Primary Small Cell Lung Carcinoma Research Articles

Novel Mouse Cell Lines and In Vivo Models for Human High-Grade Neuroendocrine Lung Carcinoma, Small Cell Lung Carcinoma (SCLC), and Large Cell Neuroendocrine Carcinoma (LCNEC).

There is a clear need to expand the toolkit of adequate mouse models and cell lines available for preclinical studies of high-grade neuroendocrine lung carcinoma (small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC)). SCLC and LCNEC are two highly aggressive tumor types with dismal prognoses and few therapeutic options. Currently, there is an extreme paucity of material, particularly in the case of LCNEC. Given the lack of murine cell lines and transplant models of LCNEC, the need is imperative. In this study, we generated and examined new models of LCNEC and SCLC transplantable cell lines derived from our previously developed primary mouse LCNEC and SCLC tumors. RNA-seq analysis demonstrated that our cell lines and syngeneic tumors maintained the transcriptome program from the original transgenic primary tumor and displayed strong similarities to human SCLC or LCNEC. Importantly, the SCLC transplanted cell lines showed the ability to metastasize and mimic this characteristic of the human condition. In summary, we generated mouse cell line tools that allow further basic and translational research as well as preclinical testing of new treatment strategies for SCLC and LCNEC. These tools retain important features of their human counterparts and address the lack of LCNEC disease models.

Tumor-derived Vimentin as a novel biomarker for distinct subtypes predicting adjuvant chemotherapy resistance and T-cell-inflamed phenotype in small cell lung cancer.

Despite recent progress in subtype classification for small cell lung carcinoma (SCLC), little is known about the biomarker for triple-negative (ASCL1, NEUROD1, and POU2F3 negative) tumors. The long-term survival, adjuvant chemotherapy (ACT) response, and immune milieu in different SCLC subtypes have also not been well established. Here, we retrospectively collected a large cohort of 192 primary SCLC tumors and reported that ASCL1-, NEUROD1- and POU2F3-dominant subtypes counted for 61.38%, 19.31%, and 6.21%, respectively. Subtype intra-tumoral heterogeneity and co-expression at the single-cell level existed substantially. The expression of tumor-derived Vimentin (VIM) was nearly restricted to triple-negative SCLC tumors (15/19, 78.9%) while YAP1 expression was distributed widely in other subtypes. The SCLC subtyping model was independently prognostic of OS and RFS (p < 0.001 and p=0.043). In particular, patients with ASCL1-positive SCLC tumors can benefit more from ACT, and VIM-positive tumors did the opposite. Compared with other subtypes, the VIM-dominant SCLC subtype was associated with abundant but functionally impaired CD4+ and CD8+ T-cells, which highly expressed inhibitory checkpoints and potentially benefit from PD-L1 blockade therapy. Our study showed that tumor-derived SCLC-V subtype could independently predict ACT response. The distinct immune landscape between subtypes may help inform personalized immune therapeutic approaches.

Bioinformatics analysis identifies key genes of prognostic value in lung cancer

Lung cancer is the most common human malignancy worldwide and can be divided into different types of carcinomas depending on their pathological features. Advances in medical science and technology have led to the identification of some lung cancer-related marker genes, including EGFR (epidermal growth factor receptor), BRAF (B-Raf proto-oncogene), RAS (RAS proto-oncogene, GTPase) and HER2 (human epidermal growth factor receptor 2). However, the underlying biomarker and key genes associated with different types of lung cancer are still poorly understood. In this study, we analyzed a GEO (Gene Expression Omnibus) dataset and identified 28 upregulated intersection DEGs (different expression genes) and 125 downregulated intersection DEGs among AC (adenocarcinoma), PTC (primary typical carcinoid), PLCC (primary large cell carcinoma), PLCNC (primary large cell lung carcinoma) and PSCLC (primary small cell lung carcinoma). Through PPI (protein-protein interaction) network analysis, we identified 14 genes among the DEGs, namely MFAP4 (microfibril-associated protein 4), PDZD2 (PDZ domain containing 2), FBLN1 (fibulin 1), FBLN5 (fibulin 5), EFEMP1 (EGF containing fibulin extracellular matrix protein 1), KDR (kinase insert domain receptor), S1PR1 (sphingosine-1-phosphate receptor 1), CAV1 (caveolin 1), GRK5 (G protein-coupled receptor kinase 5), EDNRA (endothelin receptor type A), EDNRB (endothelin receptor type B), CALCRL (calcitonin receptor-like receptor), PTGER4 (prostaglandin E receptor 4), and ADRB1 (adrenoceptor beta 1), which were found to be downregulated in different subtypes of lung cancer and associated with poor survival outcomes. In addition, most of the screened DEGs demonstrated good predictive ability in LUAD (lung adenocarcinoma) and LUSC (lung squamous cell carcinoma). Among them, MFAP4 was found to promote cell proliferation while also suppressing cell migration and angiogenesis. In summary, we propose MFAP4, PDZD2, FBLN1, FBLN5, EFEMP1, KDR, S1PR1, CAV1, GRK5, EDNRA, EDNRB, CALCRL, PTGER4 and ADRB1 as potential prognostic markers in lung cancer patients.

