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Abstract Lung cancer remains the leading cause of cancer death. Treatment with immune checkpoint inhibitor (ICI) alone or combination with chemotherapy served as first-line therapy in non-small cell lung cancer (NSCLC). However, only 20–50% of NSCLC patients respond to ICI. Necroptosis, an inflammatory form of cell death plays an important role in the regulation of tumor immune microenvironment which may affect prognosis and ICI response but its clinical significance in NSCLC patients has remained largely unknown. Therefore, we aimed to analyze the correlation between key necroptotic proteins and necroptosis and clinical outcomes, the status of tumor-infiltrating immune cells, and response to ICI in NSCLC patients. The expression of receptor-interacting protein kinase 3 (RIPK3), mixed lineage kinase domain-like protein (MLKL) and phosphorylated MLKL (pMLKL) were immunolocalized in 125 surgically resected NSCLC patients and 23 NSCLC patients administered with ICI therapy. CD8 + and FOXp3 + T cells and CD163 + M2 macrophages were also immunolocalized. High RIPK3 status was positively correlated with survival of the patients and RIPK3 turned out an independent favorable prognostic factor of the patients. RIPK3 was negatively correlated with CD8 + T cells, while MLKL positively correlated with CD163 + M2 macrophages, suggesting the possible involvement of RIPK3 and MLKL in formulating immunosuppressive microenvironment. In addition, high RIPK3 status tended to be associated with clinical resistance to ICI therapy (P-value = 0.057). Furthermore, NSCLC cells-expressing RIPK3 suppressed T cells response to ICI therapy in vitro. Therefore, RIPK3 and MLKL could induce an immunosuppressive microenvironment, resulting in low response to ICI therapy in NSCLC.

Abstract Background Primary adrenal leiomyosarcoma is a rare and aggressive mesenchymal tumor derived from the smooth muscle wall of a central adrenal vein or its tributaries; therefore, tumors tend to invade the inferior vena cava and cause thrombosis. The great majority of tumors grow rapidly, which makes the disease difficult to diagnose in its early clinical stages and needs differentiation from adrenocortical carcinomas for the selection of chemotherapy including mitotane which causes adrenal insufficiency. Case presentation We presented two patients with adrenal leiomyosarcoma who were referred to our hospital with abdominal pain and harboring large adrenal tumors and inferior vena cava thrombosis. The endocrine findings, including serum catecholamine levels, were unremarkable. These two patients were considered clinically inoperable, and CT-guided core needle biopsy was performed to obtain the definitive histopathological diagnosis and determine the modes of therapy. The masses were subsequently diagnosed as primary adrenal leiomyosarcoma based on the histological features and positive immunoreactivity for SMA (smooth muscle actin), desmin, and vimentin. Conclusions Adrenal leiomyosarcoma derived from the smooth muscle wall of a central adrenal vein or its tributaries is rare but should be considered a differential diagnosis in the case of nonfunctioning adrenal tumors extending directly to the inferior vena cava. CT-guided biopsy is considered useful for histopathological diagnosis and clinical management of patients with inoperable advanced adrenal tumors without any hormone excess.

Abstract Adrenal venous sampling (AVS) is crucial for subtyping primary aldosteronism (PA) to explore the possibility of curing hypertension. Because AVS availability is limited, efforts have been made to develop strategies to bypass it. However, it has so far proven unsuccessful in applying clinical practice, partly due to heterogeneity and missing values of the cohorts. For this purpose, we retrospectively assessed 210 PA cases from three institutions where segment-selective AVS, which is more accurate and sensitive for detecting PA cases with surgical indications, was available. A machine learning-based classification model featuring a new cross-center domain adaptation capability was developed. The model identified 102 patients with PA who benefited from surgery in the present cohort. A new data imputation technique was used to address cross-center heterogeneity, making a common prediction model applicable across multiple cohorts. Logistic regression demonstrated higher accuracy than Random Forest and Deep Learning [(0.89, 0.86) vs. (0.84, 0.84), (0.82, 0.84) for surgical or medical indications in terms of f-score]. A derived integrated flowchart revealed that 35.2% of PA cases required AVS with 94.1% accuracy. The present model enabled us to reduce the burden of AVS on patients who would benefit the most.

BACKGROUND: The checkpoint kinase 1 (CHEK1) and glutamine-fructose-6-phosphate aminotransferase 1 (GFPT1) genes have been reported to have a crucial role in carcinogenesis in colorectal cancer (CRC). However, their association with the pathogenesis of CRC remains unclear. This study was conducted to study the expression of CHEK1 and GFPT1 genes in adenoma and adenocarcinoma CRC patients’ whole blood samples compared to the healthy controls. METHODS: A comparative cross‑sectional study to examine the expression of CHEK1 and GFPT1 genes were conducted in 6 colorectal adenoma and 6 colorectal adenocarcinoma patients along with 6 healthy controls. Blood samples were taken from subjects, and CHEK1 and GFPT1 genes were analyzed by using reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS: CHEK1 gene expression in blood samples has the highest in adenoma patients meanwhile for the GFPT1 gene expression has the highest in adenocarcinoma patients. Higher CHEK1 gene expression were also found in adenoma patients compared to healthy controls (p=0.040) and adenocarcinoma patients (p=0.025). Besides, at the 5% level of significance, the median GFPT1 gene expression was higher in colorectal adenocarcinoma patients compared to colorectal adenoma patients and healthy controls. CONCLUSION: CHEK1 and GFPT1 may function as potential regulators in adenoma and adenocarcinoma and measuring their expression might be a potential tool to determine the CRC progression and could be further explored as blood diagnostic biomarkers for CRC patients. KEYWORDS: CHEK1, GFPT1, colorectal cancer, adenoma, adenocarcinoma

Double somatic mutations in CTNNB1 and GNA11/Q have recently been identified in a small subset of aldosterone-producing adenomas (APAs). As a possible pathogenesis of APA due to these mutations, an association with pregnancy, menopause, or puberty has been proposed. However, because of its rarity, characteristics of APA with these mutations have not been well characterized. A 46-year-old Japanese woman presented with hypertension and hypokalemia. She had two pregnancies in the past but had no history of pregnancy-induced hypertension. She had regular menstrual cycle at presentation and was diagnosed as having primary aldosteronism after endocrinologic examinations. Computed tomography revealed a 2 cm right adrenal mass. Adrenal venous sampling demonstrated excess aldosterone production from the right adrenal gland. She underwent right laparoscopic adrenalectomy. The resected right adrenal tumor was histologically diagnosed as adrenocortical adenoma and subsequent immunohistochemistry (IHC) revealed diffuse immunoreactivity of aldosterone synthase (CYP11B2) and visinin like 1, a marker of the zona glomerulosa (ZG), whereas 11β-hydroxylase, a steroidogenic enzyme for cortisol biosynthesis, was mostly negative. CYP11B2 IHC-guided targeted next-generation sequencing identified somatic CTNNB1 (p.D32Y) and GNA11 (p.Q209H) mutations. Immunofluorescence staining of the tumor also revealed the presence of activated β-catenin, consistent with features of the normal ZG. The expression patterns of steroidogenic enzymes and related proteins indicated ZG features of the tumor cells. PA was clinically and biochemically cured after surgery. In conclusion, our study indicated that CTNNB1 and GNA11-mutated APA has characteristics of the ZG. The disease could occur in adults with no clear association with pregnancy or menopause.

Summary: Triple-negative breast cancer (TNBC) is an aggressive subtype with no targeted therapeutics. The luminal androgen receptor (LAR) subtype constitutes 15% of TNBC and is enriched for androgen receptor (AR) and AR target genes. Here, we show that a cohort of TNBC not only expresses AR at a much higher rate (∼80%) but also expresses AR splice variants (AR-SVs) (∼20%), further subclassifying LAR-TNBC. Higher AR and AR-SV expression and corresponding aggressive phenotypes are observed predominantly in specimens obtained from African American women. LAR TNBC specimens are enriched for interferon, Janus kinase (JAK)-signal activator and transducer (STAT), and androgen signaling pathways, which are exclusive to AR-expressing epithelial cancer cells. AR- and AR-SV-expressing TNBC cell proliferation and xenograft and patient-tumor explant growth are inhibited by AR N-terminal domain-binding selective AR degrader or by a JAK inhibitor. Biochemical analysis suggests that STAT1 is an AR coactivator. Collectively, our work identifies pharmacologically targetable TNBC subtypes and identifies growth-promoting interaction between AR and JAK-STAT signaling.

Abstract Background Germline TP53 mutations have been frequently reported in patients with Li–Fraumeni syndrome (LFS), resulting in a predisposition to various malignancies. Mutations other than germline TP53 mutations can also cause LFS-associated malignancies, but their details remain unclear. We describe a novel c-myc amplification in a unique liposarcoma in a patient with LFS. Case presentation A female patient with LFS developed breast cancer twice at the age of thirty; both were invasive ductal carcinomas harboring HER2 amplifications. Computed tomography revealed an anterior mediastinal mass, which was surgically resected. Histological analysis revealed three different lesions corresponding to myxoid liposarcoma-, pleomorphic liposarcoma-, and well-differentiated liposarcoma-like lesions. Fluorescence in-situ hybridization (FISH) analysis did not detect MDM2 amplification, Rb1 deletion, break apart signals of EWS, FUS, DDIT3, or c-myc, or c-myc-IGH fusion signals, but it did detect more c-myc signals. Further FISH analysis and comprehensive genomic profiling revealed c-myc amplification. We considered two differential diagnoses, dedifferentiated liposarcoma lacking MDM2 amplification and myxoid pleomorphic liposarcoma (MPLPS), and determined that this case is most likely MPLPS. However, definite diagnosis could not be made because a clear-cut differentiation of the case from liposarcomas was not possible. Conclusions A previous study demonstrated that c-myc amplification could not be detected in various liposarcomas, but the present unique liposarcoma showed c-myc amplification, so the c-myc amplification may indicate that the present liposarcoma is an LFS-related tumor. The present case further clarifies the pathological features of MPLPS and LFS-related liposarcomas by broadening their histopathological and genetic diversities.

