. Eighteen cases were also studied by electron microscopy. Twenty-three of the twenty-five cases showed immunostaining for NSE; 24 cases displayed immunostaining for at least two of the hormones tested for; the single case that failed to show hormonal immuno-reactivity was however positive for NSE and had granules by electron microscopy. Serotonin was the most frequently demonstrated hormone followed by bombesin, vasoactive intestinal peptide, gastrin, leu-enkephalin , alpha-melanocyte stimulating hormone, somatostatin, substance P, and calcitonin. In several cases, adjacent-step sections stained for different hormones strongly indicated immunoreactivity for more than one hormone in single neoplastic cells. By electron microscopy, all 18 cases studied showed generally abundant neurosecretory granules, which, however, displayed considerable heterogeneity in their size, shape, and density. Twelve of these eighteen cases displayed evidence of squamous differentiation and 10 showed characteristic exocrine lumina. The capability of single neuroendocrine tumors and single neuroendocrine tumor cells to produce more than one immunoreactive hormone is hereby amply confirmed; these broad capabilities are certainly reflected in the heterogeneous granule populations seen by electron microscopy. The synchronous presence of squamous and exocrine features in broncho-pulmonary carcinoids indicates that they too are capable of multidirectional differentiation, which should not detract from their being regarded basically as well-differentiated neuroendocrine neoplasms. The clinical significance of strictly defining bronchial carcinoids is underscored by the fact that of 25 cases followed for 2-13 years, only one developed metastases 9 years postoperatively.

Fibrin was detected by immunospecific techniques associated with both intravascular and extravascular tumor deposits in renal cell carcinoma. In addition, coagulation factors VII and X were demonstrated in intercellular spaces within tumor tissue and adjacent to the surface of tumor cells. Scant accumulation of platelets on intravascular tumor masses was observed. These data suggest that tumor cells in renal cell carcinoma may induce fibrin formation locally by a factor VII-mediated (and thus tissue factor-initiated) pathway of blood coagulation. This mechanism may also account for the hypercoagulable state that exists with this tumor type. We postulate that local fibrin formation may contribute to the growth and spread of this particular type of cancer and that the course of renal cell carcinoma may be ameliorated by anticoagulant therapy.

The carcinogenic potential of 22 orthopedic prosthetic materials was studied by implantation in bone in Sprague-Dawley rats and evaluated by complete autopsy examination performed at the time of death or at the end of the 30 month experimental period. When the number of malignancies in these rats was compared with control and sham-operated animals, a small increase in sarcomata was observed in animals bearing metal alloy implants containing high contents of cobalt, chromium, or nickel. An increased incidence of lymphomata with bone involvement was also observed in rats bearing metal implants when compared with control and sham-operated animals.

Seventy-six consecutively resected primary pulmonary tumors were assessed first by routine light microscopy and subsequently by electron microscopy and immunohistochemical staining techniques to precisely identify features of differentiation. In 66% of the cases, this assessment provided information that modified or revised the histologic diagnosis provided by light microscopy alone. The following conclusions were reached: (1) The term “large cell undifferentiated carcinoma” has been applied to a heterogenous group of tumors, most of which have ultrastructural and immunohistochemical features of differentiation not identifiable by routine light microscopy. (2) Forty percent of the tumors previously called “large cell undifferentiated carcinoma” have predominantly neuroendocrine differentiation and appear to have a clinical course comparable to that of small cell neuroendocrine carcinoma. (3) The majority of pulmonary carcinomas (especially those previously called “poorly differentiated” or “undifferentiated”) may simultaneously demonstrate more than one pattern of differentiation when studied by electron microscopy and immunohistochemistry. (4) The frequency of neuroendocrine neoplasms of the lung, as determined by these and previous studies, is considerably greater than suspected on the basis of light microscopic studies alone. These comprise a clinical and morphologic spectrum ranging from bronchial carcinoid to small cell carcinoma, all of which have immunohistochemically demonstrable hormone production, although paraneoplastic hormonal syndromes are manifested in only a small minority of cases.

