Differential expression of the c-kit proto-oncogene in germ cell tumours

The c-kit proto-oncogene product and its ligand stem cell factor play an important role in haematopoiesis, spermatogenesis, and melanogenesis. Using an anti-c-kit antiserum raised against a synthetic peptide, we studied the immunohistochemical expression of the c-kit gene product in 60 germ cell tumours (GCTs) (53 testicular, 7 extragonadal), derived from primary GCTs in 45 cases and metastatic tumours in 15 cases. Twenty-eight out of 28 seminomas showed c-kit membranous staining in the majority of cells. A similar pattern of expression was seen in intratubular germ cell neoplasia. Nine out of 29 (32 per cent) non-seminomas displayed cytoplasmic, but not membranous, c-kit immunoreactivity in occasional cells. In three mixed GCTs, c-kit expression was limited to the seminoma component. In normal testis, c-kit expression was observed in some basal tubular cells, corresponding to undifferentiated spermatogonia. These results suggest a role for c-kit in the oncogenesis of GCT, where down-regulation of c-kit might be a critical step during progression from seminomas to non-seminomas. Immunohistochemical analysis of c-kit should be considered as a diagnostic aid for GCT and in particular may be helpful in the identification of certain extragonadal seminomas.     

Expression of the c-kit gene product in normal and neoplastic mast cells but not in neoplastic basophil/mast cell precursors from chronic myelogenous leukaemia

The expression of the c-kit gene product has been examined in normal mast cells, mast cell neoplasms, and basophil/mast cell precursors obtained from patients with chronic myelogenous leukaemia (CML). Formalin-fixed, paraffin-embedded sections or smears fixed with formalin vapour were studied by immunohistochemical methods, using a polyclonal antibody against the c-kit gene product. Normal and neoplastic mast cells showed a positive immunoreaction for c-kit gene product, but neoplastic basophil/mast cell precursors from CML patients lacked c-kit gene product by immunohistochemical and flow cytometric methods, even in cells having mast cell granules, together with or without basophil granules. Mast cell tryptase was, however, expressed in normal and neoplastic mast cells and basophil/mast cell precursors containing mast cell granules. In addition, cells of monocyte/macrophage lineage lacked c-kit gene product. These findings indicate that the c-kit gene product may play an important role in the development and function of mast cell but not of cell of basophil and monocyte/macrophage lineage.     

Heterogeneity of gonadoblastoma germ cells: similarities with immature germ cells, spermatogonia and testicular carcinoma in situ cells

Gonadoblastoma is defined as a neoplasm containing nests of germ cells and cells resembling Sertoli cells or granulosa cells. Gonadoblastomas arise almost exclusively in dysgenetic gonads. They are associated with an increased risk of developing germ cell tumours. Testicular germ cell tumours in adults are preceded by carcinoma in situ cells, which are characterized by their morphology, by their immunohistochemical expression of placental-like alkaline phosphatase, the proto oncogene c-kit and/or epitopes for the monoclonal antibodies M2A, 43–9F and TRA-1–60, and by their aneuploid DNA content. In order to elucidate if gonadoblastomas are in situ neoplasms from the beginning, showing similarities with carcinoma in situ cells in otherwise normal testes, we investigated the germ cells in gonadoblastomas for their expression of the immunohistochemical markers of carcinoma in situ cells from six patients aged 8¹₂ months to 20 years and 4 months. In addition, the DNA content of the germ cells from five of the six patients was also determined by densitometric measurement on Feulgen stained specimens. The germ cell populations were heterogeneous both within the same patient and between the patients. Expression of the testicular carcinoma in situ markers was detected in specimens from all the patients and germ cells with an aneuploid DNA distribution pattern in accordance with testicular carcinoma in situ cells were detected. However, apparently normal immature germ cells were also present in four of the patients of whom two also had germ cells with a morphology similar to normal spermatogonia. Thus, gonadoblastoma is most likely an in situ germ cell neoplasia from the beginning. It seems probable that the germ cell tumours associated with gonadoblastomas originate from the carcinoma in situ cells inside the gonadoblastoma. Our findings of carcinoma in situ cells in gonadoblastomas from children support the theory that the cells arose prenatally.     

