Tissue-specific gelatin bioink as a rheology modifier for high printability and adjustable tissue properties.

Decellularized extracellular matrix (dECM) has emerged as an exceptional biomaterial that effectively recapitulates the native tissue microenvironment for enhanced regenerative potential. Although various dECM bioinks derived from different tissues have shown promising results, challenges persist in achieving high-resolution printing of flexible tissue constructs because of the inherent limitations of dECM's weak mechanical properties and poor printability. Attempts to enhance mechanical rigidity through chemical modifications, photoinitiators, and nanomaterial reinforcement have often compromised the bioactivity of dECM and mismatched the desired mechanical properties of target tissues. In response, this study proposes a novel method involving a tissue-specific rheological modifier, gelatinized dECM. This modifier autonomously enhances bioink modulus pre-printing, ensuring immediate and precise shape formation upon extrusion. The hybrid bioink with GeldECM undergoes a triple crosslinking system-physical entanglement for pre-printing, visible light photocrosslinking during printing for increased efficiency, and thermal crosslinking post-printing during tissue culture. A meticulous gelatinization process preserves the dECM protein components, and optimal hybrid ratios modify the mechanical properties, tailoring them to specific tissues. The application of this sequential multiple crosslinking designs successfully yielded soft yet resilient tissue constructs capable of withstanding vigorous agitation with high shape fidelity. This innovative method, founded on mechanical modulation by GeldECM, holds promise for the fabrication of flexible tissues with high resilience.

Laminin-Augmented Decellularized Extracellular Matrix Ameliorating Neural Differentiation and Neuroinflammation in Human Mini-Brains.

Non-neural extracellular matrix (ECM) has limited application in humanized physiological neural modeling due to insufficient brain-specificity and safety concerns. Although brain-derived ECM contains enriched neural components, certain essential components are partially lost during the decellularization process, necessitating augmentation. Here, it is demonstrated that the laminin-augmented porcine brain-decellularized ECM (P-BdECM) is xenogeneic factor-depleted as well as favorable for the regulation of human neurons, astrocytes, and microglia. P-BdECM composition is comparable to human BdECM regarding brain-specificity through the matrisome and gene ontology-biological process analysis. As augmenting strategy, laminin 111 supplement promotes neural function by synergic effect with laminin 521 in P-BdECM. Annexin A1(ANXA1) and Peroxiredoxin(PRDX) in P-BdECM stabilized microglial and astrocytic behavior under normal while promoting active neuroinflammation in response to neuropathological factors. Further, supplementation of the brain-specific molecule to non-neural matrix also ameliorated glial cell inflammation as in P-BdECM. In conclusion, P-BdECM-augmentation strategy can be used to recapitulate humanized pathophysiological cerebral environments for neurological study.

3D bioprinted vascularized lung cancer organoid models with underlying disease capable of more precise drug evaluation.

Despite encouraging progress in the development ofin vitrocancer models,in vitrocancer models that simultaneously recapitulate the complexity of the tumor microenvironment and its diverse cellular components and genetic properties remain lacking. Here, an advanced vascularized lung cancer (LC) model is proposed, which includes patient-derived LC organoids (LCOs), lung fibroblasts, and perfusable vessels using 3D bioprinting technology. To better recapitulate the biochemical composition of native lung tissues, a porcine lung-derived decellularized extracellular matrix (LudECM) hydrogel was produced to offer physical and biochemical cues to cells in the LC microenvironment. In particular, idiopathic pulmonary fibrosis-derived lung fibroblasts were used to implement fibrotic niches similar to actual human fibrosis. It was shown that they increased cell proliferation and the expression of drug resistance-related genes in LCOs with fibrosis. In addition, changes in resistance to sensitizing targeted anti-cancer drugs in LCOs with fibrosis were significantly greater in LudECM than in that Matrigel. Therefore, assessment of drug responsiveness in vascularized LC models that recapitulate lung fibrosis can help determine the appropriate therapy for LC patients accompanied by fibrosis. Furthermore, it is expected that this approach could be utilized for the development of targeted therapies or the identification of biomarkers for LC patients accompanied by fibrosis.

3D cell-printing of gradient multi-tissue interfaces for rotator cuff regeneration.

