Histopathological analysis potential for unveiling hormone signaling in endocrine-related tumors.

Elucidating the mechanisms of action of steroid hormones will contribute to the development of therapeutic strategies for hormone-dependent tumors. Recent advances in genetic engineering have revealed the complex and diverse mechanisms of steroid hormone signaling; however, these techniques are limited to in vitro or animal experiments. It is believed that verifying hormone signals elucidated using human pathological tissue specimens will directly aid in treatment and diagnosis. However, pathological tissue specimens are generally formalin-fixed paraffin-embedded (FFPE), and protein/gene analyses of FFPE tissues are limited. Protein detection using immunohistochemistry with specific antibodies in FFPE tissues is a classical technique essential for diagnosis and treatment decisions in various types of cancer. In steroid hormone signaling, the expression and localization of receptors, hormone-related enzymes, and proteins encoded by response genes can be clarified using immunohistochemistry. Although protein-protein interactions such as receptor dimers and DNA-binding proteins are mainly detected in vitro, they can be examined in FFPE tissues using in situ proximity ligation assays and southwestern histochemistry, respectively. Using these detection methods, including immunohistochemistry, it is possible to analyze each hormone signaling pathway in hormone-related tumors histopathologically. Although FFPE tissues still suffer from gene and protein denaturation, their advantages include the ability to retrospectively study target factors/signals and obtain spatial information through microscopy. This review describes a visualization method for elucidating steroid hormone signaling in hormone-dependent tumors using FFPE tissues.

Development of aldosterone biosynthesis during fetal and pediatric periods; Histological analysis of CYP11B2-positive cell distribution in the zona glomerulosa of human adrenal.

The distribution of CYP11B2-positive or aldosterone producing adrenocortical cells in human fetuses and children and their age-dependent changes has not been studied. We aimed to explore the changes of aldosterone biosynthesis and age-related histological alterations of the zona glomerulosa in human adrenal gland during fetal and pediatric periods. We first reviewed 125 fetal and pediatric autopsy cases and retrieved 78 adrenals from 70 cases. CYP11B2 immunohistochemistry and quantitative image analysis of its results were performed in all adrenal glands. The ratio of the definitive zone (DZ) or zona glomerulosa (ZG) / the whole adrenocortical areas started to increase in the 2nd trimester, subsequently decreased in the 3rd, increased after birth, peaked in infancy, and then gradually decreased. The ratio of CYP11B2-positive / whole adrenocortical areas remained low during the fetal period but increased after birth, peaked at infancy, and then decreased. The ratio of CYP11B2-positive / DZ or ZG areas and CYP11B2-positive areas / depth of DZ or ZG demonstrated a distinctive bimodal pattern, with one peak in the fetal period and another in the neonatal period to infancy. This is the first study to perform quantitative analysis of the distribution of CYP11B2-positive cells, the histological DZ or ZG, and the development of aldosterone biosynthesis in human adrenal glands during fetal and pediatric periods.

Surgical and irradiated case of early breast cancer in a patient with Ehlers-Danlos syndrome.

BACKGROUND: Ehlers-Danlos syndrome (EDS) is a rare inherited connective tissue disease characterized by hyperextensibility of the skin and joints and tissue fragility of the skin and blood vessels, Vascular EDS is the most severe form of EDS, with abnormal arterial fragility. There have been no reports of breast cancer occurring in patients with vascular EDS. Here, we report here a very rare case of breast cancer in a patient with vascular EDS. CASE PRESENTATION: A 46-year-old woman with vascular EDS underwent partial left mastectomy and sentinel lymph node biopsy for left breast cancer (cStage 0) detected by medical examination. The final pathological diagnosis was invasive ductal carcinoma of the breast (pStage IA) [hormone receptor-positive, HER2 score 2 equivocal (FISH-positive), Ki-67LI 18%, luminal-HER2 type]. BluePrint was submitted as an aid in determining the postoperative treatment strategy, BluePrint Molecular Subtype HER2-type. However, the 10-year breast cancer mortality risk using Predict was low (5%). After consultation with the patient, the decision was made to administer postoperative radiation to the preserved breast along with hormone therapy only. There was no delay in postoperative wound healing, and the patient was free of metastatic recurrence for 9 months after surgery. CONCLUSION: We performed surgery, postoperative radiotherapy, and hormonal therapy in a breast cancer patient with vascular EDS without major complications.

