Kunihito Gotoh

Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. We sequenced and analyzed the whole genomes of 27 HCCs, 25 of which were associated with hepatitis B or C virus infections, including two sets of multicentric tumors. Although no common somatic mutations were identified in the multicentric tumor pairs, their whole-genome substitution patterns were similar, suggesting that these tumors developed from independent mutations, although their shared etiological backgrounds may have strongly influenced their somatic mutation patterns. Statistical and functional analyses yielded a list of recurrently mutated genes. Multiple chromatin regulators, including ARID1A, ARID1B, ARID2, MLL and MLL3, were mutated in ∼50% of the tumors. Hepatitis B virus genome integration in the TERT locus was frequently observed in a high clonal proportion. Our whole-genome sequencing analysis of HCCs identified the influence of etiological background on somatic mutation patterns and subsequent carcinogenesis, as well as recurrent mutations in chromatin regulators in HCCs.

Inhibition of autophagy prevents hippocampal pyramidal neuron death after hypoxic-ischemic injury.

Neonatal hypoxic/ischemic (H/I) brain injury causes neurological impairment, including cognitive and motor dysfunction as well as seizures. However, the molecular mechanisms regulating neuron death after H/I injury are poorly defined and remain controversial. Here we show that Atg7, a gene essential for autophagy induction, is a critical mediator of H/I-induced neuron death. Neonatal mice subjected to H/I injury show dramatically increased autophagosome formation and extensive hippocampal neuron death that is regulated by both caspase-3-dependent and -independent execution. Mice deficient in Atg7 show nearly complete protection from both H/I-induced caspase-3 activation and neuron death indicating that Atg7 is critically positioned upstream of multiple neuronal death executioner pathways. Adult H/I brain injury also produces a significant increase in autophagy, but unlike neonatal H/I, neuron death is almost exclusively caspase-3-independent. These data suggest that autophagy plays an essential role in triggering neuronal death execution after H/I injury and Atg7 represents an attractive therapeutic target for minimizing the neurological deficits associated with H/I brain injury.

A novel image-guided surgery of hepatocellular carcinoma by indocyanine green fluorescence imaging navigation.

The clear delineation between tumor and normal tissue is ideal for real‐time surgical navigation imaging. We present a novel indocyanine green (ICG) fluorescence imaging technique to visualize hepatocellular carcinoma (HCC).

Whole-genome mutational landscape and characterization of noncoding and structural mutations in liver cancer

Liver cancer, which is most often associated with virus infection, is prevalent worldwide, and its underlying etiology and genomic structure are heterogeneous. Here we provide a whole-genome landscape of somatic alterations in 300 liver cancers from Japanese individuals. Our comprehensive analysis identified point mutations, structural variations (STVs), and virus integrations, in noncoding and coding regions. We discovered mutational signatures related to liver carcinogenesis and recurrently mutated coding and noncoding regions, such as long intergenic noncoding RNA genes (NEAT1 and MALAT1), promoters, CTCF-binding sites, and regulatory regions. STV analysis found a significant association with replication timing and identified known (CDKN2A, CCND1, APC, and TERT) and new (ASH1L, NCOR1, and MACROD2) cancer-related genes that were recurrently affected by STVs, leading to altered expression. These results emphasize the value of whole-genome sequencing analysis in discovering cancer driver mutations and understanding comprehensive molecular profiles of liver cancer, especially with regard to STVs and noncoding mutations.

Hepatocellular carcinoma arising from non-cirrhotic nonalcoholic steatohepatitis

PurposeCharacteristics of hepatocellular carcinoma (HCC) complicating nonalcoholic steatohepatitis (NASH) are still controversial. Most NASH related HCCs are believed to develop from cirrhotic liver, but case reports about HCC arising from non-cirrhotic NASH have been accumulating recently. This study is designed to elucidate characteristics of NASH related HCC diagnosed with high accuracy by using surgically resected specimens that contain larger areas to validate than biopsy specimens.MethodsFor this study, 1168 patients who underwent hepatic resection at Osaka Medical Center for Cancer and Cardiovascular Diseases were enrolled. Patients who had clinically obvious causes of chronic liver dysfunction, such as viral and alcoholic hepatitis, were excluded. Histological diagnosis of NASH was confirmed according to Brunt’s criterion.ResultsEight (1%) patients were diagnosed with NASH. Six (75%) of them showed non-cirrhosis in non-cancerous areas. Stages of fibrosis in the non-cirrhotic patients were mild fibrosis (F2) in five and moderate fibrosis (F3) in one. All patients complicated with metabolic diseases. Although all these patients without cirrhosis were evaluated pathologically at tumor-node-metastasis stages I or II, three (50%) had multiple recurrences of tumor within a short time after surgery.ConclusionThis study indicates HCC might arise frequently from non-cirrhotic NASH. While further studies are needed to confirm this observation, both cirrhotic and non-cirrhotic NASH warrant regular screening for HCC.

