Vimentin-positive invasive breast carcinoma of no special type: A breast carcinoma with lethal biological characteristics.

Vimentin is a stable mesenchymal immunohistochemical marker and is widely recognized as a major marker of mesenchymal tumors. The purpose of the present study was to investigate if the vimentin expression status might serve as a significant predictor of outcomes in patients with invasive breast carcinoma of no special type (IBC-NST) and to investigate, by comprehensive RNA sequencing analyses, the mechanisms involved in the heightened malignant potential of vimentin-positive IBC-NSTs. This study, conducted using the data of 855 patients with IBC-NST, clearly identified vimentin expression status as a very important independent biological parameter for accurately predicting the outcomes in patients with IBC-NST. RNA sequence analyses clearly demonstrated significant upregulation of coding RNAs known to be closely associated with cell proliferation or cellular senescence, and significant downregulation of coding RNAs known to be closely associated with transmembrane transport in vimentin-positive IBC-NSTs. We conclude that vimentin-positive IBC-NSTs show heightened malignant biological characteristics, possibly attributable to the upregulation of RNAs closely associated with proliferative activity and cellular senescence, and downregulation of RNAs closely associated with transmembrane transport in IBC-NSTs.

[Clinical Outcomes of Five Cases of Occult Breast Cancer in Which the Primary Site Was Not Detected in the Breast-by-Breast MRI].

Occult breast cancer is rare in practice. We studied the clinical outcomes of 5 occult breast cancers, including 2 with Luminal and 3 with non-Luminal subtypes, for which the primary site was not detected in the breast-by-breast MRI. The percentage of occult breast cancers that we encountered at our hospital was 0.11%. The mean age was 54 years. The Ki-67 labeling index value was 30% or higher for all the patients except one. Four patients were administered neoadjuvant chemotherapy and all but one patient received non-mastectomy and axillary dissection plus radiotherapy. We observed recurrent cases in one example each of the Luminal and HER2 subtypes, and both patients were less than 40 years old. The estimates of the probability of 5 year recurrence-free survival and 5 year overall survival were 40.0% and 66.7%, respectively. One recurrence case was a patient negative for ER and positive for HER2 wherein a breast cancer lesion appeared in the breast during post-treatment follow-up. Intrabreast relapse, which is itself rare in occult breast cancer, was observed 4 years postoperatively after standard treatment. Although there was no deviation according to subtype rate, the Ki-67 labeling index value was high and the prognosis was poor in our 5 cases.

Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal.

PURPOSE: Retinal organoids generated from human pluripotent stem cells exhibit considerable variability during differentiation. Our goals are to assess developmental maturity of the neural retina in vitro and design improved protocols based on objective criteria. METHODS: We performed transcriptome analyses of developing retinal organoids from human embryonic and induced pluripotent stem cell lines and utilized multiple bioinformatic tools for comparative analysis. Immunohistochemistry, immunoblotting and electron microscopy were employed for validation. RESULTS: We show that the developmental variability in organoids was reflected in gene expression profiles and could be evaluated by molecular staging with the human fetal and adult retinal transcriptome data. We also demonstrate that the addition of 9-cis retinal, instead of the widely used all-trans retinoic acid, accelerated rod photoreceptor differentiation in organoid cultures, with higher rhodopsin expression and more mature mitochondrial morphology evident by day 120. CONCLUSION: Our studies provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids and for comparisons across different stem cell lines and platforms, which should facilitate disease modeling and evaluation of therapies in vitro.

Impact of Comorbidities on Survival in Gastric, Colorectal, and Lung Cancer Patients.

