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Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder caused by germline mutations in the folliculin gene (FLCN). It is characterized by skin tumors, multiple lung cysts, and renal tumors. Active genetic testing and appropriate periodic examinations of family lines of patients with BHD syndrome have not been widely performed. In this report, we present our experience regarding the diagnosis of asymptomatic family members with BHD syndrome. The proband was a 65-year-old female with a family history of colorectal cancer and spontaneous pneumothorax that affected her father. Computed tomography revealed an approximately 10 cm-sized tumor protruding from the upper pole of the left kidney, a buried tumor approximately 1.5 cm in length in the right kidney, and multiple pulmonary cysts. The patient underwent laparoscopic radical left nephrectomy. Pathological examination indicated that the resected tumor was a chromophobe renal cell carcinoma. After the surgery, there was no evidence of local recurrence or metastasis. The size of the tumor in the right kidney was monitored, but it did not increase. On FLCN genetic examination, targeted next generation sequencing revealed a partial deletion of exon 14, thus confirming the diagnosis of the patient to be BHD syndrome that caused the previously unreported pathogenic variant. Three years after the surgery, we conducted genetic counseling for the proposita and her three children. Genetic examination, performed at the request of the second daughter, confirmed that she carried the same genetic variant as her mother. This diagnosis prompted the second daughter to begin managing her health via periodic imaging tests.

The tumor microenvironment plays a key role in cancer progression, drug resistance, metastasis, etc. To establish a new therapeutic strategy based on control of the tumor microenvironment, I have developed a lipid nanoparticle (LNP)-based in vivo small interfering RNA (siRNA) delivery system equipped with a targeting ligand. First, I established an LNP that induces membrane fusion in response to acidification after internalization by cells using the original pH-sensitive cationic lipid YSK05. A modification of polyethylene glycol to YSK05-containing LNPs allowed significant gene silencing in the human renal cell carcinoma model. Then, I attempted to establish a tumor vasculature-targeting LNP because the vasculature is responsible for the tumor microenvironment. Cyclic RGD peptide is known to be a ligand against integrin αVβ3, which is highly expressed on tumor endothelial cells (TECs). Optimized cyclic RGD peptide-modified LNP (RGD-LNP) suppressed gene expression in TECs to 50%. The inhibition of vascular endothelial cell growth factor receptor 2 (VEGFR2), which is a dominant factor in angiogenesis, by the injection of RGD-LNP significantly delayed tumor growth. Finally, I examined the effect of RGD-LNP on the tumor microenvironment. The suppression of VEGFR2 increased pericyte coverage and endothelial junctions, which indicate maturation of the vasculature. In RGD-LNP-treated mice, systemically administered nanoparticles encapsulating doxorubicin were distributed in a larger area than in untreated mice. Moreover, the therapeutic effect of doxorubicin-loaded liposomes was significantly enhanced by RGD-LNP. In conclusion, I succeeded in developing a new therapy based on regulation of the tumor microenvironment.

A 72-year-old woman was referred to our hospital with complaints of macro-hematuria. The radiographic evaluation including computed tomography (CT) and magnetic resonance imaging (MRI) suggested it to be renal cell carcinoma (RCC) in her right kidney. She underwent laparoscopic nephrectomy. We diagnosed her with renal cell carcinoma associated with Xp11.2 translocation/TFE3 gene fusion, based on pathological findings and break apart of transcription factor E3 (TFE3)by fluorescence in situ hybridization. She was free of recurrence at 8 months postoperatively.

Clear cell renal cell carcinoma(ccRCC)with a von Hippel-Lindau(VHL)gene alteration is the most frequent histological phenotype of renal cell cancer(RCC). This gene alteration suppresses ubiquitination and increases hypoxia-inducible factor (HIF)-alpha accumulation, resulting in the upregulation of HIF-alpha target genes, which in turn leads to hypervascular tumor formation. Sorafenib, sunitinib, axitinib, and pazopanib are kinase inhibitors that block vascular endothelial growth factor receptor(VEGFR). Other families of targeted drugs for RCC are antibodies against VEGF, such as bevacizumab, and mammalian target of rapamycin inhibitors, such as temsirolimus and everolimus, which exert anti-angiogenic effects by downregulating HIF-alpha expression. Thus, the newly developed targeted drugs for RCC are classified as anti-angiogenic drugs. However, the frequency of a sustained complete response(CR)in response to these drugs is quite low, indicating that the exploration of other modalities of treatment, besides anti-angiogenic drugs, is warranted.