Deep Learning Facilitates Distinguishing Histologic Subtypes of Pulmonary Neuroendocrine Tumors on Digital Whole-Slide Images.

Simple SummaryChallenges persist in diagnosing pulmonary neuroendocrine tumors. Our case study shows that deep learning combined with convolutional neural networks has the potential to assist in the diagnosis of pulmonary neuroendocrine tumors from digital whole-slide images.The histological distinction of lung neuroendocrine carcinoma, including small cell lung carcinoma (SCLC), large cell neuroendocrine carcinoma (LCNEC) and atypical carcinoid (AC), can be challenging in some cases, while bearing prognostic and therapeutic significance. To assist pathologists with the differentiation of histologic subtyping, we applied a deep learning classifier equipped with a convolutional neural network (CNN) to recognize lung neuroendocrine neoplasms. Slides of primary lung SCLC, LCNEC and AC were obtained from the Laboratory of Clinical and Experimental Pathology (University Hospital Nice, France). Three thoracic pathologists blindly established gold standard diagnoses. The HALO-AI module (Indica Labs, UK) trained with 18,752 image tiles extracted from 60 slides (SCLC = 20, LCNEC = 20, AC = 20 cases) was then tested on 90 slides (SCLC = 26, LCNEC = 22, AC = 13 and combined SCLC with LCNEC = 4 cases; NSCLC = 25 cases) by F1-score and accuracy. A HALO-AI correct area distribution (AD) cutoff of 50% or more was required to credit the CNN with the correct diagnosis. The tumor maps were false colored and displayed side by side to original hematoxylin and eosin slides with superimposed pathologist annotations. The trained HALO-AI yielded a mean F1-score of 0.99 (95% CI, 0.939–0.999) on the testing set. Our CNN model, providing further larger validation, has the potential to work side by side with the pathologist to accurately differentiate between the different lung neuroendocrine carcinoma in challenging cases.

NeuN, a DNA-binding neuron-specific protein expressed by Merkel cell carcinoma: analysis of 15 cases.

SummaryAimMerkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine carcinoma, with an increasing worldwide incidence. It presents as a painless red to purple nodule on sun-exposed skin. MCC is presumed to arise from resident cutaneous Merkel cells. The pathogenesis of MCC is likely multifactorial with immunosuppression, UV-induced skin damage, and Merkel cell polyomavirus contributing to the development. The diagnosis of MCC relies upon characteristic morphologic features and use of immunohistochemical stains. Histologically, the differential diagnosis of Merkel cell carcinoma includes the ‘small round cell’ tumor group, particularly metastatic small cell carcinoma and hematological malignancies. This study investigates the expression of NeuN antibody, which recognizes the protein NeuN, normally present in most neuronal cell types and neuronal tumors, in Merkel cell carcinomas.Methods and resultsFifteen cases of Merkel cell carcinoma (7 men and 7 women; mean age 74 years) were retrieved from the institute database between the years 2011-2020. The immunohistochemical profile was investigated: CK20 (14/14), Neurofilament, (12/12), Synaptophysin (14/14); Chromogranin A (11/13), PAX5 (10/12), TDT (5/12), CK7 (1/14), TTF1 (0/14). Infection by Polyoma virus was detected in 11 of 14 patients. Most tumors showed middle/strong expression of NeuN. No cutaneous structures, or epidermal Merkel cells, showed expression of NeuN. The expression of NeuN was investigated in 17 primary small cell lung carcinomas: 2 cases were positive for Neu-N.ConclusionsAwareness of the staining pattern of Neu-N could aid in diagnosis of Merkel cell carcinoma, avoiding misinterpretation and erroneous diagnosis with other tumors.

