Guidelines for Handling of Cytological Specimens in Cancer Genomic Medicine.

Rapid advances are being made in cancer drug therapy. Since molecularly targeted therapy has been introduced, personalized medicine is being practiced, pathological tissue from malignant tumors obtained during routine practice is frequently used for genomic testing. Whereas cytological specimens fixed mainly in alcohol are considered to be more advantageous in terms of preservation of the nucleic acid quality and quantity. This article is aimed to share the information for the proper handling of cytological specimens in practice for genomic medicine based on the findings established in "Guidelines for Handling of Cytological Specimens in Cancer Genomic Medicine (in Japanese)" published by the Japanese Society of Clinical Cytology in 2021. The three-part practical guidelines are based on empirical data analyses; Part 1 describes general remarks on the use of cytological specimens in cancer genomic medicine, then Part 2 describes proper handling of cytological specimens, and Part 3 describes the empirical data related to handling of cytological specimens. The guidelines indicated proper handling of specimens in each fixation, preparation, and evaluation.

Metabolic Oscillations and Glycolytic Phenotypes of Cancer Cells.

Cancer cells show several metabolic phenotypes depending on the cancer types and the microenvironments in tumor tissues. The glycolytic phenotype is one of the hallmarks of cancer cells and is considered to be one of the crucial features of malignant cancers. Here, we show glycolytic oscillations in the concentrations of metabolites in the glycolytic pathway in two types of cancer cells, HeLa cervical cancer cells and DU145 prostate cancer cells, and in two types of cellular morphologies, spheroids and monolayers. Autofluorescence from nicotinamide adenine dinucleotide (NADH) in cells was used for monitoring the glycolytic oscillations at the single-cell level. The frequencies of NADH oscillations were different among the cellular types and morphologies, indicating that more glycolytic cancer cells tended to exhibit oscillations with higher frequencies than less glycolytic cells. A mathematical model for glycolytic oscillations in cancer cells reproduced the experimental results quantitatively, confirming that the higher frequencies of oscillations were due to the higher activities of glycolytic enzymes. Thus, glycolytic oscillations are expected as a medical indicator to evaluate the malignancy of cancer cells with glycolytic phenotypes.

Insulinoma-associated protein 1 expression in pancreatic neuroendocrine tumours in endoscopic ultrasound-guided fine-needle aspiration cytology: An analysis of 14 patients.

BACKGROUND: Insulinoma-associated protein 1 (INSM1) has been reported to be a useful marker for diagnosing pancreatic neuroendocrine tumours (PNETs). However, INSM1 expression in endoscopic ultrasound-guided fine needle aspiration cytology has not been examined. We evaluated INSM1 expression in the cytology of cases diagnosed with PNETs. METHODS: We immunocytochemically stained INSM1 and Ki-67 in 14 PNET cases, and according to the 2017 World Health Organisation criteria, seven PNET Grade 1 cases, four Grade 2 cases and three Grade 3/neuroendocrine carcinoma cases were identified. As a control for INSM1 and Ki-67 expression, we used cytological specimens from 15 cases of pancreatic ductal adenocarcinoma. RESULTS: In PNET cases, INSM1-expressing tumour cells were identified in all cytological specimens of PNET. The median INSM1 expression rate in Grade 1 cases was 49.8% (mean ± standard deviation: 55.1 ± 12.5%, min: 39.3%, max: 74.1%), and in Grade 2 and Grade 3/neuroendocrine carcinoma cases was 81.1% (mean ± standard deviation: 77.6 ± 18.6%, min: 50.3%, max: 100%). However, there was no correlation between INSM1 and Ki-67 expression (r = -0.15). The median expression rate in PNET cases was 64.3%, which was significantly higher than that in pancreatic ductal adenocarcinoma cases (P < 0.0001). CONCLUSION: INSM1 immunocytochemistry of cytological specimens obtained from endoscopic ultrasound-guided fine needle aspiration cytology can accurately diagnose PNETs; therefore, INSM1 could be an important diagnostic tool in assessing therapeutic strategies, including molecular-targeted therapy.