Therapeutic options for metastatic renal cell carcinoma (mRCC) have been evolving rapidly, with the approval of a variety of effective molecular targeted agents. With increasing choices of treatment modalities that may potentially improve survival, it is of great clinical benefit to be able to predict the clinical outcome of mRCC patients treated with molecular targeted agents. In addition, predictors of severe adverse events are warranted since those molecular targeted agents present a toxicity profile that is very different from that of conventional chemotherapeutic agents. Their use requires knowledge of a large number of possible adverse events. Although validation of previous criteria, such as the Motzer criteria, is important, a further implementation of RCC molecular biology knowledge would be ideal for finding a new predictive marker in this new age of molecular targeted therapies. The most prominent biomarker is vascular endothelial-derived growth factor (VEGF). Those patients who presented low serum VEGF showed prolonged progression-free survival when treated with sunitinib. Genetic markers such as single nucleotide polymorphisms (SNPs) are continuously being validated as markers, predicting molecular targeted therapy response as well as severe adverse events. The identification of biomarkers that predict response or severe adverse events from molecular targeted agents will aid in patient selection and guide therapy development.

Xp11.2/TFE3 translocation renal cell carcinoma (RCC), a recently classified distinct subtype, is a rare tumor that usually affects children and adolescents. The morphology and biological behavior are not widely recognized, Xp11.2 translocation RCC is suggestive of early metastases despite the small tumor size. The definitive diagnosis requires the evidence of several different reciprocal translocations involving the TFE3 gene located on chromosome Xp11.2. Here, we present a case of Xp11.2 translocation RCC in an 18-yearold male. He was referred to our hospital because of a right renal tumor with macroscopic hematuria and right flank colic. The radiographic evaluation including magnetic resonance imaging (MRI) suggested it to be a typical papillary renal cell carcinoma or benign renal tumor. He underwent laparoscopic nephrectomy against the repeat symptom in spite of small tumor (3.5 cm in diameter). The immunohistochemical study revealed nuclear staining for TFE3 protein in the cancer cells. The urologic and radiologic outcomes were satisfactory after more than 1 year of follow-up.

In this review, pathogenesis and genetic alterations of urologic malignancies and their therapeutic target molecule are summarized briefly. In bladder cancer, only a little has been revealed. Loss of heterozygosity of 9p/q is frequently observed in low grade, low stage tumors. In invasive or carcinoma in situ tumors, alteration of p53 and Rb tumor suppressor gene is frequently found. In prostate cancer, the process of carcinogenesis from normal epithelium to cancer hypothesized by Nelson et al. (N Engl J Med 24; 349 : 366-381) seems to be logic. Androgen independency of tumor cells is associated with androgen receptor gene mutation and amplification, however, the mechanism is not well clarified. It is a turning point, therapeutic strategy is changing from cytokine immunotherapy to molecular targeting therapy in metastatic renal cell carcinoma. The pathway from growth factors such as vascular endothelial growth factor and platelet derived growth factor, and their receptors to mTOR is a central controller of tumor angiogenesis and proliferation.

Renal Cell Carcinoma (RCC) is classified into six cell pathological types by the Thoenes classification (5). Deletion of DNA (loss of heterozeigosity: LOH) is seen with a high frequency in human RCC of all 6 types at chromosome 3p 14-25. The presence of at least three tumor suppressor genes at this domain has been pointed out. The VHL gene, one of the tumor suppressor genes (TSG), was identified in 1993 at chromosome 3p25-26 as the gene responsible for VHL disease. As a consequence, it was demonstrated that inactivation of the von Hippel-Lindau (VHL) gene is responsible for sporadic clear cell RCC. Activating mutations of c-Met receptor type tyrosine kinase has been demonstrated in papillary renal cell carcinoma families. Possible involvement of the FHIT tumor suppressor gene, located at the fragile site (FRA3B) of chromosome 3p14, has been detected in sporadic RCC. Recently, methylation of RASSF1A at chromosome 3p21.3 was pointed out in sporadic RCC. Thus, it has become apparent that chromosome 3p14-25 3 has possible TSGs for RCC. Furthermore, it was pointed out in April that germline mutation of fumarate hydratase, a Krebs cycle enzyme (FH), is present in multiple cutaneous and uterine leiomyomatosis families that develop papillary RCC. The functional significance in these genes for the development of RCC is still not apparent, except for the VHL gene. Thus, there is still a long way to go before we find all responsible TSGs in all pathological subtypes in sporadic RCC.

