Terumi Mizuno

RNA from decades-old archival tissue blocks for retrospective studies.

The validity of molecular studies using DNA and RNA extracted from decades-old formalin-fixed and paraffinembedded tissue blocks has been demonstrated. The quality and usability of DNA and RNA from archival tissues are modified by various factors, such as the fixative, the fixation time, and the postmortem time. However, in contrast to DNA, there are no comprehensive studies quantitatively addressing the feasibility of RNA from old (more than 10 years) archival samples. This study examined the integrity of RNA extracted from 738 autopsy liver and 63 autopsy thyroid cancer tissue blocks procured during a span of nearly four decades, beginning in 1952 and ending in 1989, from the atomic bomb survivors. The integrity of RNA was assessed by amplification of c-BCR messenger RNA (mRNA) between two sequential exons with an intervening intron by reverse-transcription polymerase chain reaction (RT-PCR). The integrity of RNA was influenced by the age of the samples and the postmortem time, but not by the formalin-fixation period. It was possible to amplify more than 60% of the samples. Using these RNAs, the HCV genome in liver cancers and the H4-RET gene in thyroid cancers were detectable. This study illustrates the possibility of molecular studies using RNA from routinely prepared paraffin blocks stored for long periods and provides the statistics and critical factors to consider in assessing the feasibility of such contemplated studies.

Preferential induction of RET/PTC1 rearrangement by X-ray irradiation.

Ionizing radiation is a well known risk factor of thyroid cancer development, but the mechanism of radiation induced carcinogenesis is not clear. The RETPTC oncogene, an activated form of the RET proto-oncogene, is frequently observed in papillary thyroid carcinoma (PTC); RETPTC1, -2 and -3 are known to be the three major forms. High frequencies of RETPTC rearrangements have been observed in radiation-associated PTC, such as those appearing post-Chernobyl or post-radiotherapy, but the rearrangement types differ between these two populations. We investigated whether a specific type of RETPTC rearrangement was induced by X-rays in vivo and in vitro. In human normal thyroid tissues transplanted in scid mice, the RETPTC1 rearrangement was predominantly detected throughout the observation period (up to 60 days) after X-ray exposure of 50 Gy. On the other hand, RETPTC3 was detected only 7 days after X-irradiation, and no transcript of RETPTC2 was detected. These results are supported by the results of an in vitro study. The RETPTC1 rearrangement was preferentially induced in a dose-dependent manner by X-rays within a high dose range (10, 50 and 100 Gy) in four cell lines. On the other hand, RETPTC3 was induced at a much lower frequency, and no induction of RETPTC2 was observed. These results suggest that the preferential induction of the RETPTC1 rearrangement may play an important role in the early steps of thyroid carcinogenesis induced by acute X-irradiation.

Genetic alterations in thyroid tumor progression: association with p53 gene mutations.

To identify the genetic events that must be involved in thyroid tumor progression, we initially investigated p53 gene alterations in 10 papillary adenocarcinomas, 4 follicular adenocarcinomas, and 8 undifferentiated carcinomas. Base substitutional mutations in exons 5 to 8 and loss of heterozygosity (LOH) of the p53 gene were not detected in papillary or follicular adenocarcinomas. However, 7 of 8 undifferentiated carcinomas were carrying base substitutional mutations, and LOH was detected in 3 of 5 informative cases. Furthermore, to verify that the p53 gene alterations are truly involved in tumor progression, DNA from individual foci of the four undifferentiated carcinomas coexisting with a differentiated focus and from one follicular adenocarcinoma with an undifferentiated focus was analyzed by direct sequencing and polymerase‐chain‐reaction‐restriction‐fragment‐length polymorphism (PCR‐RFLP). Base substitutional mutations in the p53 gene from exons 5 to 8 were identified exclusively in the undifferentiated foci, but not in the differentiated foci. LOH was observed in 3 of 4 informative undifferentiated foci. In one of these positive cases, LOH was observed in both papillary adenocarcinoma and undifferentiated carcinoma. However, a p53 gene mutation at codon 248 was detected in the undifferentiated carcinoma but not in the papillary adenocarcinoma. The results imply that LOH occurs first in papillary adenocarcinoma followed by a p53 mutation during the transition from papillary adenocarcinoma to undifferentiated carcinoma. Maintenance of LOH during tumor progression excludes the possibility that these different histological foci are derived from different origins and represents molecular evidence that undifferentiated carcinoma is very likely derived from preexisting papillary adenocarcinoma. Furthermore, these results strongly suggest that the mutated p53 gene plays a crucial role in de‐differentiation during the progression of thyroid tumors.