Modeling studies of heterogeneities in glycolytic oscillations in HeLa cervical cancer cells.

Previous experiments demonstrated that a population of HeLa cells starved of glucose or both glucose and serum exhibited a strong heterogeneity in the glycolytic oscillations in terms of the number of oscillatory cells, periods of oscillations, and duration of oscillations. Here, we report numerical simulations of this heterogeneous oscillatory behavior in HeLa cells by using a newly developed mathematical model. It is simple enough that we can apply a mathematical analysis, but capture the core of the glycolytic pathway and the activity of the glucose transporter (GLUT). Lognormal distributions of the values of the four rate constants in the model were obtained from the experimental distributions in the periods of oscillations. Thus, the heterogeneity in the periods of oscillations can be attributed to the difference in the rate constants of the enzymatic reactions. The activity of GLUT is found to determine whether the HeLa cells were oscillatory or non-oscillatory under the same experimental conditions. Simulation with the log-normal distribution of the maximum uptake velocity of glucose and the four randomized rate constants based on the log-normal distributions successfully reproduced the time-dependent number of oscillatory cells (oscillatory ratios) under the two starving conditions. The difference in the initial values of the metabolites has little effect on the simulated results.

Effect of tranexamic acid in improving the lifespan of naturally aging mice.

An effective method to improve lifespan is not known. Therefore, in this study, we examined the lifespan-extending effect of tranexamic acid in normal mice. We bred hairless mice without exposure to ultraviolet radiation and psychical stress until they died naturally. During the study period, the mice were orally administered tranexamic acid (12 mg/kg/day) three times weekly. An increase in the lifespan of mice was observed by tranexamic acid administration. Furthermore, age-related diseases of the skin were ameliorated by tranexamic acid administration. Moreover, the blood level of tumor necrosis factor-α, interleukin-6, reactive oxygen species (ROS), and matrix metalloproteinase (MMP)-9 was decreased by tranexamic acid administration. These results indicate that tranexamic acid suppresses the secretion of inflammatory cytokines, MMP-9, and ROS induced by natural aging, ameliorating age-related diseases, and, consequently, extending the lifespan.

The expression of programed death ligand-1 could be related with unfavorable prognosis in salivary duct carcinoma.

BACKGROUND: Salivary duct carcinoma (SDC) is a rare tumor occurring in the salivary gland. SDC is a highly aggressive tumor and its prognosis is extremely poor. Effective treatments in advanced SDC have not yet been established. Recently, immune checkpoint inhibitors have paved the way for the treatment of various malignancies. We examined the expressions of programed death ligand (PD-L) 1/PD-L2 and programed death (PD-1), and the correlation of clinicopathological findings. METHODS: We examined 18 cases of SDC and conducted immunohistochemical staining using formalin-fixed paraffin-embedded full-face sections. RESULTS: The expression of PD-L1 and PD-L2 in tumor cells was observed in nine cases (50%) and 14 cases (78%), respectively. Cases with a high expression of PD-L1 and PD-L2 were found in four (22%) and seven cases (39%), respectively. The cases with a high expression of PD-L1 showed significantly shorter overall survival compared to those with low PD-L1 expression and null expression. We also examined the expression of PD-L1/PD-L2 and PD-1 of tumor-infiltrating mononuclear cells (TIMC) in stroma. The expressions of PD-L1 in tumor cells and stroma had a significant correlation. Association between the expressions of PD-L1 in tumor cells and those of PD-1 in stroma was significant. However, PD-L2 expression in the tumor had no significant correlation with expression in TIMCs. PD-L1, PD-L2 and PD-1 expressions in stroma were not associated with patient prognosis. CONCLUSIONS: High PD-L1 expression in SDC was strongly associated with unfavorable prognosis, indicating that PD-1/PD-L1 inhibitors could be effective in SDC.

Primordial oscillations in life: Direct observation of glycolytic oscillations in individual HeLa cervical cancer cells.