MN/CA IX is a carbonic anhydrase (CA) isoenzyme expressed in normal alimentary tract in a tissue-specific manner. This antigen is activated in the majority of renal cell carcinomas (RCC) but not in normal kidney tissues. Although the exact role of CA activity in carcinogenesis and metastasis has not been established, MN/CA9 has been suggested to be implicated in acidification of extracellular milieu surrounding the cancer cells and thus create a microenvironment conductive to tumor growth and spread. Mutations in the von Hippel-Lindau (VHL) gene cause the familial syndrome and are also found in the majority of sporadic RCC. Wild-type VHL was recently described to down-regulate MN/CA9 in RCC cell lines, and the molecular mechanism of MN/CA9 and VHL in renal carcinogenesis is of interest. To investigate the mechanism of MN/CA9 activation in RCC, we examined the methylation status of this gene in RCC cell lines and human tissue samples and found that hypomethylation in the promoter region may play an important role in the expression of MN/CA9. RT-PCR analysis of blood samples from RCC patients revealed the presence of circulating MN-positive cells in the blood. This antigen may be a potential therapeutic target as well as diagnostic marker for RCC. Therefore, we are currently investigating whether or not MN/CA IX peptide could be an appropriate molecule for use antigen specific immunotherapy on RCC patients.

Birt-Hogg-Dubé (BHD) syndrome is a rare dermatological condition appearing with an autosomal dominant mode of inheritance. It was first reported in 1977 by Birt et al. and 28 cases have been reported since then. BHD syndrome is characterized by asymptomatic dome-shaped, skin-colored papules on the face and upper trunk. Recently, various neoplasms have been reported to associate with BHD syndrome, including three familial and one sporadic cases of renal tumors. We report another sporadic case with renal tumor. A 53-year-old woman complained of gross hematuria and visited our institute on November 1996. She visited the Department of Dermatology, Wakayama Medical College because of skin lesions on the face and upper trunk at her age of 44. These skin lesions were present since her mid twenties. Her daughter also had similar skin lesions and visited the same Department. There was no family history of renal tumor. The patient was diagnosed to have a right renal tumor, and radical nephrectomy was performed. Pathological diagnosis was renal cell carcinoma, papillary type. She underwent interferon injection therapy postoperatively, but died because of lung metastases on April 1997. This is the first reported case of renal tumor occurring in BHD syndrome in Japan.

The oncogenic mechanisms of renal cell carcinoma(RCC) are becoming elucidated with recent advances in molecular biology. von Hipple-Lindau disease(VHL) tumor suppressor gene is mutated and inactivated frequently in clear cell type RCCs. The VHL protein forms a complex which shows a ubiquitin ligase activity. The lost of the ubiquitin ligase activity of VHL protein may be a key step for clear cell tumorigenesis. Papillary renal cell carcinomas are caused by activating mutation in the tyrosine kinase domain of the MET gene. This tumorigenic pathway is regulated by c-Src. Immunogene therapies have been started for the patients with advanced RCC. The information based on microarray and Serial Analysis of Gene Expression(SAGE) will provide novel diagnosis and therapy which focus on the tumorigenic mechanism of RCC in the near future.

Chromophobe cell renal carcinoma is an uncommon subtype of renal cell carcinoma and the number of cases studied is still limited in Japan. We here report a case of chromophobe cell renal carcinoma in a 41-year-old Mexican male. He visited our branch hospital with the symptom of upper abdominal pain. Ultrasound examination showed a left renal mass. He was admitted to our hospital for treatment of a left renal mass. Radiological examinations revealed a hypervascular tumor in the left kidney. Under the clinical diagnosis of possible renal cell carcinoma, left radical nephrectomy was performed. This tumor was diagnosed as chromophobe cell renal carcinoma with a microscopic examination of H & E stained specimens, histochemical staining using Hale's colloidal iron and an ultrastructural study.