Heterogeneity of expression of immunohistochemical tumour markers in testicular carcinoma in situ: pathogenetic relevance

Testicular carcinoma in situ (CIS) is the precursor of germ cell tumours in adults, except for spermatocytic seminoma. The mechanism of the progression from premalignant CIS to invasive and overt tumours is largely unknown. There are currently two main hypotheses: one is that CIS can progress directly to either seminoma or nonseminoma; according to the other, seminoma is the intermediate stage between CIS and nonseminoma. CIS cells express several tumour antigens, such as placental-like alkaline phosphatase (PLAP), TRA-1-60, or the c-kit proto-oncogene protein product (Kit), which are present to varying degrees in the invasive germ cell tumours. In this study, CIS cells adjacent to either pure or combined tumours were examined by double immunohistochemical staining for simultaneous expression of TRA-1-60 (typical for embryonal carcinoma) and either Kit (expressed by seminomas) or PLAP (found mainly in seminomas, but also in some cases of nonseminoma). Marked differences in the expression of these antigens were found among CIS cells, especially those adjacent to mixed tumours. We conclude that CIS is a phenotypically heterogeneous lesion, and that the CIS cells, despite identical morphology and close spatial localization, may be in different stages of progression. The results lend support to the hypothesis that CIS can progress directly to both seminomatous and nonseminomatous tumours.A preliminary part of this study was presented as a poster at the XXII Meeting of ISOBM, Groningen, The Netherlands, 18–22 September 1994     

Immunohistochemical localization of c-erbB-2 protein and epidermal growth factor receptor in normal surface epithelium,surface inclusion cysts,and common epithelial tumours of the ovary

Summary The c-erbB-2 (HER-2/neu) protein is a membrane glycoprotein growth factor receptor showing molecular homology with the epidermal growth factor receptor (EGFR). We examined the immunohistochemical reactivity of monoclonal antibodies against both of these proteins in normal surface epithelium, surface inclusion cysts, and common epithelial tumours of the ovary. The ovarian tumours were classified as benign (16), borderline malignant (2), and malignant (19). Normal surface ovarian epithelium was weakly positve for both c-erbB-2 protein and EGFR. In surface inclusion cysts, however, the epithelial cells lining the lumen exhibited stronger staining for c-erbB-2 protein, but no staining for EGFR. All 16 benign ovarian tumours and the 2 borderline malignant ovarian tumours were positive for c-erbB-2 protein and negative for EGFR. Of the ovarian carcinomas, 13 of the 19 (68.4%) were positive for c-erbB-2 protein and negative for EGFR, while 4 showed positivity for both c-erbB-2 protein and EGFR. Two cases were negative for both proteins. Expression of both c-erbB-2 protein and EGFR was found in endometrioid carcinoma with squamous differentiation and in clinically advanced poorly differentiated serous carcinomas. Expression of c-erbB-2 protein appears to be increased and that of EGFR is reduced in the early stage of epithelial ovarian oncogenesis. The expression of EGFR with c-erbB-2 protein in ovarian carcinoma is related both to histological differentiation and/or advanced clinical stage.     

Pregnancy: The expression of c-kit protein in human adult and fetal tissues

The c-kit proto-oncogene encodes a tyrosine kinase receptorand is allelic with the dominant white-spotting (W) locus ofthe mouse. In this study we investigated the expression of humanc-kit protein in various adult and fetal human tissues immunohistochemicallyusing anti-human c-kit monoclonal antibody. To discriminatec-kit+ cells from mast cells expressing c-kit, mast cells wereidentified by staining with Toluidine blue. In oogonia, spermatogoniaand skin melanocytes of the fetus and in oocytes of adult ovary,c-kit expression was detected. In adult uterus, c-kit+ cellswere widely distributed in the basal layer of the endometrium,myometrium and cervix, the number and distribution being almostidentical to those of mast cells. In fetal uterus, c-kit+ non-mastcells clustered beneath the epithelium and a few mast cellswere observed in the myometrium and subserosal layer. In bothadult and fetus, c-kit+ non-mast cells were detected withinsmooth muscle layers of the intestine, colon and oesophagus,while mast cells were observed in the mucosal and submucosallayers of these organs. In contrast to mice, no expression ofc-kit protein was detected in the human placenta and decidua.Thus, the distribution of c-kit+ cells in various tissues issimilar but not identical between adult and fetus and betweenhuman and mouse.     