Multivalent state transitions regulate the intratumoral composition of small cell lung carcinoma.

e20587 Background: Small cell lung carcinoma (SCLC) is an aggressive, tobacco-associated tumor with neuroendocrine features characterized by rapid growth, metastatic progression, and initial response followed by almost invariable resistance to therapy. Studies to date have not resolved the extent that diverse transcriptional programs drive SCLC and contribute to its lethality. Methods: We combined one of the largest and most diverse inventories of patient-derived xenograft models of SCLC with an ex vivo culture system that maintains transcriptional fidelity with matched primary SCLC tumor to identify transcriptional state heterogeneity. Using the expression of the Ascl1, NeuroD1, and Yap1 as markers of well-conserved SCLC states, we developed a state-of-the-art fluorescent platform that can directly measure single-cell state transitions in a multi-layered ecosystem using tandemly integrated reporters. We modeled population dynamics using a discrete time Markov chain and directly measure single-cell state transitions. Results: We show significant cell-state heterogeneity in several SCLC primary tumors, patient-derived xenografts (PDX), and ex vivo cultures. These states comprise distinct subpopulations marked by the master regulatory transcription factors (TFs) Ascl1, NeuroD1, and Yap1. Ex vivo, the 3 TFs are associated with suspension aggregates of small neuroendocrine cells, pre-suspension (loosely adherent) aggregates, and large mesenchymal cells with visible cytoplasm and spindle-like membrane extensions, respectively. We have observed equilibria in cell-state proportions in SCLC tumors both in vivo (PDX) and ex vivo. In addition, we have shown that the “elasticity” of SCLC responses, measured as the extent of clinical response during chemotherapy followed by the time to relapse from the end of therapy, is dependent on tumor TF levels. These observations suggest that mechanistic modeling of intra-tumoral state dynamics is of high clinical relevance. Conclusions: Our integrative approach is poised to formulate and validate a unified model of cellular states and program diversity in SCLC. If successful, the characterization of malignant cell ontogenic programs, their plasticity, and the advancement of new therapies designed to combat plasticity by epigenetic reprogramming will create a new scientific canvas for the study of this highly lethal disease.

Clinical Values of Platelet Parameters in Small Cell Lung Carcinoma with Pleural Effusion.

Currently, there are few studies on the correlation between platelet counts (PLT), plateletcrit (PCT), and platelet-to-lymphocyte ratio (PLR) in small cell lung carcinoma (SCLC) with and without pleural effusion. This study is to investigate their likely correlation and to evaluate the potential diagnostic or prognostic applications of these platelet parameters. A total of 218 each of patients with primary SCLC and healthy controls were included. Hematological indicators and other clinically relevant information were collected. Comparisons of the differences between groups were applied to the independent samples t-test or the chi-squared test. ROC curve analysis was used to access the diagnostic performance of PLT, PCT, and PLR. Compared with healthy controls, PLT, PCT, and PLR in SCLC were significantly higher. On the other hand, mean platelet volume, lymphocytes, and hemoglobin were significantly lower. The levels of PLT, PCT, and PLR were related to malignant pleural effusion, while not related to lymph node or distant metastasis. The incidence of pleural effusion in patients with SCLC was positively correlated with the levels of PLT, PCT, and PLR. ROC curve analysis showed that PLT, PCT, and PLR were valuable markers for SCLC, and the combination of the three has higher diagnostic efficacy. Platelet parameters were significantly different between SCLC and controls. PLT, PCT, and PLR could be used to assess the presence of pleural effusion.