BACKGROUND: The presence of comorbidities in cancer patients may influence treatment decisions and prognoses. This study aimed to examine the impact of comorbidities on overall survival in Japanese patients diagnosed with major solid tumors. METHODS: To obtain patient-level information on clinical conditions and vital status, we performed a record linkage of population-based cancer registry data from Osaka Prefecture, Japan and administrative data produced under the Diagnosis Procedure Combination (DPC) system. The study population comprised patients who received a primary diagnosis of gastric, colorectal, or lung cancer between 2010 and 2012 at any of five cancer centers. We employed the Charlson Comorbidity Index (CCI) score to quantify the impact of comorbidities on survival. The association between CCI score and survival for each cancer site was analyzed using Cox proportional hazards regression models for all-cause mortality, after adjusting for patient sex, age at cancer diagnosis, and cancer stage. RESULTS: A total of 2,609 patients with a median follow-up duration of 1,372 days were analyzed. The most frequent CCI score among the patients was 0 (77.7%), followed by 2 (14.3%). After adjusting for the covariates, we detected a significant association between CCI score and all-cause mortality. The hazard ratios per one-point increase in CCI score were 1.12 (95% confidence interval [CI], 1.02-1.23), 1.20 (95% CI, 1.08-1.34), and 1.14 (95% CI, 1.04-1.24) for gastric, colorectal, and lung cancer, respectively. CONCLUSIONS: Comorbidities have a negative prognostic impact on overall survival in cancer patients, and should be assessed as risk factors for mortality when reporting outcomes.

Intracellular hypoxia measured by 18F-fluoromisonidazole positron emission tomography has prognostic impact in patients with estrogen receptor-positive breast cancer.

BACKGROUND: Hypoxia is a key driver of cancer progression. We evaluated the prognostic impact of 18F-fluoromisonidazole (FMISO) prior to treatment in patients with breast cancer. METHODS: Forty-four patients with stage II/III primary breast cancer underwent positron emission tomography/computed with 18F-fluorodeoxyglucose (FDG-PET/CT) and FMISO. After measurement by FDG-PET/CT, the tissue-to-blood ratio (TBR) was obtained using FMISO-PET/CT. FMISO-TBR was compared for correlation with clinicopathological factors, disease-free survival (DFS), and overall survival (OS). Multiplex cytokines were analyzed for the correlation of FMISO-TBR. RESULTS: Tumors with higher nuclear grade and negativities of estrogen receptor (ER) and progesterone receptor had significantly higher FMISO-TBR than other tumors. Kaplan-Meier survival curves showed that patients with a higher FMISO-TBR (cutoff, 1.48) had a poorer prognosis of DFS (p = 0.0007) and OS (p = 0.04) than those with a lower FMISO-TBR. Multivariate analysis indicated that higher FMISO-TBR and ER negativity were independent predictors of shorter DFS (p = 0.01 and 0.03). Higher FMISO-TBR was associated with higher plasma levels of angiogenic hypoxic markers such as vascular endothelial growth factor, transforming growth factor-α, and interleukin 8. CONCLUSIONS: FMISO-PET/CT is useful for assessing the prognosis of patients with breast cancer, but it should be stratified by ER status. TRIAL REGISTRATION: UMIN Clinical Trials Registry, UMIN000006802 . Registered on 1 December 2011.

Correction to: Intracellular hypoxia measured by F-18 fluoromisonidazole positron emission tomography has prognostic impact in patients with estrogen-receptor positive breast (BRCR-D17-00693).

After the publication of this article [1], we noticed that in Fig. 2, the survival curve images (C and D, lower panel) were incorrect. The corrected Fig. 2 is presented below. The correction does not affect in any our results and conclusions.

Fibrotic focus: An important parameter for accurate prediction of a high level of tumor-associated macrophage infiltration in invasive ductal carcinoma of the breast.

Our group and others have previously reported that a fibrotic focus is a very useful histological factor for the accurate prediction of the outcome of patients with invasive ductal carcinoma of the breast. We classified 258 cases of invasive ductal carcinoma into those with and those without a fibrotic focus to investigate whether the presence of a fibrotic focus was significantly associated with the degree of tumor-associated macrophage (CD68, CD163 or CD204-positive) infiltration or whether the presence of tumor-associated macrophage infiltration heightened the malignant potential of invasive ductal carcinoma with a fibrotic focus. Multiple regression analyses demonstrated that a fibrotic focus was the only factor that was significantly associated with a high level of CD68-, CD163- or CD204-positive tumor-associated macrophage infiltration. The combined assessment of the presence or absence of a fibrotic focus and a high or a low level of CD204-positive tumor-associated macrophage infiltration clearly demonstrated that CD204-positive tumor-associated macrophage infiltration had a significant prognostic power only for patients with invasive ductal carcinoma with a fibrotic focus in multivariate analyses; CD204-positive tumor-associated macrophages might only exert a significant effect on tumor progression when a fibrotic focus is present within the invasive ductal carcinoma of the breast.

