Masayoshi Kage

Pathologic diagnosis of early hepatocellular carcinoma : a report of the international consensus group for hepatocellular neoplasia

Advances in imaging techniques and establishment of surveillance protocols for high-risk populations have led to the detection of small hepatic nodules in patients with chronic liver diseases, particularly those with cirrhosis or chronic hepatitis caused by hepatitis B or C viruses. These nodules, comprising a broad range of diagnostic entities—some benign and some with malignant potential—are currently defined histologically, and their clinical management often depends on the ability to make a reliable histologic diagnosis. Evidence accumulated in the last two decades strongly favors the existence of a sequence of events in hepatic nodules that precedes the emergence of hepatocellular carcinoma (HCC),1-10 and these lesions are recognized as precursors of HCC. However, from the beginning of their recognition, there has been considerable confusion concerning nomenclature and diagnostic approaches to these hepatic nodules. To clarify these issues, an International Working Party (IWP) of the World Congresses of Gastroenterology proposed a consensus nomenclature and diagnostic criteria for hepatocellular nodular lesions in 1995.11 The IWP classified nodular lesions found in chronic liver disease into large regenerative nodule, lowgrade dysplastic nodule (L-DN), high-grade dysplastic nodule (H-DN), and HCC; this nomenclature has been widely adopted. In addition, the IWP introduced the concept of dysplastic focus as a cluster of hepatocytes with features of early neoplasia (in particular small cell change or iron-free foci in a siderotic background) measuring less than 0.1 cm, and defined small HCC as a tumor measuring less than 2 cm. More recent studies support the division of small HCC into two clinico-pathological groups that have been termed early HCC and progressed HCC. Early HCC has a vaguely nodular appearance and is well differentiated. Progressed HCC has a distinctly nodular pattern and is mostly moderately differentiated, often with evidence of microvascular invasion.12 Early HCC has a longer time to recurrence and a higher 5-year survival rate compared with progressed HCC.13 Small lesions with malignant potential have only subtle differences from the surrounding parenchyma, making them difficult to assess reproducibly. Differences in the application of diagnostic criteria between Western and Eastern pathologists has been a persistent difficulty in research and clinical management of these lesions.14 In order to obtain a refined and up-to-date international consensus on the histopathologic diagnosis of nodular lesions, such as dysplastic nodules and early HCC, the International Consensus Group for Hepatocellular Neoplasia (ICGHN) was convened in April 2002 in Kurume, Japan. The group has met several times up to July 2007 under the auspices of the Laennec Liver Pathology Society. The ICGHN is currently comprised of 34 pathologists and two clinicians from 13 countries. It includes most members of the original IWP who are still active and all the participants from the first ICGHN meeting. This consensus document summarizes the results of our meetings.

Association of PD-L1 overexpression with activating EGFR mutations in surgically resected nonsmall-cell lung cancer

BACKGROUND Recent clinical trials have shown that immune-checkpoint blockade yields a clinical response in a subset of individuals with advanced nonsmall-cell lung cancer (NSCLC). We examined whether the expression of programmed death-ligand 1 (PD-L1) is related to clinicopathologic or prognostic factors in patients with surgically resected NSCLC. PATIENTS AND METHODS The expression of PD-L1 was evaluated by immunohistochemical analysis in 164 specimens of surgically resected NSCLC. Cell surface expression of PD-L1 in NSCLC cell lines was quantified by flow cytometry. RESULTS Expression of PD-L1 in tumor specimens was significantly higher for women than for men, for never smokers than for smokers, and for patients with adenocarcinoma than for those with squamous cell carcinoma. Multivariate analysis revealed that the presence of epidermal growth factor receptor gene (EGFR) mutations and adenocarcinoma histology were significantly associated with increased PD-L1 expression in a manner independent of other factors. Cell surface expression of PD-L1 was also significantly higher in NSCLC cell lines positive for activating EGFR mutations than in those with wild-type EGFR. The EGFR inhibitor erlotinib downregulated PD-L1 expression in the former cell lines but not in the latter, suggesting that PD-L1 expression is increased by EGFR signaling conferred by activating EGFR mutations. A high level of PD-L1 expression in resected tumor tissue was associated with a significantly shorter overall survival for NSCLC patients. CONCLUSIONS High expression of PD-L1 was associated with the presence of EGFR mutations in surgically resected NSCLC and was an independent negative prognostic factor for this disease.