BackgroundThe number of patients with prolonged critical illness (PCI) has been increasing in many countries, and the adrenal gland plays an important role in maintaining homeostasis during PCI. Chronic disease burden is reportedly associated with shorter telomere lengths in human tissues. Telomere shortening in human somatic cells is largely dependent on cell divisions, and critically short telomeres lead to cellular dysfunction and aging. However, the association between PCI and telomere lengths in human adrenal cells is poorly understood. In this study, we investigated this association to assess whether the burden of PCI could accelerate the aging process in adrenal cells.MethodsAdrenocortical tissues from patients who died after PCI usually show a diffuse pattern of intracellular cholesterol ester depletion (i.e., lipid depletion). This study examined near-normal adrenal glands obtained from autopsied patients who died suddenly (control group) and lipid-depleted adrenal glands obtained from autopsied patients who died after PCI (PCI group). The control group included 7 men aged 80 to 94 years (mean age: 85.3 years) and 7 women aged 84 to 94 years (mean age: 87.7 years). The PCI group included 10 men aged 71 to 88 years (mean age: 78.8 years) and 8 women aged 77 to 95 years (mean age: 85.6 years). By using quantitative fluorescence in situ hybridization, relative telomere lengths (RTLs) were determined in the parenchymal cells of the three adrenocortical zones (zona glomerulosa, zona fasciculata, and zona reticularis [ZR]) and in the chromaffin cells of the medulla. The number of adrenal parenchymal cells was determined by immunohistochemistry and digital image analysis.ResultsRTLs in ZR cells were significantly shorter in the PCI group than in the control group for both men and women (P = 0.0001 for men and P = 0.0012 for women). However, RTLs in the remaining three types of adrenal cells did not differ between the control and PCI groups for both men and women. The number of ZR cells was higher in the PCI group than in the control group for both men and women (P < 0.0001 for both men and women). The proportion of the number of ZR cells to the total number of adrenocortical parenchymal cells was also higher in the PCI group than in the control group (P < 0.0001 for both men and women). The Ki-67 proliferation index in ZR cells was higher in the PCI group than in the control group (P = 0.0039 for men and P = 0.0063 for women).ConclusionsThis study demonstrated ZR cell-specific telomere shortening in patients with adrenal lipid depletion who died after PCI. Our results suggest that the reactive proliferation of ZR cells accelerates the telomere shortening and aging process in ZR cells in these patients. The results of our study may contribute to the understanding of adrenal aging during PCI.

Abstract Objective/Hypothesis Middle ear cholesteatoma is characterized by abnormal growth of the keratinizing squamous epithelium of the temporal bone. d‐β‐aspartic acid is the major isomer of d‐aspartic acid found in elderly tissue. We assessed the immunoreactivity to k‐β‐aspartic acid of congenital and acquired middle ear cholesteatomas. Study Design Case–control studies. Material and Methods Tissue samples were collected from 21 patients comprising 21 ears with congenital middle ear cholesteatoma and 26 patients comprising 29 ears with acquired type. Their clinical and histopathological features were investigated. We divided the middle ear cholesteatoma samples into three layers: the perimatrix, matrix, and cystic contents. The patterns of immunoreactivity to d‐β‐aspartic acid expression were then assessed immunohistochemically. Results Two patterns of immunoreactivity to d‐β‐aspartic acid were detected in middle ear cholesteatoma: infiltrative and diffuse. In congenital middle ear cholesteatoma, d‐β‐aspartic acid expression was observed throughout all the layers (perimatrix, matrix, and cystic contents), and immunoreactivity to d‐β‐aspartic acid was dramatically strong in all layers. The expression levels of d‐β‐aspartic acid to the cystic content and perimatrix were significantly higher in congenital middle ear cholesteatoma than in the acquired type. Conclusions This study showed the expression levels of d‐β‐aspartic acid in middle ear cholesteatoma to differ significantly between congenital and acquired middle ear cholesteatoma. Our results indicate that overexpression of d‐β‐aspartic acid is likely to be involved in the pathogenesis of cholesteatoma, and we speculate that d‐β‐aspartic acid could be a novel biomarker for, and a therapeutic target in, congenital and acquired middle ear cholesteatoma. Level of Evidence 4

Abstract Background Pulmonary carcinoid tumors rarely coexist with non-small cell lung carcinoma, and only nine cases have been reported previously. The pathogenesis and origin of these combined tumors remain unclear because of its rarity. Case presentation We examined two cases of adenocarcinoma coexisting with a typical or atypical carcinoid tumor: Case 1 was a 77-year-old woman and Case 2 was an 83-year-old woman. Both of these cases had no respiratory symptoms, and underwent pulmonary lobectomies due to incidentally detected lung nodules. Recurrence and metastases were not detected after the surgery. Histologically, carcinoid and adenocarcinoma components were present in both cases. The two components coexisted without mixing with each other. Next-generation sequencing was performed on the two components in these cases. In each case, no common genetic variants were detected. Conclusion We considered that our cases could histologically and genetically represent collision tumors that did not share common progenitor cells. Comprehensive analyses such as whole genome sequencing could provide important information for elucidating the pathogenesis of adenocarcinoma and carcinoid components.

Abstract Background Programmed cell death ligand 1 (PD‐L1) status has been reported to be different between metastatic and primary lesions in some cases. Therefore, the interaction between carcinoma and immune cells could influence their expression in the tumor microenvironment. PD‐L1 is known to bind not only to Programmed cell death 1 (PD‐1) but also to B7‐1 (CD80). In this study, we examined the interaction between lung carcinoma cell lines and peripheral blood mononuclear cells (PBMCs) in vitro. We then examined the significance of B7‐1 expression non‐small cell lung cancer (NSCLC) microenvironment. Methods The interaction of lung carcinoma cell lines and PBMC through the soluble factors was analyzed using a co‐culture system. The changes in expression of immune checkpoint‐related factors in PBMC were examined by PD‐1/PD‐L1 Checkpoint Pathway qPCR Array Kit. B7‐1 expression in NSCLC tissues was examined by immunohistochemistry. Results B7‐1 was upregulated following the co‐culture with the lung carcinoma cell lines. B7‐1 expression in NSCLC tissues was significantly higher in smokers and squamous cell carcinomas and was significantly positively correlated with PD‐L1 status in primary cancer. However, B7‐1 and PD‐1 were not correlated between primary and metastatic diseases in the same patients. Conclusion PD‐1 inhibitors inhibited PD‐1/PD‐L1 binding but not PD‐L1/B7‐1 binding. These results demonstrated that the intratumoral ratio of B7‐1 positive T cells in NSCLC tissue could be involved in the therapeutic efficacy of PD‐L1 inhibitors. This study focused on lymph node metastasis but other sites of distant metastases should be explored by further analysis.

Breast cancer is a hormone-dependent cancer, and sex steroids play a pivotal role in breast cancer progression. Estrogens are strongly associated with breast cancers, and the estrogen receptor (estrogen receptor α; ERα) is expressed in 70–80% of human breast carcinoma tissues. Although antiestrogen therapies (endocrine therapies) have significantly improved clinical outcomes in ERα-positive breast cancer patients, some patients experience recurrence after treatment. In addition, patients with breast carcinoma lacking ERα expression do not benefit from endocrine therapy. The androgen receptor (AR) is also expressed in >70% of breast carcinoma tissues. Growing evidence supports this novel therapeutic target for the treatment of triple-negative breast cancers that lack ERα, progesterone receptor, and human EGF receptor 2, and ERα-positive breast cancers, which are resistant to conventional endocrine therapy. However, the clinical significance of AR expression is still controversial and the biological function of androgens in breast cancers is unclear. In this review, we focus on the recent findings concerning androgen action in breast cancers and the contributions of androgens to improved breast cancer therapy.

In hormone-dependent cancers, the activation of hormone receptors promotes the progression of cancer cells. Many proteins exert their functions through protein– protein interactions (PPIs). Moreover, in such cancers, hormone–hormone receptor binding, receptor dimerization, and cofactor mobilization PPIs occur primarily in hormone receptors, including estrogen, progesterone, glucocorticoid, androgen, and mineralocorticoid receptors. The visualization of hormone signaling has been primarily reported by immunohistochemistry using specific antibodies; however, the visualization of PPIs is expected to improve our understanding of hormone signaling and disease pathogenesis. Visualization techniques for PPIs include Förster resonance energy transfer (FRET) and bimolecular fluorescence complementation analysis; however, these techniques require the insertion of probes in the cells for PPI detection. Proximity ligation assay (PLA) is a method that could be used for both fo rmalin-fixed paraffin-embedded (FFPE) tissue as well as immunostaining. It can also v isualize hormone receptor localization and post-translational modifications of hormone receptors. This review summarizes the results of recent studies on visualization techniques for PPIs with hormone receptors; these techniques include FRET and PLA. In addition, super-resolution microscopy has been recently reported to be applicable to their visualization in both FFPE tissues and living cells. Super-resolution microscopy in conjunction with PLA and FRET could also contribute to the visualization of PPIs and subsequently provide a better understanding of the pathogenesis of hormone-dependent cancers in the future.