A spectrum of 24 benign and malignant nerve sheath tumors and 10 non-neural spindle-cell tumors were studied by light microscopy for the presence of glial fibrillary acidic protein (GFAP) immunoreactivity by the peroxidase-antiperoxidase (PAP) technique. In 8 cases, these results were compared to their electron microscopic appearances. Seventy percent (7 of 10) of benign schwannomas and 50% (4 of 8) of benign neurofibromas demonstrated focal to diffuse GFAP immunoreactivity. None of the malignant nerve sheath tumors nor any of the non-neural spindle-cell neoplasms contained demonstrable GFAP immunoreactivity. Similarly, no GFAP immunoreactivity could be detected in Schwann cells in normal peripheral nerves. The solitary benign schwannoma available for electron microscopic study demonstrated diffuse and abundant cytoplasmic intermediate filaments, and this tumor displayed diffuse and intense GFAP immunoreactivity. Two benign neurofibromas showed a more variable content of intermediate filaments ultrastructurally, and their GFAP immunoreactivity was variable. All five malignant nerve sheath tumors studied by electron microscopy displayed a variable complement of intermediate filaments; however, none of these tumors possessed GFAP immunoreactivity, suggesting that these intermediate filaments are either members of a different class of intermediate filaments or may perhaps represent "altered" GFAP not recognized by these antisera.

Twenty-two breast carcinomas with membrane bound granules by electron microscopy were tested for the presence of neuron specific enolase (NSE), neuropeptides and serotonin by immunohistochemistry. By light microscopy the cases studied included infiltrating ductal carcinomas, intraductal carcinomas, apocrine carcinomas, infiltrating lobular carcinomas of both classical and alveolar types, mixed lobular/colloid carcinomas, carcinoid growth pattern and one unclassified carcinoma. Ten cases showed immunoreactivity for 1 or 2 neuropeptides in scattered cells whereas all cases were positively and rather diffusely stained with anti-NSE. Immunohistochemical staining at the ultrastructural level was carried out; the presence of neuropeptides could not be confirmed. Scattered granules were marked with gold particles when antiserum against casein was used. We conclude that neither argyrophilia, nor NSE immunoreactivity nor membrane bound granules seen by electron microscopy constitute at present sufficient evidence to designate a breast carcinoma as neuroendocrine. However, our study indicates that certain breast carcinomas of several types do include cells with neuroendocrine features demonstrable convincingly by light microscopic immunohistochemistry. We have no evidence that these breast carcinomas with neuroendocrine features behave differently from their counterparts lacking such features. The intriguing speculation is that neuropeptides produced by certain breast carcinomas may act as local modulators of tumor growth and differentiation.

Immunoperoxidase staining for Regan isoenzyme of alkaline phosphatase was performed on cryostat sections of five human tumor tisssues. With a direct immunoperoxidase staining for the localization of Regan isoenzyme at the light and electron microscope levels, sections previously fixed with 0.05 M phosphate-buffered 4% paraformaldehyde were reacted with rabbit antisera to human placenta alkaline phosphatase conjugated to horseradish peroxidase. Comparison of conventional histochemistry and immunohistochemistry for Regan isoenzyme indicated that strong specific immunoperoxidase staining appeared on the cell membrane surface, and a diffuse one, in the cytoplasm of lung and colon cancer tissue cells showing L-phenylalanine-sensitive alkaline phosphatase. No immunoperoxidase reaction was obtained in tumor cells showing sensitivity to L-homoarginine or lacking aklaline phosphatase activity.

Eighty-one primary pulmonary neuroendocrine neoplasms were assessed by the classification of Gould and associates. The neuroendocrine features of these tumors were studied by a combination of conventional light microscopy, electron microscopy, and immunohistochemical staining for hormonal substances and neuron-specific enolase. In each case, clinical follow-up was obtained to test the prognostic value of this new pathological classification. This study indicated that bronchial carcinoids are very low-grade neuroendocrine neoplasms that are locally invasive and only occasionally metastasize late in their course. Well-differentiated neuroendocrine carcinomas are relatively low-grade carcinomas that either present with or subsequently develop nodal or distant metastases in 73% of patients. Intermediate cell neuroendocrine carcinomas are highly aggressive tumors often mistakenly called "large cell undifferentiated carcinoma." Their clinical course is comparable to that of small cell neuroendocrine carcinomas, which has a mean survival of 9 months. The different clinical courses of these tumors demonstrate the predictive value of the proposed classification. It appears particularly valuable to identify well-differentiated neuroendocrine carcinoma as a low-grade carcinoma, distinct from true bronchial carcinoids. This classification may resolve some discrepancies regarding the therapy for and prognosis of "carcinoids" and their presumed variants.