Induction of PD-L1 Expression by the EML4–ALK Oncoprotein and Downstream Signaling Pathways in Non–Small Cell Lung Cancer

Purpose: Therapies targeted to the immune checkpoint mediated by PD-1 and PD-L1 show antitumor activity in a subset of patients with non–small cell lung cancer (NSCLC). We have now examined PD-L1 expression and its regulation in NSCLC positive for the EML4–ALK fusion gene. Experimental Design: The expression of PD-L1 at the protein and mRNA levels in NSCLC cell lines was examined by flow cytometry and by reverse transcription and real-time PCR analysis, respectively. The expression of PD-L1 in 134 surgically resected NSCLC specimens was evaluated by immunohistochemical analysis. Results: The PD-L1 expression level was higher in NSCLC cell lines positive for EML4–ALK than in those negative for the fusion gene. Forced expression of EML4–ALK in Ba/F3 cells markedly increased PD-L1 expression, whereas endogenous PD-L1 expression in EML4–ALK–positive NSCLC cells was attenuated by treatment with the specific ALK inhibitor alectinib or by RNAi with ALK siRNAs. Furthermore, expression of PD-L1 was downregulated by inhibitors of the MEK–ERK and PI3K–AKT signaling pathways in NSCLC cells positive for either EML4–ALK or activating mutations of the EGFR. Finally, the expression level of PD-L1 was positively associated with the presence of EML4–ALK in NSCLC specimens. Conclusions: Our findings that both EML4–ALK and mutant EGFR upregulate PD-L1 by activating PI3K–AKT and MEK–ERK signaling pathways in NSCLC reveal a direct link between oncogenic drivers and PD-L1 expression. Clin Cancer Res; 21(17); 4014–21. ©2015 AACR.

A serum “sweet-doughnut” protein facilitates fibrosis evaluation and therapy assessment in patients with viral hepatitis

Although liver fibrosis reflects disease severity in chronic hepatitis patients, there has been no simple and accurate system to evaluate the therapeutic effect based on fibrosis. We developed a glycan-based immunoassay, FastLec-Hepa, to fill this unmet need. FastLec-Hepa automatically detects unique fibrosis-related glyco-alteration in serum hyperglycosylated Mac-2 binding protein within 20 min. The serum FastLec-Hepa counts increased with advancing fibrosis and illustrated significant differences in medians between all fibrosis stages. FastLec-Hepa is sufficiently sensitive and quantitative to evaluate the effects of PEG-interferon-α/ribavirin therapy in a short post-therapeutic interval. The obtained fibrosis progression is equivalent to -0.30 stages/year in patients with sustained virological response, and 0.01 stages/year in relapse/nonresponders. Furthermore, long-term follow-up of the severely affected patients found hepatocellular carcinoma developed in patients after therapy whose FastLec-Hepa counts remained above a designated cutoff value. FastLec-Hepa is the only assay currently available for clinically beneficial therapy evaluation through quantitation of disease severity.

General rules for recording endoscopic findings of esophagogastric varices (2ND EDITION)

General rules for recording endoscopic findings of esophageal varices were initially proposed in 1980 and revised in 1991. These rules have widely been used in Japan and other countries. Recently, portal hypertensive gastropathy has been recognized as a distinct histological and functional entity. Endoscopic ultrasonography can clearly depict vascular structures around the esophageal wall in patients with portal hypertension. Owing to progress in medicine, we have updated and slightly modified the former rules. The revised rules are simpler and more straightforward than the former rules and include newly recognized findings of portal hypertensive gastropathy and a new classification for endoscopic ultrasonographic findings.