Abstract Background It is important to confirm CD30 expression in T-cell lymphoma cases, but immunohistochemical staining for CD30 is not commonly performed and no comparison has been done between the results of flow cytometry (FCM) and immunohistochemical staining for CD30. Therefore, we devised a notation that we termed proportion of immunoreactivity/expression for FCM (PRIME-F notation), based on the cellular proportion showing different antigen-antibody reactivity. Methods We retrospectively compiled 211 cases of T-cell lymphoma, assessed via FCM, from major hospitals in Miyagi Prefecture from January 2012 to January 2019, and compared 52 of these cases with the immunohistochemical immunoreactive (IR) pattern of CD30 (PRIME-I notation). The PRIME-F notation was divided into five levels: notations starting with “-” followed by 3, 2, and 1 “>” correspond to level-I, level-II, or level-III; notations starting with “(dim)+” correspond to level-IV; and those starting with “+” or “(bright)+” correspond to level-V. Results The 52 cases of PRIME-F notation with “+” included 16 cases of peripheral T-cell lymphoma (PTCL/NOS), 3 of follicular T-cell lymphoma (FTL), 3 of angioimmunoblastic T-cell lymphoma (AITL), 6 of extranodal NK/T-cell lymphoma/nasal type (ENKL), 18 of adult T-cell lymphoma (ATL), and 6 cases of anaplastic large cell lymphoma (ALCL). Eight of the 52 cases were immunohistochemically CD30-negative. In the PRIME-F level-I to III group (excluding false-positive cases), 21.7% (5 out of 23 cases) were 1/3 upon IHC, while in level-V, only 7.1% (one out of 14 cases) did. The CD30 negative rate tended to be low (p = 0.0877) in level-V. Conclusions To our knowledge, this is the first report describing the correspondence between FCM and immunohistochemistry findings for CD30 through newly proposed notations. The PRIME-F and PRIME-I notations for CD30 showed a minor positive correlation. The PRIME notation is considered universally applicable to antibodies, and notations of both FCM and IHC show great potential for big data.

This review presents several aspects of the innovative concept of sebaceous immunobiology, which summarizes the numerous activities of the sebaceous gland including its classical physiological and pathophysiological tasks, namely sebum production and the development of seborrhea and acne. Sebaceous lipids, which represent 90% of the skin surface lipids in adolescents and adults, are markedly involved in the skin barrier function and perifollicular and dermal innate immune processes, leading to inflammatory skin diseases. Innovative experimental techniques using stem cell and sebocyte models have clarified the roles of distinct stem cells in sebaceous gland physiology and sebocyte function control mechanisms. The sebaceous gland represents an integral part of the pilosebaceous unit and its status is connected to hair follicle morphogenesis. Interestingly, professional inflammatory cells contribute to sebocyte differentiation and homeostasis, whereas the regulation of sebaceous gland function by immune cells is antigen-independent. Inflammation is involved in the very earliest differentiation changes of the pilosebaceous unit in acne. Sebocytes behave as potent immune regulators, integrating into the innate immune responses of the skin. Expressing inflammatory mediators, sebocytes also contribute to the polarization of cutaneous T cells towards the Th17 phenotype. In addition, the immune response of the perifollicular infiltrate depends on factors produced by the sebaceous glands, mostly sebaceous lipids. Human sebocytes in vitro express functional pattern recognition receptors, which are likely to interact with bacteria in acne pathogenesis. Sex steroids, peroxisome proliferator-activated receptor ligands, neuropeptides, endocannabinoids and a selective apoptotic process contribute to a complex regulation of sebocyte-induced immunological reaction in numerous acquired and congenital skin diseases, including hair diseases and atopic dermatitis.

Abstract Necroptosis, a regulated form of necrosis, has emerged as a novel therapeutic strategy that could enhance cancer immunotherapy. However, its role in tumorigenesis is still debated because recent studies have reported both anti- and pro-tumoral effects. Here, we aimed to systematically evaluate the associations between tumor necroptosis (mixed lineage kinase domain-like protein, MLKL; phosphorylated MLKL, pMLKL; and receptor-interacting protein kinase 1–receptor-interacting protein kinase 3, RIPK1–RIPK3 interaction) and tumor-infiltrating immune cells (CD8+ and FOXp3+ T cells and CD163+ M2 macrophages) and tumor PD-L1 by immunohistochemistry in 88 cholangiocarcinoma (CCA) patients who had undergone surgical resection. Their associations with clinicopathological characteristics, survival data, and prognosis were evaluated. MLKL was found to be an unfavorable prognostic factor (p-value = 0.023, HR = 2.070) and was inversely correlated with a clinically favorable immune cell signature (high CD8+/high FOXp3+/low CD163+). Both pMLKL and RIPK1–RIPK3 interaction were detected in CCA primary tissues. In contrast to MLKL, pMLKL status was significantly positively correlated with a favorable immune signature (high CD8+/high FOXp3+/low CD163+) and PD-L1 expression. Patients with high pMLKL-positive staining were significantly associated with an increased abundance of CD8+ T cell intratumoral infiltration (p-value = 0.006). Patients with high pMLKL and PD-L1 expressions had a longer overall survival (OS). The results from in vitro experiments showed that necroptosis activation in an RMCCA-1 human CCA cell line selectively promoted proinflammatory cytokine and chemokine expression. Jurkat T cells stimulated with necroptotic RMCCA-1-derived conditioned medium promoted PD-L1 expression in RMCCA-1. Our findings demonstrated the differential associations of necroptosis activation (pMLKL) and MLKL with a clinically favorable immune signature and survival rates and highlighted a novel therapeutic possibility for combining a necroptosis-based therapeutic approach with immune checkpoint inhibitors for more efficient treatment of CCA patients.

Abstract Background The prognostic values of inflammation-based markers in well-differentiated pancreatic neuroendocrine neoplasms, diagnosed according to the new 2017 World Health Organization classification, have remained unclear. Therefore, we assessed the ability to predict the recurrence of such markers after curative resection in patients with these neoplasms. Methods Circulating/systemic neutrophil–lymphocyte, monocyte–lymphocyte, platelet–lymphocyte, and platelet–white cell ratios were evaluated in 120 patients who underwent curative resection for well-differentiated pancreatic neuroendocrine neoplasms without synchronous distant metastasis between 2001 and 2018. Recurrence-free-survival and overall survival were compared using Kaplan–Meier analysis and log-rank tests. Univariate or multivariate analyses, using a Cox proportional hazards model, were used to calculate hazard ratios with 95% confidence intervals. Results Univariate analysis demonstrated that preoperative neutrophil–lymphocyte ratio, tumor size, European Neuroendocrine Tumor Society TMN classification, 2017 World Health Organization classification, and venous invasion were associated with recurrence. The optimal preoperative neutrophil–lymphocyte ratio cut-off value was 2.62, based on receiver operating characteristic curve analysis. In multivariate analysis, a higher preoperative neutrophil–lymphocyte ratio (HR = 3.49 95% CI 1.05–11.7; P = 0.042) and 2017 World Health Organization classification (HR = 8.81, 95% CI 1.46–168.2; P = 0.015) were independent recurrence predictors. Conclusions The circulating/systemic neutrophil–lymphocyte ratio is a useful and convenient preoperative prognostic marker of recurrence in patients with well-differentiated pancreatic neuroendocrine neoplasm based on the 2017 World Health Organization classification.

Primary aldosteronism (PA) is the most common cause of secondary hypertension, and is associated with an increased incidence of cardiovascular events. PA itself is clinically classified into the following two types: unilateral PA, mostly composed of aldosterone-producing adenoma (APA); and bilateral hyperaldosteronism, consisting of multiple aldosterone-producing micronodules (APMs) and aldosterone-producing diffuse hyperplasia. Histopathologically, those disorders above are all composed of compact and clear cells. The cellular morphology in the above-mentioned aldosterone-producing disorders has been recently reported to be closely correlated with patterns of somatic mutations of ion channels including KCNJ5, CACNA1D, ATP1A1, ATP2B3, and others. In addition, in non-pathological adrenal glands, APMs are frequently detected regardless of the status of the renin-angiotensin-aldosterone system (RAAS). Aldosterone-producing nodules have been also proposed as non-neoplastic nodules that can be identified by hematoxylin and eosin staining. These non-neoplastic CYP11B2-positive nodules could represent possible precursors of APAs possibly due to the presence of somatic mutations. On the other hand, aging itself also plays a pivotal role in the development of aldosterone-producing lesions. For instance, the number of APMs was also reported to increase with aging. Therefore, recent studies indicated the novel classification of PA into normotensive PA (RAAS-independent APM) and clinically overt PA.

