[A Case of Metachronous Multiple Liver Metastases of AFP and PIVKA-â ¡ Producing Gastric Cancer, Responding to Transcatheter Arterial Chemoembolization].

We report a case of a 75-year-old man with a-fetoprotein(AFP)-producing gastric cancer accompanied by multiple large liver metastases. The patient underwent a total gastrectomy for gastric cancer(p-T3N3H0P0M0, fStage ⅢB). The patient then underwent chemotherapy(TS-1 80m g/day)following the radical operation. However, 5 months after the radical operation, he presented with multiple large liver tumors, which were subsequently biopsied. Based on immunohistochemical examination, the liver tumors were negative for AFP protein, but were similar to hepatoid adenocarcinoma, and no fibrosis was observed in the background liver. Therefore, we diagnosed the tumors as liver metastases of AFP producing gastric cancer and metachronous liver metastasis. The patient underwent transcatheter arterial chemoembolization(TACE). TACE decreased the AFP and PIVKA-Ⅱ levels and reduced the multiple huge liver metastases. Due to the increase in AFP and the multiple liver metastases, despite intensive hepatic infusion chemotherapy, he died 5 months after admission.

[A Case of Melena Caused by Peritoneal Dissemination, Treated with Interventional Radiology].

We reported a case of melena caused by perineal dissemination and treated with radiologic intervention. The patient was a 67-year-old woman, who underwent a partial duodenectomy for duodenal(4th portion)cancer in 2013. The pathological examination revealed that the tumor was tub2>por2 adenocarcinoma, SE, n+(10/20), M0. The patient received 2 courses of cisplatin(CDDP)plus S-1 and 8 courses of S-1 monotherapy. About 2 years postoperatively, the patient was hospitalized due to unauthorized bleeding. Metrorrhagia was diagnosed as intrapelvic dissemination based on abdominal computed tomography in April 2016. The patient underwent sigmoid colostomy because she developed bowel obstruction. Postoperatively, the patient received 6 courses of capecitabine plus oxaliplatin(CapeOX)plus bevacizumab. Three months later, a reduction in the recurrent lesion was observed. However, after 6 months, the patient was again hospitalized due to melena. Her condition improved after receivinga blood transfusion and infusinga hemostat. In order to control the hemorrhage, radiation therapy of 50 Gy/25 fractions to the intrapelvic dissemination was conducted. Bleedingcould not be controlled by conservative treatment with blood transfusion. Therefore, radiologic intervention was performed for melena caused by peritoneal dissemination. Neither rebleedingnor symptoms of possible ischemic complications were observed after the intervention until she died 3 months later.

Natural history of thyroid cancer [Review].

Thyroid cancers have long been considered to arise in middle age and, after their repeated proliferation, resulting in further damage to the genome, they progress to more aggressive and lethal cancers. However, in 2014, some studies were reported that might lead to a marked change in our understanding of the natural history of thyroid cancer. A high prevalence of papillary carcinoma in the young suggested that the first initiation of thyroid cancer is likely to occur in the infantile period. Such a conclusion was also supported by a very slow growth rate of papillary microcarcinomas (PMCs) in an observation trial. The proliferation rate of PMCs was negatively correlated with the age, and surgery to remove PMCs did not contribute to reduce mortality from thyroid cancer. These findings strongly suggested the existence of self-limiting cancers, which are truly malignant but do not progress to lethal cancers, for the first time in human history. The early detection of self-limiting cancers results in overdiagnosis. Ultrasonographic screening of the thyroid in the young should be avoided. Lethal thyroid cancers, whose origin is still unknown, appear suddenly after middle age. In the elderly, thyroid cancers are a mixture of self-limiting and lethal cancers; thus, when thyroid cancer is detected, careful follow-up with examination of its growth rate is required.

Fetal cell carcinogenesis of the thyroid: a modified theory based on recent evidence.