Background: Clinical significance of polymorphism of tumour necrosis factor (TNF) genes encoded on the short arm of the 6th chromosome in the patients with renal cell carcinoma (RCC) has not been evaluated well so far. We studied on the TNF genes polymorphism of RCC focusing on the relationship between the genetic polymorphism and the prognosis., Methods: The subjects were seventy-three patients with RCC treated at our hospitals during the past 20 years. The genomic DNA was examined by the methods of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) from frozen peripheral blood of these patients. The items examined were the genetic polymorphisms of TNF-alpha (alpha 1, alpha 2) and TNF-beta (beta 1, beta 2), and we tried to study on the prognostic outcome of RCC based upon each zygote of TNF., Results: 1) The proportion of TNF-alpha and TNF-beta polymorphisms: We observed TNF-alpha 1/1 homozygote in 71 patients (97.3%). As to TNF-beta polymorphism, we observed TNF-beta 2/2 homozygote in 33 patients (45.2%), TNF-beta 1/2 heterozygote in 31 (42.5%) and TNF-beta 1/1 homozygote in 9 (12.3%). The proportion of TNF-beta polymorphism was almost the same as that of healthy Japanese. 2) PROGNOSIS: Regarding the 17-year survival, all patients with TNF-beta 1/1 homozygote were alive, and we observed a significantly favourable prognosis in the patients with TNF-beta 1/1 homozygote compared with other zygotes of TNF-beta polymorphism. The reasons for these favourable prognosis were thought that the patients with TNF-beta 1/1 homozygote showed much lower stage and/or grade than those of other zygotes., Conclusion: We conclude that the TNF-beta gene polymorphism is a useful marker for understanding the prognosis of RCC and a part of cellular immunity related to the tumour and its host.

Seventy one patients with renal cell carcinomas were examined for a variety of markers associated with tumor malignancy: nuclear DNA ploidy, AgNORs, PCNA and c-erbB-2. Usefulness of the markers in predicting the prognosis was studied by analyzing the relationship between each of these markers and the prognosis of the patients with renal cell carcinomas. DNA ploidy was analyzed by flow cytometry. AgNORs were stained by the silver colloid method. PCNA and c-erbB-2 were detected by immunohistochemistry. In all the patients examined, DNA ploidy, AgNORs, PCNA and c-erbB-2 were significant predictors of the prognosis. Of the patients with grade 2 carcinomas, the survival rate was significantly higher in the patients with the PCNA-positive cells of lower than 35.0% than in those with the positive cells of more than 35.0%. The patients without the expression of c-erbB-2 exhibited a significantly higher survival rate than those with the expression. In the patients with grade 2 carcinomas, however, neither DNA ploidy nor AgNoRs was a significant predictor of the prognosis. These findings suggest that PCNA and c-erbB-2 provide more accurate information than the others to understand the biological characteristics of the grade 2 carcinomas and are useful in predicting the prognosis of the patients with grade 2 renal cell carcinomas.

Identification of numerical abberations of chromosome #3, #7, #11, #17, #18, X and Y were evaluated using biotinylated probes specific for the alpha satellite region on paraffin embedded sections from formalin-fixed materials of renal cell carcinoma utilizing in situ hybridization (ISH). The copy number of each chromosome did not show any correlation with tumor grade, clinical (Robson) stage, pT, pN, pV or the presence of metastasis. Copy numbers of #3 chromosome were highly correlated with the patient prognosis (p < 0.01). Since numerical abberations of #3 chromosome are suggested to be correlated with the prognosis of renal cell carcinoma, this technique can now be applied to the detection of biological activity in renal cell carcinoma.

Two renal cell carcinoma (RCC) cell lines, JMSU2 and JMSU3, derived from the primary sites of mixed cell type and spindle cell type RCC, respectively, have been established and maintained for 31 and 22 months. Karyotypic analysis revealed human karyotypes with modal numbers of 84 and 55, respectively. Consistent chromosomal abnormalities were 1p+, 3p-, 6q- or 8p- in the JMSU2 cells and 1p-, inv (5p + q-) or loss of sex chromosome in the JMSU3 cells. Electron microscopy revealed abundant glycogen granules, lipid droplets and microvilli. The JMSU3 cells transplanted to nude mice produced tumors with a spindle cell pattern similar to that of the original tumor. High concentrations of cytokines, such as interleukin-6 (145,000 pg/ml), interleukin-8 (35,300 pg/ml) and granulocyte-colony stimulating factor (6,340 pg/ml), were detected in the culture supernatant of the JMSU3 cells. Interleukin-1 beta (IL-1 beta) dose-dependently inhibited the proliferation of the JMSU2 and JMSU3 cells in culture. Tumor cytotoxic factor/hepatocyte growth factor (TCF/HGF) dose-dependently enhanced JMSU3 cell proliferation, but suppressed JMSU2 cell proliferation. These findings suggest that IL-1 beta and TCF/HGF have regulatory roles in the proliferation of RCC.