Continued expression of a tissue specific activated oncogene in the early steps of radiation-induced human thyroid carcinogenesis.

Ionizing radiation is a well-known risk factor of cancer development, but the mechanism of radiation induced carcinogenesis is not clear. Chromosomal rearrangements induced by radiation most likely are one of the principal genetic alterations resulting in malignant transformation. The chimeric BCR-ABL associated with chronic myelogenous leukemia (CML) and H4-RET oncogenes associated with thyroid papillary carcinoma are the result of a translocation and inversion, respectively. In vitro studies showed these genes were induced by high-doses of X-irradiation in cell lines. Studies also show that therapeutic external X-ray doses as high as 60 Gy for treatment of various childhood cancers including Hodgkins disease significantly increase the risk of thyroid cancer. Therefore, we examined the induction and persistence of these chimeric genes in human thyroid tissues transplanted in scid mice after 50 Gy exposure as a function of time for 2 months to elucidate the early events of thyroid carcinogenesis. The H4-RET genes were detected on day 2 and throughout the 2 month period. On the other hand, BCR-ABL genes were detected on day 2 and were undetectable subsequently. These results suggest that ionizing radiation causes various oncogene activations, but cells with only specific gene alteration uniquely associated with thyroid carcinogenesis are selectively retained demonstrating one of the early events in the beginnings of radiation carcinogenesis in human thyroid tissues.

Relationship of hepatocellular carcinoma to soya food consumption: A cohort-based, case-control study in Japan

To determine if the risk of hepatocellular carcinoma (HCC) is reduced by consumption of soya foods, we conducted a case‐control study within a cohort of Japanese A‐bomb survivors. We compared the prediagnosis consumption of isoflavone‐rich miso soup and tofu to HCC risk, adjusting for hepatitis B (HBV) and C (HCV) viral infections, the major HCC risk factors in this population. The study included 176 pathologist‐confirmed cases of HCC diagnosed in 1964–1988 and 560 controls who died of diseases other than liver cancer. We examined dietary information collected at least 2 years before diagnosis or death and tissue‐based measures of viral hepatitis. Using logistic regression, crude ORs were 0.5 (95% CI 0.29–0.95) and 0.5 (95% CI 0.20–0.99) for high vs. low miso soup and tofu intake, respectively. Adjusting for year of birth, sex, HBV, HCV and other factors, the OR for miso soup was unchanged at 0.5 (95% CI 0.14–1.55), and miso results were similar when ORs were recalculated separately for earlier and later birth cohorts to assess consistency of results. The adjusted OR for tofu was 0.9 (95% CI 0.20–3.51). We also found a statistically significant (p < 0.0001) interaction between sex and HCV, with risk of HCC being substantially higher for women. We conclude that consumption of miso soup and other soya foods may reduce HCC risk.

Hepatocellular carcinoma among atomic bomb survivors: significant interaction of radiation with hepatitis C virus infections.