Thyroid cancer cells were believed to be generated by multi-step carcinogenesis, in which cancer cells are derived from thyrocytes, via multiple incidences of damage to their genome, especially in oncogenes or anti-oncogenes that accelerate proliferation or foster malignant phenotypes, such as the ability to invade the surrounding tissue or metastasize to distant organs, until a new hypothesis, fetal cell carcinogenesis, was presented. In fetal cell carcinogenesis, thyroid tumor cells are assumed to be derived from three types of fetal thyroid cell which only exist in fetuses or young children, namely, thyroid stem cells (TSCs), thyroblasts and prothyrocytes, by proliferation without differentiation. Genomic alternations, such as RET/PTC and PAX8-PPARγ1 rearrangements and a mutation in the BRAF gene, play an oncogenic role by preventing thyroid fetal cells from differentiating. Fetal cell carcinogenesis effectively explains recent molecular and clinical evidence regarding thyroid cancer, including thyroid cancer initiating cells (TCICs), and it underscores the importance of identifying a stem cells and clarifying the molecular mechanism of organ development in cancer research. It introduces three important concepts, the reverse approach, stem cell crisis and mature and immature cancers. Further, it implies that analysis of a small population of cells in a cancer tissue will be a key technique in establishing future laboratory tests. In the contrary, mass analysis such as gene expression profiling, whole genomic scan, and proteomics analysis may have definite limitations since they can only provide information based on many cells.

Hypovascular hepatic nodules showing hypointense on the hepatobiliary-phase image of Gd-EOB-DTPA-enhanced MRI to develop a hypervascular hepatocellular carcinoma: a nationwide retrospective study on their natural course and risk factors.

OBJECTIVE: We aimed to investigate the natural outcome of nonhypervascular lesions detected in the hepatobiliary phase of gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced MRI by performing a longitudinal study retrospectively enrolled in a nationwide manner. METHODS: Between February 2008 and March 2011, 224 patients with 504 nodules that were diagnosed as nonhypervascular by imaging were recruited from institutions that participated in the present study. We examined the natural outcome of nonhypervascular lesions and evaluated the risk factors. RESULTS: Of the 504 nodules, 173 (34.3%) showed hypervascular transformation. The overall cumulative incidence of hypervascular transformation was 14.9% at 12 months and 45.8% at 24 months. Multivariate analysis using the Cox regression model revealed previous treatment history for hepatocellular carcinoma (HCC; relative risk = 1.498; p = 0.036, 95% CI 1.03-2.19) and hyperintensity on T2-weighted images (relative risk = 1.724; p = 0.015, 95% CI 1.11-2.67) were identified as independent factors for hypervascular transformation. CONCLUSIONS: Patients who have a previous treatment history for HCC and with hypointense nodules showing hyperintensity on T2-weighted images need careful follow-up because of the high incidence of hypervascular transformation.

Preparation of thyroid follicular cells for mRNA quantification after fluorescence-activated cell sorting.

We established a novel method to analyze cells collected by fluorescence-activated cell sorting (FACS) named mRNA quantification after FACS (FACS-mQ) in which cells are labeled with a fluorescence dye in a manner that minimizes RNA degradation, and then cells sorted by FACS are examined by analyzing their gene expression profile. In order to analyze cells using FACS-mQ, it is essential to prepare single-cell suspensions without RNA degradation. We found that a new tissue preservation medium, ThelioKeep™, which contains epigallocatechin-3-gallate (EGCG), was suitable for preservation of thyroid tissues. The aim of this study was to establish a cell dispersion method of thyroid follicular cells using ThelioKeep™. We compared the efficiency of cell dispersion between the two methods, the conventional cold pre-incubation method and the ThelioKeep™ method; then we determined if cells obtained by the ThelioKeep™ method were suitable for FACS-mQ analysis. We found that a larger number of cells were recovered using ThelioKeep™ than using the conventional cold pre-incubation method. Furthermore, cell viability was higher with the ThelioKeep™ method than with the cold pre-incubation method. Thyroid cells collected by this method were analyzed by FACS-mQ. A clear shift in flow cytometry analysis was observed when cells were stained with an anti-thyroglobulin or anti-thyroid transcription factor-1 antibody. After sorting, the same copy number of ACTB mRNA was detected in thyroid cells as in an anaplastic carcinoma cell line, 8305C. These findings imply that preparation of thyroid cells using the present method is suitable for FACS-mQ analysis.

