Training high-performance deep learning classifier for diagnosis in oral cytology using diverse annotations.

The uncertainty of true labels in medical images hinders diagnosis owing to the variability across professionals when applying deep learning models. We used deep learning to obtain an optimal convolutional neural network (CNN) by adequately annotating data for oral exfoliative cytology considering labels from multiple oral pathologists. Six whole-slide images were processed using QuPath for segmenting them into tiles. The images were labeled by three oral pathologists, resulting in 14,535 images with the corresponding pathologists' annotations. Data from three pathologists who provided the same diagnosis were labeled as ground truth (GT) and used for testing. We investigated six models trained using the annotations of (1) pathologist A, (2) pathologist B, (3) pathologist C, (4) GT, (5) majority voting, and (6) a probabilistic model. We divided the test by cross-validation per slide dataset and examined the classification performance of the CNN with a ResNet50 baseline. Statistical evaluation was performed repeatedly and independently using every slide 10 times as test data. For the area under the curve, three cases showed the highest values (0.861, 0.955, and 0.991) for the probabilistic model. Regarding accuracy, two cases showed the highest values (0.988 and 0.967). For the models using the pathologists and GT annotations, many slides showed very low accuracy and large variations across tests. Hence, the classifier trained with probabilistic labels provided the optimal CNN for oral exfoliative cytology considering diagnoses from multiple pathologists. These results may lead to trusted medical artificial intelligence solutions that reflect diverse diagnoses of various professionals.

Effectiveness of deep learning classifiers in histopathological diagnosis of oral squamous cell carcinoma by pathologists.

The study aims to identify histological classifiers from histopathological images of oral squamous cell carcinoma using convolutional neural network (CNN) deep learning models and shows how the results can improve diagnosis. Histopathological samples of oral squamous cell carcinoma were prepared by oral pathologists. Images were divided into tiles on a virtual slide, and labels (squamous cell carcinoma, normal, and others) were applied. VGG16 and ResNet50 with the optimizers stochastic gradient descent with momentum and spectral angle mapper (SAM) were used, with and without a learning rate scheduler. The conditions for achieving good CNN performances were identified by examining performance metrics. We used ROCAUC to statistically evaluate diagnostic performance improvement of six oral pathologists using the results from the selected CNN model for assisted diagnosis. VGG16 with SAM showed the best performance, with accuracy = 0.8622 and AUC = 0.9602. The diagnostic performances of the oral pathologists statistically significantly improved when the diagnostic results of the deep learning model were used as supplementary diagnoses (p-value = 0.031). By considering the learning results of deep learning model classifiers, the diagnostic accuracy of pathologists can be improved. This study contributes to the application of highly reliable deep learning models for oral pathological diagnosis.

Transient Methionine Deprivation Triggers Histone Modification and Potentiates Differentiation of Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) require high levels of methionine (Met). Met deprivation results in a rapid decrease in intracellular S-adenosyl-methionine (SAM), poising human iPSCs for differentiation and leading to the apoptosis of undifferentiated cells. Met deprivation triggers rapid metabolic changes, including SAM, followed by reversible epigenetic modifications. Here, we show that short-term Met deprivation impairs the pluripotency network through epigenetic modification in a 3D suspension culture. The trimethylation of lysine 4 on histone H3 (H3K4me3) was drastically affected compared with other histone modifications. Short-term Met deprivation specifically affects the transcription start site (TSS) region of genes, such as those involved in the transforming growth factor ß pathway and cholesterol biosynthetic process, besides key pluripotent genes such as NANOG and POU5F1. The expression levels of these genes decreased, correlating with the loss of H3K4me3 marks. Upon differentiation, Met deprivation triggers the upregulation of various lineage-specific genes, including key definitive endoderm genes, such as GATA6. Upon differentiation, loss of H3K27me3 occurs in many endodermal genes, switching from a bivalent to a monovalent (H3K4me3) state. In conclusion, Met metabolism maintains the pluripotent network with histone marks, and their loss potentiates differentiation.

Circulating tumor cells and cell-free tumor DNA analyses in urothelial cancer using the LiquidBiopsy platform.

