Yoshinori Nimura

p73 at chromosome 1p36.3 is lost in advanced stage neuroblastoma but its mutation is infrequent

p73, a novel p53 family member, is a recently identified candidate neuroblastoma (NBL) suppressor gene mapped at chromosome 1p36.33 and was found to inhibit growth and induce apoptosis in cell lines. To test the hypothesis that p73 is a NBL suppressor gene, we analysed the p73 gene in primary human NBLs. Loss of heterozygosity (LOH) for p73 was observed in 19% (28/151) of informative cases which included 92 mass-screening (MS) tumors. The high frequency of p73 LOH was significantly associated with sporadic NBLs (9% vs 34%, P<0.001), N-myc amplification (10% vs 71%, P<0.001), and advanced stage (14% vs 28%, P<0.05). Both p73α and p73β transcripts were detectable in only 46 of 134 (34%) NBLs at low levels by RT – PCR methods, while they were easily detectable in most breast cancers and colorectal cancers under the same conditions. They found no correlation between p73 LOH and its expression levels (P>0.1). We found two mutations out of 140 NBLs, one somatic and one germline, which result in amino acid substitutions in the C-terminal region of p73 which may affect transactivation functions, though, in the same tumor samples, no mutation of the p53 gene was observed as reported previously. These results suggest that allelic loss of the p73 gene may be a later event in NBL tumorigenesis. However, p73 is infrequently mutated in primary NBLs and may hardly function as a tumor suppressor in a classic Knudsons manner.

p73, a geme related to p53, is not mutated in esophageal carcinomas

A novel gene, termed p73, encodes a protein with a significant homology to p53 and has been mapped at chromosome 1p36.3, which is a locus of multiple suppressor genes for tumors including neuroblastoma and other cancers. Since the 1p36 locus is reported to be deleted and p53 is frequently mutated in esophageal carcinomas, we examined loss of heterozygosity (LOH) and mutation of the p73 gene in 48 untreated esophageal tumors, as well as mRNA expression in 8 tumors. We screened the P1 genomic library to obtain a P1 clone containing the p73 gene and found a polymorphic short tandem CT repeat site at intron 9. Intragenic sequences for 14 PCR primer sets and a primer pair flanking the repeat were also determined for the analysis of PCR single‐strand conformation polymorphism (SSCP) and LOH studies, respectively. Expression of p73 mRNA was detectable but at low levels in all 8 tumor tissues by reverse transcriptase PCR. We did not find any type of mutation other than polymorphisms in the 48 esophageal carcinomas, though aberration of the p53 gene on the PCR‐SSCP gels was observed in 15 of 38 (39%) tumors of the same set. In addition, LOH for p73 was found in only 2 of 25 (8%) tumors. These results suggest that, at least in esophageal carcinomas, allelic loss or mutation of p73 may not be a main genetic event for the tumorigenesis as it is with p53. Int. J. Cancer 78:437–440, 1998.

Cathepsin D Is a Potential Serum Marker for Poor Prognosis in Glioma Patients

Cathepsin D is an aspartyl protease involved in protein catabolism and tissue remodeling which can be secreted from cancer cells. To identify a potential serum marker for gliomas, we investigated the gene expression levels of cathepsin D in 87 tissue samples and measured the protein concentrations in sera of glioma patients. The tissue samples consisted of 43 glioblastomas, 13 anaplastic astrocytomas, 22 astrocytomas, and 9 normal brain tissues. The results of real-time quantitative reverse transcription-PCR analysis showed that cathepsin D transcript levels became significantly higher as the glioma grade advanced (P = 0.0466, glioblastoma and anaplastic astrocytoma; P = 0.0008, glioblastoma and astrocytoma; P = 0.0271, glioblastoma and normal brain tissue; unpaired t test). Immunohistochemical analysis with anti-cathepsin D antibody revealed dense and spotty staining in the tumor cells with high transcript levels. The low expression of cathepsin D significantly correlated with long survival of the glioma patients. Furthermore, the glioblastoma patients with high gene expression of cathepsin D lived significantly shorter than those with low expression (P = 0.0104, Cox-Mantel log-rank test) and frequently had leptomeningeal dissemination (P = 0.0016, chi2 test). The multivariate analysis confirmed that the cathepsin D expression level was an independent predictor for short survival (P = 0.0102, Cox proportional hazard regression model). Measurement of the serum cathepsin D concentrations by ELISA showed a significant increase in the patients with high-grade gliomas as compared with the low-grade tumors (P = 0.0081, chi2 test). These results collectively suggest that cathepsin D could be a potential serum marker for the prediction of aggressive nature of human gliomas.