Expression of the C-erbB-3 protein in gastrointestinal tract tumours determined by monoclonal antibody RTJ1

We have examined the expression of the c-erbB-3 protein in a wide range of tumours of the gastrointestinal (GI) tract using immunocytochemical staining. Two antibodies were employed, a polyclonal antibody, 49.3, and a new monoclonal antibody, RTJ1, both raised to a synthetic peptide from the cytoplasmic domain of the human c-erbB-3 protein. The antibodies recognize c-erbB-3 by immunoprecipitation and Western blotting of lysate prepared from a cell line engineered to overexpress the protein. Both antibodies also detect expression of the protein in formalin-fixed, paraffin-embedded human tissues. The RTJ1 monoclonal antibody gave superior staining, showing lower background and more pronounced cell surface immunoreactivity. The c-erbB-3 protein was found in normal epithelial cells throughout the GI tract, in squamous epithelium of the oropharanyx and oesophagus, in the parietal cells of the stomach, and in the surface enterocytes of the small and large bowel. Seventy-six tumours arising at these sites were examined for c-erbB-3 protein expression. Widely varying levels of expression were seen, from absent to intense staining with cell membrane accentuation.     

Endocrine differentiation of extra-pulmonary small cell carcinoma demonstrated by immunohistochemistry using antibodies to PGP 9.5, neuron-specific enolase and the C-flanking peptide of human pro-bombesin

Several recent studies have confirmed the endocrine nature of small cell carcinoma of the lung. In extra-pulmonary sites, small cell 'undifferentiated' carcinomas have classical morphological features similar to their pulmonary counterpart. We therefore investigated, using immunocytochemistry, the possibility that the non-pulmonary neoplasms may also be endocrine in nature. Sections of 29 small cell carcinomas from oesophagus, stomach, larynx, colon and urinary bladder were immunostained using antisera to protein gene product 9.5 (PGP 9.5), neuron-specific enolase (NSE), cytokeratin, leucocyte common antigen and peptides including bombesin, the C-flanking peptide of human probombesin, adrenocorticotrophic hormone, neurotensin, calcitonin and pancreatic polypeptide. All the tumours showed immunoreactivity for at least one of the two general endocrine markers PGP 9.5 and NSE. Twenty-three of the 29 cases were immunoreactive for PGP 9.5, 27 for NSE. All were positive for cytokeratin and negative for leucocyte common antigen. Of the regulatory peptides, immunoreactivity was obtained with antisera to bombesin (one case), the C-flanking peptide of human pro-bombesin (14 cases), adrenocorticotrophic hormone (one case) and calcitonin (three cases). No PGP 9.5-, NSE- or peptide-like immunoreactivity was detected in 25 control tumours from similar sites, including lymphomas and poorly differentiated tumours. These results suggest that non-pulmonary small cell carcinoma has an endocrine character.     