Background: Emerging data suggested that liquid biopsy such as detection of circulating tumor cells (CTCs) and cell-free tumor DNA analysis augments the management of patients with urothelial cancer (UC). We presented our pilot experience of liquid biopsy using the Ion Torrent platform to detect CTCs and genomic alterations in UC. Materials and methods: Blood or urine samples from 16 patients were subjected to CTC and plasma/urine cell-free tumor DNA isolation for next generation sequencing (NGS) using the Ion S5 system to detect mutations among 50 oncogenes on the Ion AmpliSeq Cancer Hotspot Panel. Results: The Ion Torrent platform detected a higher number of CTCs than those in previous studies using the CellSearchTM system. Overall, mutations were detected in 13/16 (81.3%) patients with a median number of 18 (range 12-25). NGS isolated 17 hotspot mutations from 11 genes and 41 novel genomic alterations from 24 genes, some of which are supposed to be clinically actionable. Conclusions: The Ion Torrent platform efficiently detected CTCs compared with previous reports. NGS with the present system also allowed for detection of gene alterations which are likely to be therapeutic targets and provided an attractive tool to guide personalized therapy for patients with advanced UC.

Structural and functional characteristics of the tripartite ABC transporter.

ATP-binding cassette (ABC) transporters are one of the largest protein superfamilies and are found in all living organisms. These transporters use the energy from ATP binding and hydrolysis to transport various substrates. In this review, we focus on the structural and functional aspects of ABC transporters, with special emphasis on type VII ABC transporters, a newly defined class possessing characteristic structures. A notable feature of type VII ABC transporters is that they assemble into tripartite complexes that span both the inner and outer membranes of Gram-negative bacteria. One of the original type VII ABC transporters, which possesses all characteristic features of this class, is the macrolide efflux transporter MacB. Recent structural analyses of MacB and homologue proteins revealed the unique mechanisms of substrate translocation by type VII ABC transporters.

Association of Nrf2 expression and mutation with Weiss and Helsinki scores in adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare malignant tumor. Genetic abnormalities that may represent therapeutic targets and prognostic factors in ACC remain unclear. Besides being one of the main cellular defense mechanisms that regulates antioxidant pathways for detoxifying reactive oxygen species (ROS), the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) promotes tumor proliferation by increasing metabolic activity. In surgical specimens from 12 cases of nonmetastatic ACCs and nine cases of benign adrenocortical adenoma (ACA), we investigated gene mutation and protein expressions for Nrf2 and the preoperative maximum standard glucose uptake (SUVmax) on [18 F]fluorodeoxy-glucose positron emission tomography. Three of five ACCs with a Weiss score of 7 to 9 were Nrf2 mutants; these ACCs had higher expression of Nrf2 and higher preoperative SUVmax. The other seven ACCs had a Weiss score of 3 to 6; these seven ACCs and all the ACAs were non-Nrf2 gene mutants. Patients with a Weiss score of 7 to 9 and Nrf2 mutant ACC had shorter overall survival. Based on Helsinki scoring, three ACCs with a Helsinki score greater than 17 had Nrf2 mutants, higher expression of Nrf2, higher preoperative SUVmax, and shorter overall survival. Our findings indicate that Nrf2 activation and the associated increase in metabolism play roles in ACC, in particular in ACC with a Weiss score of 7 to 9 and a Helsinki score of greater than 17.

Increased expression of Nrf2 and elevated glucose uptake in pheochromocytoma and paraganglioma with SDHB gene mutation.

BACKGROUND: Pheochromocytomas (PCC) and paragangliomas (PGL) are catecholamine-producing neuroendocrine tumors. According to the World Health Organization Classification 2017, all PCC/PGL are considered to have malignant potential. There is growing evidence that PCC/PGL represent a metabolic disease that leads to aerobic glycolysis. Cellular energy metabolism involves both transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and succinate dehydrogenase (SDH) subtypes, but the association of these substances with PCC/PGL is largely unknown. METHODS: We investigated SDHB gene mutation and protein expressions for SDHB and Nrf2 in surgical specimens from 29 PCC/PGL. We also assessed preoperative maximum standard glucose uptake (SUVmax) on [18F]fluorodeoxy-glucose positron emission tomography and mRNA levels for Nrf2. RESULTS: Among 5 PCC/PGL with a PASS Score ≥ 4 or with a moderately to poorly differentiated type in the GAPP Score, 4 were metastatic and found to be SDHB mutants with homogeneous deletion of SDHB protein. SDHB mutants showed a higher expression of Nrf2 protein and a higher preoperative SUVmax than non-SDHB mutants with a PASS < 4 or a well-differentiated GAPP type. Furthermore, protein expression of Nrf2 was positively associated with preoperative SUVmax. The Nrf2 mRNA level positively correlated with malignant phenotype, higher expression for Nrf2 protein and SDHB gene mutant, but negatively correlated with expression for SDHB protein. There was also a positive correlation between Nrf2 mRNA level and SUVmax. CONCLUSION: These results suggest that activation of Nrf2 and elevated metabolism play roles in PCC/PGL with malignant potential that have SDHB gene mutation and SDHB deficiency.