Tumor Growth Suppression in Pancreatic Cancer by a Putative Metastasis Suppressor Gene Cap43/NDRG1/Drg-1 through Modulation of Angiogenesis

Cap43 has been identified as a nickel- and calcium-induced gene, and is also known as N-myc downstream-regulated gene 1 (NDRG1), Drg-1 and rit42. It is also reported that overexpression of Cap43 suppresses metastasis of some malignancies, but its precise role remains unclear. In this study, we asked how Cap43 could modulate the tumor growth of pancreatic cancer. Stable Cap43 cDNA transfectants of pancreatic cancer cells with Cap43 overexpression showed similar growth rates in culture as their control counterparts with low Cap43 protein level. By contrast, Cap43 overexpression showed a marked decrease in tumor growth rates in vivo. Moreover, a marked reduction in tumor-induced angiogenesis was observed. Gelatinolytic activity by matrix metalloproteinase-9 and invasive ability in Matrigel invasion activity were markedly decreased in pancreatic cancer cell lines with high Cap43 expression. Cellular expression of matrix metalloproteinase-9 and two major angiogenic factors, vascular endothelial growth factor and interleukin-8, were also significantly decreased in cell lines with Cap43 overexpression as compared with their parental counterparts. Immunohistochemical analysis of specimens from 65 patients with pancreatic ductal adenocarcinoma showed a significant association between Cap43 expression and tumor microvascular density (P = 0.0001) as well as depth of invasion (P = 0.0003), histopathologic grading (P = 0.0244), and overall survival rates for patients with pancreatic cancer (P = 0.0062). Thus, Cap43 could play a key role in the angiogenic on- or off-switch of tumor stroma in pancreatic ductal adenocarcinoma.

Loss of PTEN Expression by Blocking Nuclear Translocation of EGR1 in Gefitinib-Resistant Lung Cancer Cells Harboring Epidermal Growth Factor Receptor–Activating Mutations

Gefitinib (Iressa) and erlotinib (Tarceva), which target the epidermal growth factor receptor (EGFR), are approved for treatment of patients with advanced non-small cell lung cancer (NSCLC). Patients whose tumors harbor mutations in the EGFR gene, including delE746-A750 in exon 19 and L858R in exon 21, may benefit in particular from gefitinib treatment. However, acquired resistance to gefitinib has been a serious clinical problem, and further optimization is needed for application of EGFR-targeted drugs in lung cancer patients. In this study, we established gefitinib-resistant NSCLC cells from PC-9 cell line, which harbors the delE746-A750 mutation, by exposing the cell line to gefitinib for over 7 months. Gefitinib-resistant PC-9/GEFs cell lines showed a marked downregulation of PTEN expression and increased Akt phosphorylation. In revertant, gefitinib-sensitive clones (PC-9/Rev) derived from PC-9/GEF1-1 and PC-9/GEF2-1, PTEN expression, as well as sensitivity to gefitinib and erlotinib, was restored. Knockdown of PTEN expression using small interfering RNA specific for PTEN in PC-9 cells resulted in drug resistance to gefitinib and erlotinib. Nuclear translocation of the EGR1 transcription factor, which regulates PTEN expression, was shown to be suppressed in resistant clones and restored in their revertant clones. Reduced PTEN expression was also seen in tumor samples from a patient with gefitinib-refractory NSCLC. This study thus strongly suggests that loss of PTEN expression contributes to gefitinib and erlotinib resistance in NSCLC. Our findings reinforce the therapeutic importance of PTEN expression in the treatment of NSCLC with EGFR-targeted drugs.

Angioarchitecture and blood circulation in focal nodular hyperplasia of the liver