Neoadjuvant chemotherapy with trastuzumab, docetaxel, and carboplatin administered every 3 weeks for Japanese women with HER2-positive primary breast cancer: efficacy and safety.

BACKGROUND: This phase II neoadjuvant study evaluated the efficacy and safety of a triweekly regimen of docetaxel and carboplatin in combination with trastuzumab (TCbH) in Japanese women with human epidermal growth factor receptor type2 (HER2)-positive primary breast cancer. METHODS: Patients with HER2-positive, stage I-III invasive breast cancer received six courses of trastuzumab (8 mg/kg loading dose, then 6 mg/kg, day 1), docetaxel (75 mg/m2, day 1), and carboplatin (area under the curve: 6, day 1) every 3 weeks. The primary endpoint was pathological complete response (pCR) of both breast and axillary lymph node disease. RESULTS: Fifty patients were enrolled in this study. Median age was 58 (range 32-75) years. All patients underwent definitive surgery. Thirty-three (66%) patients completed the chemotherapy course, while the treatment was delayed or discontinued in the other 17 (34%) patients because of adverse events (AEs). The pCR rate was 52%; the overall response rate was 66%. Grade 3/4 AEs due to nonhematological toxicity were anorexia (4%), diarrhea (2%), and rash (2%), and those due to hematological toxicity were neutropenia (36%), anemia (12%), and thrombocytopenia (2%). CONCLUSION: Although the triweekly six-course regimen of TCbH achieved a high pCR rate, hematological AEs frequently occurred during the latter part of the chemotherapy course. One-third of patients experienced delayed or discontinued chemotherapy. Clinical registration number: http://www.umin.org.au UMIN000013513.

[Choice of Adjuvant Chemotherapy Based on Breast Cancer Subtype].

Adjuvant chemotherapy targets micrometastases. If micrometastases are destroyed, patients with breast cancer will be cured. Currently, in Japan, standard breast cancer therapy is decided based on subtypes that are defined according to ER, PgR, HER2, and Ki-67 expression. We present here an outline of the recommended chemotherapy regimens based on evidence.

Safety and feasibility of adjuvant chemotherapy with S-1 in Japanese breast cancer patients after primary systemic chemotherapy: a feasibility study.

BACKGROUND: Advanced breast cancer patients have a higher risk of postoperative recurrence than early-stage breast cancer patients. Recurrence is believed to be caused by the increase in micrometases, which were not eradicated by preoperative or postoperative chemotherapy. Therefore, a new therapeutic strategy that can improve treatment efficacy is mandatory for advanced breast cancer. S-1 was shown to be effective and safe in Japanese metastatic breast cancer patients treated with previous chemotherapy, including anthracyclines. Thus, in this study, we evaluated S-1 as adjuvant chemotherapy in breast cancer patients after standard primary systemic chemotherapy. METHODS: The treatment consisted of 18 courses (a 2-week administration and a 1-week withdrawal; one year) administered at 80-120 mg/body/day. In cases judged to require postoperative radiotherapy, it was concurrently initiated on Day 1 of the study. If the estrogen receptor and/or human epidermal growth factor receptor 2 were positive, endocrine therapy and/or trastuzumab were permitted, concurrently. RESULTS: Of the 45 patients enrolled between September 2007 and September 2009 from 3 institutions, 43 patients were eligible. Thirty-two of the 43 (74.4%) patients received concurrent radiotherapy. Twenty-two of the 43 (51.2%) patients completed the scheduled courses of chemotherapy. The most common reasons for withdrawal of treatment were subjective symptoms, such as nausea, anorexia, or general fatigue during the first 9 courses of treatment in 9/43 (20.9%) patients, recurrence in 7/43 (16.3%) patients, and adverse events in 5/43 (11.6%) patients. The cumulative percentage of administration for 365 days was 66.4% (95% confidence interval: 50.8-79.1%). Although grade 3 neutropenia (9.3%), leukopenia (4.7%), and diarrhea (4.7%) were observed, they were manageable. No grade 4 adverse effects were observed. CONCLUSIONS: The percentage of Japanese breast cancer patients completing the 18-course treatment and the cumulative percentage of administration for 365 days using S-1 after standard primary systemic chemotherapy were similar with the results of another study of adjuvant chemotherapy for the Japanese gastric cancer patients with no severe adverse effects. A phase III trial investigating the usefulness of adjuvant S-1 is now ongoing in Japan, and it is expected that S-1 will have a significant survival benefit in breast cancer patients. UMIN000013469.