Multiple hemangiomas (hepatic small vessel neoplasia) in the liver with Budd-Chiari syndrome.

Hepatic small vessel neoplasia (HSVN) is a recently recognized hemangioma of the liver with uncertain malignant potential. Almost all the patients are asymptomatic. Budd-Chiari syndrome (BCS) is a rare disorder characterized by noncardiogenic hepatic venous outflow obstruction. Benign hepatocellular nodules have been acknowledged for a long time in the liver with the chronic BCS. However, there has been no case report of BCS associated with HSVN. The patient was diagnosed with BCS 13 years ago. The imaging test initially displayed multiple hepatic nodules that were suspected of benign hepatocellular nodules. They gradually increased in size and number in the course of the disease. At an autopsy, these nodules were confirmed to be multifocal HSVN. The tumor of the present case could not be proved to have GNAQ and GNQ14 mutations. We describe the case focusing on the chronological imaging changes and discuss on the relationship between BCS and HSVN.

Measurement of TFF3 mRNA in aspirates from thyroid nodules using mesh filtration: the first clinical trial in 130 cases.

Measurement of gene expression levels in thyroid tumor cells in aspirates was difficult because it is interfered with peripheral blood cells or infiltrating lymphocytes. In this study, we established a novel method to separate thyroid tumor cells from blood cells efficiently with mesh filtration. The expression level of trefoil factor 3 (TFF3) mRNA was estimated using LGALS3 mRNA as an internal control (T/G ratio) in 148 preoperative thyroid aspirates. Intra-assay coefficients of variation (CV) of T/G ratio for high, moderate, and low samples were 6.5%, 2.5%, and 9.7%, respectively, and inter-assay CV for high, moderate, and low samples were 27.7%, 21.9%, and 38.2%, respectively. Nondiagnostic samples in terms of T/G ratio and cytology were 12.2% and 16.9%, respectively. We observed no interference with the data by contaminating blood cells. Among these patients, 12 patients received more than two repeated aspirations. We did not observe a marked day-to-day variation except in two cases. All 13 preoperative aspirates diagnosed as malignant by cytology showed an extremely low T/G ratio, whereas 93 aspirates diagnosed as benign by cytology showed extremely varied T/G ratios and 21.5% of them showed a T/G ratio below the cut-off value. Eleven cases underwent surgery. All nodules showing a low T/G ratio were diagnosed as papillary carcinoma by pathological diagnosis. However, one nodule diagnosed as follicular adenoma after surgery showed a high T/G ratio. Our present method may be a promising preoperative test for measuring mRNAs in thyroid aspirates.

[Aspiration biopsy-nucleic acid diagnosis for widely used preparative testing].

Cancer is believed to be generated from normal cells via multi-step carcinogenesis. This hypothesis led researchers to perform studies utilizing genetic alternations in cancer cells in pre or post operative diagnostic tests. However, such an approach has not led to the establishment of widely used molecular-based diagnostic methods, which shows a clear contrast to conventional tumor markers. Although fine needle aspiration biopsy (FNAB) is the most accurate tool for preoperative diagnosis of thyroid malignancy, differential diagnosis between thyroid follicular adenomas and follicular carcinomas is quite difficult. Thus, a preoperative diagnostic method for follicular tumor has been anticipated for a long time. We tried to find a molecular marker to distinguish benign and malignant thyroid nodules based on a gene expression which can be used in Aspiration Biopsy-Nucleic Acid Diagnosis (ABND), and found that the decreased expression of trefoil factor 3 (TFF3) mRNA is a promising marker of thyroid malignancies, including follicular carcinoma. Furthermore, we established a novel method to separate thyroid tumor cells from blood cells using mesh filtration in order to avoid interference with peripheral blood cells that are aspirated simultaneously by FNAB. Using this method, we started a clinical trial and measured TFF3 mRNA in aspirates obtained from patients with a thyroid nodule. All preoperative aspirates diagnosed as malignant by cytology showed a low TFF3 mRNA expression. The preoperative aspirates diagnosed as benign by cytology showed extremely varied TFF3 mRNA expressions and about 20% showed a low TFF3 mRNA expression. Since ABND measuring TFF3 mRNA in aspirates covers the majority of thyroid malignancies and thyroid nodule is a very common clinical problem, it is expected to be the first widely used molecular-based screening test of cancer.