S3050 Gastric Mass as a Rare Presentation of Primary Lung Carcinoma

INTRODUCTION: Symptomatic gastrointestinal metastases are a rare initial presentation of primary lung cancer. The most common sites of lung cancer metastasis are lymph nodes, liver, adrenal glands, bone, and brain. Gastric metastases are often asymptomatic. We report a case of a gastric mass as the initial clinical manifestation of primary small cell lung cancer (SCLC). CASE DESCRIPTION/METHODS: An 82 year old woman with past medical history of hypertension and asthma presented with a complaint of abdominal pain, abdominal distention, and associated shortness of breath for one month. She also endorsed nausea and weight loss (∼25 lbs). She is a former smoker, quitting 19 years prior. Physical exam was notable for moderate abdominal distention with positive fluid wave. CT abdomen showed large volume ascites and an ill-defined mass of the stomach originating from the lesser curvature and extending into the liver hilum, with extensive peritoneal metastatic implants. Paracentesis revealed red-tinged ascitic fluid, with cytology studies suggestive of metastatic cancer of likely gastric origin. Esophagogastroduodenoscopy (EGD) demonstrated external compression of the stomach along the lesser curvature > 5 cm, and two large gastric ulcers >2 cm each with raised edges along the greater curve. Endoscopic ultrasound (EUS) revealed a mixed echogenic gastric mass along the lesser curvature to the antrum abutting the portal vein ( >8 cm longitudinally, and >5 cm vertically), with an area of necrosis (1 cm × 1.5 cm) within the lesion. No vascular flow was identified within the lesion. Additionally, hypoechoic lesions typical of lymph nodes were seen in the mediastinum, celiac axis, and porta hepatis. Pathology report of the tissue biopsy revealed small cell carcinoma, and immunohistochemistry analysis was positive for CD56, TTF-1, and CAM5.2, suggestive of gastric metastasis from primary small cell lung carcinoma. The patient was started on carboplatin/etoposide chemotherapy. DISCUSSION: Small cell lung carcinoma initially presenting with gastrointestinal symptomatology is a rare occurrence. In our patient, the ascitic fluid cytology and the radiologic finding of a large gastric mass were initially suggestive of a primary gastric carcinoma. However, on further staging efforts with EGD/EUS and gastric tissue biopsy, the true primary lesion was revealed to be of lung origin, demonstrating the importance of gastroscopy and detailed immunohistochemistry studies in establishing an accurate diagnosis.

Conjunctival metastasis as an initial sign of small cell lung cancer.

Introduction. To report a case of a conjunctival metastasis as the initial manifestation of small cell lung cancer. Methods. Observational case report. Results. A 50-year-old man without known systemic disease developed a conjunctival mass in his right eye. He underwent incisional biopsy of the tumor and systemic evaluation. Histopathologically, the conjunctival mass was a metastatic small cell carcinoma. Further evaluation revealed a primary small cell lung carcinoma with metastasis to liver and bones. The primary and metastatic tumors were treated with chemotherapy. Conclusion. Conjunctival metastasis may be the initial manifestation of lung cancer. It should be considered in the differential diagnosis of a deep conjunctival mass.

Small cell glioblastoma or small cell carcinoma: A case report and review of the literature

It is often easy to distinguish between primary brain tumors and metastases based on morphology alone. However, in some cases immunohistochemistry (IHC) is necessary to obtain a diagnosis, but, as the present case report illustrates, this is not always straightforward. A 75-year old man was admitted to the hospital with left-sided loss of motor function. A MRI revealed a 6 cm tumor in the right temporoparietal area. The histology was consistent with both glioblastoma multiforme (GBM) and small cell lung carcinoma (SCLC) but IHC was suggestive of a SCLC metastasis. PET-CT revealed no enhancement in the lung, so the tumor was treated as a GBM. Eight months after the primary diagnosis a new MRI revealed metastases in the spinal cord, but there was still no enhancement in the lungs. We reviewed the literature concerning markers used to differentiate between GBM and SCLC and found that most of these markers showed limited specificity. It is further discussed whether the case illustrates an example of spontaneous regression of primary SCLC or might be an example of a GMB metastasizing to the spinal cord. Although immunohistochemical markers are of great help in many situations, the case illustrates important limitations and the need for better diagnostic markers.

An ectopic ACTH-producing small cell lung carcinoma associated with enhanced corticosteroid biosynthesis in the peritumoral areas of adrenal metastasis

A 60-year-old Japanese male presented with swelling of bilateral cervical lymph nodes was subsequently diagnosed as the late stage of primary small cell lung carcinoma (SCLC). He was then treated with cisplatin and irinotecan as first-line chemotherapy, but hypokalemia with muscle weakness of the bilateral legs became gradually noticeable following two months of effective chemotherapy. A computed tomography (CT) scan revealed enlargement of bilateral adrenal glands and abdominal and mediastinal lymph nodes, though primary lung tumor remained the same in size. An ectopic ACTH-producing syndrome (EAS) was subsequently revealed by the following endocrinological studies. Hypokalemia was clinically improved by the treatment with metyrapone and the second-line chemotherapy with amrubicin for SCLC was started, but the patient died 12 days after the second-line chemotherapy. Post-mortem examination revealed ACTH immunoreactivity in tumor cells of all the metastatic lesions. Non-neoplastic adrenal cortex demonstrated hyperplasia associated with lipid depletion and marked expression of steroidogenic enzymes, especially in cortical cells around tumor infiltration, suggestive of paracrine ACTH stimulation of cortisol production. This is the first report evaluating expression of steroidogenic enzymes in adrenal cortex especially adjacent to the adrenal metastasis in the patients with EAS due to SCLC. These findings suggest that ACTH producing adrenal metastasis can induce EAS more frequently and severely, and that the symptoms and examination of EAS should be monitored carefully in the patients with adrenal metastasis of SCLC.