BACKGROUND/AIMS We sought to clarify important unresolved points regarding angioarchitecture and blood circulation in focal nodular hyperplasia. METHODS Twenty-nine surgically resected focal nodular hyperplasia lesions were examined histologically, immunohistochemically, and radiologically. In three autopsy cases, red- and blue-colored gelatin was injected into the hepatic artery and the portal vein, respectively, to demonstrate the vasculature in focal nodular hyperplasia. RESULTS Histologically, no orientation with respect to portal tracts and central veins was evident in any lesion. Within lesions, vessels were classified as: (i) anomalous arteries in the fibrous septa, (ii) capillaries in the fibrous septa, or (iii) venous vessels located mainly in the parenchyma. Vessels and sinusoids adjacent to fibrous septa were stained for CD 34 and von Willebrand factor. The anomalous arteries were connected to the capillaries. Capillaries in the fibrous septa were connected to sinusoids adjacent to fibrous septa. Venous vessels were connected to central or hepatic veins surrounding the lesions. Intranodular sinusoids were connected to the sinusoids in the surrounding normal liver. Red-colored gelatin, injected at autopsy into the hepatic artery, appeared not only in the anomalous arteries but also in capillaries and in sinusoids adjacent to the fibrous septa of the lesion. Angiography clearly depicted hepatic veins located near the lesions in nine cases. Computed tomography during arterial portography disclosed no portal blood flow in the lesions. CONCLUSIONS In focal nodular hyperplasia, arterial blood flows from the anomalous arteries via the capillaries into sinusoids adjacent to fibrous septa. The blood in the sinusoids drained to the hepatic vein either directly or via perinodular sinusoids.

Molecular diagnosis of activating EGFR mutations in non-small cell lung cancer using mutation specific antibodies for immunohistochemical analysis

Purpose: Therapeutic responses of non–small cell lung carcinoma (NSCLC) to epidermal growth factor receptor (EGFR)–targeted drugs, such as gefitinib and erlotinib, are closely associated with activating EGFR mutations. The most common mutations are delE746-A750 in exon 19 and L858R in exon 21, accounting for ∼90% of all EGFR mutations. Recently, EGFR mutation-specific antibodies were developed and did well in immunohistochemical analysis, giving a sensitivity of ∼90%. We have investigated whether this method detects activating EGFR mutations with sensitivity comparable with direct DNA sequencing, which is used to detect these mutations in NSCLC. Experimental Design: We used antibodies specific for the E746-A750 deletion mutation in exon 19 and the L858R point mutation in exon 21 in Western blot analysis and immunohistochemistry to determine the presence of these mutations in NSCLC cell lines. We also examined these EGFR mutations in NSCLC tumor samples from 60 patients by immunohistochemically and direct DNA sequencing. Results: We were able to identify EGFR mutations in NSCLC tumor samples immunohistochemically with a sensitivity of 79% using the anti–delE746-A750 antibody and 83% using the anti-L858R antibody. Additional DNA sequencing markedly improved the sensitivity obtained by immunohistochemistry. Conclusions: This simple and rapid assay for detecting EGFR mutations, even in the small bronchial biopsies obtained in stage IV NSCLC patients, will be useful for diagnosing responsiveness to EGFR-targeted drugs in patients with NSCLC. Combining this with DNA sequencing is recommended for the development of improved personalized EGFR-targeted therapeutics. Clin Cancer Res; 16(12); 3163–70. ©2010 AACR.

Application of a new histological staging and grading system for primary biliary cirrhosis to liver biopsy specimens: Interobserver agreement

Recently the authors proposed a new staging and grading system for primary biliary cirrhosis (PBC) that takes into account necroinflammatory activity and histological heterogeneity. Herein is proposed a convenient version of this system. Scores for fibrosis, bile duct loss, and chronic cholestasis were combined for staging: stage 1, total score of 0; stage 2, score 1–3; stage 3, score 4–6; and stage 4, score 7–9. Cholangitis activity (CA) and hepatitis activity (HA) were graded as CA0–3, and HA0–3, respectively. Analysis of interobserver agreement was then conducted. Digital images of 62 needle liver biopsy specimens of PBC were recorded as virtual slides on DVDs that were sent to 28 pathologists, including five located overseas. All participants were able to apply this version in all 62 cases. For staging, kappa was 0.385 (fair agreement) and the concordance rate was 63.9%. For necroinflammatory activity, the kappa and concordance rate were 0.110 (slight agreement) and 36.9% for CA, and 0.197 (slight agreement) and 47% for HA, respectively. In conclusion, this new staging and grading system for PBC seems to be more convenient and practical than those used at present, but more instruction and guidance are recommended for the grading of necroinflammatory activity in practice.