We studied the correlation between microvessel density (MD) and gene expression of vascular endothelial growth factor (VEGF), its receptor (KDR) and basic fibroblast growth factor (bFGF) in 30 human renal cell carcinomas (RCCs). Gene expression assessed by Northern blot hybridization was quantified by a densitometer. Tissue localization of VEGF was assessed by anti VEGF-antiserum staining. MD was measured in tumor tissue sections stained for factor VIII-related antigen. VEGF gene was overexpressed in 18 (60%) of 30 RCCs. Cytoplasm of the tumor cells was stained positively for VEGF staining. Intratumoral expression of KDR gene significantly correlated with that of VEGF gene. bFGF gene expression was very weak, and no tumor overexpressed this gene. The MD in the tumors significantly correlated with the expression intensity of VEGF gene and KDR gene. These results suggest that VEGF contributes to neovascularization of RCCs through a paracrine mechanism, and could be used as a relevant indicator of angiogenesis.

Background: Renal cell carcinomas (RCCs) develop in 8-63% of von Hippel-Lindau disease (VHL) patients, and loss of 3p segments, chromosome aberrations found in 90% of sporadic RCCs, has also been observed in RCCs associated with VHL. In fact, comparative analysis showed that the chromosome aberrations in RCCs associated with VHL are similar to those found in sporadic RCCs. VHL patients have the whole spectrum of tumors from small early lesions to large ones in the same kidney, providing a unique opportunity to analyze tumors in different stages of development. Subsequently deoxyribonucleic acid (DNA) content in RCCs of VHL patients was examined and correlated to their tumor size to gain some insight in the progression of sporadic RCCs., Methods: From 1988 to 1991, we have experienced 6 cases of RCCs associated with VHL who underwent partial or radical nephrectomy. A total number of 52 paraffin-embedded samples from 33 RCCs from 6 patients with VHL was analyzed by flow cytometry., Results: The sizes of tumors ranged from 0.2 to 8.2 cm. DNA aneuploid patterns demonstrated in none of 9 tumors less than 1.6 cm, 4 of 14 tumors (29%) as large as 1.6 to 2.5 cm, and 5 of 10 tumors (50%) larger than 2.5 cm (p < 0.05). Twelve tumors less than 1.8 cm showed DNA diploid, so the smallest size of aneuploid tumors was 1.8 cm., Conclusion: These data suggest that DNA ploidy change (diploid to aneuploid) in RCCs probably takes place as tumors grow approximately 1.8 cm in size.

We investigated the reduction in the accuracy of DNA replication and repair (i.e. genetic instability) in urinary tract malignancy using microsatellite regions. The subjects were 17 patients with renal cell carcinoma and 14 with bladder tumors. After polymerase chain reaction (PCR) was performed with FITC-labeled primers, CA repeats were analyzed. The primers used were D2S123, D3S1067, and TP53. Tissue positive for all 3 primers was considered to show a replication error (RER). Genetic instability was found in 5 out of 17 patients with renal cell carcinoma (29%) and 3 out of 14 patients with bladder tumors (21%). Among the bladder tumor patients, 2 were positive for only TP53 and 1 was considered to have RER. Among the patients with renal cell carcinoma, one was positive only for D3S1067 and the other 4 were considered to have RER.

Renal cell carcinoma is derived from renal tubule, blastematous tubule or nephrogenic rest. Most common sporadic clear cell tumors have structural changes of the chromosomes, such as the cancer suppressor genes at 3p14, 3p21 or 3p25 (VHL gene). Cytogenetically, clear cell tumor (non papillary tumor) initially develops as a cancer, while papillary tumor progresses sequentially from atypical tubule to adenoma to carcinoma. Papillary tumors demonstrate numeric changes in the chromosomes. Renal cell carcinoma complicated with acquired renal cysts in dialysis patients tends to develop from papillary tumor. However, the exact mechanism inducing these genetic changes remains to be clarified.