Analysis of c-myc DNA amplification in non-small cell lung carcinoma in comparison with small cell lung carcinoma using polymerase chain reaction

Previous studies of c-myc DNA amplification in lung cancer have focused primarily on analysis of small cell carcinoma or its tumor cell lines. There are few data about c-myc DNA amplification in histological types of lung cancer other than small cell carcinoma. Therefore the present study was conducted to investigate c-myc oncogene amplification in non-small cell lung carcinoma. We studied 46 lung tumor specimens for c-myc DNA amplification (15 adenocarcinomas, 15 squamous cell carcinomas, 6 large cell carcinomas, and 10 small cell carcinomas). Polymerase chain reaction, digoxigenin DNA labeling, and elecrophoresis were utilized to investigate the c-myc copy number in the lung tumor specimens. The c-myc copy number of non-small cell carcinoma ranged from 1.5 to more than 20.0 in adenocarcinoma and squamous cell carcinoma, and from 6.0 to 12.0 in large cell carcinoma. That of small cell carcinoma ranged from 1.8 to 12.0. The c-myc copy number of non-small cell carcinoma was significantly higher that than of small cell carcinoma (Wilcoxon rank sum test, Z=2.06, P=0.040). However, the differences in c-myc copy number among these four histological types were not statistically significant. Amplification of c-myc (more than 4 copies) was observed not only in small cell carcinoma but also in non-small cell carcinoma at similarly high frequency (12/15 in adenocarcinoma and squamous cell carcinoma, 6/6 in large cell carcinoma, and 9/10 in small cell carcinoma). Received: 17 October 2000 / Accepted: 7 May 2001     

Identity of M2A (D2-40) antigen and gp36 (Aggrus, T1A-2, podoplanin) in human developing testis, testicular carcinoma in situ and germ-cell tumours

Testicular germ-cell tumours of young adults are derived from a pre-invasive intratubular lesion, carcinoma in situ (CIS). In a recent genome-wide gene expression screening using cDNA microarrays, we found PDPN over-expressed in CIS compared to normal adult testis. PDPN encodes podoplanin (Aggrus, human gp36, T1A-2), a transmembrane glycoprotein expressed in lymphatic endothelium and various solid tumours. To examine a potential role for PDPN in testicular neoplasms and during testicular development, we investigated its expression pattern during the development of human testis and in a series of testicular CIS, gonadoblastoma and overt germ-cell tumours. We established by RT-PCR and by immunohistochemistry with a gp36 antibody that PDPN mRNA and the protein product were expressed in testes with germ-cell neoplasms but not in the normal adult testis. We also found gp36 expression in early foetal gonocytes and immature Sertoli cells, similar to the expression pattern of M2A antigen, a previously identified marker for CIS and seminoma. This reinforced our previous proposal that M2A (D2-40) antigen was identical to gp36 (podoplanin, Aggrus, T1A-2). Our findings also suggest that podoplanin has a function in developing testis, most likely at the level of cell–cell interactions among pre-meiotic germ cells and immature Sertoli cells.     

Cytomorphological,cytogenetic, and molecular biological characterization of four new human renal carcinoma cell lines of the clear cell type

Four new permanent cell lines (RCC-A, -B,-C, and -D) derived from different human renal cell carcinomas of the clear cell type were established in tissue culture. The cell lines displayed characteristic differences in cell size and shape, which allowed individual identification by phase contrast microscopy. Ultrastructurally, the cell lines exhibited varying amounts of cytoplasmatic glycogen and lipid. Immunohistochemistry revealed co-expression of vimentin and cytokeratin in all cell lines. The mean population doubling time ranged from 27 h (RCC-A) to 104 h (RCC-D). RCC-B and -C cells produced slowly growing tumours after heterotransplantation into nude mice, whereas RCC-A and RCC-D cells were non-tumorigenic. The modal chromosome number was either near-diploid (RCC-A, -B, and -C) or near triploid (RCC-D). Clonal abnormalities affecting the short arm of chromosome 3 were seen in all cell lines. Northern blot analysis revealed no expression of the proto-on-cogenes c-fos, c-ros, and c-mos, whereas c-Ki-ras expression was observed in all cell lines. Expression of c-myc was observed in RCC-A, RCC-B, and RCC-D cells, whereas c-raf expression could be detected in RCC-B and RCC-D. Tumour suppressor gene p53 mRNA was observed in the cell line RCC-D.     