Abstract Immunotherapy is considered one of the most important therapeutic strategies for patients with lung adenocarcinoma after the development of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR‐TKI) resistance. However, useful predictors of immunotherapy for these patients has not been examined well, although the status of the tumor immune microenvironment (TIME), including programmed death‐ligand 1 expression and lymphocyte infiltration, has been generally known to provide predictive markers for the efficacy of immunotherapy. This study aimed to clarify novel predictors of immunotherapy following EGFR‐TKI resistance in lung adenocarcinoma, especially regarding micro RNA (miRNA). We evaluated the correlation between EGFR‐TKI resistance and lymphocyte infiltration, before and after acquiring EGFR‐TKI resistance, in 21 cases of lung adenocarcinoma, and further explored this by in vitro studies, using miRNA PCR arrays. Subsequently, we transfected miRNA‐1 (miR‐1), the most variable miRNA in this array, into three kinds of lung cancer cells, and examined the effects of miR‐1 on EGFR‐TKI sensitivity, cytokine expression and lymphocyte migration. Histopathological examination demonstrated that infiltration levels of CD8‐positive T cells were significantly decreased after development of EGFR‐TKI resistance. In vitro studies revealed that miR‐1 significantly inhibited EGFR‐TKI effect and induction of cytokines, such as C‐C motif chemokine ligand 5 and C‐X‐C motif chemokine ligand 10, causing inhibition of monocyte migration. These results indicate that the upregulated miR‐1 might suppress the TIME, following development of EGFR‐TKI resistance. Therefore, miR‐1 could be a clinically useful marker to predict therapeutic efficacy of immunotherapy in lung adenocarcinoma patients with EGFR‐TKI resistance.

Well-differentiated papillary mesothelioma (WDPM) is an uncommon mesothelial neoplasm, which is generally regarded as benign or indolent in terms of its clinical behavior. However, details about WDPM have remained relatively unknown. Therefore, in this study, we examined six incidentally detected cases of WDPM of the peritoneum. All six cases were surgically excised, without any additional therapeutic measures. None of the cases showed recurrence. All six cases presented single lesions and the tumor sizes ranged from 2 to 10 mm. Histologically, all six cases exhibited papillary proliferation of cytologically bland mesothelial cells with a fibroconnective tissue core. One of the cases (Case 6) presented small invasive foci in the stalk. The tumor cells were immunohistochemically positive for mesothelial markers and negative for GLUT-1, p53, and CD146. The Ki-67 labeling index of the tumor cells was lower than 5% at the hot spots. All samples were BAP1-positive. None of the samples presented p16 homozygous deletion, as assessed by fluorescence in situ hybridization (FISH). None of the patients deceased due to WDPM. However, in Case 3, death occurred due to pancreatic cancer. The results of this study indicate the importance of analyzing immunohistochemical markers and p16 status to diagnose WDPM accurately.

Abstract 18F-FDG PET/CT has been used as an indicator of chemotherapy effects, but cancer cells can remain even when no FDG uptake is detected, indicating the importance of exploring other metabolomic pathways. Therefore, we explored the amino acid metabolism, including L-type amino acid transporter-1 (LAT1), in breast cancer tissues and clarified the role of LAT1 in therapeutic resistance and clinical outcomes of patients. We evaluated LAT1 expression before and after neoadjuvant chemotherapy and examined the correlation of glucose uptake using FDG-PET with the pathological response of patients. It revealed that LAT1 levels correlated with proliferation after chemotherapy, and amino acid and glucose metabolism were closely correlated. In addition, LAT1 was considered to be involved in treatment resistance and sensitivity only in luminal type breast cancer. Results of in vitro analyses revealed that LAT1 promoted amino acid uptake, which contributed to energy production by supplying amino acids to the TCA cycle. However, in MCF-7 cells treated with chemotherapeutic agents, oncometabolites and branched-chain amino acids also played a pivotal role in energy production and drug resistance, despite decreased glucose metabolism. In conclusion, LAT1 was involved in drug resistance and could be a novel therapeutic target against chemotherapy resistance in luminal type breast cancer.

Abstract Background Streptozocin (STZ) is used for treating both pancreatic (PanNET) and gastrointestinal (GI-NET) neuroendocrine tumors but its therapeutic efficacy is relatively low in GI-NETs. Therefore, it has become pivotal to select GI-NET patients who could benefit from STZ treatment. STZ is transported via the glucose transporter 2 (GLUT2) into the cells and the loss of O6-methylguanine DNA methyltransferase (MGMT) also increases its therapeutic efficacy. Therefore, GLUT2 high and MGMT low status could be the surrogate markers of STZ. Methods In this study, we examined the MGMT and GLUT2 status in gastrointestinal neuroendocrine neoplasm (NEN). We studied 84 NEN cases: 33 foregut and 37 hindgut GI-NETs and 14 gastrointestinal neuroendocrine carcinomas (GI-NECs). Results In GI-NETs, MGMT scores of ≥2 and ≥ 3 were 77% (54/70) and 56% (39/70), respectively, and GLUT2 scores of ≥4 and ≥ 6 were 30% (21/70) and 4.3% (3/70), respectively. Methylation-specific polymerase chain reaction revealed that MGMT promoter methylation was detected only in 2/14 GI-NECs but none of the included GI-NETs. GLUT2 (GLUT2 score) and MGMT immunoreactivity (MGMT and H-scores) were both significantly correlated with Ki-67 labeling index (GLUT2 score: P = 0.0045, ρ = − 0.4570; MGMT score: P = 0.0064, ρ = − 0.4399; H-score: P = 0.0110, ρ = − 0.4135) and MGMT immunoreactivity were significantly correlated with GLUT2 immunoreactivity (MGMT score: P = 0.0198; H-score, P = 0.0004, ρ = 0.5483) in hindgut NETs, but not in foregut NETs. However, discrepancies from the above correlation between GLUT2 and MGMT immunoreactivity were detected in several GI-NET cases which could be potential candidates for STZ therapy. Conclusion The evaluation of MGMT and GLUT2 status could provide an important information in planning STZ therapy in GI-NET patients.

Abstract Background Glomus tumors are subcutaneous tumors arising from glomus bodies, thermoregulatory components of the skin. These tumors could occur in visceral organs where glomus bodies are not normally present. Herein, we report a case of primary pancreatic glomus tumor with aggressive direct invasion into the superior mesenteric vein (SMV). To the best of our knowledge, this is the second case report of a glomus tumor arising in the pancreas. Case presentation A 46-year-old woman was referred to our hospital due to vomiting with epigastric and back pain. Dynamic-CT revealed a well-circumscribed hypervascular mass, measuring 37 mm in its maximal diameter involving the pancreatic head. Both CT and endoscopic ultrasonography (EUS) revealed direct invasion into the SMV and radiologically suspected tumor thrombus. Biopsy sample obtained by EUS-guided fine needle aspiration revealed proliferation of small cells, round-to-oval tumor cells with round nuclei and scant cytoplasm. A histological diagnosis of pancreatic neuroendocrine tumor, G1 was initially considered. Therefore, subtotal stomach-preserving pancreatoduodenectomy using Child-II reconstruction was subsequently performed. Her SMV was resected and reconstructed due to extensive tumor involvement. Subsequent histopathological analysis revealed solid tumor cells proliferation that comprised oval-shaped nuclei and scant cytoplasm around disorganized or slit-shaped vessels in hematoxylin–eosin-stained slides. Immunohistochemical analysis then demonstrated positive immunoreactivity for smooth muscle actin, vimentin, and CD34, but negative for chromogranin A, synaptophysin, CD56, and signal transducer and activator of transcription 6. Based on these histological findings of resected specimens, the lesion was subsequently diagnosed as a primary pancreatic glomus tumor harboring direct invasion into the SMV. Her postoperative course was uneventful and annual surveys for the following 4 years post-op detected no clinical signs of recurrence. Conclusions We report a very rare case of glomus tumor of the pancreas accompanied by venous invasion. Curative surgical resection is the best treatment option for pancreatic glomus tumors. Although pancreatic glomus tumor is rare, it should be taken into consideration in the differential diagnosis of a pancreatic solid tumor with hypervascularity.

Abstract Background Toll-like receptor 3 (TLR3) ligand which activates TLR3 signaling induces both cancer cell death and activates anti-tumor immunity. However, TLR3 signaling can also harbor pro-tumorigenic consequences. Therefore, we examined the status of TLR3 in cholangiocarcinoma (CCA) cases to better understand TLR3 signaling and explore the potential therapeutic target in CCA. Methods The expression of TLR3 and receptor-interacting protein kinase 1 (RIPK1) in primary CCA tissues was assayed by Immunohistochemical staining and their associations with clinicopathological characteristics and survival data were evaluated. The effects of TLR3 ligand, Poly(I:C) and Smac mimetic, an IAP antagonist on CCA cell death and invasion were determined by cell death detection methods and Transwell invasion assay, respectively. Both genetic and pharmacological inhibition of RIPK1, RIPK3 and MLKL and inhibitors targeting NF-κB and MAPK signaling were used to investigate the underlying mechanisms. Results TLR3 was significantly higher expressed in tumor than adjacent normal tissues. We demonstrated in a panel of CCA cell lines that TLR3 was frequently expressed in CCA cell lines, but was not detected in a nontumor cholangiocyte. Subsequent in vitro study demonstrated that Poly(I:C) specifically induced CCA cell death, but only when cIAPs were removed by Smac mimetic. Cell death was also switched from apoptosis to necroptosis when caspases were inhibited in CCA cells-expressing RIPK3. In addition, RIPK1 was required for Poly(I:C) and Smac mimetic-induced apoptosis and necroptosis. Of particular interest, high TLR3 or low RIPK1 status in CCA patients was associated with more invasiveness. In vitro invasion demonstrated that Poly(I:C)-induced invasion through NF-κB and MAPK signaling. Furthermore, the loss of RIPK1 enhanced Poly(I:C)-induced invasion and ERK activation in vitro. Smac mimetic also reversed Poly(I:C)-induced invasion, partly mediated by RIPK1. Finally, a subgroup of patients with high TLR3 and high RIPK1 had a trend toward longer disease-free survival (p = 0.078, 28.0 months and 10.9 months). Conclusion RIPK1 plays a pivotal role in TLR3 ligand, Poly(I:C)-induced cell death when cIAPs activity was inhibited and loss of RIPK1 enhanced Poly(I:C)-induced invasion which was partially reversed by Smac mimetic. Our results suggested that TLR3 ligand in combination with Smac mimetic could provide therapeutic benefits to the patients with CCA. Video abstract Graphical abstract