In the previous studies, epidermal growth factor (EGF) was found to elevate glycolipid sulfotransferase activity in a human renal cell carcinoma cell line, SMKT-R3. To elucidate whether Ras is involved in the signal transduction pathway from EGF to the expression of the sulfotransferase, effects of EGF and a tyrosine kinase inhibitor on the sulfotransferase activity were investigated in renal cancer cells stably expressing activated Ras. SMKT-R3 cells were transfected with a plasmid carrying v-H-ras (pv-H-ras). As a control, SMKT-R3 cells transfected with a mutant v-H-ras, in which glycine at position 15 was replaced by valine (pG15V), was used. The expression of v-H-ras in transfected cells was examined by RT-PCR analysis. Two clones transfected with pv-H-ras, named A1 and A6, and two clones transfected with pG15 V, termed B1 and B4, were found to express v-H-ras and G15V genes, respectively, and employed in the following experiments. Though EGF elevated the activity of glycolipid sulfotransferase in B1, B4 and SMKT-R3 cells, it did not change the activity levels in A1 and A6 cells. This result suggested that since the signal from Ras was saturated in the cells expressing activated Ras, the cells did not respond to the stimulation by EGF. To know the association between tyrosine kinase of EGF receptor and Ras, the effect of Genistein, a specific inhibitor of tyrosine kinase, on the sulfotransferase activity was examined in these cell lines. Genistein reduced the activity of glycolipid sulfotransferase in B1, B4, and SMKT-R3 cells, whereas it hardly affected the sulfotransferase activity in A1 and A6 cells. This result indicated that the signal from tyrosine kinase was dissociated from the regulation of the sulfotransferase activity in the cells expressing activated Ras. These observations suggest that Ras is involved in the signal transduction from EGF to the expression of glycolipid sulfotransferase activity in SMKT-R3 cells and acts in the downstream of tyrosine kinase of EGF receptor.

We examined serum levels of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) in 32 patients with renal cell carcinoma. Serum IL-1 beta was elevated in only one patient (3.1%), IL-6 in 17 (53.1%) and TNF-alpha in 6 (18.8%). Positive rate of serum IL-6 increased with progression of the stage (stage I and II: stage III and IV = 14.3%: 88.9%, p < 0.0001) and was higher for rapid growth type (slow and intermediate growth type: rapid growth type = 36.4%: 90.0%, p < 0.005). However, increased serum levels of IL-1 beta and TNF-alpha were not correlated with stage, grade, or growth type. Gene expressions of IL-1 beta, IL-6, IL-6 receptor and TNF-alpha in the tissues resected from 15 patients with renal cell carcinoma and 3 cell lines established from renal cancers were evaluated by Northern blot analysis. Gene expression of IL-1 beta and TNF-alpha were not detected in any of the tissues or cell cultures, but highly enhanced expressions of IL-6 were revealed in two tumor specimens (13.3%, 1 rapid and 1 slow growth type) and 2 of 3 cell lines. Expressions of IL-6 receptor in tissues and cells were weak. These results suggest that IL-6 plays an important role in the progression of renal cell carcinoma, and that measurement of IL-6 in patients with renal cell carcinoma may be useful clinically.

DNA ploidy was investigated using flow cytometry on fresh surgical specimens from 116 cases with renal cell carcinoma. DNA diploid tumor was observed in 39 cases (33.6%) and aneuploid in 77 cases (66.4%). DNA aneuploid group was further classified into 3 subgroups by DNA index (D.I.); near-diploid group (D.I.: 0.8-1.2, 21.6%), near-tetraploid group (D.I.: 1.8-2.2, 12.9%) and other aneuploid group (31.9%). DNA ploidy pattern correlated with clinical stages and histological grading, indicating significantly a higher incidence of DNA diploid in cases in stage I and grade 1. DNA clonal heterogeneity was observed in 48.1%, and homogeneously diploid was in 28.6% of 77 cases who were analyzed more than 2 specimens. Incidences of DNA heterogeneity and homogeneous diploid correlated with the number of analyzed specimens and our results showed that more than 4 specimens were necessary to diagnose the DNA ploidy patterns. The near-tetraploid group was shown to have an extremely poor prognosis compared with the diploid group and the other aneuploid group. There were no significant difference between the diploid group and the aneuploid group in terms of the early prognosis, however, the incidence of disease recurrence was significantly higher in the aneuploid group. Our results demonstrated that DNA ploidy was an useful prognostic factor in the evaluation of patients with renal cell carcinoma.