Immunohistochemical Profiling of SSTR2 and HIF-2α with the Tumor Microenvironment in Pheochromocytoma and Paraganglioma.

Metastatic pheochromocytomas and paragangliomas (PPGLs) are rare endocrine malignancies with limited effective treatment options. The association between the tumor microenvironment (TME) with somatostatin receptor 2 (SSTR2) and hypoxia-induced factor-2α (HIF-2α) in PPGLs, critical for optimizing combination therapeutic strategies with immunotherapy, remains largely unexplored. To evaluate the association of SSTR2 and HIF-2α immunoreactivity with the TME in patients with PPGLs, we analyzed the expression of SSTR2A, HIF-2α, and TME components, including tumor-infiltrating lymphocytes (CD4 and CD8), tumor-associated macrophages (CD68 and CD163), and PD-L1, using immunohistochemistry in patients with PPGLs. The primary outcome was to determine the association of the immune profiles with SSTR2A and HIF-2α expression. Among 45 patients with PPGLs, SSTR2A and HIF2α were positively expressed in 21 (46.7%) and 14 (31.1%) patients, respectively. The median PD-L1 immunohistochemical score (IHS) was 2.0 (interquartile range: 0-30.0). Positive correlations were observed between CD4, CD8, CD68, and CD163 levels. A negative correlation was found between the CD163/CD68 ratio (an indicator of M2 polarization) and SSTR2A expression (r = -0.385, p = 0.006). HIF-2α expression showed a positive correlation with PD-L1 IHS (r = 0.348, p = 0.013). The co-expression of PD-L1 (HIS > 10) and HIF-2α was found in seven patients (15.6%). No associations were observed between SDHB staining results and the CD163/CD68 ratio, PD-L1, or SSTR2A expression. Our data suggest the potential of combination therapy with immunotherapy and peptide receptor radionuclide therapy or HIF-2α inhibitors as a treatment option in selected PPGL populations.

Screening Cutoff Values for the Detection of Aldosterone-Producing Adenoma by LC-MS/MS and a Novel Noncompetitive CLEIA.

Context: Detecting patients with surgically curable aldosterone-producing adenoma (APA) among hypertensive individuals is clinically pivotal. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the ideal method of measuring plasma aldosterone concentration (PAC) because of the inaccuracy of conventional chemiluminescent enzyme immunoassay (CLEIA). However, LC-MS/MS is expensive and requires expertise. We have developed a novel noncompetitive CLEIA (NC-CLEIA) for measuring PAC in 30 minutes. Objective: This work aimed to validate NC-CLEIA PAC measurements by comparing them with LC-MS/MS measurements and determining screening cutoffs for both measurements detecting APA. Methods: We retrospectively measured PAC using LC-MS/MS and NC-CLEIA in 133 patients with APA, 100 with bilateral hyperaldosteronism, and 111 with essential hypertension to explore the accuracy of NC-CLEIA PAC measurements by comparing with LC-MS/MS measurements and determined the cutoffs for detecting APA. Results: Passing-Bablok analysis revealed that the values by NC-CLEIA (the regression slope, intercept, and correlation coefficient were 0.962, -0.043, and 0.994, respectively) were significantly correlated and equivalent to those by LC-MS/MS. Bland-Altman plot analysis of NC-CLEIA and LC-MS/MS also demonstrated smaller systemic errors (a bias of -0.348 ng/dL with limits of agreement of -4.390 and 3.694 within a 95% CI) in NC-CLEIA than LC-MS/MS. The receiver operating characteristic analysis demonstrated that cutoff values for aldosterone/renin activity ratio obtained by LC-MS/MS and NC-CLEIA were 31.2 and 31.5 (ng/dL per ng/mL/hour), with a sensitivity of 91.0% and 90.2% and specificity of 75.4% and 76.8%, respectively, to differentiate APA from non-APA. Conclusion: This newly developed NC-CLEIA for measuring PAC could serve as a clinically reliable alternative to LC-MS/MS.