Neoadjuvant triweekly nanoparticle albumin-bound paclitaxel followed by epirubicin and cyclophosphamide for Stage II/III HER2-negative breast cancer: evaluation of efficacy and safety.

OBJECTIVE: Nanoparticle albumin-bound paclitaxel (nab-PTX) is a solvent-free paclitaxel coupled to human albumin without an associated increase in toxicity. The neoadjuvant study of primary breast cancer was planned to evaluate tumor response and safety of triweekly nanoparticle albumin-bound paclitaxel. METHODS: Patients with Stage II/III HER2-negative primary breast cancer received four courses of nanoparticle albumin-bound paclitaxel 260 mg/m(2) every 3 weeks (q3w), followed by four courses of epirubicin 90 mg/m(2) plus cyclophosphamide 600 mg/m(2) q3w. Tumor response after nanoparticle albumin-bound paclitaxel was histologically evaluated. In addition, the clinical response, breast-conserving rate and safety of this treatment were monitored. RESULTS: Among 53 patients who received nanoparticle albumin-bound paclitaxel followed by epirubicin and cyclophosphamide neoadjuvant chemotherapy, pathological complete response and near-pathological complete response were confirmed in 3 (5.7%) and 7 (13.2%) patients who had surgery, respectively. The overall objective response rate was 71.7% after completion of chemotherapy. Based on Positron Emission Tomography Response Criteria in Solid Tumors using (18)F-fluorodeoxyglucose, complete metabolic response and partial metabolic response after 2-3 courses of nanoparticle albumin-bound paclitaxel were 15.1 and 52.8%, respectively. The most common significant toxicities of q3w nanoparticle albumin-bound paclitaxel were Grade 3 muscle pain, neuropathy and febrile neutropenia, each in 1 (1.9%) patient. There were no incidences of anaphylaxis or Grade 4/5 adverse events. CONCLUSION: Neoadjuvant chemotherapy using q3w nanoparticle albumin-bound paclitaxel followed by epirubicin and cyclophosphamide was feasible in breast cancer patients with acceptable clinical response and drug tolerance, but conferred a low rate of pathological complete response. Monotherapy with q3w nanoparticle albumin-bound paclitaxel could be an appropriate substitute for solvent-based taxane in terms of therapeutic and safety management.

Clinicopathological and prognostic impact of imaging of breast cancer angiogenesis and hypoxia using diffuse optical spectroscopy.

Near-infrared diffuse optical spectroscopy (DOS) imaging can non-invasively measure tumor hemoglobin concentration using high contrast to normal tissue, thus providing vascularity and oxygenation status. We assessed the clinical usefulness of DOS imaging in primary breast cancer. In all, 118 women with a histologically confirmed diagnosis of primary malignant tumor were enrolled. All participants underwent testing using time-resolved DOS before treatment initiation. Visual assessment of DOS imaging for detecting tumors was carried out by two readers blinded to the clinical data. Relative total hemoglobin (rtHb) and oxygen saturation (stO2 ) of the tumors was compared with clinicopathological variables and 10-year prognosis was calculated. Sensitivity for detecting a tumor based on the rtHb breast map was 62.7% (74/118). The sensitivity depended on T stage: 100% (7/7) for T3, 78.9% (45/57) for T2, 44.7% (17/38) for T1, and 31.3% (5/16) for Tis . Tumors showed unique features of higher rtHb with a wider range of stO2 than normal breast tissue, depending on histological type. There was a significant correlation of rtHb with tumor size, lymphatic vascular invasion, and histological grade, and of stO2 with age and tumor size. Neither rtHb nor stO2 correlated with intrinsic biomarkers such as estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2; rtHb inversely correlated with 10-year relapse-free survival and overall survival, with statistical significance. Diffuse optical spectroscopy imaging has limited utility for the early detection of breast cancer; nonetheless, the findings suggest that the degree of tumor angiogenesis and hypoxia may be associated with tumor aggressiveness and poor prognosis.

Comprehensive prognostic prediction of metastatic breast cancer treated with eribulin using blood­based parameters and ratio.