We established four renal cell carcinoma (RCC) cell lines (HANKS), namely the primary tumor (HANKS-Pr), metastasized to the lung (HANKS-Lu), liver (HANKS-Li) and lymph node (HANKS-LN) derived from a patient with advanced RCC, and analyzed their characters. Each had an epithelial morphology and exhibited multilayering. These cell lines have been maintained for more than 36 months and over 100 in vitro passages. In karyotype analysis, the common aberration in the four cell lines was marker chromosome t (3;18) (p13;q21). In soft agar culture, HANKS-Pr showed the lowest growth. Furthermore, we found high level expression of major histocompatibility complex (MHC) class I antigen on HANKS-Pr and HANKS-Lu, and low expression of MHC class II antigen on four cell lines. HANKS-LN had transplantability in nude mice. We determined the different biological properties among HANKS cell lines stemming from the same origin.

Several rodent studies based on molecular biology have suggested that accumulation of genetic alterations in cancer-associated genes is required to convert a normal cell into a malignant cell. Activation of oncogenes and inactivation of tumor suppressor genes appear to be involved in carcinogenesis. In renal cell carcinomas, we have recently implied that the presence of tumor suppressor genes at chromosome 3p13-14.3 and 21.3, the regions where are also commonly deleted in adenocarcinoma of the lung; at chromosome 5q21, the region where the MCC (mutated in colorectal cancer) gene and APC (adenomatous polyposis coli) gene are located; at chromosome 6q27; and at 10q 21-23. We have also indicated that genes on 3p is probably important for development of RCCs and genes on 5q, 6q, and 10q may be associated with progression of RCCs.

We established a new cell line (TC-1) from primary site of a renal cell carcinoma (RCC) patient. Its doubling time in tissue culture was 20 hours at 45th passage and mycoplasma contamination test was negative. The karyotypic analysis demonstrated a human karyotype with a modal number of 70. A consistent chromosomal abnormality was noted such as No. 4 monosomy, No. 7 trisomy and a loss of Y chromosome. Electron microscopic examination showed a brush border, vacuoles and abundant glycogen granules in the cytoplasm, which was compatible with RCC cells. This cell line was transplantable to nude mice and the grown tumor closely resembled the original tumor, i.e. clear cell type and hypervascularity. High titer of interleukin-6 (IL-6) was detected in the supernatant of TC-1 cell culture (approximately 5 ng/ml) as well as in sera of nude mice bearing this tumor (260 pg/ml). Exogenous IL-6 did not enhance the TC-1 cell proliferation as determined by cell count. Flow cytometric analysis could not demonstrate the existence of IL-6 receptor on the cell surface. These results suggested the produced IL-6 did not act as an autocrine growth factor in the cell line. Additional IL-1 alpha to the culture medium induced 3-4 times higher concentration of IL-6 in the culture supernatant compared with that of non-stimulating cells, while exogenous TNF alpha did not stimulate IL-6 production.

The accumulating data have shown that a single or certain combinations of proto-oncogenes are genetically altered and acquire oncogenic activities in certain tumor types, through a point mutation and/or overexpression. In the present study, Northern blotting analysis was applied to clinical samples of renal cell carcinomas (RCC) to elucidate expression levels of the c-myc, c-fos and Harvey ras genes in this tumor form. In 11 cases of 23 tumors examined (48%) was shown more than 3-fold expression level of the c-myc gene compared with the matching normal RNA, the c-fos gene in 6 cases (26%), and the Harvey ras gene in one case (4%) showing the concomitant overexpression of two other genes. Overexpression of both the c-myc and c-fos genes was detected in 5 cases (22%). Although the distinct correlation between overexpressions of any genes and the clinical background was not induced, it is considered that these aberrations of proto-oncogenes are involved in oncogenesis of a certain subgroup of RCC.