Binding of TS1, an anti-keratin 8 antibody, in small-cell lung cancer after 177Lu-DOTA-Tyr3-octreotate treatment: a histological study in xenografted mice

BackgroundSmall-cell lung carcinoma (SCLC) is an aggressive malignancy characterised by an early relapse, a tendency towards drug resistance, and a high incidence of metastasis. SCLC cells are of neuroendocrine origin and express high levels of somatostatin receptors; therefore, future treatment might involve targeting tumours with radiolabelled somatostatin analogues. This therapy induces abundant necrotic patches that contain exposed keratins; thus, keratin 8, which is one of the most abundant cytoskeletal proteins may represent an interesting secondary target for SCLC. This study aimed to investigate the effects of177Lu-DOTA-Tyr3-octerotate and the binding of the monoclonal anti-keratin 8 antibody, TS1, in vitro in treated SCLC- and midgut-xenografted mouse models.MethodsNCI-H69- and GOT1-xenotransplanted mice were treated with three doses of 30 MBq177Lu-DOTA-Tyr3-octreotate administered 24 h apart. Mice xenotransplanted with NCI-H69 were sacrificed 1, 5, 12, 20 and 150 days post-injection or when the tumour had regrown to its original size. GOT1-xenotransplanted mice were sacrificed 3 days post-injection. Immunohistochemistry was performed to evaluate TS1 staining in tumours and in seven human biopsies of primary SCLC from pulmonary bronchi. Central cell density and nucleus size were determined in NCI-H69 sections.ResultsTwelve days after177Lu-DOTA-Tyr3-octerotate treatment, the SCLC xenograft response was extensive. Twenty days after treatment, one of three analysed tumours displayed complete remission. The other two tumours showed 1/4 the cell density of untreated controls and cell nuclei were about three times larger than those of untreated controls. At 150 days after treatment, one of four mice exhibited complete remission. Treated tumours displayed increased TS1 antibody accumulation and high TS1 binding in necrotic patches. All seven human SCLC biopsies displayed necrotic areas with TS1 staining.ConclusionsRadiation treatment with three injections of 30 MBq177Lu-DOTA-Tyr3-octreotate had pronounced effects on tumour cell density and cell nuclei, which indicated mitotic catastrophe. Despite these anti-tumour effects, two of three SCLC tumours recurred. Further studies should investigate the nature of tumour cell survival and develop more effective treatments. High TS1 accumulation in tumour sections in vitro after177Lu-DOTA-Tyr3-octerotate treatment indicated that TS1 might represent a promising secondary therapeutic strategy.

Numb cheek syndrome as the first manifestation of anti–Hu paraneoplastic neuronopathy