Owing to the prevalence of rotator cuff (RC) injuries and suboptimal healing outcome, rapid and functional regeneration of the tendon-bone interface (TBI) after RC repair continues to be a major clinical challenge. Given the essential role of the RC in shoulder movement, the engineering of biomimetic multi-tissue constructs presents an opportunity for complex TBI reconstruction after RC repair. Here, we propose a gradient cell-laden multi-tissue construct combined with compositional gradient TBI-specific bioinks via 3D cell-printing technology. In vitro studies demonstrated the capability of a gradient scaffold system in zone-specific inducibility and multi-tissue formation mimicking TBI. The regenerative performance of the gradient scaffold on RC regeneration was determined using a rat RC repair model. In particular, we adopted nondestructive, consecutive, and tissue-targeted near-infrared fluorescence imaging to visualize the direct anatomical change and the intricate RC regeneration progression in real time in vivo. Furthermore, the 3D cell-printed implant promotes effective restoration of shoulder locomotion function and accelerates TBI healing in vivo. In summary, this study identifies the therapeutic contribution of cell-printed constructs towards functional RC regeneration, demonstrating the translational potential of biomimetic gradient constructs for the clinical repair of multi-tissue interfaces.

Modular assembly of bioprinted perfusable blood vessel and tracheal epithelium for studying inflammatory respiratory diseases.

In vitroorgan models allow for the creation of precise preclinical models that mimic organ physiology. During a pandemic of a life-threatening acute respiratory disease, an improved trachea model (TM) is required. We fabricated a modular assembly of the blood vessel and TMs using 3D bioprinting technology. First, decellularized extracellular matrix (dECM) were prepared using the porcine trachea and blood vessels. A trachea module was fabricated based on the tracheal mucosa-derived dECM and microporous membrane. Further, a blood vessel module was manufactured using the prepared vascular-tissue-derived dECM. By assembling each manufactured module, a perfusable vascularized TM simulating the interface between the tracheal epithelium and blood vessels was fabricated. This assembled model was manufactured with efficient performance, and it offered respiratory symptoms, such as inflammatory response and allergen-induced asthma exacerbation. These characteristics indicate the possibility of manufacturing a highly functional organ model that mimics a complex organ environment in the future.

Kidney Decellularized Extracellular Matrix Enhanced the Vascularization and Maturation of Human Kidney Organoids.

Kidney organoids derived from human pluripotent stem cells (hPSCs) have extensive potential for disease modelling and regenerative medicine. However, the limited vascularization and immaturity of kidney organoids have been still remained to overcome. Extracellular matrix (ECM) can provide mechanical support and a biochemical microenvironment for cell growth and differentiation. Here in vitro methods using a kidney decellularized extracellular matrix (dECM) hydrogel to culture hPSC-derived kidney organoids, which have extensive vascular network and their own endothelial cells, are reported. Single-cell transcriptomics reveal that the vascularized kidney organoids cultured using the kidney dECM have more mature patterns of glomerular development and higher similarity to human kidney than those cultured without the kidney dECM. Differentiation of α-galactosidase A (GLA)-knock-out hPSCs generated using CRISPR/Cas9 into kidney organoids by the culture method using kidney dECM efficiently recapitulate Fabry nephropathy with vasculopathy. Transplantation of kidney organoids with kidney dECM into kidney of mouse accelerates the recruitment of endothelial cells from the host mouse kidney and maintains vascular integrity with the more organized slit diaphragm-like structures than those without kidney dECM. The kidney dECM methodology for inducing extensive vascularization and maturation of kidney organoids can be applied to studies for kidney development, disease modeling, and regenerative medicine.

A Bioprinted Tubular Intestine Model Using a Colon-Specific Extracellular Matrix Bioink.