We report the first direct observation of glycolytic oscillations in HeLa cervical cancer cells, which we regard as primordial oscillations preserved in living cells. HeLa cells starved of glucose or both glucose and serum exhibited glycolytic oscillations in nicotinamide adenine dinucleotide (NADH), exhibiting asynchronous intercellular behaviors. Also found were spatially homogeneous and inhomogeneous intracellular NADH oscillations in the individual cells. Our results demonstrate that starved HeLa cells may be induced to exhibit glycolytic oscillations by either high-uptake of glucose or the enhancement of a glycolytic pathway (Crabtree effect or the Warburg effect), or both. Their asynchronous collective behaviors in the oscillations were probably due to a weak intercellular coupling. Elucidation of the relationship between the mechanism of glycolytic dynamics in cancer cells and their pathophysiological characteristics remains a challenge in future.

Structures of complexes of type 5 17ß-hydroxysteroid dehydrogenase with structurally diverse inhibitors: insights into the conformational changes upon inhibitor binding.

Type 5 17ß-hydroxysteroid dehydrogenase (17ß-HSD5) is an aldo-keto reductase expressed in the human prostate which catalyzes the conversion of androstenedione to testosterone. Testosterone is converted to 5α-dihydrotestosterone, which is present at high concentrations in patients with castration-resistant prostate cancer (CRPC). Inhibition of 17ß-HSD5 is therefore considered to be a promising therapy for treating CRPC. In the present study, crystal structures of complexes of 17ß-HSD5 with structurally diverse inhibitors derived from high-throughput screening were determined. In the structures of the complexes, various functional groups, including amide, nitro, pyrazole and hydroxyl groups, form hydrogen bonds to the catalytic residues His117 and Tyr55. In addition, major conformational changes of 17ß-HSD5 were observed following the binding of the structurally diverse inhibitors. These results demonstrate interactions between 17ß-HSD5 and inhibitors at the atomic level and enable structure-based drug design for anti-CRPC therapy.

Identification of N-ethylmethylamine as a novel scaffold for inhibitors of soluble epoxide hydrolase by crystallographic fragment screening.

Soluble epoxide hydrolase (sEH) is a potential target for the treatment of inflammation and hypertension. X-ray crystallographic fragment screening was used to identify fragment hits and their binding modes. Eight fragment hits were identified via soaking of sEH crystals with fragment cocktails, and the co-crystal structures of these hits were determined via individual soaking. Based on the binding mode, N-ethylmethylamine was identified as a promising scaffold that forms hydrogen bonds with the catalytic residues of sEH, Asp335, Tyr383, and Tyr466. Compounds containing this scaffold were selected from an in-house chemical library and assayed. Although the starting fragment had a weak inhibitory activity (IC50: 800µM), we identified potent inhibitors including 2-({[2-(adamantan-1-yl)ethyl]amino}methyl)phenol exhibiting the highest inhibitory activity (IC50: 0.51µM). This corresponded to a more than 1500-fold increase in inhibitory activity compared to the starting fragment. Co-crystal structures of the hit compounds demonstrate that the binding of N-ethylmethylamine to catalytic residues is similar to that of the starting fragment. We therefore consider crystallographic fragment screening to be appropriate for the identification of weak but promising fragment hits.

Collective and individual glycolytic oscillations in yeast cells encapsulated in alginate microparticles.

Yeast cells were encapsulated into alginate microparticles of a few hundred micrometers diameter using a centrifuge-based droplet shooting device. We demonstrate the first experimental results of glycolytic oscillations in individual yeast cells immobilized in this way. We investigated both the individual and collective oscillatory behaviors at different cell densities. As the cell density increased, the amplitude of the individual oscillations increased while their period decreased, and the collective oscillations became more synchronized, with an order parameter close to 1 (indicating high synchrony). We also synthesized biphasic-Janus microparticles encapsulating yeast cells of different densities in each hemisphere. The cellular oscillations between the two hemispheres were entrained at both the individual and population levels. Such systems of cells encapsulated into microparticles are useful for investigating how cell-to-cell communication depends on the density and spatial distribution of cells.

Distinct properties of telmisartan on agonistic activities for peroxisome proliferator-activated receptor γ among clinically used angiotensin II receptor blockers: drug-target interaction analyses.