Sirs: Unilateral numb cheek syndrome (NCS) has rarely been described as the first manifestation of malignancy due to neoplastic involvement of the trigeminal nerve, more specifically the maxillary branch [1, 2]. NCS as the first symptom of subacute paraneoplastic sensory neuronopathy has, to the best of our knowledge, never been reported. We present this case because its rarity led to a diagnostic delay of several months. A 47-year-old man presented with a one month history of numbness in the left cheek, both inner and outer surfaces. The area of numbness corresponded with the mandibular division of the left trigeminal nerve (V3). His medical history was unremarkable. Neurological examination was normal including the left V3 area. As the main part of the cheek is innervated by the buccal nerve we considered that our patient had a mandibular sensory neuropathy probably due to compression or infiltration of the nerve by local malignancy, as seen in other types of trigeminal mononeuropathy [3]. Ancillary investigations including contrast enhanced brain MRI, thorax CT and extensive laboratory examinations of the blood and urine were normal. Cerebrospinal fluid (CSF) examination revealed mild lymphocytic pleocytosis, slightly elevated protein of 0.69 g/l (0.2–0.5) and no malignant cells or oligoclonal bands. Although intracranial malignancy could not be ruled out an expectant policy was chosen by the patient. Two months later he complained of slightly unsteady gait. A second MRI of the brain and skull base was normal. Another 4 months later he noticed numbness of his left hand and foot. Neurological examination showed only left sided facial hypalgesia. Nerve conduction studies were consistent with a non uniformly distributed, predominantly axonal, sensorimotor neuropathy, most pronounced in the left extremities. Repeated conventional CSF examination only showed an increased protein level of 0.86 g/l. Anti-Hu antibodies in CSF were detected with indirect immunofluorescence and Western blotting techniques. A second thorax CT and whole body PET-scan eventually showed a solitary lesion in the right hilus, which proved to be a metastasis of an anaplastic small cell carcinoma on histological examination. Although the primary tumour remained unrevealed, a paraneoplastic multifocal mainly sensory neuronopathy associated with presumed primary small cell lung carcinoma was diagnosed and combined chemotherapy started. Fifteen months after the initial complaint the primary tumour remained undetected and no metastases were found. The neuropathy progressed resulting in very severe sensorimotor neuropathy with asymmetric painful paresthesias, diffuse proprioceptive loss and weakness in all extremities. In patients with paraneoplastic anti-Hu associated neuropathy (AHN) involvement of the cranial nerves may be present but is very rarely the presenting symptom. AHN most commonly presents as a subacute sensory neuropathy, less frequently as a sensorimotor neuropathy [5, 6]. These neurological symptoms typically antedate the diagnosis of cancer and most commonly involve the extremities [4–7]. In 306 patients with malignancy and associated anti-Hu antibodies, 50 % of the patients had a sensory neuropathy [4–6]. Three of them had trigeminal nerve involvement, but in no one was cheek numbness described as the only presenting symptom. As initially no primary tumour was found, we did not consider AHN as a potential cause of the unilateral cheek numbness in our patient. The final diagnosis was reached because of the extension of the sensory symptoms and eventually the evidence of an asymmetric sensorimotor neuropathy. The diagnostic delay between the NCS and final diagnosis was 6 months, which lies within the range of reported diagnostic delays in AHN patients [4–7]. As early treatment of the underlying malignancy is the mainstay of treatment, we report this patient in order to add NCS to the list of presenting symptoms of AHN.

Oxytocin receptor pattern of expression in primary lung cancer and in normal human lung

In order to assess if oxytocin- and vasopressin-induced mitogenic effects detected on small-cell lung carcinoma (SCLC) cell lines could be transposed on primary SCLC, the aim of the present work was to identify mediators of these mitogenic actions on primary tumours samples. This was addressed on normal human lung tissue, on SCLC and on non-SCLC (NSCLC). Herein, we observe, in normal human lung, that OTR is colocalized with vascular endothelial cells of the lung and is not expressed by lung cells of epithelial nature. We detected mRNA amplification of V1aR, V2R and of a V2R variant. We observed that 86% of SCLC biopsies analyzed expressed at least the OTR and that 71% expressed the OTR, the V1aR and the V2R altogether. Comparatively, 50% of NSCLC biopsies tested expressed at least the OTR and 32% expressed the OTR, the V1aR and the V2R altogether. The occurrence of the V1bR/V3R is of 28 and 18% for SCLC and NSCLC, respectively. Nevertheless, for the SCLC biopsies analyzed in this study, V1bR/V3R expression correlates, in all cases, with the expression of all the other neurohypophysial peptide receptors. Our results suggest that neurohypophysial peptide antagonists may offer promise as a potential new therapeutic modality for the treatment of lung cancer expressing at least one of the neurhypophysial peptide receptor subtypes.

Cytology applications of p63 and TTF-1 immunostaining in differential diagnosis of lung cancers