Eribulin is widely used to treat metastatic breast cancer (BC). Higher neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) are associated with higher mortality in several cancer types. However, the association between BC prognosis and peripheral immune status remains controversial. In the present study, the relative effects of NLR and PLR on survival in patients with metastatic BC were quantified and their clinical prognostic value was evaluated. This retrospective study included 156 patients with metastatic BC who received eribulin monotherapy at Saitama Medical University International Medical Center. Clinicopathological features were examined (peripheral blood findings and biochemical liver and kidney function test results) and univariate and multivariate analyses were conducted of the overall survival (OS). The 156 patients treated with eribulin had a median follow-up duration of 18.3 months. Before eribulin treatment, patients with absolute lymphocyte counts (ALC) >1,500/µl, NLR <3.0, and PLR <150 had significantly longer OS than those with lower ALC, and higher NLR and PLR (median OS, 25.5 vs. 15.5 months; P<0.01; 20.3 vs. 13.6 months, P<0.01; and 29.2 vs. 14.8 months; P<0.001, respectively). Patients with anemia [hemoglobin (Hb) <10 g/dl] or liver dysfunction [albumin-bilirubin (ALBI) grade 2/3] had significantly shorter OS than those without (P<0.001, respectively). Multivariate analysis revealed low ALBI grade (P<0.001), high Hb (P<0.01) and low PLR (P<0.05) as independent factors of longer OS after eribulin administration. Low PLR, anemia and liver dysfunction might be factors associated with prolonged OS in patients with metastatic BC on eribulin therapy, which could be clinically useful, as their evaluation requires neither new equipment nor invasive testing.

Optical imaging of tumor vascularity associated with proliferation and glucose metabolism in early breast cancer: clinical application of total hemoglobin measurements in the breast.

BACKGROUND: Near-infrared optical imaging targeting the intrinsic contrast of tissue hemoglobin has emerged as a promising approach for visualization of vascularity in cancer research. We evaluated the usefulness of diffuse optical spectroscopy using time-resolved spectroscopic (TRS) measurements for functional imaging of primary breast cancer. METHODS: Fifty-five consecutive TNM stage I/II patients with histologically proven invasive ductal carcinoma and operable breast tumors (<5 cm) who underwent TRS measurements were enrolled. Thirty (54.5%) patients underwent 18F-fluoro-deoxy-glucose (FDG) positron emission tomography with measurement of maximum tumor uptake. TRS was used to obtain oxyhemoglobin, deoxyhemoglobin, and total hemoglobin (tHb) levels from the lesions, surrounding normal tissue, and contralateral normal tissue. Lesions with tHb levels 20% higher than those present in normal tissue were defined as "hotspots," while others were considered "uniform." The findings in either tumor type were compared with clinicopathological factors. RESULTS: "Hotspot" tumors were significantly larger (P= 0.002) and exhibited significantly more advanced TNM stage (P=0.01), higher mitotic counts (P=0.01) and higher levels of FDG uptake (P=0.0004) compared with "uniform" tumors; however, other pathological variables were not significantly different between the two groups. CONCLUSIONS: Optical imaging for determination of tHb levels allowed for measurement of tumor vascularity as a function of proliferation and glucose metabolism, which may be useful for prediction of patient prognosis and potential response to treatment.

Clinicopathological prognostic characteristics and long­term outcomes of patients with breast cancer and collagen disorder in comparison to those without collagen disorder.

Collagen disorders are chronic autoimmune diseases with a complex clinical course; however, the risk of breast cancer in patients with collagen disorders remains unclear. The present study aimed to investigate long-term outcomes in women with breast cancer and collagen disorders. A total of 25 patients with breast cancer and collagen disorders who were treated between January 2004 and December 2011 were included. The clinicopathological factors, treatment, recurrence-free survival (RFS) and overall survival (OS) were reviewed. The mean age was 56.4±12.6 years, and 14, eight and three patients had cancer of clinical stages I, II and III, respectively. Regarding comorbid collagen disorders, 11 patients had rheumatoid arthritis, four had systemic lupus erythematosus, four had polymyositis/dermatomyositis, two had mixed connective tissue disease, two had Sjogren's syndrome, one had scleroderma and one had adult-onset Still's disease. The expression statuses of hormone receptors (HR) and human epidermal growth factor receptor 2 (HER2) were HR(+), HER2(+) and HR(-)HER2(-) in 20 (80.0%), four (16.0%) and four (16.0%) patients, respectively. A total of 22 (84.0%) patients received steroids or immunosuppressive drugs for collagen disorders. The collagen disorder group had a higher mean Ki-67 labeling index than the control group (41.1 vs. 20.8%; P=0.007). After median observation periods of 103 and 114 months, the RFS and OS rates were lower in the collagen group than in the control group (64.5 and 80.7% vs. 85.3 and 94.3%, respectively; P<0.01). Patients with breast cancer and collagen disorders had relatively high Ki-67 expression, and relatively low RFS and OS rates. Thorough follow-up is necessary for patients with breast cancer who also have collagen disorders and high Ki-67 values.