A 31-year-old man with Williams syndrome (WS) was referred to our hospital because of a 9-year history of hypertension, hypokalemia, and high plasma aldosterone concentration to renin activity ratio. A diagnosis of primary aldosteronism (PA) was clinically confirmed but an abdominal CT scan showed no abnormal findings in his adrenal glands. However, a 13-mm hypervascular tumor in the posterosuperior segment of the right hepatic lobe was detected. Adrenal venous sampling (AVS) subsequently revealed the presence of an extended tributary of the right adrenal vein to the liver surrounding the tumor. Segmental AVS further demonstrated a high plasma aldosterone concentration (PAC) in the right superior tributary vein draining the tumor. Laparoscopic partial hepatectomy was performed. The resected tumor histologically separated from the liver was composed of clear cells, immunohistochemically positive for aldesterone synthase (CYP11B2), and subsequently diagnosed as aldosterone-producing adrenal adenoma. After surgery, his blood pressure, serum potassium level, plasma renin activity and PAC were normalized. To the best of our knowledge, this is the first report of WS associated with PA. WS harbors a high prevalence of hypertension and therefore PA should be considered when managing the patients with WS and hypertension. In this case, the CT findings alone could not differentiate the adrenal rest tumor. Our case, therefore, highlights the usefulness of segmental AVS to distinguish adrenal tumors from hepatic adrenal rest tumors.

Renovascular hypertension (RVHT) is an important and potentially treatable form of resistant hypertension. Hypercortisolemia could also cause hypertension and diabetes mellitus. We experienced a case wherein adrenalectomy markedly improved blood pressure and plasma glucose levels in a patient with RVHT and low-level autonomous cortisol secretion. A 62-year-old Japanese man had been treated for hypertension and diabetes mellitus for 10 years. He was hospitalized because of a disturbance in consciousness. His blood pressure (BP) was 236/118 mmHg, pulse rate was 132 beats/min, and plasma glucose level was 712 mg/dL. Abdominal CT scanning revealed the presence of bilateral adrenal masses and left atrophic kidney. Abdominal magnetic resonance angiography demonstrated marked stenosis of the left main renal artery. The patient was subsequently diagnosed with atherosclerotic RVHT with left renal artery stenosis. His left adrenal lobular mass was over 40 mm and it was clinically suspected the potential for cortisol overproduction. Therefore, laparoscopic left nephrectomy and adrenalectomy were simultaneously performed, resulting in improved BP and glucose levels. Pathological studies revealed the presence of multiple cortisol-producing adrenal nodules and aldosterone-producing cell clusters in the adjacent left adrenal cortex. In the present case, the activated renin-angiotensin-aldosterone system and cortisol overproduction resulted in severe hypertension, which was managed with simultaneous unilateral nephrectomy and adrenalectomy.

Abstract Background Esophageal squamous cell carcinoma (ESCC) is a highly malignant neoplasm. The glucocorticoid (GC)-glucocorticoid receptor (GR) pathway plays pivotal roles in cellular response to various stresses of tumor cells, including chemotherapy. However, the status of the GC-GR pathway in ESCC, including its correlation with chemotherapeutic responses, is largely unknown. Methods GR, serum-and glucocorticoid-regulated kinase 1 (Sgk1), and N-myc down regulation gene 1 (NDRG1) were immunolocalized in 98 patients with ESCC who had undergone esophagectomy following neoadjuvant chemotherapy (NAC) with 2 courses of 5-fluorouracil + cisplatin. We also examined biopsy specimens before NAC in 42 cases and compared the results between those before and after NAC. Results Overall survival (OS) of the patients treated with surgery following NAC was significantly shorter in the group with high GR than that with low GR status (P = 0.0473). Both OS and disease-free survival (DFS) were significantly shorter in both Sgk1- and NDRG1-high groups than in the low groups (OS: Sgk1, P = 0.0055; NDRG1, P = 0.0021; DFS: Sgk1, P = 0.0240; NDRG1, P = 0.0086). Biopsy specimens before NAC showed significantly shorter DFS in the high Sgk1 group (P = 0.0095), while both OS and DFS were shorter in the high NDRG1 group (OS, P = 0.0233; DFS, P = 0.0006) than in the respective low groups. In the high NDRG1 group of biopsy specimens before NAC, the tumor reduction rate by NAC was significantly attenuated (P = 0.021). Conclusions High GR, Sgk1, and NDRG1 statuses in ESCC after NAC was significantly associated with an overall worse prognosis, with no significant changes in their expression levels before and after NAC. Therefore, increased activity of the GC-GR pathway with enhanced induction of Sgk1 and NDRG1 in carcinoma cells play pivotal roles in tumor progression and development of chemo-resistance in patients with ESCC undergoing NAC.

Abstract Carney complex (CNC) is a rare hereditary syndrome that involves endocrine dysfunction and the development of various types of tumors. Chromosome 2p16 and PRKAR1A on chromosome 17 are known susceptibility loci for CNC. Here we report a mother and son with CNC caused by an 8.57‐kb deletion involving the transcription start site and non‐coding exon 1 of PRKAR1A. The proband is a 28‐year‐old male with bilateral large‐cell calcified Sertoli cell testicular tumors and pituitary adenoma. Comprehensive genomic profiling for cancer mutations using Foundation One CDx failed to detect any mutations in PRKAR1A in DNA from the testicular tumor. Single‐nucleotide polymorphism array analysis of the proband’s genomic DNA revealed a large deletion in the 5′ region of PRKAR1A. Genomic walking further delineated the region an 8.57‐kb deletion. A 1.68‐kb DNA fragment encompassed by the deleted region showed strong promoter activity in a NanoLuc luciferase reporter assay. The patient’s mother, who is suffering from recurrent cardiac myxoma, a critical sign for CNC, carried an identical deletion. The 8.57‐kb deleted region is a novel lesion for CNC and will facilitate molecular diagnosis of the disease.

Abstract Background Lymph node metastasis is one of the pivotal factors of the clinical outcomes of patients with esophageal cancer receiving neoadjuvant chemoradiation therapy (NACRT). Both the nuclear factor‐erythroid 2‐related factor 2 (Nrf2) signaling pathway and heme oxygenase‐1 (HO‐1) are frequently upregulated in various human malignancies and associated with resistance to chemoradiation therapy, subsequently resulting in adverse clinical outcomes. However, the Nrf2 and HO‐1 status in lymph node metastasis and their differences between primary and metastatic lesions are unknown. Aims To examine the levels of Nrf2 signaling proteins and HO‐1 in primary and metastatic lesions of patients with esophageal squamous cell carcinoma using immunohistochemistry. Methods and Results We immunolocalized Nrf2 signaling proteins in 69 patients with lymph node metastases, who received NACRT with 5‐fluorouracil and cisplatin before esophagectomy. We also compared the findings between primary and metastatic lesions. Residual lymph node metastases were detected in 30 patients and among them, both primary and metastatic lesions were available for evaluation in 25 patients. Subsequently, we correlated the results with patients' survival. Nrf2, HO‐1, and the Ki‐67 labeling index were all significantly lower in the patients with lymph node metastases than in those with primary tumors. Carcinoma cells with high HO‐1 levels were significantly associated with pathological resistance to NACRT. These results suggested that overall and disease‐free survival of esophageal squamous cell carcinoma were significantly associated with both pN2 and high HO‐1 levels, respectively. Conclusions Protein expression in the Nrf2 pathway was significantly lower in patients with lymph node metastases than in those with primary lesions. HO‐1 levels in lymph node metastases could be used to predict the eventual clinical outcome of patients with esophageal cancer receiving NACRT.

Due to its rarity, adrenal hemorrhage is difficult to diagnose, and its precise etiology has remained unknown. One of the pivotal mechanisms of adrenal hemorrhage is the thrombosis of the adrenal vein, which could be due to thrombophilia. However, detailed pathological evaluation of resected adrenal glands is usually required for definitive diagnosis. Here, we report a case of a cortisol-secreting adenoma with concomitant foci of hemorrhage due to antiphospholipid syndrome diagnosed both clinically and pathologically. In addition, the tumor in this case was pathologically diagnosed as cortisol-secreting adenoma, although the patient did not necessarily fulfill the clinical diagnostic criteria of full-blown Cushing or sub-clinical Cushing syndrome during the clinical course, which also did highlight the importance of detailed histopathological investigations of resected adrenocortical lesions.