[FACS-mQ as a powerful clinical test in the cancer stem cell era].

Stem cells are pluripotent and self renewing, and possess an ability to differentiate into the various cell types of a particular tissue. In cancer tissues, existence of cells showing biological similarities with stem cells, named cancer stem cells (CSC), are known to regulate the growth of the tissue and determine its prognosis. Stem cells and CSCs usually exist as minor populations of cells in a tissue. Detection and analysis of these cells are usually laborious even using fluorescence activated cell sorting (FACS) with the conventional protocols. Considering these drawbacks, we developed a novel analytical method named mRNA quantification after FACS (FACS-mQ). In FACS-mQ, cells are labeled with a fluorescent dye in a manner that minimizes RNA degradation, then cells sorted by FACS are examined by analyzing their gene expression profile. We established protocols to obtain single cells from clinical samples for flow cytometry analysis. Further, we performed FACS-mQ analysis using fluorescence-labeled antibodies, cRNA probes and locked nucleic acid (LNA) probes. Evident decrease of intracellular RNAs did not observed in FACS-mQ using immunocytochemistry. Approximately 60% of intracellular RNA was preserved after in situ hybridization using cRNA probes. These RNAs from a small number of sorted cells were suitable for quantitative analysis to establish gene expression profiles. FACS-mQ does not require laborious and time-consuming procedures; thus, it is expected to facilitate research on stem cells or cancer stem cells.

2022 European Thyroid Association Guidelines for the management of pediatric thyroid nodules and differentiated thyroid carcinoma.

At present, no European recommendations for the management of pediatric thyroid nodules and differentiated thyroid carcinoma (DTC) exist. Differences in clinical, molecular, and pathological characteristics between pediatric and adult DTC emphasize the need for specific recommendations for the pediatric population. An expert panel was instituted by the executive committee of the European Thyroid Association including an international community of experts from a variety of disciplines including pediatric and adult endocrinology, pathology, endocrine surgery, nuclear medicine, clinical genetics, and oncology. The 2015 American Thyroid Association Pediatric Guideline was used as framework for the present guideline. Areas of discordance were identified, and clinical questions were formulated. The expert panel members discussed the evidence and formulated recommendations based on the latest evidence and expert opinion. Children with a thyroid nodule or DTC require expert care in an experienced center. The present guideline provides guidance for healthcare professionals to make well-considered decisions together with patients and parents regarding diagnosis, treatment, and follow-up of pediatric thyroid nodules and DTC.

Evaluation of the efficacy of LAMP-based SARS-CoV-2 detection with simple RNA extraction from nasopharyngeal swabs: A prospective observational study.

The Loopamp SARS-CoV-2 Detection Kit is used for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Loop-mediated isothermal amplification (LAMP) is based on a measurement principle that can be used with a relatively simple device. Detection using this kit requires viral RNA extraction from samples with the QIAGEN QIAamp Viral Mini Kit (QIAGEN extraction) or the Loopamp Viral RNA Extraction Kit (Eiken extraction), which are recommended by the manufacturer. However, the efficacy of LAMP-based SARS-CoV-2 detection using these extraction methods has not been compared. In this study, we aimed to compare the results of genome extraction and detection from nasopharyngeal swab samples using the QIAGEN and Eiken extraction kits. The present study involved patients who presented to the Rinku General Medical Center with suspected COVID-19 (25 positive and 26 negative cases). A comparison of the results obtained using each extraction method with those obtained via PCR showed that the positive, negative, and overall concordance rates between QIAGEN extraction and PCR were 96.0% (24/25 samples), 100% (26/26), and 98.0% (50/51; κ = 0.96, 95% CI = 0.69-1.00), respectively. Results with Eiken extraction were also favorable, with positive, negative, and overall concordance rates of 88.0% (22/25), 100% (26/26), and 94.1% (48/51; κ = 0.88, 95% CI = 0.61-1.00), respectively. Favorable results were obtained using both QIAGEN and Eiken extraction kits. Since Eiken extraction can be completed in a few minutes, it enables prompt and reliable testing for SARS-CoV-2 detection.