Tremendous advances have been made toward accurate recapitulation of the human intestinal system in vitro to understand its developmental process, and disease progression. However, current in vitro models are often confined to 2D or 2.5D microarchitectures, which is difficult to mimic the systemic level of complexity of the native tissue. To overcome this problem, physiologically relevant intestinal models are developed with a 3D hollow tubular structure using 3D bioprinting strategy. A tissue-specific biomaterial, colon-derived decellularized extracellular matrix (Colon dECM) is developed and it provides significant maturation-guiding potential to human intestinal cells. To fabricate a perfusable tubular model, a simultaneous printing process of multiple materials through concentrically assembled nozzles is developed and a light-activated Colon dECM bioink is employed by supplementing with ruthenium/sodium persulfate as a photoinitiator. The bioprinted intestinal tissue models show spontaneous 3D morphogenesis of the human intestinal epithelium without any external stimuli. In consequence, the printed cells form multicellular aggregates and cysts and then differentiate into several types of enterocytes, building junctional networks. This system can serve as a platform to evaluate the effects of potential drug-induced toxicity on the human intestinal tissue and create a coculture model with commensal microbes and immune cells for future therapeutics.

A 3D bioprinted hybrid encapsulation system for delivery of human pluripotent stem cell-derived pancreatic islet-like aggregates.

Islet transplantation is a promising treatment for type 1 diabetes. However, treatment failure can result from loss of functional cells associated with cell dispersion, low viability, and severe immune response. To overcome these limitations, various islet encapsulation approaches have been introduced. Among them, macroencapsulation offers the advantages of delivering and retrieving a large volume of islets in one system. In this study, we developed a hybrid encapsulation system composed of a macroporous polymer capsule with stagger-type membrane and assemblable structure, and a nanoporous decellularized extracellular matrix (dECM) hydrogel containing pancreatic islet-like aggregates using 3D bioprinting technique. The outer part (macroporous polymer capsule) was designed to have an interconnected porous architecture, which allows insulin-producingß-cells encapsulated in the hybrid encapsulation system to maintain their cellular behaviors, including viability, cell proliferation, and insulin-producing function. The inner part (nanoporous dECM hydrogel), composed of the 3D biofabricated pancreatic islet-like aggregates, was simultaneously placed into the macroporous polymer capsule in one step. The developed hybrid encapsulation system exhibited biocompatibilityin vitroandin vivoin terms of M1 macrophage polarization. Furthermore, by controlling the printing parameters, we generated islet-like aggregates, improving cell viability and functionality. Moreover, the 3D bioprinted pancreatic islet-like aggregates exhibited structural maturation and functional enhancement associated with intercellular interaction occurring at theß-cell edges. In addition, we also investigated the therapeutic potential of a hybrid encapsulation system by integrating human pluripotent stem cell-derived insulin-producing cells, which are promising to overcome the donor shortage problem. In summary, these results demonstrated that the 3D bioprinting approach facilitates the fabrication of a hybrid islet encapsulation system with multiple materials and potentially improves the clinical outcomes by driving structural maturation and functional improvement of cells.

3D Bioprinting-Based Vascularized Tissue Models Mimicking Tissue-Specific Architecture and Pathophysiology for in vitro Studies.

A wide variety of experimental models including 2D cell cultures, model organisms, and 3D in vitro models have been developed to understand pathophysiological phenomena and assess the safety and efficacy of potential therapeutics. In this sense, 3D in vitro models are an intermediate between 2D cell cultures and animal models, as they adequately reproduce 3D microenvironments and human physiology while also being controllable and reproducible. Particularly, recent advances in 3D in vitro biomimicry models, which can produce complex cell structures, shapes, and arrangements, can more similarly reflect in vivo conditions than 2D cell culture. Based on this, 3D bioprinting technology, which enables to place the desired materials in the desired locations, has been introduced to fabricate tissue models with high structural similarity to the native tissues. Therefore, this review discusses the recent developments in this field and the key features of various types of 3D-bioprinted tissues, particularly those associated with blood vessels or highly vascularized organs, such as the heart, liver, and kidney. Moreover, this review also summarizes the current state of the three categories: (1) chemical substance treatment, (2) 3D bioprinting of lesions, and (3) recapitulation of tumor microenvironments (TME) of 3D bioprinting-based disease models according to their disease modeling approach. Finally, we propose the future directions of 3D bioprinting approaches for the creation of more advanced in vitro biomimetic 3D tissues, as well as the translation of 3D bioprinted tissue models to clinical applications.

Development of a Tongue Immobilization Device Using a 3D Printer for Intensity Modulated Radiation Therapy of Nasopharyngeal Cancer Patients.