Feasibility and Efficacy of Combination Therapy With Preoperative Full-Dose Gemcitabine, Concurrent Three-Dimensional Conformal Radiation, Surgery, and Postoperative Liver Perfusion Chemotherapy for T3-Pancreatic Cancer

Objective:To evaluate both the feasibility and efficacy of our combined therapy, which consisted of preoperative chemoradiation, surgery, and postoperative liver perfusion chemotherapy (LPC) for patients with T3 (extended beyond the pancreatic confines) cancer of the pancreas. Summary Background Data:Because of the high incidence of local recurrence and liver metastasis, long-term outcomes for patients after resection of T3-pancreatic cancer are extremely poor. Methods:During the period from 2002 to 2007, 38 patients with T3-pancreatic cancers consented to receive a combination of preoperative chemoradiation, surgery, and postoperative LPC. With the aid of 3D radiation planning, irradiation fields were constructed that included both the primary pancreatic tumor and retropancreatic tissues while taking care to exclude any section of the gastrointestinal tract. The total dose of radiation was 50 Gy (2 Gy × 25 fractions/5 weeks) and was administered in combination with gemcitabine treatments (1000 mg/m2/week × 9/3 months). Preoperative restaging via computerized tomography and intraoperative inspection were used to determine if pancreatectomy was indicated. For respected cases, one catheter was placed into the gastroduodenal artery and another one into the superior mesenteric vein. Postoperatively, 5-FU (125 mg/day × 28 days) was infused via each of these 2 routes. Results:Preoperative chemoradiation was completed for all 38 patients, including 3 patients who required gemcitabine-dose reduction. Seven patients (18%) did not undergo surgical resection because either distant metastases or progressive local tumors had been detected after chemoradiation. The remaining 31 patients (82%) underwent pancreatectomy plus postoperative LPC, without postoperative or in-hospital mortality. The 5-year survival rate after pancreatectomy was 53%, with low incidences of both local recurrence (9%) and liver metastasis (7%). Postoperative histopathologic study revealed a marked degenerative change in cancer tissue, showing negative surgical margins (R0) for 30 patients (96%) and negative nodal involvement for 28 patients (90%). Conclusion:Results of this trial suggest that a combination of preoperative full-dose gemcitabine, concurrent 3D-conformal radiation, surgery, and postoperative LPC is feasible for the treatment of T3-pancreatic cancer. Using the method described in this article, we were able to effectively reduce the incidence of both local and liver recurrence. Therefore, this type of combination therapy seems promising for improving long-term outcomes for patients with T3-cancers of the pancreas. This study is registered with University hospital Medical information Network clinical trials Registry number, UMIN000001804.

Whole-genome mutational landscape of liver cancers displaying biliary phenotype reveals hepatitis impact and molecular diversity

Intrahepatic cholangiocarcinoma and combined hepatocellular cholangiocarcinoma show varying degrees of biliary epithelial differentiation, which can be defined as liver cancer displaying biliary phenotype (LCB). LCB is second in the incidence for liver cancers with and without chronic hepatitis background and more aggressive than hepatocellular carcinoma (HCC). To gain insight into its molecular alterations, we performed whole-genome sequencing analysis on 30 LCBs. Here we show, the genome-wide substitution patterns of LCBs developed in chronic hepatitis livers overlapped with those of 60 HCCs, whereas those of hepatitis-negative LCBs diverged. The subsequent validation study on 68 LCBs identified recurrent mutations in TERT promoter, chromatin regulators (BAP1, PBRM1 and ARID2), a synapse organization gene (PCLO), IDH genes and KRAS. The frequencies of KRAS and IDHs mutations, which are associated with poor disease-free survival, were significantly higher in hepatitis-negative LCBs. This study reveals the strong impact of chronic hepatitis on the mutational landscape in liver cancer and the genetic diversity among LCBs.

Preoperative gemcitabine-based chemoradiation therapy for resectable and borderline resectable pancreatic cancer.