Nodular skin lesions consisting of eosinophilic cell infiltrates developed in a 45-year-old man with metastatic islet cell amphicrine carcinoma of the pancreas. These infiltrates did not fit previously described eosinophilic skin conditions. Although the tumor responded to conventional chemotherapy, the skin lesions did not. They did, however, respond completely to radiation therapy. This clinical constellation has not been reported previously and may represent a new paraneoplastic syndrome.

Two primary neuroendocrine skin carcinomas, one bronchial carcinoma, and one typical medullary thyroid carcinoma have been shown by immunohistochemical and electron microscopy to contain two or more immunoreactive peptides as well as melanosomes and premelanosomes. Of the battery of antisera tested, somatostatin was the only material present in all four tumors; calcitonin and ACTH were present in three of the four neoplasms. Neurosecretory granules on the one hand and melanosomes on the other were more readily identifiable in separate cells. However, in at least 2 cases, individual cells were thought to contain both types of structures. Of the various explanations possible, we choose to interpret these tumors as complex neuroendocrine carcinomas comprising cellular populations capable of synchronous production of immunoreactive peptides and melanin. since melanocytes are members of the dispersed neuroendocrine (APUD) system, these observations may not appear entirely surprising. Note-worthy, however, is that melanin synthesis seemingly remains a synchronously available option to neoplastic neuroendocrine cell populations of possibly diverse histogenesis that are actively engaged in the production of multiple hormonal peptides. We consider that these phenomena represent yet another facet of neoplastic multidirectional differentiation. It is possible that the determination of immunoreactive peptides as "markers" may play a future role in the diagnosis, therapy, and prognosis of these and similar tumors and in the study of the dysplasias that may precede them.

Interest in the mechanisms of interaction of mesothelioma with the coagulation mechanism stems from its common association with thrombocytosis (3,5,6) and hypercoagulability manifested by a high incidence of venous thromboembolism (3). The results of studies on the mechanism of coagulation activation in mesothelioma (using immunohistochemical techniques) are the subject of this report

Ten consecutive cases of basal cell carcinomas were reviewed. Nine of these displayed the typical histology of basal cell carcinoma, the other case was composed of small spindle to ovoid cells with scant cytoplasm and a high mitotic rate, resembling an “oat cell” carcinoma. These were studied using the immunoperoxidase technique for tissue localization of calcitonin, insulin, glucagon, somatostatin, ACTH, gastrin and nerve growth factor. Three cases were negative for all hormones tested. Three cases were focally positive for a single hormone; one each for calcitonin, somatostatin, and ACTH. Two cases were focally positive for ACTH and somatostatin and two cases were focally positive for calcitonin, somatostatin and ACTH. None of the other hormones displayed activity. The positive staining was eliminated after absorption by the specific antigen. This immunohistochemical study illustrated neuroendocrine differentiation in basal cell carcinomas as has previously been suggested by the Grimelius stain and electron microscopy. Thus, as demonstrated in other epithelial neoplasms, basal cell carcinoma may also display neuroendocrine differentiation. This illustrates the potential multidirectional differentiation in neoplastic epithelial cells.

The binding of pure human serum albumin and pure human hemoglobin to L-phenylalinine Sepharose and aniline Sepharose columns, two chromatographic columns of differing hydrophobicity, has been investigated for various concentrations of ammonium sulfate salts. The binding of hemoglobin at a lower ammonium sulfate concentration than albumin in both hydrophobic support systems parallels the solubility and precipitation characteristics of these two proteins in solution and mirrors the phenomenon of salting out of proteins in solution. Both hemoglobin and albumin bind at lower concentrations on aniline Sepharose than on L-phenylalanine Sepharose, reflecting the greater efficiency of binding by the more hydrophobic support matrix.