PURPOSE: This study aimed to reduce radiation doses to the tongue, a patient-specific semi-customized tongue immobilization device (SCTID) was developed using a 3D printer for helical tomotherapy (HT) of nasopharyngeal cancer (NPCa). Dosimetric characteristics and setup stability of the SCTID were compared with those of a standard mouthpiece (SMP). MATERIALS AND METHODS: For displacement and robust immobilization of the tongue, the SCTID consists of four parts: upper and lower tooth stoppers, tongue guider, tongue-tip position guide bar, and connectors. With the SCTID and SMP, two sets of planning computed tomography and HT plans were obtained for 10 NPCa patients. Dosimetric and geometric characteristics were compared. Position reproducibility of the tongue with SCTID was evaluated by comparing with planned dose and adaptive accumulated dose of the tongue and base of the tongue based on daily setup mega-voltage computed tomography. RESULTS: Using the SCTID, the tongue was effectively displaced from the planning target volume compared to the SMP. The median mucosa of the tongue (M-tongue) dose was significantly reduced (20.7 Gy vs. 27.8 Gy). The volumes of the M-tongue receiving a dose of 15 Gy, 30 Gy, and 45 Gy and the volumes of the mucosa of oral cavity and oropharynx (M-OC/OP) receiving a dose of 45 Gy and 60 Gy were significantly lower than using the SMP. No significant differences was observed between the planned dose and the accumulated adaptive dose in any dosimetric characteristics of the tongue and base of tongue. CONCLUSION: SCTID can not only reduce the dose to the M-tongue and M-OC/OP dramatically, when compared to SMP, but also provide excellent reproducibility and easy visual verification.

Effect of the Duration of Meteorological Data Collection on the Prospective Assessment of Long-Term Atmospheric Dispersion Factors.

Assessing the environmental dispersion of radioactive effluents from a nuclear facility under normal operation is a prerequisite in applying for the construction permit and operation license of the facility. In Korea, the Nuclear Safety and Security Commission requires at least 1- and 2-year periods of site-specific meteorological data to assess the atmospheric dispersion of the effluents from a nuclear reactor facility for a construction permit and operating license, respectively. In this study, we investigated if an extended period of meteorological data would be certainly preferable for the prospective assessment of dispersion of the atmospheric effluents under current data handling approaches. For test estimation, the meteorological data collected in the Kori nuclear power plant site in 2001 to 2008 were utilized. We suggest that the accumulated meteorological data be reflected in assessing the atmospheric dispersion in the form of individual annual data rather than the averaged data over the whole period.

Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain.

Cucumber mosaic virus (CMV) is a destructive pathogen affecting Capsicum annuum (pepper) production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase). Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum 'Bukang' cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP). Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic) leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.

Extensive structural variations between mitochondrial genomes of CMS and normal peppers (Capsicum annuum L.) revealed by complete nucleotide sequencing.

BACKGROUND: Cytoplasmic male sterility (CMS) is an inability to produce functional pollen that is caused by mutation of the mitochondrial genome. Comparative analyses of mitochondrial genomes of lines with and without CMS in several species have revealed structural differences between genomes, including extensive rearrangements caused by recombination. However, the mitochondrial genome structure and the DNA rearrangements that may be related to CMS have not been characterized in Capsicum spp. RESULTS: We obtained the complete mitochondrial genome sequences of the pepper CMS line FS4401 (507,452 bp) and the fertile line Jeju (511,530 bp). Comparative analysis between mitochondrial genomes of peppers and tobacco that are included in Solanaceae revealed extensive DNA rearrangements and poor conservation in non-coding DNA. In comparison between pepper lines, FS4401 and Jeju mitochondrial DNAs contained the same complement of protein coding genes except for one additional copy of an atp6 gene (ψatp6-2) in FS4401. In terms of genome structure, we found eighteen syntenic blocks in the two mitochondrial genomes, which have been rearranged in each genome. By contrast, sequences between syntenic blocks, which were specific to each line, accounted for 30,380 and 17,847 bp in FS4401 and Jeju, respectively. The previously-reported CMS candidate genes, orf507 and ψatp6-2, were located on the edges of the largest sequence segments that were specific to FS4401. In this region, large number of small sequence segments which were absent or found on different locations in Jeju mitochondrial genome were combined together. The incorporation of repeats and overlapping of connected sequence segments by a few nucleotides implied that extensive rearrangements by homologous recombination might be involved in evolution of this region. Further analysis using mtDNA pairs from other plant species revealed common features of DNA regions around CMS-associated genes. CONCLUSIONS: Although large portion of sequence context was shared by mitochondrial genomes of CMS and male-fertile pepper lines, extensive genome rearrangements were detected. CMS candidate genes located on the edges of highly-rearranged CMS-specific DNA regions and near to repeat sequences. These characteristics were detected among CMS-associated genes in other species, implying a common mechanism might be involved in the evolution of CMS-associated genes.