A proportion of angiotensin II type 1 receptor blockers (ARBs) improves glucose dyshomeostasis and insulin resistance in a clinical setting. Of these ARBs, telmisartan has the unique property of being a partial agonist for peroxisome proliferator-activated receptor γ (PPARγ). However, the detailed mechanism of how telmisartan acts on PPARγ and exerts its insulin-sensitizing effect is poorly understood. In this context, we investigated the agonistic activity of a variety of clinically available ARBs on PPARγ using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) system. Based on physicochemical data, we then reevaluated the metabolically beneficial effects of telmisartan in cultured murine adipocytes. ITC and SPR assays demonstrated that telmisartan exhibited the highest affinity of the ARBs tested. Distribution coefficient and parallel artificial membrane permeability assays were used to assess lipophilicity and cell permeability, for which telmisartan exhibited the highest levels of both. We next examined the effect of each ARB on insulin-mediated glucose metabolism in 3T3-L1 preadipocytes. To investigate the impact on adipogenesis, 3T3-L1 preadipocytes were differentiated with each ARB in addition to standard inducers of differentiation for adipogenesis. Telmisartan dose-dependently facilitated adipogenesis and markedly augmented the mRNA expression of adipocyte fatty acid-binding protein (aP2), accompanied by an increase in the uptake of 2-deoxyglucose and protein expression of glucose transporter 4 (GLUT4). In contrast, other ARBs showed only marginal effects in these experiments. In accordance with its highest affinity of binding for PPARγ as well as the highest cell permeability, telmisartan superbly activates PPARγ among the ARBs tested, thereby providing a fresh avenue for treating hypertensive patients with metabolic derangement.

Nuclear Y-box-binding protein-1 is a poor prognostic marker and related to epidermal growth factor receptor in uterine cervical cancer.

OBJECTIVE: Y-box binding protein-1 (YB-1) is a member of the cold shock protein family and functions in transcription and translation. Many studies indicate that YB-1 is strongly expressed in tumor cells and is considered a marker of tumor aggressiveness and clinical prognosis. Overexpression of epidermal growth factor receptor (EGFR) has been associated with poor outcomes in cervical cancer. Clinical trials of EGFR family-base therapy are currently being initiated in cervical cancer. Nuclear YB-1 expression correlates with EGFR expression in various types of cancer. However, the clinical significance of nuclear YB-1 expression in different settings, the correlation with EGFR, and the prognostic implications of YB-1 expression in cervical cancer remain elusive. PATIENTS AND METHODS: Nuclear YB-1 expression was immunohistochemically analyzed in tissue specimens obtained from 204 patients with cervical cancer who underwent surgery. Associations of nuclear YB-1 expression with clinicopathological factors such as survival, EGFR expression, and human epidermal growth factor receptor 2 (HER2) expression were investigated. RESULTS: Nuclear YB-1 expression was found in 41 (20.2%) of 204 cases of cervical cancer and correlated with disease stage, tumor diameter, stromal invasion, and lymph-node metastasis. Nuclear YB-1 expression also correlated with EGFR expression (P=0.0114) as well as HER2 expression (P=0.0053). Kaplan-Meier survival analysis showed that nuclear YB-1 expression was significantly associated with poor progression-free survival (P=0.0033) and overall survival (P=0.0003), respectively. CONCLUSION: Nuclear YB-1 expression is a prognostic marker and correlates with EGFR expression in cervical cancer.

Structural insights into binding of inhibitors to soluble epoxide hydrolase gained by fragment screening and X-ray crystallography.