Primary aldosteronism (PA) usually accompanies suppressed plasma renin activity (PRA) through a negative feedback mechanism. While some cases of PA with unsuppressed PRA were reported, there have been no studies about the characteristics of PA with unsuppressed PRA; thus, these characteristics were examined herein. Nine patients with unsuppressed PRA and 86 patients with suppressed PRA were examined. All patients underwent segmental adrenal venous sampling (sAVS) and adrenalectomy, and were pathologically confirmed to have cytochrome P450 11B2 (CYP11B2)-positive aldosterone-producing adenoma according to international histopathology consensus criteria. Unsuppressed and suppressed PRA were defined as PRA levels of > 1.0 and ≤ 1.0 ng/mL/hr, respectively, in multiple blood samples obtained in the resting position. The unsuppressed PRA group had higher morning cortisol levels (12.6 [8.5, 13.5] vs. 8.5 [7.1, 11.0] μg/dL, P = 0.03) and higher cortisol levels after a 1 mg dexamethasone suppression test (DST) (2.2 [1.6, 2.5] vs. 1.3 [1.0, 1.9] μ g/dL, P = 0.004) than the suppressed PRA group. The unsuppressed PRA group also showed higher aldosterone levels on the non-surgical side during sAVS (P = 0.02 before adrenocorticotropic hormone (ACTH) stimulation, P = 0.002 after ACTH stimulation), a higher intensity of CYP17 expression in the resected adrenal gland (P = 0.02), and a lower clinical complete success rate 1 year after surgery (P = 0.04) compared with those in the suppressed PRA group. These findings suggest that PA should not be ruled out by unsuppressed PRA among patients with hypertension, particularly when their cortisol levels remain unsuppressed in the 1 mg DST. Meanwhile, it should be acknowledged that patients with unsuppressed PRA have higher aldosterone levels on the non-surgical side, and a lower likelihood of postoperative complete clinical success is to be expected.

Non-small cell lung cancer (NSCLC) accounts for the majority (80-85%) of all lung cancers. All current available treatments have limited efficacy. The epidermal growth factor receptor (EGFR) plays a critical role in the development and progression of NSCLC, with high EGFR expression associated with increased cell proliferation and poor prognosis. Thus, interfering with EGFR signaling has been shown to effectively reduce cell proliferation and help in the treatment of NSCLC. We previously demonstrated that the progesterone receptor (PR) contains a polyproline domain (PPD) that directly interacts with Src homology 3 (SH3) domain-containing molecules and expression of PR-PPD peptides inhibits NSCLC cell proliferation. In this study, we investigated whether the introduction of PR-PPD by cell-penetrating peptides (CPPs) could inhibit EGF-induced cell proliferation in NSCLC cells. PR-PPD was attached to a cancer-specific CPP, Buforin2 (BR2), to help deliver the PR-PPD into NSCLC cells. Interestingly, addition of BR2-2xPPD peptides containing two PR-PPD repeats was more effective in inhibiting NSCLC proliferation and significantly reduced EGF-induced phosphorylation of Erk1/2. BR2-2xPPD treatment induced cell cycle arrest by inhibiting the expression of cyclin D1 and CDK2 genes in EGFR-wild type A549 cells. Furthermore, the combination treatment of EGFR-tyrosine kinase inhibitors (TKIs), including Gefitinib or Erlotinib, with BR2-2xPPD peptides further suppressed the growth of NSCLC PC9 cells harboring EGFR mutations as compared to EGFR-TKIs treatment alone. Importantly, BR2-2xPPD peptides mediated growth inhibition in acquired Gefitinib- and Erlotinib- resistant lung adenocarcinoma cells. Our data suggests that PR-PPD is the minimal protein domain sufficient to inhibit NSCLC cell growth and has the potential to be developed as a novel NSCLC therapeutic agent.

Summary: Sustained treatment of estrogen receptor (ER)-positive breast cancer with ER-targeting drugs results in ER mutations and refractory unresponsive cancers. Androgen receptor (AR), which is expressed in 80%–95% of ER-positive breast cancers, could serve as an alternate therapeutic target. Although AR agonists were used in the past to treat breast cancer, their use is currently infrequent due to virilizing side effects. Discovery of tissue-selective AR modulators (SARMs) has renewed interest in using AR agonists to treat breast cancer. Using translational models, we show that AR agonist and SARM, but not antagonist, inhibit the proliferation and growth of ER-positive breast cancer cells, patient-derived tissues, and patient-derived xenografts (PDX). Ligand-activated AR inhibits wild-type and mutant ER activity by reprogramming the ER and FOXA1 cistrome and rendering tumor growth inhibition. These findings suggest that ligand-activated AR may function as a non-canonical inhibitor of ER and that AR agonists may offer a safe and effective treatment for ER-positive breast cancer. : Molecular Biology; Molecular Mechanism of Gene Regulation; Cancer Subject Areas: Molecular Biology, Molecular Mechanism of Gene Regulation, Cancer

Abstract Our aim was to investigate the efficacy and safety of initial neoadjuvant endocrine therapy with exemestane alone followed by tailored treatment, either continued exemestane monotherapy or exemestane plus docetaxel–cyclophosphamide (TC) combination therapy, in postmenopausal patients with primary invasive estrogen receptor–positive, human epidermal growth factor receptor 2–negative, stage I‐IIIA breast cancer and Ki67 labeling index ≤30%. In this open‐label phase II study, patients initially received exemestane 25 mg/d for 12 weeks. Responders were defined as patients who achieved complete response (CR), partial response (PR) with Ki67 labeling index ≤5% after treatment, or stable disease with Ki67 labeling index ≤5% both before and after treatment. For the subsequent 12 weeks, exemestane monotherapy was continued for responders (group A), whereas nonresponders received exemestane plus four cycles of TC (docetaxel 75 mg/m2 and cyclophosphamide 600 mg/m2 every 3 weeks) (group B). Clinical response rate (ie the proportion of patients with CR or PR) at 24 weeks was the primary endpoint. Of 64 patients provisionally enrolled between December 2010 and May 2016, 58 (median age 60 years) started the study treatment. Five patients discontinued treatment in the initial exemestane monotherapy period, and 39 completed the study treatment. Clinical response rates at 8‐12 and 24 weeks were 71% (10/14, 95% confidence interval [CI] 41.9%‐91.6%) and 57% (8/14, 95% CI 28.9%‐82.3%), respectively, in group A, and 16% (4/25, 95% CI 4.5%‐36.1%) and 56% (14/25, 95% CI 34.9%‐75.6%), respectively, in group B. Grade ≥3 adverse events were reported in 8% (1/15) and 53% (20/38) in group A and group B, respectively. The tailored treatment maintained the favorable clinical response to exemestane alone in responders and improved clinical response in nonresponders. Trial number UMIN000004752 (UMIN Clinical Trials Registry).

Introduction: Estrogen receptors (ER) (ERα, ERβ) and aromatase (key enzyme for estrogen synthesis) are expressed in most human NSCLCs. High intratumoral estrogen levels and elevated aromatase expression in NSCLC predict poor outcome. This open-label, phase 1b, single-center study evaluated the safety and tolerability of escalating doses of the aromatase inhibitor, exemestane, in combination with carboplatin and pemetrexed in postmenopausal women with stage IV nonsquamous NSCLC. Methods: Patients received exemestane (starting 1-wk before chemotherapy) at 25 mg orally (PO) daily (cohort 1) or 50 mg PO daily (cohort 2) combined with carboplatin (area under the curve 6 mg × min/mL) and pemetrexed (500 mg/m2) intravenously every 3 weeks for four cycles. Thereafter, patients were eligible for continued therapy with exemestane and pemetrexed or pemetrexed alone. Results: A total of 10 patients consented for therapy, and two patients failed in the screening. Four patients completed the therapy in cohort 1 and four patients in cohort 2. The median number of cycles administered was 15 (range: 1–54). Maximum tolerated dose was exemestane 50 mg PO daily with combination chemotherapy. Intention-to-treat analysis revealed an objective response rate (ORR) of 62.5% (five of eight patients with partial response) and a clinical benefit rate of 87.5% (seven of eight patients with either stable disease or partial response). ORR was associated with aromatase expression (p = 0.02). Circulating estrogen levels decreased with exemestane use, and quality of life measurements did not significantly change during the treatment. There were no adverse events. Conclusions: The combination of carboplatin, pemetrexed, and exemestane in postmenopausal women with metastatic NSCLC is safe and well tolerated. Biomarker studies revealed that ORR correlates with tumor aromatase expression. These findings support future clinical trials to confirm the antitumor efficacy with this combination therapy.

A 67-year-old Japanese woman who had end-stage renal disease was referred to our hospital for kidney transplantation. Abdominal CT revealed a large adrenal mass with inhomogeneity. She had a history of hospitalization for stroke and heart failure and exhibited prominent hyporeninemic hyperaldosteronism. Histological examination of the resected tumor with anti-CYP11B2 antibody indicated that she had a vascular endothelial cyst with primary aldosteronism (PA) due to multiple adrenocortical micronodules. This report implicates the pathological interaction between adrenal vascular cysts and PA-mediated vascular damage of the adrenal vein.