Epithelial-mesenchymal transition increases during the progression of in situ to invasive basal-like breast cancer.

Epithelial-mesenchymal transition (EMT) is known to play an important role in breast cancer invasion and metastatic progression. However, the pattern of expression of EMT markers in the progression from in situ to invasive breast carcinoma is not clear. To investigate this, we performed immunohistochemical analyses of EMT markers (expression of vimentin, smooth muscle actin, osteonectin, and N-cadherin; loss of E-cadherin; alteration of ß-catenin), breast cancer stem cell (CSC) markers (CD44(+)/CD24(-), ALDH1), and CD146, an EMT inducer, in invasive carcinomas and ductal carcinoma in situ (DCIS) of the breast. Expression of EMT markers was closely associated with the basal-like subtype and CSC phenotype in invasive carcinoma but not in pure DCIS, except for vimentin. The expression of smooth muscle actin and N-cadherin, loss of E-cadherin, and alteration of ß-catenin were significantly higher in invasive carcinomas than in pure DCIS (P = .015, P = .029, P = .001, and P = .007, respectively). Subgroup analyses revealed greater loss of E-cadherin and alteration of ß-catenin in invasive carcinoma than in pure DCIS in basal-like subtype (P = .001) but not in non-basal-like subtypes. Moreover, expression of EMT markers and CD146 was higher in the invasive than in the DCIS component of basal-like cancers. Our study confirmed that EMT is an intrinsic characteristic of basal-like subtype and is associated with CSC phenotype. Furthermore, we showed higher expression of EMT markers in invasive carcinomas than in pure DCIS, especially in basal-like subtype, and in the invasive component of basal-like breast cancers, suggesting that EMT may be involved in the progression from in situ to invasive basal-like breast cancers.

Invasive cribriform carcinoma arising in malignant phyllodes tumor of breast: a case report.

Phyllodes tumor is an uncommon fibroepithelial neoplasm of the breast. And it is characterized by expanded stroma with increased cellularity and elongated epithelium-lined clefts. Mammary carcinomas within phyllodes tumors have been rarely reported. To date, however, no reports have described the invasive cribriform carcinoma arising in malignant phyllodes tumor. Here, we report a 62-year-old woman who presented with a large breast mass. Microscopically, the mass was a typical malignant phyllodes tumor showing well developed leaf-like architecture and stromal overgrowth with high cellularity and nuclear pleomorphism. In a portion of the tumor, however, the epithelial component showed a cribriform pattern of proliferation in the absence of myoepithelial cells, suggestive of the invasive cribriform carcinoma. To our knowledge, this is rare and it is difficult to make a differential diagnosis of it. Here, we report our case with a review of literatures.

FGFR1 is amplified during the progression of in situ to invasive breast carcinoma.