Soluble epoxide hydrolase (sEH) is a component of the arachidonic acid cascade and is a candidate target for therapies for hypertension or inflammation. Although many sEH inhibitors are available, their scaffolds are not structurally diverse, and knowledge of their specific interactions with sEH is limited. To obtain detailed structural information about protein-ligand interactions, we conducted fragment screening of sEH, analyzed the fragments using high-throughput X-ray crystallography, and determined 126 fragment-bound structures at high resolution. Aminothiazole and benzimidazole derivatives were identified as novel scaffolds that bind to the catalytic triad of sEH with good ligand efficiency. We further identified fragment hits that bound to subpockets of sEH called the short and long branches. The water molecule conserved in the structure plays an important role in binding to the long branch, whereas Asp496 and the main chain of Phe497 form hydrogen bonds with fragment hits in the short branch. Fragment hits and their crystal structures provide structural insights into ligand binding to sEH that will facilitate the discovery of novel and potent inhibitors of sEH.

Feasibility study of personalized peptide vaccination for recurrent ovarian cancer patients.

CONTEXT: To develop a personalized peptide vaccine (PPV) for recurrent ovarian cancer patients and evaluate its efficacy from the point of view of overall survival (OS), Phase II study of PPV was performed. PATIENTS AND METHODS: Forty-two patients, 17 with platinum-sensitive and 25 with platinum-resistant recurrent ovarian cancer, were enrolled in this study and received a maximum of four peptides based on HLA-A types and IgG responses to the peptides in pre-vaccination plasma. RESULTS: Expression of 13 of the 15 parental tumor-associated antigens encoding the vaccine peptides, with the two prostate-related antigens being the exceptions, was confirmed in the ovarian cancer tissues. No vaccine-related systemic severe adverse events were observed in any patients. Boosting of cytotoxic T lymphocytes or IgG responses specific for the peptides used for vaccination was observed in 18 or 13 of 42 cases at 6th vaccination, and 19 or 29 of 30 cases at 12th vaccination, respectively. The median survival time (MST) values of the platinum-sensitive- and platinum-resistant recurrent cases were 39.3 and 16.2 months, respectively. The MST of PPV monotherapy or PPV in combination with any chemotherapy during the 1st to 12th vaccination of platinum-sensitive cases was 39.3 or 32.2 months, and that of platinum-resistant cases was 16.8 or 16.1 months, respectively. Importantly, lymphocyte frequency and epitope spreading were significantly prognostic of OS. DISCUSSION AND CONCLUSION: Because of the safety and possible prolongation of OS, a clinical trial of PPV without chemotherapy during the 1st to 12th vaccination in recurrent ovarian cancer patients is merited.

N-Myc Downstream Regulated Gene-1 May Play an Important Role in the Prognosis of Ovarian Cancer, in Its Association with Beta-Catenin.

NDRG1 is a nickel- and calcium-inducible gene that plays important roles in the primary growth of malignant tumors, as well as in invasion and metastasis. This study investigated the associations of NDRG1 expression with cell adhesion and other clinicopathological factors in ovarian cancer. The clinical records of 123 women who underwent surgery for ovarian cancer in our institute were reviewed retrospectively. The expression of NDRG1, E-cadherin, and beta-catenin in surgical specimens were evaluated immunohistochemically. The NDRG1 expression level was significantly associated with beta-catenin expression, peritoneal metastasis outside the pelvic cavity, lymph node metastasis, and FIGO stages. The Kaplan-Meier analysis showed a significant association between the NDRG1 expression level and progression-free survival: high NDRG1 expression was related to poor survival. Our results suggest that the increased expression of NDRG1 is associated with cell adhesion and may be a poor prognostic indicator in women with ovarian cancer.

Simulation of metal-ligand self-assembly into spherical complex M6L8.

Molecular dynamics simulations were performed to study the self-assembly of a spherical complex through metal-ligand coordination interactions. M(6)L(8), a nanosphere with six palladium ions and eight pyridine-capped tridentate ligands, was selected as a target system. We successfully observed the spontaneous formation of spherical shaped M(6)L(8) cages over the course of our simulations, starting from random initial placement of the metals and ligands. To simulate spontaneous coordination bond formations and breaks, the cationic dummy atom method was employed to model nonbonded metal-ligand interactions. A coarse-grained solvent model was used to fill the gap between the time scale of the supramolecular self-assembly and that accessible by common molecular dynamics simulation. The simulated formation process occurred in the distinct three-stage (assembly, evolution, fixation) process that is well correlated with the experimental results. We found that the difference of the lifetime (or the ligand exchange rate) between the smaller-sized incomplete clusters and the completed M(6)L(8) nanospheres is crucially important in their supramolecular self-assembly.