Molecular analysis of bovine leukemia virus in early epidemic phase in Japan using archived formalin fixed paraffin embedded histopathological specimens.

Bovine leukemia virus (BLV) is an important pathogen associated with enzootic bovine leukosis. In this study, we performed PCR and sequencing analysis to characterize BLVgp51 sequences from formalin-fixed paraffin-embedded (FFPE) specimens made from 1974 to 2000 and successfully obtained BLV proviral genome sequences from 94% of the analyzed samples. Furthermore, from these samples, we reconstructed eight full-length and nearly full-length BLVgp51 sequences. These sequences were classified as BLV genotype 1, implying that genotype1 has already been circulating in Japan since the 1970s. In our results, the proviral DNA was detected in the 1970s, 1980s, and 1990s in the same manner, indicating that the detection of BLV proviral genome depends on storage conditions rather than storage period. The sequences obtained in this study provide direct insights into BLV sequences before 2000, which serves as a good calibrator for inferring ancient BLV diversity.

A Successful Case of Switching Treatment from Ketamine to Methadone for Complex Neuropathic Pain.

Background: Methadone is frequently used for the management of complex pain at the end of life by palliative care specialists. It is also used in low doses as an add-on therapy to chronic opioid treatment of cancer-related pain, usually with good effect, and without any reported severe adverse effects. However, there are few reports of switching from ketamine to methadone. Case: We report a case of a patient with rectal cancer and intractable pain. Switching from ketamine to methadone to maintain analgesia was successfully carried out without impacting activities of daily living. Established measurement tools, such as numerical rating scale, Douleur Neuropathique, Functional Independence Measure, and Barthel Index, were used. Conclusion: Switching from ketamine to methadone may be beneficial in relieving refractory cancer-related neuropathic pain without decreasing functioning.

Mammaglobin protein localization and gene expression in the salivary glands.

PURPOSE: This study aims to delve deeper into the hypothesis that normal salivary gland tissue expresses both protein and mRNA of mammaglobin (MGB). METHODS: Formalin-fixed paraffin-embedded samples of submandibular (10), parotid (5), palatal (5) and labial glands (30) salivary glands were immunohistochemically investigated. The labial samples were used to examine the MGB positive ratio (MGB-PR), and localize MGB by double immunofluorescence staining and quantitative mRNA gene expression. Mann-Whitney U and Kruskal Wallis rank-sum test for group comparison, and Spearman's rank correlation coefficient for correlation analysis were used. RESULTS: The distribution of MGB-positive cells was variable throughout samples with significantly higher MGB-PR of acini than ducts (P = 0.00376), and there was no difference when compared based on age (P = 0.0646) and gender (P = 0.245). Besides acinar cells, a number of myoepithelial cells and ductal cells also demonstrated strong MGB reactivity with varying MGB mRNA expression levels in 6 of the 7 samples (with MGB-PR > 20%) tested. CONCLUSION: This novel study shows that unlike aberrant protein expression in some carcinomas, MGB expression in salivary gland neoplasms represents the nature of original cells, giving a better insight into the neoplasms expressing MGB.

Single nucleotide variants of succinate dehydrogenase A gene in renal cell carcinoma.

Succinate dehydrogenase (SDH)-deficient renal cell carcinoma (RCC) is mainly associated with a mutation in the SDHB gene and sometimes with mutations in the SDHC or SDHD genes. However, only three cases of succinate dehydrogenase A (SDHA)-deficient RCC have been reported, and the relation between SDHA mutations and RCC has not been clarified. This study assessed the role of SDHA gene mutations in human RCC. We investigated SDHA/B/C/D gene mutations in 129 human RCCs. Targeted next-generation sequencing and direct Sanger sequencing revealed single nucleotide variants (SNVs) of the SDHA gene with amino acid sequence variations in 11/129 tumors, while no SDHB/C/D gene mutations were found. Tumor cells with SNVs of the SDHA gene were characterized by eosinophilic cytoplasm and various patterns of proliferation. Immunohistochemistry examination found that the 11 tumors with SNVs of the SDHA gene showed significant reduction of SDHA protein and SDHB protein expression compared to the 19 tumors without SDHA or SDHB mutations (both P < .0001). Western blotting showed a greater decrease in the expression of SDHA and SDHB proteins in the 11 tumors with SNVs of the SDHA gene than in the 19 tumors without (both P < .0001). There was a positive correlation between SDHA and SDHB protein levels (P < .0001). On immunohistochemistry and Western blotting, the 11 tumors with SNVs of the SDHA gene had higher protein expression for nuclear factor E2-related factor 2 (Nrf2) compared to the 19 tumors without the mutation (P < .01). These observations suggest that SDHA gene mutations might be associated with a subset of RCC.