Messenger RNA quantification after fluorescence activated cell sorting using intracellular antigens.

Recent studies using stem cells or cancer stem cells have revealed the importance of detecting minor populations of cells in blood or tissue and analyzing their biological characteristics. The only possible method for carrying out such procedures is fluorescence activated cell sorting (FACS). However, FACS has the following limitations. First, cells without an appropriate cell surface marker cannot be sorted. Second, the cells have to be kept alive during the sorting process in order to analyze their biological characteristics. If an intracellular antigen that was specific to a particular cell type could be stained with a florescent dye and then the cells can be sorted without causing RNA degradation, a more simple and universal method for sorting and analyzing cells with a specific gene expression pattern could be established since the biological characteristics of the sorted cells could then be determined by analyzing their gene expression profile. In this study, we established a basic protocol for messenger RNA quantification after FACS (FACS-mQ) targeting intracellular antigens. This method can be used for the detection and analysis of stem cells or cancer stem cells in various tissues.

Messenger RNA quantification after fluorescence-activated cell sorting using in situ hybridization.

Recent studies using stem cells or cancer stem cells have revealed the importance of detecting minor populations of cells in blood or tissue and analyzing their biological characteristics. The only possible method for carrying out such procedures is fluorescence-activated cell sorting (FACS). However, FACS has the following two limitations. First, cells without an appropriate cell surface marker cannot be sorted. Second, some laborious procedures such as rapid sorting or treatment under sterilized conditions may require in order to analyze their biological characteristics. If a specific mRNA in a particular cell type can be stained with a florescent dye and then the cells can be sorted without causing RNA degradation, a more simple and universal method for sorting and analyzing cells with a specific gene expression pattern could be established since the biological characteristics of the sorted cells could then be determined by analyzing their gene expression profile. In this study, we established a basic protocol for messenger RNA quantification after FACS (FACS-mQ) using a cRNA probe. This method could be used for the detection and analysis of stem cells or cancer stem cells in various tissues.

Overdiagnosis of Juvenile Thyroid Cancer.

Overdiagnosis is the detection of a disease that does not do any harm to the patient throughout the lifetime. Thyroid cancer in children is a rare disease; however, since 2011, many children in Fukushima, Japan, have been diagnosed with it, and the number has shown a steady increase to over 200 cases at present. Some experts have stated that this phenomenon is due to overdiagnosis caused by thyroid ultrasound (US)-based thyroid screening detecting self-limiting thyroid cancer, which will not lead to clinical symptoms in the future. Harm caused by overdiagnosis of thyroid cancer is more serious in the young, since it is difficult to perform active surveillance and children diagnosed with cancer are likely to suffer from stigma. Thus, overdiagnosis of thyroid cancer in the young is not only a health problem but also a problem of human rights. Conflicts of interest among people related to screening programs and some experts with incomplete knowledge on overdiagnosis help to spread misleading opinions together with fear of radiation exposure among residents, which has led to their erroneous understanding of the nature of US-based thyroid screening. Scientific and honest discussions among experts to enhance education of residents and consideration of medical ethics are crucial to prevent the expansion of overdiagnosis.

Trefoil factor 3 (TFF3): a promising indicator for diagnosing thyroid follicular carcinoma.

Since the introduction of fine needle aspiration biopsy (FNAB) in the 1970's, a preoperative diagnostic technique for thyroid follicular carcinoma has long been awaited. Many markers that distinguish follicular carcinomas from adenomas have been reported; however, most of them have not been confirmed to be beneficial for clinical use. Trefoil factor 3 (TFF3) is a relatively new family of peptides that bears the three-loop trefoil domain. Several groups have reported that the suppression of TFF3 mRNA expression is related to malignant characteristics of thyroid follicular cell-derived tumors and the expression level of TFF3 mRNA is the most promising indicator for diagnosing follicular carcinoma. Development of TFF3-based diagnostic methods is now ongoing and it may not be long before thyroid follicular carcinoma can be diagnosed preoperatively using an aspirated sample from the tumor.