Reserpine maintains photoreceptor survival in retinal ciliopathy by resolving proteostasis imbalance and ciliogenesis defects.

Ciliopathies manifest from sensory abnormalities to syndromic disorders with multi-organ pathologies, with retinal degeneration a highly penetrant phenotype. Photoreceptor cell death is a major cause of incurable blindness in retinal ciliopathies. To identify drug candidates to maintain photoreceptor survival, we performed an unbiased, high-throughput screening of over 6000 bioactive small molecules using retinal organoids differentiated from induced pluripotent stem cells (iPSC) of rd16 mouse, which is a model of Leber congenital amaurosis (LCA) type 10 caused by mutations in the cilia-centrosomal gene CEP290. We identified five non-toxic positive hits, including the lead molecule reserpine, which maintained photoreceptor development and survival in rd16 organoids. Reserpine also improved photoreceptors in retinal organoids derived from induced pluripotent stem cells of LCA10 patients and in rd16 mouse retina in vivo. Reserpine-treated patient organoids revealed modulation of signaling pathways related to cell survival/death, metabolism, and proteostasis. Further investigation uncovered dysregulation of autophagy associated with compromised primary cilium biogenesis in patient organoids and rd16 mouse retina. Reserpine partially restored the balance between autophagy and the ubiquitin-proteasome system at least in part by increasing the cargo adaptor p62, resulting in improved primary cilium assembly. Our study identifies effective drug candidates in preclinical studies of CEP290 retinal ciliopathies through cross-species drug discovery using iPSC-derived organoids, highlights the impact of proteostasis in the pathogenesis of ciliopathies, and provides new insights for treatments of retinal neurodegeneration.

Leber congenital amaurosis (LCA) is an inherited disease that affects the eyes and causes sight loss in early childhood, which generally gets worse over time. Individuals with this condition have genetic mutations that result in the death of light-sensitive cells, known as photoreceptors, in a region called the retina at the back of the eye. Patients carrying a genetic change in the gene CEP290 account for 20-25% of all LCA. At present, treatment options are only available for a limited number of patients with LCA. One option is to use small molecules as drugs that may target or bypass the faulty processes within the eye to help the photoreceptors survive in many different forms of LCA and other retinal diseases. However, over 90% of new drug candidates fail the first phase of clinical trials for human diseases. This in part due to the candidates having been developed using cell cultures or animal models that do not faithfully reflect how the human body works. Recent advances in cell and developmental biology are now enabling researchers to use stem cells derived from humans to grow retina tissues in a dish in the laboratory. These tissues, known as retinal organoids, behave in a more similar way to retinas in human eyes than those of traditional animal models. However, the methods for making and maintaining human retinal organoids are time-consuming and labor-intensive, which has so far limited their use in the search for new therapies. To address this challenge, Chen et al. developed a large-scale approach to grow retinal organoids from rd16 mutant mice stem cells (which are a good model for LCA caused by mutations to CEP290) and used the photoreceptors from these organoids to screen over 6,000 existing drugs for their ability to promote the survival of photoreceptors. The experiments found that the drug reserpine, which was previously approved to treat high blood pressure, also helped photoreceptors to survive in the diseased organoids. Reserpine also had a similar effect in retinal organoids derived from human patients with LCA and in the rd16 mice themselves. Further experiments suggest that reserpine may help patients with LCA by partially restoring a process by which the body destroys and recycles old and damaged proteins in the cells. The next steps following on from this work will be to perform further tests to demonstrate that this use of reserpine is safe to enter clinical trials as a treatment for LCA and other similar eye diseases.

Human PSCs determine the competency of cerebral organoid differentiation via FGF signaling and epigenetic mechanisms.