Objective: To evaluate the outcome of preoperative gemcitabine-based chemoradiation therapy (CRT) for resectable and borderline resectable pancreatic cancer (PC), with a focus on the differences in surgical outcomes and patterns of recurrence between these 2 categories. Background: Various multimodal treatment strategies have been proposed to improve the surgical outcomes of PC. Preoperative CRT and subsequent surgery is one of the promising strategies for resectable (PC-R) and borderline resectable (PC-BR) PC. Methods: A total of 268 patients with PC-R and PC-BR received preoperative gemcitabine-based CRT. The numbers of PC-R and PC-BR cases were 188 and 80, respectively. We evaluated the following comparisons between patients with PC-R and those with PC-BR: (1) resection rate, (2) rate of margin-negative resection, (3) survival, and (4) pattern of the treatment failure, including local recurrence, peritoneal dissemination, and distant metastasis. Results: The resection rate of patients with PC-R (87%) was higher than that of patients with PC-BR (54%) (P < 0.001). Pathological margin-negative resection was achieved in 99% and 98% of the patients with PC-R and PC-BR, respectively. The 5-year survival rates of the PC-R and PC-BR cases were 57% and 34%, respectively (P = 0.029). Although the 5-year cumulative incidence of local recurrence was comparable in both groups (15% and 13%, respectively; P = 0.508), the 5-year cumulative incidence of peritoneal and distant recurrence was significantly higher in the patients with PC-BR (43 and 76%) than in the patients with PC-R (17% and 43%). Conclusions: In the resected cases, the locoregional control was comparable between patients with PC-R and PC-BR after preoperative CRT. The survival rate for the patients with PC-BR was lower than the rate for those with PC-R due to a higher incidence of peritoneal and distant recurrence in the patients with PC-BR. (UMIN000001804)

Perineural invasion and lymph node involvement as indicators of surgical outcome and pattern of recurrence in the setting of preoperative gemcitabine-based chemoradiation therapy for resectable pancreatic cancer.

Objective:To analyze the histopathological indicators significantly associated with surgical outcome and the pattern of recurrence in the setting of preoperative gemcitabine-based chemoradiation therapy (CRT) and subsequent pancreatectomy. Background:Clinicopathological assessment of the resected specimen is an indispensable tool for predicting patient prognosis and localizing high-risk sites for tumor relapse. This procedure is also essential for the establishment of efficient postoperative follow-up protocols in the setting of a preoperative CRT strategy. Methods:In a prospective phase II clinical trial at our hospital, 110 patients received preoperative CRT and subsequent resection. All 110 resected cases were included in this study. We employed disease-free survival (DFS) as a surgical outcome, and the pattern of recurrence was divided into 2 categories: (1) recurrence in the abdominal cavity (RAC), defined as either a locoregional or a peritoneal recurrence; or (2) distant recurrence (DR), defined as cancer recurrence in a distant organ. Clinicopathological variables were analyzed in association with DFS, RAC, and DR. Results:Positive nodal involvement and perineural invasion were independent factors that were significantly associated with an unfavorable DFS (P = 0.021 and P = 0.026, respectively). The presence of perineural invasion was the single independent variable significantly associated with an increased risk of RAC (P = 0.002), whereas the status of nodal involvement was the single independent variable significantly associated with an increased risk of DR (P = 0.013). Conclusions:The status of nodal involvement and perineural invasion in resected specimens are significantly associated with DFS and clearly predict the pattern of recurrence in the setting of a preoperative gemcitabine-based CRT strategy. This study is registered at UMIN-CTR and carries the ID number UMIN000001804.

Participation of autophagy in the initiation of graft dysfunction after rat liver transplantation

Better ways to prevent the cold ischemia-warm reperfusion (CI/WR) injury associated with liver transplantation are needed, and many investigations have focused on the molecular mechanisms of this injury. However, the mechanisms reported to date are controversial and no improvement in therapy has resulted. Here, using prolonged CI and orthotopic transplantation of rat liver grafts, we found that the CI/WR injury was closely associated with autophagy. By 15 min after the start of WR, small masses of hepatocytes that possessed abundant autophagosomes and autolysosomes frequently dissociated from the hepatic cords and obstructed the sinusoid, causing massive necrosis of hepatocytes within 2 hours. The cell masses included TUNEL-positive nuclei without caspase-3 and -7 activation. Autophagy suppression with the phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin or LY294002, reduced both liver damage and the mortality rate of recipient rats. To elucidate the downstream mechanisms of this autophagic pathway, liver grafts were treated with aspartic and cysteine proteinase inhibitors, pepstatin and leupeptin. This treatment also significantly improved the survival rate of recipient rats. These data suggest that autophagy-associated hepatocyte death triggers liver graft dysfunction. The protective effects of suppressing autophagy may suggest new ways to prevent CI/WR injury of the liver.