INTRODUCTION: Gene amplification is an important mechanism for activating oncogenes in malignant tumors. Although amplification of HER2, C-MYC, CCND1 and FGFR1 has been reported in breast cancers, their role in the progression of in situ to invasive breast carcinoma is unclear. To investigate this question we compared the amplification frequencies of these genes in pure ductal carcinoma in situ (DCIS), DCIS associated with invasive carcinoma, and invasive carcinoma. METHODS: We performed fluorescence in situ hybridization of the selected genes on tissue microarrays composed of 179 pure DCIS and 438 invasive carcinomas. Two hundred and sixteen of the latter had DCIS components, and in those cases we compared gene amplification in the intraductal and invasive components of each carcinoma. RESULTS: The rate of amplification of FGFR1 was higher in invasive carcinomas than in the pure DCIS, but the opposite was true for HER2 amplification. These findings applied consistently to high-grade tumors, but not to low/intermediate-grade tumors. The amplification status of HER2, C-MYC, CCND1 and FGFR1 was generally similar in the matched invasive and DCIS components of the same tumors. However, FGFR1 amplification was more common in the invasive components than in the DCIS components. In survival analyses, FGFR1 amplification was found to be an independent prognostic factor for poor disease-free survival for all patients with invasive carcinoma and for the hormone receptor-positive subgroup. CONCLUSION: Amplification of HER2, C-MYC and CCND1 seems to play a role in the early development of breast cancer, but not in its progression. However, the increased frequency of FGFR1 amplification in invasive carcinomas compared with pure DCIS and in the invasive components of individual tumors, and its association with decreased disease-free survival, suggests a role for FGFR1 amplification in the progression of breast cancer including in situ-to-invasive transition, as well as initiation.

Intratumoral heterogeneity of HER2 gene amplification in breast cancer: its clinicopathological significance.

Intratumoral heterogeneity of human epidermal growth factor receptor 2 (HER2) gene amplification has been reported to occur with variable frequencies in breast cancers. However, there have been few studies of its clinicopathological significance. We used tissue microarrays to evaluate two aspects of intratumoral heterogeneity of HER2 gene amplification: regional heterogeneity and genetic heterogeneity. We examined 96 invasive breast cancers in which HER2 amplification had been diagnosed in whole sections, and determined the clincopathological characteristics of those tumors. HER2 regional heterogeneity, defined as the existence of amplification/negative or amplification/equivocal patterns in different tissue microarray cores of a tumor, was present in 17 (18%) of the 96 cases. HER2 genetic heterogeneity, defined as the presence of tumor cells with a HER2/chromosome enumeration probe 17 ratio higher than 2.2 in 5-50% of the tumor cells, was found in 11 cases (11%), all of which showed HER2 regional heterogeneity. The cases with intratumoral heterogeneity of HER2 gene amplification were characterized by low grade or equivocal HER2 amplification and equivocal (2+) HER2 expression in whole sections. The patients with intratumoral heterogeneity of HER2 gene amplification had significantly shorter disease-free survival times than those with homogeneous HER2 gene amplification, and this effect was also evident in subgroup analysis by hormone receptor status. In multivariate analysis, intratumoral HER2 heterogeneity retained its status as an independent prognostic factor for disease-free survival. In conclusion, intratumoral heterogeneity of HER2 gene amplification is present in a subset of HER2-amplified breast cancers, especially in cases with low-grade HER2 amplification and equivocal HER2 expression, indicating a need for HER2 testing on more representative, larger tumor samples for accurate assessment of HER2 status in such cases. The patients with this heterogeneity have decreased disease-free survival, suggesting that genetic instability, and hence aberrant HER2 amplification in subclones of such tumors, may be associated with breast cancer progression.

The hormone receptor, human epidermal growth factor receptor 2, and molecular subtype status of individual tumor foci in multifocal/multicentric invasive ductal carcinoma of breast.

Multifocal/multicentric breast cancers are common. However, investigations of biomarkers such as estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 in individual tumor foci of such cancers are rare. This study was designed to evaluate the status of the hormone receptors, human epidermal growth factor receptor 2, and its molecular subtypes in individual foci of multifocal/multicentric invasive ductal carcinoma of the breast and to identify the factors associated with the different phenotypes of individual foci. We performed immunohistochemical analyses of the estrogen receptor, progesterone receptor, cytokeratin 5/6, epidermal growth factor receptor, and p53 and fluorescence in situ hybridization of human epidermal growth factor receptor 2 in individual foci of 65 cases of multifocal/multicentric invasive ductal carcinoma and the associated ductal carcinoma in situ components using tissue microarrays. The estrogen receptor status differed in 2 (3%) of the 65 invasive ductal carcinomas, progesterone receptor status in 7 (11%), human epidermal growth factor receptor 2 status in 4 (6%), and molecular subtypes in 5 (8%). The presence of different molecular subtypes in the invasive tumor foci was associated with differences in histologic features (P = .005), high histologic and nuclear grade (P = .012 and P = .021, respectively), p53 overexpression (P = .006), and mixed molecular subtypes in the ductal carcinoma in situ components (P = .011). Multifocal/multicentric invasive ductal carcinomas usually have a single phenotype in terms of hormone receptors, human epidermal growth factor receptor 2, and molecular subtypes; and thus, immunohistochemical analyses of the index tumor may be sufficient in routine practice. However, if multifocal/multicentric invasive ductal carcinomas are of high grade, of different histologic features, or of heterogeneous ductal carcinoma in situ component, biomarkers of the various foci need to be evaluated separately.