BRAF mutation in papillary thyroid carcinoma in a Japanese population: its lack of correlation with high-risk clinicopathological features and disease-free survival of patients.

Recent studies have demonstrated that BRAF(V600E) mutation is a common event in papillary thyroid carcinoma and a majority of these lesions have shown a direct relationship between BRAF(V600E) mutation and aggressive characteristics, including a worse patient prognosis. However, there are no studies from Japan regarding this issue in a large series with adequate postoperative follow-up periods. We investigated BRAF(V600E) mutation in 631 patients with papillary carcinoma having median follow-up periods of 83 months. The prevalence of BRAF(V600E) mutation was 38.4%, and the rate was higher in carcinoma larger than 1.0 cm but did not successively increase with tumor size. Furthermore, the prevalence did not significantly increase in cases demonstrating high-risk biological features such as clinically apparent lymph node metastasis, massive extrathyroid extension, advanced age, distant metastasis at surgery, and advanced Stage. The disease-free survival of patients with BRAF(V600E) mutation did not differ from that of those without BRAF(V600E) mutation. These findings indicate that, although BRAF(V600E) mutation may play some roles in local carcinoma development, there is no evidence that BRAF(V600E) mutation significantly reflects the aggressive characteristics and poor prognosis of patients with papillary carcinoma in Japan.

[Fetal cell carcinogenesis hypothesis and the prospect of future laboratory tests].

A novel hypothesis of carcinogenesis, the "fetal cell carcinogenesis" hypothesis, was established based on molecular evidence of thyroid carcinoma. In this hypothesis, cancer cells are derived directly from the remnants of fetal cells, instead of well-differentiated somatic cells by de-differentiation. For example, thyroid cancer cells are generated from three types of fetal thyroid cell, namely, thyroid stem cells (TSCs), thyroblasts, and prothyrocytes by proliferation without differentiation, which results in producing anaplastic, papillary, and follicular carcinoma, respectively. Genomic alternations, such as RET/PTC and PAX8-PPARgamma1 rearrangements and a mutation in the BRAF gene, play an oncogenic role by preventing thyroid fetal cells from differentiating. Fetal cell carcinogenesis effectively explains recent molecular evidence regarding cancer, including cancer stem cells, and it underscores the importance of identifying a stem cells and clarifying the molecular mechanism of organ development in cancer research. Further, it introduces two important concepts, the reverse approach and stem cell crisis. Analysis of the molecular behavior of a single cell will be a key technique in establishing future laboratory tests. On the other hand, mass analyses such as gene expression profiling, whole genomic scan, and proteomics analysis have definite limitations since they can only provide information based on many cells. In light of these aspects, we started a project to establish FACS-mQ (mRNA quantification after Fluorescence Activated Cell Sorting). In FACS-mQ, cells are sorted by a specific gene expression pattern, and the gene expression profile in sorted cells can be easily analyzed.

[A case of hepatocellular carcinoma with liver cirrhosis type C surviving over 2 years by repeated transarterial infusion therapy using cisplatinum].

A 58-year-old man with hepatocellular carcinoma (HCC) with liver cirrhosis type C was admitted to our division in April, 2004 to receive transarterial chemo-embolization (TACE) as a first-line chemotherapy for HCC. Thereafter, he had TACE twice for multicentric HCC. In December, 2005, TACE was thought to have become ineffective since his multicentric HCC progressed. So, we employed transarterial injection therapy (TAI) with fine powder cisplatinum (IAC) as a second-line chemotherapy. Stable disease was accomplished by the 22 sessions of TAI with IAC. After February 2008, IAC was given in combination with degradable starch microspheres (DSM) to reinforce the anti-cancer effect. The total therapeutic sessions using IAC became 26 in number. Total IAC amount used reached to more than 2,200 mg without any serious adverse reaction. In conclusion, TAI using IAC is thought to be effective in the chemotherapy of HCC, safely controlling the tumor progression.