We conducted a nested case‐control study within the cohort of Japanese survivors of the 1945 atomic bombings to study the joint effects of HBV and HCV with radiation on the risk of HCC. Among subjects who received autopsies during 1954–1988, we analyzed archival tissue samples for 238 pathologically confirmed HCC cases and 894 controls who died from diseases other than liver cancer. Using logistic regression and adjusting for potential confounders and other factors, we found a statistically significant, supermultiplicative interaction between A bomb radiation and HCV in the etiology of HCC. Compared to subjects who were negative for HCV and radiation, ORs of HCC for HCV‐positive subjects showed a statistically significant, greater than multiplicative increase for liver irradiation exposures in the second (>0.018–0.186 Sv, p = 0.04) and third (>0.186 Sv, p = 0.05) tertiles of non‐zero radiation exposure but not for first tertile exposure (>0–0.018 Sv, p = 0.86). Limiting analysis to subjects without cirrhosis, HCV‐infected subjects were at 58.0‐fold (95% CI 1.99–∞) increased risk of HCC per Sv of radiation exposure (p = 0.017), a supermultiplicative interaction between radiation and HCV that was not found among subjects with cirrhosis (p = 0.67). We found no evidence of interaction between HBV infection and radiation exposure in the etiology of HCC, regardless of cirrhosis status (p = 0.58). We conclude that among survivors of the nuclear bombings of Hiroshima and Nagasaki, subjects who were both HCV‐positive and radiation‐exposed were at a significantly, supermultiplicatively increased risk of HCC without concurrent cirrhosis.

Induction of BCR-ABL fusion genes by in vitro X-irradiation.

The Philadelphia chromosome consists of a reciprocal translocation between the ABL oncogene at chromosome 9q34 and the BCR gene at chromosome 22q1l, resulting in the expression of chimeric BCR‐ABL mRNAs specific to chronic myelogenous leukemia (CML). Presence of the fusion gene can be detected with high specificity and sensitivity by means of reverse transcription and polymerase chain reaction. Using this assay, it was possible to detect BCR‐ABL fusion genes induced among HL60 cells after 100 Gy of X‐irradiation in vitro. In total, five fusion gene transcripts were obtained among 108 cells examined. These fusion genes contained not only CML‐specific BCR‐ABL rearrangements, but also other forms of BCR‐ABL fusions. These latter genes had junctions of BCR exon 4/ABL exon 2 intervened by a segment of DNA of unknown origin, BCR exon 5/ABL exon 2, and BCR exon 4/ABL exon 2. The results appear to be direct evidence for the induction of the BCR‐ABL fusion gene by X‐irradiation. In terms of leukemogenesis, it appears that only those cells bearing certain CML‐related BCR‐ABL fusion genes are positively selected by virtue of a growth advantage in vivo.

HaeIII polymorphism in intron 1 of the human p53 gene

A new HaeIII polymorphism, which is found in the first intron of the human p53 gene, provides a genetic marker for tumor suppressor p53 gene alterations.

Multiple, unique, and common p53 mutations in a thorotrast recipient with four primary cancers

Four primary cancers found at autopsy of a patient who received the thorium-based contrast agent Thorotrast 50 years ago and who was healthy up until a few months before his death from liver failure were analyzed for p53 mutations. The data suggest that the chronic alpha-irradiation may be a large causative factor. Multiple mutations were found in all the cancer tissues: two foci of a cholangiocellular carcinoma, a tubular adenocarcinoma of the stomach, a squamous cell carcinoma of the lung, and an adenocarcinoma of Vaters ampulla. The total number of point mutations detected were 13. Moreover, homozygous aberrations were detected in a large area of normal small intestine and noncancer liver tissues suggesting that nontumor cells which harbored p53 abnormalities gained a survival advantage and clonally expanded.

The usefulness of severe combined immunodeficiency (SCID) mice to study human carcinogenesis

In the present study, we engrafted normal colonic epithelial and histologically diagnosed colonic adenomas from a familial adenomatous polyposis (FAP) patient into severe combined immunodeficient (SCID) mice and subsequently examined them histologically and molecular biologically. Successful engraftment and metastasis was observed. The facts that human normal colonic epithelium and adenomatous polyps can take in SCID mice indicates the possibility that this human SCID mouse system will be useful for investigating the dynamics of human carcinogenesis in various tissues.