Toshiyuki Fukuhara

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.

Immunohistochemical staining of Reg IV and claudin-18 is useful in the diagnosis of gastrointestinal signet ring cell carcinoma.

Signet-ring cell carcinoma (SRCC) is a unique subtype of adenocarcinoma that is characterized by abundant intracellular mucin accumulation and a crescent-shaped nucleus displaced toward one end of the cell. Identification of an SRCCs primary site is important for better planning of patient management because the treatment and prognosis differs markedly depending on the origin of the SRCC. In the present study, we analyzed the immunohistochemical characteristics of 94 cases of SRCC, including 21 cases of gastric SRCC, 16 of colorectal SRCC, 10 of breast SRCC, and 47 of pulmonary SRCC, with antibodies against Reg IV and claudin-18, which we previously identified as gastric cancer-related genes. We also tested known markers cytokeratin 7, cytokeratin 20, MUC2, MUC5AC, caudal-related homeobox gene 2 (CDX2), thyroid transcription factor-1, mammaglobin, gross cystic disease fluid protein 15, and estrogen receptor. All 21 cases of gastric SRCC and 16 cases of colorectal SRCC were positive for Reg IV, and the remaining SRCCs were negative. Eighteen of 21 (86%) gastric SRCCs and 6 of 16 (38%) colorectal SRCCs were positive for claudin-18, whereas another SRCCs were negative. In conclusion, Reg IV staining and claudin-18 staining can aid in diagnosis of gastrointestinal SRCC.

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.

Effects of Radiation on Incidence of Primary Liver Cancer among Atomic Bomb Survivors

We describe the radiation risk for primary liver cancers between 1958 and 1987 in a cohort of atomic bomb survivors in Hiroshima and Nagasaki, Japan. The analysis is based on a comprehensive pathology review of known or suspected liver neoplasms that generated 518 incident, first primary cases, mostly hepatocellular carcinoma. Excess relative risk from atomic bomb radiation was linear: 0.81 per sievert weighted liver dose (95% CI [0.32, 1.43]; P < 0.001). Males and females had similar relative risk so that, given a threefold higher background incidence in males, the radiation-related excess incidence was substantially higher in males. Excess risk peaked for those with age at exposure in the early 20s; there was essentially no excess risk in those exposed before age 10 or after age 45. Whether this was due to a difference in sensitivity or possible confounding by other factors could not be addressed retrospectively in the full cohort. A paucity of cholangiocarcinoma and hemangiosarcoma cases suggested that they are not significantly associated with whole-body radiation exposure, as they are with the internal alpha-particle-emitting radiological contrast medium Thorotrast. Because most of the radiation-related excess cases occurred among males, it is important to ascertain what factors put men at greater risk of radiation-related liver cancer.

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.

Temporal changes in liver cancer incidence rates in Japan: Accounting for death certificate inaccuracies and improving diagnostic techniques

Primary liver cancer (PLC) rates have risen dramatically during the past few decades in some regions, particularly in Japan, where PLC is now the third major cause of cancer death. PLC is one of the most difficult tumors to diagnose correctly, because (i) the liver is a frequent site of cancer metastasis and (ii) death from PLC is often attributed to cirrhosis or chronic hepatitis. Also, because the disease is often rapidly fatal, a large proportion of liver cancer cases are identified based on death certificates alone without confirmation by clinical records. Thus, worldwide differences in published incidence rates for this disease reflect regional or national differences in both the accuracy of death certificates and the sensitivity of diagnostic methods. By comparing death certificate causes of death with those based on pathology review, we were able to adjust 1958–1994 incidence rates for a large Japanese cohort for these errors. Although the death certificate false‐positive error rate declined, the false‐negative error rate remained high throughout the study. The introduction of improved liver cancer diagnostic methods in Japan in the early 1980s was associated with a sharp increase in PLC incidence. We conclude that errors in death certificate causes of death and changes in liver cancer diagnostic techniques have had an important impact on the reported incidence of this disease. Taking these factors into account, rates of hepatocellular carcinoma rose between 2.4‐ and 4.3‐fold in our Japanese cohort from 1960 to 1985, peaked about 1993 and declined thereafter. Incidence rates of cholangiocarcinoma remained stable through 1987.

Characteristic gene expression in stromal cells of gastric cancers among atomic‐bomb survivors

To elucidate the mechanism of radiation‐induced cancers, molecular analysis of cancers in atomic‐bomb survivors is important. In our study, we developed a custom oligonucleotide array of 208 genes. We analyzed gene expression profiles of gastric cancers (GCs) from atomic‐bomb survivors and identified 9 genes with significantly lower expression in GCs from exposed patients than in GCs from nonexposed patients. Among these 9 genes, expression of versican and osteonectin was investigated in greater detail using immunohistochemistry in 116 GCs from 64 exposed and 52 nonexposed patients who developed GC after the bombing. In the Stage I/II GCs, the clinicopathologic, phenotypic and proliferative characteristics of GCs from exposed and nonexposed patients did not differ significantly; however, versican and osteonectin were expressed at much lower levels in the area of tumor‐associated stroma of exposed patients than in nonexposed patients (p = 0.026 and p = 0.024, respectively). These results suggest that the characteristics of tumor‐associated stromal cells differ between GCs from exposed and nonexposed patients.

Large-cell neuroendocrine carcinoma of the distal bile duct

Large-cell neuroendocrine carcinoma (LCNEC) in the distal bile duct is very rare and different from common distal bile duct adenocarcinoma. A 77-year-old man was admitted with obstructive jaundice. Severe stenosis of the distal bile duct was revealed by percutaneous transhepatic cholangiography. Subtotal stomach-preserving pancreaticoduodenectomy was performed. A tumor measuring 1.8xa0cm in diameter was located in the distal bile duct. Both histopathological and immunohistochemical examination of the resected specimen revealed features of LCNEC of the bile duct. The patient developed multiple liver metastases, lung metastases, and local recurrence and died of disease 3xa0months after the operation. The clinical behavior of LCNEC in the distal bile duct appears to be highly aggressive with early metastases and a fatal outcome.

Lack of association between acute exposure to ionizing radiation and liver cirrhosis.

Purpose: Although previous studies have shown significantly increased risks of liver cirrhosis and chronic liver disease for acute radiation exposure among survivors of the atomic bombings of Hiroshima and Nagasaki, Japan, these studies have not taken into account hepatitis B virus (HBV) infections. Because HBV is associated with both A-bomb radiation and liver cirrhosis, our goal was to investigate the relationship of acute ionizing radiation to liver cirrhosis adjusting for HBV, co-occurring primary liver cancer (PLC), and other potential confounders. Materials and methods: Using a cross-sectional design and pathology review of a cohort of Japanese atomic-bomb survivors, we found that 213 of 335 (63.6%) subjects with PLC and 55 of 776 (7.1%) subjects without PLC had cirrhosis. Results: We found no association between acute exposure to A-bomb radiation and liver cirrhosis. The adjusted odds ratio of cirrhosis per Sv liver irradiation was 0.59 (95% confidence interval: 0.27 – 1.27). Cirrhosis risks for the highest tertile of radiation exposure (mean exposure 0.7 Sv) were also not elevated (0.8, 0.26 – 2.12 and 0.2, 0.03 – 0.98 among subjects with and without PLC. Conclusions: Acute exposure to liver irradiation does not increase risks of liver cirrhosis, regardless of PLC status.