Oscillations and Dynamic Symbiosis in Cellular Metabolism in Cancer.

The grade of malignancy differs among cancer cell types, yet it remains the burden of genetic studies to understand the reasons behind this observation. Metabolic studies of cancer, based on the Warburg effect or aerobic glycolysis, have also not provided any clarity. Instead, the significance of oxidative phosphorylation (OXPHOS) has been found to play critical roles in aggressive cancer cells. In this perspective, metabolic symbiosis is addressed as one of the ultimate causes of the grade of cancer malignancy. Metabolic symbiosis gives rise to metabolic heterogeneities which enable cancer cells to acquire greater opportunities for proliferation and metastasis in tumor microenvironments. This study introduces a real-time new imaging technique to visualize metabolic symbiosis between cancer-associated fibroblasts (CAFs) and cancer cells based on the metabolic oscillations in these cells. The causality of cellular oscillations in cancer cells and CAFs, connected through lactate transport, is a key point for the development of this novel technique.

Glycolytic oscillations in HeLa cervical cancer cell spheroids.

Previous studies have unravelled glycolytic oscillations in cancer cells, such as HeLa cervical and DU145 prostate cancer cells, using a monolayer culture system. Here, we demonstrate glycolytic oscillations in HeLa cervical cancer cell spheroids. Experiments revealed that a small number of HeLa cells in spheroids exhibited heterogeneous oscillations with a higher frequency than those in monolayers. Model analyses and our previous experiments indicated that the higher frequencies of oscillations in spheroids were mostly due to the increase in glycolytic enzyme activity in the cells, and to the decrease in glucose concentration by diffusional transport of glucose from the surface to inside the spheroids, as well as the increase in cell density through spheroid formation. These results and our previous studies imply that more malignant cancer cells tend to exhibit glycolytic oscillations with higher frequencies than less malignant cells. Adjacent cells in spheroids oscillated within a 10% difference in frequency, but did not synchronize with each other. This suggests that weak cell-to-cell interactions might exist among HeLa cells connected with cadherins in the spheroid microenvironment; however, the interactions were not strong enough to induce synchronization of glycolytic oscillations.

Nuclear ß-catenin expression in basal cell adenomas of salivary gland.

BACKGROUND: Nuclear localization of ß-catenin is known in a wide variety of human neoplasms; however, there are few reports in basal cell adenoma of the salivary gland. Our objective was to confirm the nuclear localization of ß-catenin in basal cell adenoma and to examine whether nuclear ß-catenin expression could be a useful marker in the diagnosis of basal cell adenoma. METHODS: To evaluate the nuclear localization of ß-catenin in basal cell adenomas, immunohistochemistry (IHC) and mutation analysis of CTNNB1 were performed in 22 and 21 cases, respectively. Mutation analysis of CTNNB1 in exon 3 was performed by DNA direct sequencing. In a comparative study, IHC for ß-catenin was also performed in 157 other salivary gland tumors. RESULTS: Nuclear ß-catenin expression was examined in 22 basal cell adenomas; scores were 2+ in 18 cases (81.8%), 1+ in three cases (13.6%), and 0 in one case (4.5%). Expression was localized in the basaloid myoepithelial cells. CTNNB1 mutation analysis was performed in 21 basal cell adenomas; mutations, including I35T and T41P, were detected in 11/21 (52%) cases. In comparison with other salivary gland tumors, one of three basal cell adenocarcinomas showed nuclear ß-catenin expression, whereas there was no nuclear ß-catenin expression in 154 other salivary gland tumors. CONCLUSIONS: We demonstrated nuclear ß-catenin expression and activation of the CTNNB1 gene in basal cell adenoma. Although nuclear ß-catenin expression may be unable to distinguish basal cell adenoma from basal cell adenocarcinoma, it should be a helpful marker in the diagnosis of basal cell adenoma.