Distinct patterns of promoter CpG island methylation of breast cancer subtypes are associated with stem cell phenotypes.

Although DNA methylation profiles in breast cancer have been connected to breast cancer molecular subtype, there have been no studies of the association of DNA methylation with stem cell phenotype. This study was designed to evaluate the promoter CpG island methylation of 15 genes in relation to breast cancer subtype, and to investigate whether the patterns of CpG island methylation in each subtype are associated with their cancer stem cell phenotype represented by CD44+/CD24- and ALDH1 expression. We performed MethyLight analysis of the methylation status of 15 promoter CpG island loci involved in breast cancer progression (APC, DLEC1, GRIN2B, GSTP1, HOXA1, HOXA10, IGF2, MT1G, RARB, RASSF1A, RUNX3, SCGB3A1, SFRP1, SFRP4, and TMEFF2) and determined cancer stem cell phenotype by CD44/CD24 and ALDH1 immunohistochemistry in 36 luminal A, 33 luminal B, 30 luminal-HER2, 40 HER2 enriched, and 40 basal-like subtypes of breast cancer. The number of CpG island loci methylated differed significantly between subtypes, and was highest in the luminal-HER2 subtype and lowest in the basal-like subtype. Methylation frequencies and levels in 12 of the 15 genes differed significantly between subtypes, and the basal-like subtype had significantly lower methylation frequencies and levels in nine of the genes than the other subtypes. CD44+/CD24- and ALDH1+ putative stem cell populations were most enriched in the basal-like subtype. Methylation of promoter CpG islands was significantly lower in CD44+/CD24-cell (+) tumors than in CD44+/CD24-cell (-) tumors, even within the basal-like subtype. ALDH1 (+) tumors were also less methylated than ALDH1 (-) tumors. Our findings showed that promoter CpG island methylation was different in relation to breast cancer subtype and stem cell phenotype of tumor, suggesting that breast cancers have distinct patterns of CpG island methylation according to molecular subtypes and these are associated with different stem cell phenotypes of the tumor.

Usefulness of a break-apart FISH assay in the diagnosis of Xp11.2 translocation renal cell carcinoma.

Xp11.2 translocation renal cell carcinoma (RCC) is a rare subtype of RCC predominantly reported in young patients. It results from gene fusions between the transcription factor E3 (TFE3) gene, which is located on chromosome Xp11.2, and various fusion partners. Recently, a dual color, break-apart fluorescence in situ hybridization (FISH) assay to detect Xp11.2 translocation was reported. We performed this study to evaluate the usefulness of the FISH assay in the diagnosis of Xp11.2 translocation RCC using a commercially available TFE3 break-apart probe. We immunohistochemically analyzed TFE3 nuclear expression in 809 cases of RCCs using 14 tissue microarray blocks and selected nine cases those showed moderate to strong positive nuclear immunoreactivity for TFE3. The extent of TFE3 nuclear expression was variable. The TFE3 FISH assay was performed in these 9 selected cases and 44 negative control cases. Only four out of nine selected cases showed the TFE3 break-apart signal. TFE3 FISH-positive cases mainly showed diffuse and strong TFE3 immunopositivity, but one case revealed focal and moderate TFE3 staining. On the contrary, TFE3 FISH-negative cases mainly revealed focal and moderate TFE3 immunoreactivity, however, one FISH-negative case revealed diffuse and strong TFE3 nuclear immunopositivity. All negative control cases revealed normal TFE3 FISH results. Our results reveal that TFE3 immunohistochemistry can show false-positive results, and that the TFE3 break-apart FISH assay is a useful complementary method for confirming the diagnosis of Xp11.2 translocation RCC.