The pathologic distinction of small cell from non-small cell-lung carcinoma is of considerable therapeutic significance. In particular, the ability to distinguish poorly differentiated non-small-cell lung cancer from small-cell lung carcinoma (SCLC) is at times difficult based upon morphology alone; available immunohistochemical markers such as neuroendocrine markers are of limited utility. We have demonstrated the role of p63 and thyroid transcription factor-1 (TTF-1) in the differential diagnosis of poorly differentiated squamous-cell carcinoma (PDSCC) versus SCLC, mostly in biopsy samples (Wu et al., American Journal of Clinical Pathology 2003;119:696-702). Here, we examine further the utility of this panel in cytologic cell-block samples of lung cancers including both primary and metastatic cancers of pulmonary origin, and cases of nonpulmonary cancers metastatic to lung in which differential diagnoses included a lung primary.Four-micron thick sections of 30 alcohol-fixed paraffin-embedded cell blocks from 14 lung FNAs, 6 liver FNAs, 3 bronchial washings, 1 subcarinal lymph node FNA, 1 iliac lymph node FNA, 1 pelvic mass FNA, 1 neck lymph node FNA, 1 adrenal FNA, and 1 pleural effusion were deparaffinized and stained with monoclonal antibodies reactive to p63 (1:800, Santa Cruz Biotechnology) and TTF-1 (1:50, Dako). Slides were stained for p63 using a streptavidin-biotin kit (BioGenex) and diaminobenzidine as chromagen, and counterstained with hematoxylin. Slides were stained for TTF-1 using a Dako Autostainer. Thirty cases were examined, including 8 primary SCLCs, 8 extra-pulmonary metastases of lung SCLCs, 4 PDSCCs and 4 primary pulmonary adenocarcinomas, and 6 nonpulmonary adenocarcinomas metastatic to lung or other sites. Fifteen out of 16 (94%) SCLC cases were p63-/TTF-1+, ranging in intensity from focal-weak to diffuse-strong; 1/16 SCLCs from a bronchial washing was p63-/TTF-1- but synaptophysin was positive. All 4 primary lung adenocarcinoma cases were p63-/TTF-1+; contrasting with nonpulmonary adenocarcinomas that were all p63-/TTF-1-. All 4 PDSCC cases were p63+/TTF-1-. The panel of p63 and TTF-1 appears to be useful in the diagnostic evaluation of cytologic cell-block samples of pulmonary malignancy.

1028. Virus-Mediated RNAi for Skp-2 in Human Small Cell Lung Carcinoma Cells Resulted in the Inhibition of Tumor Cell Growth through the Increase of p27Kip1 and p21

In many cancers, low expression of p27Kip1, a cyclin-dependent kinase (cdk) inhibitor, has been reported to associate with the poor prognosis and highly aggressive nature of the tumors. Since the level of p27Kip1 protein is mainly regulated by ubiquitin-proteosomal proteolysis, the enhanced degradation of p27Kip1 is considered to be an important process of the reduced expression of p27Kip1 in cancers. Skp-2, a member of the F-box protein family, is the specific substrate-recognition subunit of an SCF ubiquitin-protein ligase complex and is involved in p27Kip1 degradation. Increased expression of Skp-2 has been reported in many cancers including small cell lung carcinoma (SCLC) and indicates that Skp-2 may be involved in the carcinogenesis of tumors with reduced p27Kip1 levels. In SCLC, Skp-2 gene was amplified and consequently overexpressed in 44% of primary SCLC cases with the reduced expression of p27Kip1. We therefore attempted to evaluate the role of Skp-2 in SCLC and whether the Skp-2 could be a good molecular target for cancers with enhanced Skp-2 expression by using virus-mediated RNA interference (RNAi) for Skp-2.

Molecular and immunological evaluation of the transcription factor SOX-4 as a lung tumor vaccine antigen.

The developmental transcription factor SOX-4 has been shown to be highly and differentially overexpressed in primary small cell lung carcinomas (SCLC). To examine the potential of SOX-4 for broad use as a lung cancer vaccine, we have evaluated the expression of SOX-4 in a panel of primary adenocarcinoma, squamous, and large cell tumor samples as well as in a panel of established small cell and non-small cell lung carcinoma tumor cell lines. SOX-4 mRNA is shown to be overexpressed in a substantial fraction of each of these lung tumor types. To examine the immunological potential of SOX-4, we have evaluated the presence of SOX-4-specific CD4 and CD8 T cells in PBMC of healthy donors and the presence of SOX4-specific Abs in sera from SCLC patients. We demonstrate the presence of both CD4 and CD8 T cells that recognize naturally processed epitopes derived from SOX-4 as well as the presence of SOX-4-specific Abs in sera from SCLC patients, but not in sera from healthy donors. The lung tumor-specific overexpression and demonstration of a comprehensive Ag-specific immune response specific for SOX-4 support the use of this molecule in the development of whole gene-, peptide-, or protein-based vaccination strategies against lung cancer. Furthermore, the identification of naturally processed T cell and Ab epitopes from SOX-4 provides valuable tools for the development of peptide-based vaccination strategies against lung cancer as well as to monitor SOX-4-specific responses in vaccinated patients.