Characterization of various types of mast cells derived from model mice of familial gastrointestinal stromal tumors with KIT-Asp818Tyr mutation

Sporadic mast cell neoplasms and gastrointestinal stromal tumors (GISTs) often have various types of somatic gain-of-function mutations of the c-kit gene which encodes a receptor tyrosine kinase, KIT. Several types of germline gain-of-function mutations of the c-kit gene have been detected in families with multiple GISTs. All three types of model mice for the familial GISTs with germline c-kit gene mutations at exon 11, 13 or 17 show development of GIST, while they are different from each other in skin mast cell number. Skin mast cell number in the model mice with exon 17 mutation was unchanged compared to the corresponding wild-type mice. In the present study, we characterized various types of mast cells derived from the model mice with exon 17 mutation (KIT-Asp818Tyr) corresponding to human familial GIST case with human KIT-Asp820Tyr to clarify the role of the c-kit gene mutation in mast cells. Bone marrow-derived cultured mast cells (BMMCs) derived from wild-type mice, heterozygotes and homozygotes were used for the experiments. Immortalized BMMCs, designated as IMC-G4 cells, derived from BMMCs of a homozygote during long-term culture were also used. Ultrastructure, histamine contents, proliferation profiles and phosphorylation of various signaling molecules in those cells were examined. In IMC-G4 cells, presence of additional mutation(s) of the c-kit gene and effect of KIT inhibitors on both KIT autophosphorylation and cell proliferation were also analyzed. We demonstrated that KIT-Asp818Tyr did not affect ultrastructure and proliferation profiles but did histamine contents in BMMCs. IMC-G4 cells had an additional novel c-kit gene mutation of KIT-Tyr421Cys which is considered to induce neoplastic transformation of mouse mast cells and the mutation appeared to be resistant to a KIT inhibitor of imatinib but sensitive to another KIT inhibitor of nilotinib. IMC-G4 cells might be a useful mast cell line to investigate mast cell biology.     

Immunocytochemical localization of c-erbB-2 protein in transitional cell carcinoma of the urinary bladder

The level of expression and cellular localization of the c-erbB-2 gene product in transitional cell carcinoma of the urinary tract is controversial. Analysis of the c-erbB-2 gene structure and comparison of its expression in the same cells by Southern, Northern and immunoblotting, and by immunocytochemistry minimize the errors of interpretation inherent in one technique. Such a ‘correlative study’ has been performed on tumours from 82 patients, c-erbB-2 gene amplification was detected in 14 per cent of initial tumours and was associated with grade (.P< 0·001). Raised levels of mRNA were seen in those tumours with increased gene copy number and in 13 per cent of the remainder. Immunoblotting detected the expected 185 kD immunoreactive protein and a 155 kD piotein associated with high gene copy number. Immunocytochemistry localized c-erbB-2 immunoreactivity to the cell membrane and cytoplasm, and the latter predominated. Four antibodies to c-erbB-2 (AB-3, 21N, pAb 1, and NCL CB11) were compared on contiguous sections of the same tumour and showed the same pattern of immunoreactivity. Similarly, analyses carried out in three independent laboratories identified the same cellular localization. Membrane and cytoplasmic immunoreactivity was demonstrated in all tumours with gene amplification or increased mRNA levels and in 40 per cent of the remaining tumours. We showed that immunocytochemistry requires careful standardization of techniques and quantitation between different groups. However, despite variations in the intensity of immunoreactivity, the total number of positive cells remained constant. Therefore quantitation must be based on the number of positive cells and, ideally, their immunoreactive content relative to normal and positive tissue controls.     

Suppression of serum-induced c-jun expression by activated Ki-ras in human colon cancer cells

Summary Through gene-targeting, we have established human colon cancer cell lines, HK2-6 and HKe-3, with and without activated Ki-ras, respectively, derived from a human colon cancer cell line HCT116, and we have reported that activated Ki-ras is involved in the deregulation of c-myc expression. To further examine the relation between Ki-ras-mediated signals and other immediate early genes, c-jun was analyzed on these cells stimulated by serum. Rapid and strong induction of c-jun was observed in HKe-3, but not in HCT116 or HK2-6. To elucidate the regulatory mechanisms of c-jun expression by Ki-ras, protein kinase C (PKC) and c-Raf were examined at serum-starved and serum-stimulated conditions. Phosphorylations of c-Raf were same among these cells, however, the cytosolic PKC activity in HKe-3 was two times higher than that in HCT116 on serum-starved and serum-stimulated conditions. These results suggested that serum responsiveness of c-jun may be suppressed by activated Ki-ras through PKC rather than c-Raf pathway in colon cancer cells.     