Investigating the cut-off values of captopril challenge test for primary aldosteronism using the novel chemiluminescent enzyme immunoassay method: a retrospective cohort study.

The measurement evolution enabled more accurate evaluation of aldosterone production in hypertensive patients. However, the cut-off values for novel assays have been not sufficiently validated. The present study was undertaken to validate the novel chemiluminescent enzyme immunoassay for aldosterone in conjunction with other methods. Moreover, we also aimed to establish a new cut-off value for primary aldosteronism in the captopril challenge test using the novel assay. First, we collected 390 plasma samples, in which aldosterone levels measured using liquid chromatography-mass spectrometry ranged between 0.18 and 1346 ng/dL. The novel chemiluminescent enzyme immunoassay showed identical correlation of plasma aldosterone with liquid chromatography-mass spectrometry, in contrast to conventional radioimmunoassay. Further, we enrolled 299 and 39 patients with primary aldosteronism and essential hypertension, respectively. Plasma aldosterone concentrations measured using the novel assay were lower than those measured by radioimmunoassay, which resulted in decreased aldosterone-to-renin ratios. Subsequently, positive results of the captopril challenge test based on radioimmunoassay turned into "negative" based on the novel assay in 45% patients with primary aldosteronism, using the conventional cut-off value (aldosterone-to-renin activity ratio > 20 ng/dL per ng/mL/h). Receiver operating characteristic curve analysis demonstrated that aldosterone-to-renin activity ratios > 8.2 ng/dL per ng/mL/h in the novel assay was compatible with the conventional diagnosis (sensitivity, 0.874; specificity, 0.980). Our study indicates the great measurement accuracy of the novel chemiluminescent enzyme immunoassay for aldosterone, and the importance of measurement-adjusted cut-offs in the diagnosis of primary aldosteronism.

Double somatic mutations in CTNNB1 and GNA11 in an aldosterone-producing adenoma.

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 11ß-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 ß-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.

Mitochondrial dynamics as a novel treatment strategy for triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC), recognized as the most heterogeneous type of breast cancer (BC), exhibits a worse prognosis than other subtypes. Mitochondria dynamics play a vital role as mediators in tumorigenesis by adjusting to the cell microenvironments. However, the relationship between mitochondrial dynamics and metabophenotype exhibits discrepancies and divergence across various research and BC models. Therefore, this study aims to explore the role of mitochondrial dynamics in TNBC drug resistance and tumorigenesis. METHODS: The Wst-8 test was conducted to assess doxorubicin sensitivity in HCC38, MDA-MB-231 (TNBC), and MCF-7 (luminal). Confocal microscopy and FACS were used to quantify the mitochondrial membrane potential (ΔφM), mitophagy, and reactive oxygen species (ROS) production. Agilent Seahorse XF Analyzer was utilized to measure metabolic characteristics. Dynamin-related protein-1 (DRP1), Parkin, and p62 immunohistochemistry staining were performed using samples from 107 primary patients with BC before and after neoadjuvant chemotherapy (NAC). RESULTS: MDA-MB-231, a TNBC cell line with reduced sensitivity to doxorubicin, reduced ΔφM, and enhanced mitophagy to maintain ROS production through oxidative phosphorylation (OXPHOS)-based metabolism. HCC38, a doxorubicin-sensitive cell line, exhibited no alterations in ΔφM or mitophagy. However, it demonstrated an increase in ROS production and glycolysis. Clinicopathological studies revealed that pretreatment (before NAC) expression of DRP1 was significant in TNBC, as was pretreatment expression of Parkin in the hormone receptor-negative group. Furthermore, low p62 levels seem to be a risk factor for recurrence-free survival. CONCLUSION: Our findings indicated that the interplay between mitophagy, linked to a worse clinical prognosis, and OXPHOS metabolism promoted chemotherapy resistance in TNBC. Mitochondrial fission is prevalent in TNBC. These findings suggest that targeting the unique mitochondrial metabolism and dynamics in TNBC may offer a novel therapeutic strategy for patients with TNBC.