Various culture methods have been developed for maintaining human pluripotent stem cells (PSCs). These PSC maintenance methods exhibit biased differentiation; for example, feeder-dependent PSCs efficiently yield cerebral organoids, but it is difficult to generate organoids from feeder-free PSCs. It remains unknown how PSC maintenance conditions affect differentiation. In this study, we identified fibroblast growth factor (FGF) signaling in feeder-free PSC maintenance as a key factor that determines the differentiation toward cerebral organoids. The inhibition of FGF signaling in feeder-free PSCs rescued organoid generation to the same level in feeder-dependent cultures. FGF inhibition induced DNA methylation at the WNT5A locus, and this epigenetic change suppressed the future activation of non-canonical Wnt signaling after differentiation, leading to reliable cerebral organoid generation. This study underscores the importance of PSC culture conditions for directed differentiation into cerebral organoids, and the epigenetic status regulated by FGF signaling is involved in the underlying mechanisms.

In vitro modeling and rescue of ciliopathy associated with IQCB1/NPHP5 mutations using patient-derived cells.

Mutations in the IQ calmodulin-binding motif containing B1 (IQCB1)/NPHP5 gene encoding the ciliary protein nephrocystin 5 cause early-onset blinding disease Leber congenital amaurosis (LCA), together with kidney dysfunction in Senior-Løken syndrome. For in vitro disease modeling, we obtained dermal fibroblasts from patients with NPHP5-LCA that were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into retinal pigment epithelium (RPE) and retinal organoids. Patient fibroblasts and RPE demonstrated aberrantly elongated ciliary axonemes. Organoids revealed impaired development of outer segment structures, which are modified primary cilia, and mislocalization of visual pigments to photoreceptor cell soma. All patient-derived cells showed reduced levels of CEP290 protein, a critical cilia transition zone component interacting with NPHP5, providing a plausible mechanism for aberrant ciliary gating and cargo transport. Disease phenotype in NPHP5-LCA retinal organoids could be rescued by adeno-associated virus (AAV)-mediated IQCB1/NPHP5 gene augmentation therapy. Our studies thus establish a human disease model and a path for treatment of NPHP5-LCA.

A next-generation iPSC-derived forebrain organoid model of tauopathy with tau fibrils by AAV-mediated gene transfer.

It is known that the human cellular models of Alzheimer's disease (AD) and tauopathy can only recapitulate the very early stage of the disease. To overcome these limitations, we developed a technology to make forebrain organoids (FBOs) from feeder-free induced pluripotent stem cells (iPSC)s by regulating a FGF2 concentration and applied this method to generate FBOs from patients with familial AD (fAD FBOs). The obtained fAD FBOs recapitulated the amyloid-ß pathology and increased tau phosphorylation but not tau aggregates. To fully induce the tau pathology, FBOs were injected with adeno-associated virus (AAV)-expressing P301L mutant tau. In these Tau-P301L FBOs, tau fibrils were observed in the neuronal cell body and neurites with immunoelectron microscopy, in addition to the sarkosyl-insoluble and thioflavin S-positive phospho-tau aggregates. Collectively, this model can be used as a platform for investigating pathogenetic mechanisms and evaluation of target molecules for drug discovery for tauopathy.

Tumor budding and fibrotic focus-proposed grading system for tumor budding in invasive carcinoma no special type of the breast.

Tumor budding grade is a very useful histological prognostic indicator for colorectal cancer patients. Recently, it has been also reported as a significant prognostic indicator in invasive breast carcinoma patients. Our group and others have previously reported that the presence of a fibrotic focus in the tumor is a very useful histological finding for accurately predicting the prognosis in patients with invasive carcinoma of no special type (ICNST) of the breast. The purpose of the present study was to investigate whether a grading system incorporating tumor budding in a fibrotic focus is superior to the conventional grading system for tumor budding to accurately predict outcomes in patients with ICNST. According to our new grading system, we classified the tumors into grade I (164 cases), grade II (581 cases), and grade III (110 cases), and the results clearly demonstrated the significant superiority of the new grading system over that of conventional tumor budding alone for accurately predicting outcomes in patients with ICNST. Our findings strongly suggest that tumor cells and tumor-stromal cells interaction play very important roles in tumor progression rather than tumor cells alone.