Non-receptor tyrosine kinase Etk is involved in the apoptosis of small cell lung cancer cells

Epithelial and endothelial tyrosine kinase (Etk), also known as Bmx (bone marrow X kinase) plays an important role in apoptosis of epithelial cells. The goal of this study was to investigate whether Etk is involved in apoptosis of small cell lung cancer (SCLC) cells and correlated with the expression levels of apoptosis-associated proteins such as Bcl-2, Bcl-X_L and p53. One hundred and seventy-one cases of lung cancer specimens including seventy-one SCLCs and one hundred NSCLCs were immunostained for Etk, Bcl-2, Bcl-X_L and p53. Parental SCLC H446 cell line, and its subline (H446-Etk) that overexpresses Etk, were used to study the role of Etk in apoptosis induced by doxorubicin. It was found that high expression of Etk occurs in 74.6% of SCLC cases, but only in 40% of NSCLC cases, and there is marked difference in the expression levels of Bcl-2, Bcl-X_L and p53 between Etk-positive and Etk-negative SCLC cases. Furthermore, the levels of Bcl-2 and Bcl-X_L significantly increased in H446-Etk cells than that in H446 cells after doxorubicin treatment, and were positively associated with Etk expression. However, p53 did not correspond with Etk expression although its expression decreased greatly with apoptosis both in H446-Etk and H446 cells. After doxorubicin treatment, the cell viability was significantly higher in H446-Etk cells than in parental H446 cells. Downregulation of Etk by Etk siRNA sensitized H446 cells to doxorubicin. Our results indicate that upregulation of tyrosine kinase Etk may be a new mechanism involved in protection of SCLC cells from apoptosis. Bcl-2 and Bcl-X_L but not p53 may contribute to doxorubicin-induced apoptosis through Etk pathway.     

MOC-31/Ep-CAM immunoreactivity in Merkel cells and Merkel cell carcinomas

AIMS: To evaluate the monoclonal antibody MOC-31 in Merkel cell carcinomas and normal Merkel cells. Merkel cell carcinoma is a rare and aggressive tumour that occurs mainly in elderly individuals. The histological diagnosis of Merkel cell carcinoma can be difficult because it looks similar to other small blue cell tumours, particularly skin metastases of small-cell lung carcinomas. This antibody recognizes the epithelial cell adhesion molecule (Ep-CAM), that has been assigned to the small cell lung cancer cluster 2 of antibodies. To the best of our knowledge, immunostaining for MOC-31/Ep-CAM has not been previously described in Merkel cells or Merkel cell carcinomas. METHODS AND RESULTS: Thirty-one cases of Merkel cell carcinoma and three samples of normal human fingertip were selected to analyse the expression of MOC-31/Ep-CAM by immunohistochemistry. A high number of Merkel cell carcinomas (21/31, 67.7%) showed intense and readily interpretable positivity. Immunostaining was diffuse or focal and always localized to the plasma membrane. Normal Merkel cells of human fingertip also showed plasma membrane immunoreactivity for MOC-31/Ep-CAM. CONCLUSION: The demonstration of positivity for MOC-31/Ep-CAM in Merkel cell carcinomas precludes the use of this immunohistochemical marker to distinguish between tumours and skin metastases of small-cell lung carcinoma.     