Mutational analysis of the p73 gene in human breast cancers.

In primary breast cancer, mutations of the p53 tumor suppressor gene lead to loss of growth‐suppressive properties and poor outcome. Recently, a p53‐related gene, termed p73, has been cloned and its gene product possesses a function similar to p53. p73 has been mapped at chromosome 1p36.3, a region frequently deleted in breast cancer, neuroblastoma and other malignancies. To elucidate the functional significance of p73 in the oncogenesis of breast cancer, we have studied genetic alterations of p73 in tissue specimens obtained from 87 patients with primary breast cancer. Thirteen percent of informative cases showed loss of heterozygosity (LOH) at the p73 gene. However, there was no correlation between the p73 LOH and clinical features such as histo‐pathological types, metastatic behavior or expression of estrogen or progesterone receptor. The levels of p73 transcript in primary breast cancer were not significantly different from those in normal breast tissue. Moreover, PCR‐SSCP analysis failed to detect any missense or frameshift mutations in the p73 gene. Our observations suggest that allelic loss, expression levels and mutations of the p73 gene may not contribute to oncogenesis of primary breast cancers. Int. J. Cancer (Pred. Oncol.) 84:321–325, 1999.

Absence of mutation of the p73 gene localized at chromosome 1p36.3 in hepatocellular carcinoma.

SummaryAccumulating evidence has demonstrated that aberration of the p53 tumour-suppressor gene is one of the pivotal genetic events in hepatocellular carcinogenesis. Recent reports suggest that the product of hepatitis B virus (HBV) interacts with p53 and that the hepatitis C virus (HCV) core protein reduces p53 expression. A novel p73 gene, which is related to p53, has recently been identified and mapped to chromosome 1p36.3, which is a locus of multiple tumour-suppressor genes for many cancers, including hepatocellular carcinoma (HCC) and neuroblastoma. Here, we investigated mRNA expression, allelotype and mutation of p73 in 48 HCCs obtained from untreated patients. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that p73 mRNA was expressed ubiquitously at low levels in all the tumour tissues, as well as in the adjacent normal liver tissues. The frequency of p73 loss of heterozygosity was observed in 20% of HCCs, but PCR-single strand conformation polymorphism (SSCP) analysis showed no mutations in the 48 tumours except for three types of polymorphisms. These results suggest that p73 may play a role in hepatocellular carcinogenesis in a different manner from a Knudson two-hit model. The regulatory mechanism of interaction between p73 and hepatitis viruses remains to be determined.

Downregulation of hASH1 is associated with the retinoic acid-induced differentiation of human neuroblastoma cell lines

BACKGROUND MASH1, a transcription factor with basic helix-loop-helix domain, has a pivotal function to promote differentiation of neural crest cells into autonomic neurons. PROCEDURE To investigate the functional significance of human MASH1 (hASH1) in the pathogenesis of neuroblastoma, which is originated from autonomic precursor cells, we studied hASH1 gene expression in primary neuroblastomas and human nueroblastoma cell lines. RESULTS The follovving results were obtained: (i) hASH1 was expressed in 40 out of 61 (66%) primary neuroblastomas, (ii) hASH1 transcripts were downregulated in several cell lines prior to differentiation induced by all-trans retinoic acid (RA), (iii) a neuroblasotma cell line without expression of endogenous hASH1 did not respond to RA at all, and (iv) the analysis of the hASH1 genomic DNA revealed two possible transcription initiation sites, which may correspond to 3.0 kb and 3.5 kb transcripts. CONCLUSIONS Our observations suggest that, although hASH1 may not preserve the growth capacity of neuroblastomas, downregulation of hASH1 may be necessary to promote neuronal differentiation of neuroblastoma.