Background. Adrenocortical carcinoma (ACC) is a rare and highly aggressive malignancy. ACCs often secrete adrenal steroid hormones including cortisol and androgens; however, aldosterone-producing ACC is very rare. Although adrenal production of aldosterone is assessed by adrenal venous sampling, the use of sampling from the relevant vein to assess aldosterone production from a tumor arising from ACC metastasis has not been previously reported. Case Presentation. We report the case of a 69-year-old Japanese man with aldosterone-producing ACC with hepatic metastasis. He presented with a history of treatment-resistant hypertension and hypokalemia. Endocrinological examination showed markedly increased plasma aldosterone concentration and suppressed plasma renin activity. Serum cortisol concentration was not suppressed by administration of dexamethasone 1 mg, and normal circadian variation of cortisol secretion was disrupted. Abdominal computed tomography showed a large tumor in the left adrenal gland and multiple tumors in the liver. Together, these results strongly suggested ACC with multiple liver metastases causing primary aldosteronism and subclinical Cushing syndrome. Adrenal and hepatic venous sampling showed markedly increased aldosterone concentration in the left adrenal vein but no increase in the hepatic vein, despite a pathological diagnosis of ACC with hepatic metastasis, with immunohistochemical investigation showing both primary and secondary tumors to have synthetic capability for aldosterone. The patient received mitotane but declined combination chemotherapy and died 2 months later. Conclusion. This is the first report of adrenal and hepatic venous sampling in a case of aldosterone-producing ACC with hepatic metastasis. The case suggests that hepatic venous sampling is unable to detect aldosterone production from liver metastases arising from ACC.

Dysgerminoma is a malignant ovarian germ cell tumor, and unlike sex-cord stromal tumors, endocrine manifestation is considered rare. Here, we report the first case of dysgerminoma presenting precocious puberty. The patient is a 7-year-old girl who presented with a breast development in Tanner stage 3. Serum estradiol (E2) was markedly elevated while luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were suppressed below the detection limit. Microscopically, the right ovarian mass displayed nests of large polygonal cells and fibrous septa which were focally concentrated by theca-like plump spindle cells. Immunohistochemistry revealed that the spindle cells expressed various steroidogenic enzymes involved in estrogen biosynthesis including P450 aromatase. The tumor was diagnosed with pure dysgerminoma with estrogen-producing functioning stroma. After the operation, serum E2 declined below the detection limit; LH and FSH returned within the normal range. This case demonstrates that even a conventional dysgerminoma can present endocrine manifestation through functioning stroma.

Pheochromocytomas (PHEOs) are relatively rare catecholamine-producing tumors derived from adrenal medulla. Tumor microenvironment (TME) including neoangiogenesis has been explored in many human neoplasms but not necessarily in PHEOs. Therefore, in this study, we examined tumor infiltrating lymphocytes (CD4 and CD8), tumor associated macrophages (CD68 and CD163), sustentacular cells (S100p), and angiogenic markers (CD31 and areas of intratumoral hemorrhage) in 39 cases of PHEOs in the quantitative fashion. We then compared the results with pheochromocytoma of the adrenal gland scaled score (PASS), grading system for pheochromocytoma and paraganglioma (GAPP) and the status of intra-tumoral catecholamine-synthesizing enzymes (TH, DDC, and PNMT) as well as their clinicopathological factors. Intratumoral CD8 (p = 0.0256), CD31 (p = 0.0400), and PNMT (p = 0.0498) status was significantly higher in PHEOs with PASS

Pheochromocytomas and paragangliomas (PHEO/PGL) are rare but occasionally life-threatening neoplasms, and are potentially malignant according to WHO classification in 2017. However, it is also well known that histopathological risk stratification to predict clinical outcome has not yet been established. The first histopathological diagnostic algorithm for PHEO, “PASS”, was proposed in 2002 by Thompson et al. Another algorithm, GAPP, was then proposed by Kimura et al. in 2014. However, neither algorithm has necessarily been regarded a ‘gold standard’ for predicting post-operative clinical behavior of tumors. This is because the histopathological features of PHEO/PGL are rather diverse and independent of their hormonal activities, as well as the clinical course of patients. On the other hand, recent developments in wide-scale genetic analysis using next-generation sequencing have revealed the molecular characteristics of pheochromocytomas and paragangliomas. More than 30%–40% of PHEO/PGL are reported to be associated with hereditary genetic abnormalities involving > 20 genes, including SDHXs, RET, VHL, NF1, TMEM127, MAX, and others. Such genetic alterations are mainly involved in the pathogenesis of pseudohypoxia, Wnt, and kinase signaling, and other intracellular signaling cascades. In addition, recurrent somatic mutations are frequently detected and overlapped with the presence of genetic alterations associated with hereditary diseases. In addition, therapeutic strategies specifically targeting such genetic abnormalities have been proposed, but they are not clinically applicable at this time. Therefore, we herein review recent advances in relevant studies, including histopathological and molecular analyses, to summarize the current status of potential prognostic factors in patients with PHEO/PGL.

To characterize the expression of steroidogenic enzymes implicated in the development of ovarian steroid cell tumors, not otherwise specified (SCT-NOS). We present 4 ovarian SCT-NOS evaluated by immunohistochemical staining of steroidogenic enzymes as an approach to define this entity pathologically. All 4 ovarian SCT-NOS showed increased expression for cholesterol side-chain cleavage enzyme (CYP11A1), 17α-hydroxylase (CYP17A1), 17β-hydroxysteroid dehydrogenase 1 (HSD17B1), aldo-ketoreductase type 1 C3 (AKR1C3), 3β-hydroxysteroid dehydrogenase 2 (HSD3B2), 5α-reductase type 2 (SRD5A2), steroid sulfatase (SULT2A1), estrogen sulfotransferase (EST), and aromatase (CYP19A1). Expression was negative for 21-hydroxylase (CYP21A2) and 17β-hydroxysteroid dehydrogenase 2 (HSD17B2). 17β-hydroxysteroid dehydrogenase 3 (HSD17B3) and 5α-reductase type 1 (SRD5A1) showed variable expression. Our analysis reveals a novel finding of increased expression of AKR1C3, HSD17B1, SRD5A2, SULT2A1, and EST in ovarian SCT-NOS, which is clinically associated with androgen excess and virilization. Further studies are needed to validate these enzymes as new markers in the evaluation of hyperandrogenic ovarian conditions.

BACKGROUND:Many genomic analyses of cortisol-producing adrenocortical carcinoma (ACC) have been reported, but very few have come from East Asia. The first objective of this study is to verify the genetic difference with the previous reports by analyzing targeted deep sequencing of 7 Japanese ACC cases using next-generation sequencing (NGS). The second objective is to compare the somatic variant findings identified by NGS analysis with clinical and pathological findings, aiming to acquire new knowledge about the factors that contribute to the poor prognosis of ACC and to find new targets for the treatment of ACC. METHOD:DNA was extracted from ACC tissue of seven patients and two reference blood samples. Targeted deep sequencing was performed using the MiSeq system for 12 genes, and the obtained results were analyzed using MuTect2. The hypothesis was obtained by integrating the somatic variant findings with clinical and pathological data, and it was further verified using The Cancer Genome Atlas (TCGA) dataset for ACC. RESULTS:Six possible pathogenic and one uncertain significance somatic variants including a novel PRKAR1A (NM_002734.4):c.545C>A (p.T182K) variant were found in five of seven cases. By integrating these data with pathological findings, we hypothesized that cases with TP53 variants were more likely to show atypical mitotic figures. Using TCGA dataset, we found that atypical mitotic figures were associated with TP53 somatic variant, and mRNA expression of CCNB2 and AURKA was significantly high in TP53 mutated cases and atypical mitotic figure cases. CONCLUSION:We believe this is the first report that discusses the relationship between atypical mitotic figures and TP53 somatic variant in ACC. We presumed that overexpression of CCNB2 and AURKA mRNA may cause atypical mitosis in TP53 somatic mutated cases. Because AURKA is highly expressed in atypical mitotic cases, it may be an appropriate indicator for AURKA inhibitors.

A 47-year-old woman with a history of diabetes mellitus (DM) and obesity was admitted to our hospital for glucose control. She was detected to have hypertension (HT) and diagnosed with primary aldosteronism (PA) based on the high level of aldosterone to renin ratio and the results of the upright furosemide-loading test according to the criteria of the Japanese Society of Hypertension (JSH) guidelines. Computed tomography revealed left renal tumor and adrenocortical adenoma. She underwent left nephrectomy and adrenalectomy. The pathological findings were clear-cell renal cell carcinoma (RCC) and nonfunctional adrenocortical adenoma. Her nonneoplastic adrenal tissue histologically revealed CYP11B2-positive multiple adrenocortical micronodules (MNs) and concomitant paradoxical hyperplasia of the zona glomerulosa. Therefore, MNs were thought to be responsible for PA in this patient. After surgery, HT was improved, and the result of upright furosemide-loading test after 12 months of surgery did not fulfill the criteria of PA according to the JSH guidelines. However, the adrenocorticotrophic hormone stimulation test was positive; considering the possibility of slight aldosterone overproduction from the right adrenal gland, the administration of spironolactone was started. Herein, we report a rare case of RCC in conjunction with PA histologically associated with MNs.