Decreased endothelin receptor B expression in large primary uveal melanomas is associated with early clinical metastasis and short survival.

The most devastating aspect of cancer is the metastasis of tumour cells to organs distant from the original tumour site. The major problem facing oncologists treating uveal melanoma, the most common cancer of the eye, is metastatic disease. To lower mortality, it is necessary to increase our understanding of the molecular genetic alterations involved in this process. Using suppression subtractive hybridisation, we have analysed differential gene expression between four primary tumours from patients who have developed clinical metastasis and four primary tumours from patients with no evidence of metastasis to date. We have identified endothelin receptor type B as differentially expressed between these tumours and confirmed this observation using comparative multiplex RT–PCR. In a further 33 tumours, reduced endothelin receptor type B expression correlated with death from metastatic disease. Reduced expression also correlated with other known prognostic indicators, including the presence of epithelioid cells, chromosome 3 allelic imbalance and chromosome 8q allelic imbalance. Endothelin receptor type B expression was also reduced in four out of four primary small cell lung carcinomas compared to normal bronchial epithelium. We also show that the observed down-regulation of endothelin receptor type B in uveal melanoma was not due to gene deletion. Our findings suggest a role for endothelin receptor type B in the metastasis of uveal melanoma and, potentially, in the metastasis of other neural crest tumours.British Journal of Cancer (2002) 87, 1308–1313. doi:10.1038/sj.bjc.6600620 www.bjcancer.com© 2002 Cancer Research UK

The CpG island of the novel tumor suppressor gene RASSF1A is intensely methylated in primary small cell lung carcinomas.

Loss of heterozygosity at 3p21.3 occurs in more than 90% of small cell lung carcinomas (SCLCs). The Ras association domain family 1 (RASSF1) gene cloned from the lung tumor suppressor locus 3p21.3 consists of two major alternative transcripts, RASSF1A and RASSF1C. Epigenetic inactivation of isoform A (RASSF1A) was observed in 40% of primary non-small cell lung carcinomas and in several tumor cell lines. Transfection of RASSF1A suppressed the growth of lung cancer cells in vitro and in nude mice. Here we have analysed the methylation status of the CpG island promoters of RASSF1A and RASSF1C in primary SCLCs. In 22 of 28 SCLCs (=79%) the promoter of RASSF1A was highly methylated at all CpG sites analysed. None of the SCLCs showed evidence for methylation of the CpG island of RASSF1C. The results suggest that hypermethylation of the CpG island promoter of the RASSF1A gene is associated with SCLC pathogenesis.

Two Cases of Gastric metastasis from Small Cell Lung Cancer

This is a report of gastric metastases secondary from a primary small cell carcinoma of the lung in two men. Blood-borne metastatic involvement of the stomach by cancer is a rare entity. According to the reports in the literature the prevalence of metastasis to the stomach occurs in 0.4% and the most common cell type of the primary lung carcinoma is large cell type(3.7%) followed by adenocarcinoma(2.4%), small cell carcinoma(1.7%) and squamous cell carcinoma(0.7%). The most common tumors that spread to the stomach through the blood stream are malignant melanoma, breast carcinoma and lung carcinoma. Most of the gastrointestinal tract metastases had no specific symptoms because of its submucosal involvement. The prognosis was poor and the mean survival period from the onset of symptoms was 49 days. The first patient was a 56-year-old man who had primary lung carcinoma with brain metastasis. Gastroscopic findings showed two elevated mass lesions in the anterior wall of the mid body with central ulcer and the posterior wall of the fundus with intact surface mucosa. Pathologic examination of stomach tissue revealed small cell type tumor cells infiltrate in the stomach wall segmentally without destruction of the glands. The second patient was a 67-year-old man who had no other evidence of the distant metastasis. Gastroscopic findings showed a huge, oval shaped, ulcerofungating mass with deep penetrating central ulcer coated with dirty exudate in the anterior wall from mid to upper body of the stomach, and thickened elevated rugal folds in the posterior wall of the fundus. Pathologic examination of stomach tissues revealed the small cell type tumor cells showing small smudged nucleus infiltrate into the mucosa of the stomach and the architecture of mucosa intact. We report the two cases of metastatic gastric cancer from the primary small cell lung carcinoma with the literature review.