Histopathological analysis of tumor microenvironment in adrenocortical carcinoma: Possible effects of in situ disorganized glucocorticoid production on tumor immunity.

Adrenocortical carcinoma (ACC) patients with glucocorticoid excess have been reported to be associated with decreased tumor-infiltrating immune cells, but the effects of in situ glucocorticoid production on tumor immunity have remained unknown. In addition, ACC was also known to harbor marked intra-tumoral heterogeneity of steroidogenesis or disorganized steroidogenesis. Therefore, in this study, we immune-profiled tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) and pivotal steroidogenic enzymes of glucocorticoid biosynthesis (CYP17A and CYP11B1) to explore the potential effects of in situ glucocorticoid production and intra-tumoral heterogeneity/disorganized steroidogenesis on tumor immunity of ACC. We also studied the correlations of the status of tumor immunity with that of angiogenesis and tumor grade to further explore the tumor tissue microenvironment of ACC. TILs (CD3, CD4, CD8, and FOXP3), TAMs (CD68 and CD163), key steroidogenic enzymes of glucocorticoid (CYP17A and CYP11B1), angiogenesis (CD31 and vasohibin-1 (VASH-1)), tumor grade (Ki-67 and Weiss score) were immunohistochemically evaluated in 34 ACCs. Increased CYP17A immunoreactivity in the whole tumor area was significantly positively correlated with FOXP3-positive TILs (p = 0.021) and negatively with CD4/CD3 ratio (p = 0.001). Increased CYP11B1 immunoreactivity in the whole tumor area was significantly positively correlated with CD8/CD3 (p = 0.039) and CD163/CD68 ratios (p = 0.006) and negatively with CD4-positive TILs (p = 0.036) and CD4/CD3 ratio (p = 0.001). There were also significant positive correlations between CYP17A and CD8 (r = 0.334, p < 0.001) and FOXP3-positive TILs (r = 0.414, p < 0.001), CD8/CD3 ratio (r = 0.421, p < 0.001), and CD68-positive TAMs (r = 0.298, p < 0.001) in randomly selected areas. Significant positive correlations were also detected between CYP11B1 and CD8/CD3 ratio (r = 0.276, p = 0.001) and negative ones detected between CYP11B1 and CD3- (r = -0.259, p = 0.002) and CD4-positive TILs (r = -0.312, p < 0.001) in those areas above. Increased micro-vessel density (MVD) -VASH-1 was significantly positively correlated with CD68- (p = 0.015) and CD163-positive TAMs (p = 0.009) and CD163/CD68 ratio and the high VASH-1 with CD163-positive TAMs (p = 0.042). Ki-67 labeling index was significantly positively correlated with MAD-VASH-1 (p = 0.006) and VASH-1 (p = 0.006) status. Results of our present study indicated that in situ glucocorticoid production did influence the status of tumor immunity in ACC. In particular, increased levels of CYP17A and CYP11B1, both involved in glucocorticoid producing immunoreactivity played different effects on tumor immunity, i.e., reflecting the involvement of intra-tumoral heterogeneity and disorganized steroidogenesis of ACC, which also did indicate the importance of in situ approaches when analyzing tumor immunity of ACC.

Identifying primary aldosteronism patients who require adrenal venous sampling: a multi-center study.

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.

Challenges in the diagnosis of the enigmatic primary adrenal leiomyosarcoma: two case reports and review of the literature.

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.

Intratumoral cortisol associated with aromatase in the endometrial cancer microenvironment.