The effect of fibroblast growth factor 7 on human dental pulp stem cells for differentiation to AQP5-positive and αSMA-positive cells in vitro and in vivo.

OBJECTIVES: Transplantation of stem cells into wounds has become popular in regeneration therapies. As stem cells for transplantation, human dental pulp stem cells (hDPSCs) are known to be pluripotent cells that are relatively easy to collect from the pulp of deciduous or wisdom teeth. The purpose of this study was to investigate whether hDPSCs treated with fibroblast growth factor 7 (FGF7) would contribute to the regeneration of wounded rat submandibular glands (SMGs). MATERIALS AND METHODS: In in vitro studies, hDPSCs were treated with or without FGF7 and mRNA expression levels were examined at days 3, 7 and 14 using qRT-PCR. The target genes analyzed were BMI1 as an undifferentiated marker, AQP5 as an acinar cell marker, CK19 as a ductal epithelial cell marker, αSMA as a myoepithelial cell marker and VIMENTIN as a fibroblast marker. In in vivo studies, hDPSCs treated with or without FGF7 for 14 days were mixed with type I collagen gels and were transplanted into wounded rat SMGs. Hematoxylin-Eosin and immunohistochemical staining were performed at days 3 and 7, and the numbers of positive cells were counted. The primary antibodies used were against BMI1, AQP5, αSMA, PanCK and VIMENTIN. RESULTS: In the in vitro studies, mRNA levels of BMI1 were decreased and αSMA were increased at days 3, 7 and 14, while AQP5 was increased at day 14 in the FGF7 group. In the in vivo studies, the proliferation of hDPSCs and cell islands was observed at day 7 in the FGF7 group. Few BMI1-positive cells were observed, while numbers of AQP5-positive and αSMA-positive cells were increased at days 3 and 7 in the FGF7 group. Moreover, cell islands were AQP5-positive. CONCLUSION: These results suggest that FGF7-treated hDPSCs differentiate into AQP5-positive and αSMA-positive cells. Moreover, AQP5-positive cell aggregations were formed.

Factors Associated with Dose Modification of Lenalidomide Plus Dexamethasone Therapy in Multiple Myeloma.

Long-term combination treatment with lenalidomide and low-dose dexamethasone is important to achieve a curative effect in patients with multiple myeloma (MM). In this study, the plasma concentration of lenalidomide was measured at 3 h after oral administration, when the drug is in the elimination phase and can be easily measured in outpatients, to identify factors that may lead to the discontinuation of this combination therapy. Patients were assigned to continuation or discontinuation of therapy groups, and the baseline characteristics of patients, lenalidomide concentration, and concentration/dose (C/D) ratios reflecting oral clearance were compared between the two groups. The efficacy and severity of adverse events were also compared. The results showed that patients who discontinued or modified treatment had low plasma concentrations of lenalidomide and C/D ratios, indicating high oral clearance of lenalidomide. The estimated creatinine clearance rate was negatively correlated with the C/D ratio. The plasma concentrations of lenalidomide were independent from kidney function and differed significantly among patients. Taken together, the results indicate that low plasma concentrations of lenalidomide and low C/D ratios may lead to discontinuation of combination therapy in patients with MM. This suggests that early measurement of lenalidomide plasma continuation would help to prevent discontinuation of therapy or a delay in modifying the dose of lenalidomide.

A retrospective analysis of treatment outcomes in 45 patients with cardiac light-chain amyloidosis: a single-center experience in Japan.

The prognosis of cardiac light-chain (AL) amyloidosis is considered to be very poor. We studied the treatment efficacy and outcomes by retrospectively analyzing the clinical results of 45 patients with cardiac AL amyloidosis treated at our hospital between September 2008 and March 2016. The group of patients analyzed included 29 males and 16 females with a median age of 68 years. Their baseline median NT-proBNP, cTnT, and dFLC were 3167 pg/ml, 0.080 ng/ml, and 286.17 mg/l, respectively. Twenty-eight patients were in Cardiac Stage (CS) III and 17 patients were in Revised Prognostic Stage (RPS) IV. At the median follow-up of 10 months, the median overall survival (OS) was 16 months and 3-year OS was 35.9%. The patients in CS III showed significantly poorer survival rate than those in CS I or II (3-year OS: 12.2% vs. 65.8%, p = 0.0115) and the patients in RPS IV showed significantly poorer survival rate than those in RPS I, II, or III (3-year OS: 11.0% vs. 53.3%, p = 0.000914). Regardless of the therapeutic approaches, patients who achieved hematological CR or cardiac organ response demonstrated significantly improved prognosis. Therefore, achievement of hematological and organ responses is important in the treatment of cardiac AL amyloidosis.