We have characterized seven human renal cell carcinoma cell lines established from primary sites of five patients between 1987 and 1989. Two lines, OUR-20P and OUR-20S, were derived from the OUR-20 cells by cloning with a dilution method 3 months after the primary culture. These three cell lines were tumorigenic in athymic nude mice when inoculated subcutaneously. Examined by a dye uptake method, OUR-20 was highly sensitive to interferon-alpha (IFN-alpha); OUR-20P, OUR-20S and OUR-30 showed slight sensitivities, while the other three cell lines were insensitive. All seven cell lines have been maintained for more than 2 years and over 50 passages in vitro. Cytogenetic analyses performed 1.5 to 3 years after the starts of primary cultures indicated that all seven cell lines, which exhibited different morphologies in phase-contrast micrographs, were aneuploid with modal chromosome numbers 41 to 89.

We report a 63-year-old woman with renal cell carcinoma associated with von Hippel-Lindau disease. The patient was referred to the department of neurosurgery at our hospital, complaining of gait disturbance. There was a history of retinal hemangioma. After further examination, von Hippel-Lindau disease was the conclusion with evidence of cerebellar hemangioblastoma. An abdominal CT-scan and arteriography revealed multiple hypervascular tumors in the right kidney, but not in the left. She underwent a right radical nephrectomy and lymphadenectomy 2 months after resection of a brain tumor. Von Hippel-Lindau disease is generally recognized as an autosomal-dominant inherited disorder. The patient had a positive family history, seen in both her younger brother and son. Both were diagnosed with renal cell carcinoma with central nervous system involvement before any sign of disease was found in the patient. Twenty one cases of von Hippel-Lindau disease associated with renal tumors have been reported in the Japanese literature. The clinical findings of these cases are discussed.

Of 8 tumors surgically extirpated from 8 patients with renal cell carcinoma, 7 were successfully processed in short-term culture by collagenase method, and 130 metaphases were extracted from these 7 tumors and were subjected to chromosomal analysis according to the G banding technique. As for karyotype, 4, 2 and 1 cases were mainly diploid, hypodiploid and aneuploid, respectively. The highest incidence of chromosome aberration was marked by #3 chromosome (5 of 7 cases of which 4 and 1 showed monosomy as clonal aberration and trisomy as non-clonal aberration, respectively). The commonest gain of chromosome was noted for #7 trisomy (4 of 7 cases). Two cases had already had metastases at the time of surgery, each showing #7 trisomy. Marker chromosome was noted in 6 of 7 cases. Structural chromosome aberration had a lesser incidence compared with numerical aberration; only clonal aberrations were long-arm aberrations (2q+, 6q+) of #2 and #6 chromosomes. Short-arm aberrations (3p-, 8p-) of #3 and #8 chromosomes were noted in one case each. 3p deletion, which is reported to be predominant in the literature, was noted only in one case. This was observed in triploid cells. However, all of metaphases showing 2q+ and 6q+ had concomitant #3 monosomy, and excess portions of #2 and #6 chromosomes were in good agreement with the band pattern of #3 chromosomal long-arm. Therefore, translocation of 3q12-qter in #2 and #6 chromosomes is supposed to lead to 3p deletion. A similar mechanism seems to be involved in the formation of marker chromosomes, suggesting the involvement of deleted #3 chromosome in marker chromosomes.

For the purpose of elucidating the role of oncogenes like c-myc in renal cell carcinoma, the methods of introducing exogenous genes into human cells might be powerful tools. In the present study, the electroporation gene transfer method was investigated for its application to renal cancer cells. A mixture of ACHN cells (human renal cancer cell line, which cannot grow in the presence of neomycin) and DNA of neomycin-resistant genes with SV 40 promoters was exposed to electric pulses from an electroporated (Bio-Pulser 101, UNISOKU). And the cells were cultured in a medium containing Geneticin (neomycin analog) for 3 weeks. Then, the number of formed neomycin-resistant neoR colonies was counted. In the cells of neoR colonies, the existence of neoR genes and their expression were confirmed by Southern and Northern blot gene analyses. The transformation efficiency (number of neoR colonies/inoculated cells) was positively correlated with cell densities, DNA concentrations, and discharged voltages under our experimental conditions. The transformation efficiency was 1.3 - 3.3 x 10(-4)/cell in the condition of 1 x 10(7) cells/ml, 1 microgram DNA/10(6) cells, and 2 kV/cm. These results suggest that the electroporation gene transfer method is applicable for the study of phenotypic alterations after introducing oncogenes into human renal cancer cells.