Genetic analysis of p73 localized at chromosome 1p36.3 in primary neuroblastomas

BACKGROUND Human p73, a novel homolog of p53, has recently been cloned and mapped at chromosome 1p36.3, the locus for putative tumor suppressor gene(s) of neuroblastoma (NBL) and other cancers. p73, like p53, inhibits growth and induces apoptosis in neuroblastoma and osteosarcoma cell lines. PROCEDURE To test the hypothesis that p73 is a NBL suppressor gene, we examined expression, allelo-typing, and mutation of the p73 gene in primary human neuroblastomas. Loss of heterozygosity (LOH) for p73 was performed in 272 primary NBLs using a CT repeat polymorphic marker, which we found in intron 9 of the p73 gene. RESULTS p73 LOH was observed in 28 out of 151 (19%) informative cases. The high frequency of p73 LOH was significantly associated with sporadic neuroblastomas (P< 0.001), MYCN amplification (P< 0.001), and advanced stages (P< 0.05). Mutational analyses by PCR-SSCP (single strand conformation polymorphism) revealed two mis-sense mutations in 140 NBLs, one somatic and one germline. CONCLUSION Thus, the present results have shown that mutation of p73 is infrequent in NBLs, although the p73 locus is frequently lost in advanced stage tumors. These suggest that p73 may not be a tumor suppressor in the classic Knudson manner.

The influence of excess body weight on the surgical treatment of patients with gastric cancer

Sixty-two overweight gastric cancer patients were compared with 201 normal-weight patients to clarify the influences of excessive weight on the surgical treatment of gastric cancer. The frequencies of hypertension and diabetes mellitus were significantly higher in the overweight group (P<0.01), but no pathologic differences in the resected tumor were found between the two groups. The operative times were longer (P<0.01) and the number of lymph nodes extirpated and examined was smaller (P<0.01) in the overweight group. The incidence of postoperative complications was not higher in the overweight group. The postoperative survival rate of patients with nodal metastasis was statistically lower in the overweight group (P<0.05). Regarding the causes of death in patients with nodal metastasis, 61.1% of overweight patients and 43.8% of normal-weight patients died of recurrence of gastric cancer. In conclusion, surgical treatment of overweight patients with gastric cancer was found to be technically more difficult and the prognosis of such patients with nodal metastasis may thus be worse than that of their normal-weight counterparts.

RERESECTION FOR LOCAL RECURRENCE OF RECTAL CANCER

Local recurrence is one of the major reasons that rectal cancer surgery is unsuccessful. The aim of this study was to investigate the surgical characteristics of patients undergoing reresection for local recurrence of rectal cancer. A total of nine patients were enrolled in this study, six of whom underwent total pelvic exenteration, one, posterior exenteration, one, abdominoperineal resection with sacral resection, and one, lymph node dissection alone. The mean operative time was 8h 15min, and the mean operative blood loss was 2 325 ml. Although major postoperative complications occurred in four patients (44%), there were no postoperative or hospital deaths. Lateral lymph node metastasis was detected in all four patients whose lateral lymph nodes were dissected or extirpated at the reresection. Two patients survived for more than 5 years without rerecurrence, and the cumulative 5-year survival rate was 26%. The para-aortic lymph nodes were the most common site of first rerecurrence. The results of this study indicate that patients who undergo reresection for local recurrence of rectal cancer are at high risk of developing lateral or para-aortic nodal metastasis. Nevertheless, reresection may be a therapeutic option for the local recurrence of rectal cancer.

Hepatic resection of giant cavernous hemangioma of the liver.

Surgical treatment of giant hemangioma of the liver is still controversial. The aim of this study is to examine the efficacy of hepatic resection for giant hemangioma of the liver. Twenty patients with giant cavernous hemangioma of the liver were treated by hepatic resection. The mean diameter of the hemangiomas was 13.9 cm (range, 6.5-30 cm). The surgical outcome was reviewed retrospectively. Major hepatectomy was performed in 14 patients and minor hepatectomy in 6 patients. Complications occurred in 7 of the 20 patients treated by hepatic resection. At a mean follow-up of 79 months (range, 12-173 months), 18 patients were symptom free whereas 2 patients had died--one died of pneumonia at 2 years and the other died of gastric cancer 6 years after surgery. Mean intraoperative hemorrhage and blood transfusion in all patients was 4,343 mL (range, 270-24,000 mL) and 1,860 mL (range, 0-8,800 mL) respectively. In the seven patients with preoperative high levels of fibrin degradation products (FDP), mean intraoperative hemorrhage and blood transfusion were markedly higher (9,371 mL and 3,714 mL respectively) than in the 13 patients without abnormal FDP (1,603 mL and 900 mL respectively). Preoperative hematologic status returned to normal after operation in all patients. Hepatic resection is a useful treatment for giant cavernous hemangioma of the liver. More careful management to reduce intraoperative hemorrhage is recommended to increase the safety of surgery, particularly in patients with preoperative abnormal FDP.