Significant high expression of cytokeratins 7, 8, 18, 19 in pulmonary large cell neuroendocrine carcinomas,compared to small cell lung carcinomas

The aim of the present study was to clarify protein profiling in small cell lung carcinoma (SCLC) and pulmonary large cell neuroendocrine carcinoma (LCNEC). The proteomic approach was used, and involved cell lysate from two cell lines (N231 derived from SCLC and LCN1 derived from LCNEC), with 2‐D gel electrophoresis (2‐DE). In the present study, 25 protein spots with greater than twofold quantitative differences between LCN1 and N231 cells on 2‐DE gels were confirmed. Within the 25 identified proteins, cytokeratins (CK) 7, 8, 18 and 19 were upregulated in LCN1 cells compared with N231 cells. The expression of CK7, 8, 18, and 19 was further studied on immunohistochemistry with 81 formalin‐fixed and paraffin‐embedded pulmonary carcinomas, which included 27 SCLC, 30 LCNEC, 14 adenocarcinomas, and 10 squamous cell carcinomas. Although the expression of CK7, 8, 18, and 19 was observed in all histological types, the mean immunostaining scores of CK7, 8, 18, and 19 were significantly higher in LCNEC than in SCLC (P < 0.001, P < 0.001, P < 0.01 and P < 0.001, respectively). These data suggest that the biological characteristics of LCNEC and SCLC may be different and the expression of CK may serve as differential diagnostic markers.     

Investigation of the genes for RET and its ligand complex,GDNF/GFRα-1, in small cell lung carcinoma

RET is a receptor tyrosine kinase expressed in neuroendocrine cells and in tumors of these cell types. RET activation may be mediated by a ligand complex comprising glial cell line-derived neurotrophic factor (GDNF) and GDNF family receptor alpha-1 (GFRα-1). Activating RET mutations are found in the inherited cancer syndrome multiple endocrine neoplasia type 2 and in a subset of the related sporadic tumors, medullary thyroid carcinoma and pheochromocytoma, both being derived from neuroendocrine tissues. In one small study, mutations were identified in another tumor with neuroendocrine features, small cell lung carcinoma (SCLC). To determine whether RET mutations contribute to the pathogenesis of SCLC, we examined a panel of 54 SCLC cell lines. No mutations were identified in RET exons 10, 11, and 13–16, regions previously implicated in SCLC or other neuroendocrine tumors. We further examined the expression pattern of RET and the genes encoding the components of its ligand complex GDNF and GFRα-1 , in 21 SCLC lines by using RT-PCR. Although we found no consistent pattern of expression for these three genes, RET was expressed in 57% of SCLC lines. Thus, although RET mutations appear unlikely to be an important step in the tumorigenesis of SCLC, the frequent expression of this gene suggests that RET may have a mitogenic role in a subset of SCLC cell lines. Genes Chromosomes Cancer 21:326–332, 1998. © 1998 Wiley-Liss, Inc.     

Expression of c-myc protein is related to cell proliferation and expression of growth factor receptors in transitional cell bladder cancer

Archival biopsy specimens from transitional cell bladder tumours (n=185) were analysed immunohistochemically for expression of c-myc protein. The results were compared with compared with histopathological and clinical parameters and survival. Forty-three per cent of the tumours were negative for c-myc protein and weak, moderate, or strong cytoplasmic expression was found in 34, 14, and 9 per cent of cases, respectively. Nuclear positivity for c-myc protein was detected in 35 per cent of tumours and nuclear opositivity was related to overexpression of c-erb B-2 (P=0.01) and a high proportion of nuclei were also positive for p53 oncoprotein (p<0.05). Cytoplasmic expression of c-myc protein was related to histological grade (P=0.005), papillary status (P=0.007), the S-phase fraction (P=0.008), the mitotic index (P=0.021), overexpression of epidermal growth factor receptor (P=0.045), and c-erb B-2 (P=0.17). Expression of c-myc protein was not significantly related to the progression of tumours and it had no prognostic value in survival analysis. Independent predictors were the T-category (P<0.001), papillary status. (P=0.001), and S-phase fraction (P=0.061). The results show that while c-myc gene product participates in growth regulation of human bladder cancer cells, it has no independent prognostic significance.