Farnesoid X receptor and liver X receptors regulate Oct3/4 expression by multiple feedback regulating system in normal renal-derived cells and renal adenocarcinoma cells.

In this study, we found that nuclear receptors FXR and LXR (originally characterized as regulatory factors involved in cholesterol/bile acid homeostasis) regulate the expression of Oct3/4, a marker for cell differentiation, in both normal renal-derived cell line HK-2 and renal adenocarcinoma cell line ACHN. Down-regulation of Oct3/4 expression by activating FXR and LXR occurs only in normal renal cell-derived HK-2 cells. We also found that the RNA-binding protein, ELAVL2, oppositely regulates Oct3/4 expressions in HK-2 and ACHN cells. Moreover, we revealed that LXR-alpha and LXR-beta regulate each other's expression. Although an LXR-beta-specific agonist is assumed to be the basis for an anti-arteriosclerotic drug that only stimulates reverse cholesterol transport, our findings show that the development of such an anti-arteriosclerotic drug would require further elucidation of the complex mechanism of LXR-alpha and LXR-beta regulation.

[Spontaneous regression of primary gastric EBV-positive diffuse large B-cell lymphoma].

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) in the elderly was revised from the category EBV-positive DLBCL not otherwise specified in WHO 2017. The prognosis of this lymphoma is very poor. We report a case of an 82-year-old woman diagnosed with gastric EBV-positive DLBCL (WHO 2008). Gastroduodenoscopy revealed multiple ulcers and fold thickening. She was followed-up without any treatment because of her old age. Repeat endoscopy one year and eight months later revealed a single ulcer with no lymphoma cells found in a biopsy specimen. Two years later, the lesion had spontaneously disappeared.

Comparison between autologous and allogeneic stem cell transplantation as salvage therapy for multiple myeloma relapsing/progressing after autologous stem cell transplantation.

Allogeneic stem cell transplantation (allo-SCT) offers a clinical option to young patients with multiple myeloma (MM) relapsing/progressing after autologous SCT (ASCT); however, this claim remains debatable. Thus, in this retrospective study, we analyzed 526 patients with MM who underwent SCT for MM relapsing/progressing after the prior ASCT using the registry data of the Japan Society for Hematopoietic Cell Transplantation (2001-2015) and compared overall survival (OS) between allo-SCT (n = 192) and autologous stem cell retransplantation groups (ReASCT; n = 334) based on risk factor points. Significant adverse factors for OS in all patients were (1) male sex, (2) less than partial response to SCT, (3) performance status of 2 to 4, and (4) short duration from the prior ASCT. We scored factor 2 as 1 point, factor 3 as 2 points, and factor 4 as 0, 1, or 2 points for more than 30, 9 to 30, or less than 9 months, respectively. We categorized patients into three risk subgroups based on their total points (0, 1-3, and 4-5 points), indicating the usefulness of this scoring system for prognosis prediction and treatment selection. Subgroup comparison revealed OS after ReASCT to be higher than that after allo-SCT in the intermediate-risk subgroup comprising the largest population (28.2% vs 21.5%, P < .004). We observed no significant advantages of allo-SCT over ReASCT in the low- and high-risk subgroups. These findings suggest that ReASCT is more advantageous than allo-SCT in many patients with MM relapsing/progressing after the prior ASCT. However, long-term survival patients were noted only in the allo-SCT group, and allo-SCT could exhibit clinical efficacy, particularly in the low-risk group. While further examination is warranted, allo-SCT could be a potential tool for a specific population with MM relapsing/progressing after the prior ASCT.

[Successful treatment of relapse/refractory multiple myeloma with carfilzomib, lenalidomide, and dexamethasone combination therapy following allogeneic bone marrow transplantation].

A 50-year-old male was diagnosed with multiple myeloma (MM) and treated by high-dose melphalan followed by autologous stem cell transplantation in April 2014. However, he relapsed and received non-myeloablative bone marrow transplantation from an unrelated HLA-matched donor (UR-BMT) in July 2016. After 100 days of UR-BMT, the disease remained stable disease and the patient was treated with carfilzomib, lenalidomide, and dexamethaonse (KRd) therapy. After 10 cycles of KRd, he obtained stringent complete response without exacerbation of graft-versus-host disease. We concluded that KRd after allogeneic stem cell transplantation is one of the useful treatment regimens for relapsed refractory MM.