Abstract Patients with estrogen receptor (ER)‐positive breast cancer are less likely to achieve a pathological complete response (pCR) with neoadjuvant chemotherapy. Neoadjuvant endocrine therapy may be more appropriate than neoadjuvant chemotherapy in these hormone‐sensitive patients. Most patients with ER‐positive breast cancer are postmenopausal, and therefore, generally older and less able to tolerate chemotherapy. We aimed to investigate the efficacy and safety of tailored neoadjuvant endocrine and chemoendocrine therapy for postmenopausal breast cancer patients. Untreated patients with primary invasive ER‐positive, HER2‐negative, stage I‐IIIA breast cancer, and Ki67 index ≤30% were enrolled. Patients received exemestane 25 mg/d for 12 weeks. Based on clinical response and change in Ki67 index, assessed at 8‐12 weeks, patients with complete response (CR), partial response (PR) with Ki67 index ≤5% after treatment, or stable disease (SD) with Ki67 index ≤5% before and after treatment were defined as responders. For the subsequent 24 weeks, responders continued exemestane monotherapy (group A), and nonresponders received exemestane 25 mg/d plus cyclophosphamide 50 mg/d (group B). The primary endpoint was clinical response at weeks 24 and 36. A total of 59 patients (median age, 69 years) started initial exemestane monotherapy. After exclusion of three patients who discontinued during this period, 56 remained enrolled to receive subsequent treatment. Clinical response rates (CR and PR) and 95% CI at weeks 24 and 36 were 85% (12/14; 57.2%‐98.2%) and 71% (10/14; 41.9%‐91.6%), respectively, in group A; and 54% (23/42; 38.7%‐70.2%) and 71% (30/42; 55.4%‐84.3%), respectively, in group B. At week 36, no significant difference was found in median Ki67 index between the groups (3.5% and 4.0%). There were no treatment‐related deaths. We found that clinical response comparable to that of responders was achieved in nonresponders after addition of cyclophosphamide to the initial endocrine therapy.

Abstract Background Post-transplant lymphoproliferative disorder (PTLD) is a life-threatening complication that can be difficult to treat; moreover, determination of the pathophysiological type is difficult. We report a rare case of a patient who developed two types of Epstein–Barr virus (EBV)-negative PTLD following living donor liver transplantation (LDLT). Case presentation A 64-year-old man underwent LDLT for acute fulminant hepatitis B. Sixty-five months later, he developed EBV-negative monomorphic B cell PTLD. Reduction of immunosuppressive therapy and chemotherapy with rituximab resulted in a partial response. He received radioimmunotherapy with yttrium-90-ibritumomab tiuxetan, which was effective for all lesions, except for the splenic hilar lesion, which enlarged and seemed to penetrate the stomach. Therefore, he underwent resection of the pancreatic tail with splenectomy and partial gastrectomy. The pathological diagnosis was EBV-negative classical Hodgkin lymphoma (cHL)-type PTLD. Conclusions This patient showed an unexpected course of PTLD, from both a clinical and pathological perspective. There are no prior reports of an adult case of EBV-negative cHL-type PTLD coexisting with EBV-negative monomorphic B cell PTLD. When a strange and refractory lesion persists despite effective therapy for PTLD, we must consider the possibility of another type of PTLD within the residual lesion.

Abstract Recent positron emission tomography (PET) studies have demonstrated the accumulation of tau PET tracer in the affected region of progressive supranuclear palsy (PSP) cases. To confirm the binding target of radiotracer in PSP, we performed an imaging-pathology correlation study in two autopsy-confirmed PSP patients who underwent [18F]THK5351 PET before death. One patient with PSP Richardson syndrome showed elevated tracer retention in the globus pallidus and midbrain. In a patient with PSP-progressive nonfluent aphasia, [18F]THK5351 retention also was observed in the cortical areas, particularly the temporal cortex. Neuropathological examination confirmed PSP in both patients. Regional [18F]THK5351 standardized uptake value ratio (SUVR) in antemortem PET was significantly correlated with monoamine oxidase-B (MAO-B) level, reactive astrocytes density, and tau pathology at postmortem examination. In in vitro autoradiography, specific THK5351 binding was detected in the area of antemortem [18F]THK5351 retention, and binding was blocked completely by a reversible selective MAO-B inhibitor, lazabemide, in brain samples from these patients. In conclusion, [18F]THK5351 PET signals reflect MAO-B expressing reactive astrocytes, which may be associated with tau accumulation in PSP.

The adrenal cortex produces steroid hormones as adrenocortical hormones in the body, secreting mineralocorticoids, glucocorticoids, and adrenal androgens, which are all considered essential for life. Adrenocortical tumors harbor divergent hormonal activity, frequently with steroid excess, and disrupt homeostasis of the body. Aldosterone-producing adenomas (APAs) cause primary aldosteronism (PA), and cortisol-producing adenomas (CPAs) are the primary cause of Cushing’s syndrome. In addition, adrenocortical carcinoma (ACC) is a highly malignant cancer harboring poor prognosis. Various genetic abnormalities have been reported, which are associated with possible pathogenesis by the alteration of intracellular signaling and activation of transcription factors. In particular, somatic mutations in APAs have been detected in genes encoding membrane proteins, especially ion channels, resulting in hypersecretion of aldosterone due to activation of intracellular calcium signaling. In addition, somatic mutations have been detected in those encoding cAMP-PKA signaling-related factors, resulting in hypersecretion of cortisol due to its driven status in CPAs. In ACC, mutations in tumor suppressor genes and Wnt-β-catenin signaling-related factors have been implicated in its pathogenesis. In this article, we review recent findings on the genetic characteristics and regulation of intracellular signaling and transcription factors in individual tumors.

Abstract Background We report a rare case of a juxta-adrenal schwannoma that could not be discriminated from an adrenal tumor before surgical resection and was complicated by bilateral hyperaldosteronism. To the best of our knowledge, this is first case in which both a juxta-adrenal schwannoma and hyperaldosteronism co-existed. Case presentation A 69-year-old male treated for hypertension was found to have a left supra-renal mass (5.8 × 5.2 cm) by abdominal computed tomography. His laboratory data showed that his plasma aldosterone concentration (PAC) was within the normal range, but his plasma renin activity (PRA) was reduced, resulting in an increased aldosterone/renin ratio (ARR). Load tests of captopril or furosemide in the standing position demonstrated autonomous aldosterone secretion and renin suppression. Adrenal venous sampling (AVS) with ACTH stimulation indicated bilateral hypersecretion of aldosterone. A left supra-renal tumor was resected because of the possibility of malignancy and was found to be a benign schwannoma arising from the juxta-adrenal region together with an adrenal gland. The dissected left adrenal gland was morphologically hyperplastic in the zona glomerulosa, but was immunohistochemically negative for CYP11B2 (aldosterone synthase). Multiple CYP11B2-positive adrenocortical micronodules were detected in the adrenal gland, indicating micronodular hyperplasia. Although bilateral aldosteronism was indicated by AVS before the operation, the PRA, PAC and ARR values were within their respective reference ranges after resection of the unilateral tumor, suggesting that the slight increase in hormone secretion from the remaining right-sided lesion could not be detected after resection. Conclusion A clinical and morphologic diagnosis of juxta-adrenal schwannoma is difficult, particularly in a case of hyperaldosteronism, as shown in this case. These data suggest the complexity and difficulty diagnosing adrenal incidentaloma.

Abstract Background Adrenocoricotrophic hormone (ACTH) – independent bilateral adrenocortical macronodular hyperplasia (AIMAH) is a rare cause of Cushing’s syndrome, and is characterized by bilateral adrenal hyperplasia. However, Primary aldosteronism (PA) is a relatively common adrenal disease. Case presentation A 56-year-old man who has been treated hypertension and diabetes mellitus was detected low plasma potassium level with an elevated level of plasma aldosterone concentration and bilateral adrenal swelling. Endocrinological examinations showed autonomous secretion of cortisol and aldosterone, with suppression of plasma ACTH level and renin activity. A selective adrenal venous sampling demonstrated that left adrenal gland was responsible for aldosterone hypersecretion. He was diagnosed preclinical Cushing’s syndrome due to ACTH – independent bilateral adrenocortical macronodular hyperplasia (AIMAH) associated with aldosterone producing adenoma of the left adrenal gland. A laparoscopic left adrenalectomy was performed. Conclusion The resected adrenal specimen histologically consisted with a diagnosis of AIMAH. Moreover, tiny cell clusters positive immunostaining for aldosterone synthase was revealed. This is a rare case of AIMAH accompanied by preclinical Cushing’s syndrome and primary aldosteronism.

Abstract Background Gangliocytic paraganglioma (GP) is a rare neuroendocrine neoplasm, which occurs mostly in the periampullary portion of the duodenum; the majority of the reported cases of duodenal GP has been of benign nature with a low incidence of regional lymph node metastasis. GP arising from the pancreas is extremely rare. To date, only three cases have been reported and its clinical characteristics are largely unknown. Case presentation A nodule located in the pancreatic head was incidentally detected in an asymptomatic 68-year-old woman. Computed tomography revealed 18-, 8-, and 12-mm masses in the pancreatic head, the pancreatic tail, and the left adrenal gland, respectively. Subsequent genetic examination revealed an absence of mutations in the MEN1 and VHL genes. Macroscopically, the tumor located in the pancreatic head was 22 mm in size and displayed an ill-circumscribed margin along with yellowish-white color. Microscopically, it was composed of three cell components: epithelioid cells, ganglion-like cells, and spindle cells, which led to the diagnosis of GP. The tumor was accompanied by a peripancreatic lymph node metastasis. The tumor in the pancreatic tail was histologically classified as a neuroendocrine tumor (NET) G1 (grade 1, WHO 2010), whereas the tumor in the left adrenal gland was identified as an adrenocortical adenoma. The patient was disease-free at the 12-month follow-up examination. Conclusions Pancreatic GP is associated with a higher incidence of metastasis and larger tumor size than duodenal GPs, suggesting that the primary organ of GP is an important prognostic factor.