Glucocorticoids bind to glucocorticoid receptors (GR). In the peripheral tissues, active cortisol is produced from inactive cortisone by 11ß-hydroxysteroid dehydrogenase (HSD)1. 11ß-HSD2 is responsible for this reverse catalysis. Although GR and 11ß-HSDs have been reported to be involved in the malignant behavior of various cancer types, the concentration of glucocorticoids in cancer tissues has not been investigated. In this study, we measured glucocorticoids in serum and cancer tissues using liquid chromatography-tandem mass spectrometry and clarified, for the first time, the intratumoral "intracrine" production of cortisol by 11ß-HSD1/2 in endometrial cancer. Intratumoral cortisol levels were high in the high-malignancy type and the cancer proliferation marker Ki-67-high group, suggesting that cortisol greatly contributes to the malignant behavior of endometrial cancer. A low expression level of the metabolizing enzyme 11ß-HSD2 is more important than a high expression level of the synthase 11ß-HSD1 for intratumoral cortisol action. Intratumoral cortisol was positively related to the expression/activity of estrogen synthase aromatase, which involved GR expressed in fibroblastic stromal cells but not in cancer cells. Blockade of GR signaling by hormone therapy is expected to benefit patients with endometrial cancer.

Accelerated telomere shortening in adrenal zona reticularis in patients with prolonged critical illness.

Background: The 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. Methods: Adrenocortical 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. Results: RTLs 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). Conclusions: This 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.

Somatic SLC30A1 mutations altering zinc transporter ZnT1 cause aldosterone-producing adenomas and primary aldosteronism.

Primary aldosteronism (PA) is the most common form of endocrine hypertension and is characterized by inappropriately elevated aldosterone production via a renin-independent mechanism. Driver somatic mutations for aldosterone excess have been found in approximately 90% of aldosterone-producing adenomas (APAs). Other causes of lateralized adrenal PA include aldosterone-producing nodules (APNs). Using next-generation sequencing, we identified recurrent in-frame deletions in SLC30A1 in four APAs and one APN (p.L51_A57del, n = 3; p.L49_L55del, n = 2). SLC30A1 encodes the ubiquitous zinc efflux transporter ZnT1 (zinc transporter 1). The identified SLC30A1 variants are situated close to the zinc-binding site (His43 and Asp47) in transmembrane domain II and probably cause abnormal ion transport. Cases of PA with SLC30A1 mutations showed male dominance and demonstrated increased aldosterone and 18-oxocortisol concentrations. Functional studies of the SLC30A151_57del variant in a doxycycline-inducible adrenal cell system revealed pathological Na+ influx. An aberrant Na+ current led to depolarization of the resting membrane potential and, thus, to the opening of voltage-gated calcium (Ca2+) channels. This resulted in an increase in cytosolic Ca2+ activity, which stimulated CYP11B2 mRNA expression and aldosterone production. Collectively, these data implicate zinc transporter alterations as a dominant driver of aldosterone excess in PA.

Morphometric analysis of nuclear shape irregularity as a novel predictor of programmed death-ligand 1 expression in lung squamous cell carcinoma.

Immune checkpoint inhibitor (ICI) therapy has been established as one of the key treatment strategies for lung squamous cell carcinoma (LUSQ). The status of programmed death-ligand 1 (PD-L1) in tumor cells and/or immune cells using immunohistochemistry has been primarily used as a surrogate marker for determining ICI treatment; however, when the tissues to be examined are small, false-negative results could be unavoidable due to the heterogeneity of PD-L1 immunoreactivity. To overcome this practical limitation, we attempted to explore the status of nuclear atypia evaluated using morphometry as a potential predictor of PD-L1 status in LUSQ. We correlated the parameters related to nuclear atypia with PD-L1 status using two different cohorts of LUSQ patients (95 cases from The Cancer Genome Atlas database and 30 cases from the Miyagi Cancer Center). Furthermore, we studied the gene mutation status to elucidate the genetic profile of PD-L1 predictable cases. The results revealed that nuclear atypia, especially morphometric parameters related to nuclear shape irregularity, including aspect ratio, circularity, roundness, and solidity, were all significantly associated with PD-L1 status. Additionally, LUSQ cases with high PD-L1 expression and pronounced nuclear atypia were significantly associated with C10orf71 and COL14A1 mutations compared with those with low PD-L1 expression and mild nuclear atypia. We demonstrated for the first time that nuclear shape irregularity could represent a novel predictor of PD-L1 expression in LUSQ. Including the morphometric parameters related to nuclear atypia in conjunction with PD-L1 status could help determine an effective ICI therapeutic strategy; however, further investigation is required.

Coexistence of Pheochromocytoma and Primary Aldosteronism due to Multiple Aldosterone-producing Micronodules in the Ipsilateral Adrenal Gland.

A 46-year-old woman was referred for hypertension and a right adrenal tumor. Primary aldosteronism (PA) was suspected because of the high plasma aldosterone concentration-to-plasma renin activity ratio. However, a subsequent evaluation revealed coexistent PA and pheochromocytoma. We performed laparoscopic right adrenalectomy. Histology of the resected adrenal gland confirmed pheochromocytoma and multiple aldosterone-producing adrenocortical micronodules. Following adrenalectomy, the urinary catecholamine levels normalized, and hyperaldosteronism improved but persisted. Hypertension also improved but persisted and was normalized with spironolactone. The clinical course indicated that the PA lesions were likely bilateral. This was a histologically proven case of coexistent pheochromocytoma and PA due to multiple aldosterone-producing micronodules.

Node-by-node diagnosis for multiple ipsilateral nodules by segmental adrenal venous sampling in primary aldosteronism.

OBJECTIVES: In patients with primary aldosteronism (PA), multiple adrenocortical nodules may be present on the surgical side. The aim of this study was to clarify the pathological diagnosis and the node-by-node diagnostic capability of segmental adrenal venous sampling (sAVS). DESIGN: Retrospective study. PATIENTS: A total of 162 patients who underwent adrenalectomy following sAVS were studied. MEASUREMENTS: Multiple nodules on the surgical side were extracted while referring to contrast-enhanced computed tomography images. We also performed a detailed histopathological analysis of the resected specimens from patients undergoing sAVS, which included immunohistochemistry for CYP11B2. RESULTS: In 11 (6.8%) patients, two to three nodules were detected on the surgical side. All patients were diagnosed by sAVS with at least one aldosterone-producing adenoma (APA) for localized aldosterone elevation in tributaries. Seven patients showed a lateralization index value of ≥4 after ACTH stimulation. Histopathologically and clinically, two patients had two or three CYP11B2-positive APAs, and the other nine patients both APAs and non-APAs. The positive predictive value of the most suspected APA, that is, the drainer that showed the highest aldosterone level by sAVS, was 11/11 (100%, 95% confidence interval [CI]: 71.5%-100%), while that for the second and third suspected APA was 3/7 (42.9%, 95% CI: 9.9%-81.6%), and they were significantly different (p = .01). Further, the positive predictive value of non-APA was 4/4 (100%, 95% CI: 39.8%-100%). CONCLUSIONS: The sAVS could correctly diagnose the aldosterone production in multiple ipsilateral adrenal nodules.

YM750, an ACAT Inhibitor, Acts on Adrenocortical Cells to Inhibit Aldosterone Secretion Due to Depolarization.

Primary aldosteronism (PA) is considered the most common form of secondary hypertension, which is associated with excessive aldosterone secretion in the adrenal cortex. The cause of excessive aldosterone secretion is the induction of aldosterone synthase gene (CYP11B2) expression by depolarization of adrenocortical cells. In this study, we found that YM750, an Acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor, acts on adrenocortical cells to suppress CYP11B2 gene expression and aldosterone secretion. YM750 inhibited the induction of CYP11B2 gene expression by KCl stimulation, but not by angiotensin II and forskolin stimulation. Interestingly, YM750 did not inhibit KCl-stimulated depolarization via an increase in intracellular calcium ion concentration. Moreover, ACAT1 expression was relatively abundant in the zona glomerulosa (ZG) including these CYP11B2-positive cells. Thus, YM750 suppresses CYP11B2 gene expression by suppressing intracellular signaling activated by depolarization. In addition, ACAT1 was suggested to play an important role in steroidogenesis in the ZG. YM750 suppresses CYP11B2 gene expression and aldosterone secretion in the adrenal cortex, suggesting that it may be a potential therapeutic agent for PA.