Cell cycle length governs heterochromatin reprogramming during early development in non-mammalian vertebrates.

Heterochromatin marks such as H3K9me3 undergo global erasure and re-establishment after fertilization, and the proper reprogramming of H3K9me3 is essential for early development. Despite the widely conserved dynamics of heterochromatin reprogramming in invertebrates and non-mammalian vertebrates, previous studies have shown that the underlying mechanisms may differ between species. Here, we investigate the molecular mechanism of H3K9me3 dynamics in medaka (Japanese killifish, Oryzias latipes) as a non-mammalian vertebrate model, and show that rapid cell cycle during cleavage stages causes DNA replication-dependent passive erasure of H3K9me3. We also find that cell cycle slowing, toward the mid-blastula transition, permits increasing nuclear accumulation of H3K9me3 histone methyltransferase Setdb1, leading to the onset of H3K9me3 re-accumulation. We further demonstrate that cell cycle length in early development also governs H3K9me3 reprogramming in zebrafish and Xenopus laevis. Together with the previous studies in invertebrates, we propose that a cell cycle length-dependent mechanism for both global erasure and re-accumulation of H3K9me3 is conserved among rapid-cleavage species of non-mammalian vertebrates and invertebrates such as Drosophila, C. elegans, Xenopus and teleost fish.

Enhancement of cytotoxic effects with ALA-PDT on treatment of radioresistant cancer cells.

Radiation therapy is a lower invasive local treatment than surgery and is selected as a primary treatment for solid tumors. However, when some cancer cells obtain radiotherapy tolerance, cytotoxicity of radiotherapy for cancer cells is attenuated. Photodynamic therapy (PDT) is a non-invasive cancer therapy combined with photosensitizers and laser irradiation with an appropriate wavelength. PDT is carried out for recurrent esophageal cancer patients after radiation chemotherapy and is an effective treatment for radiation-resistant tumors. However, it is not clear why PDT is effective against radioresistant cancers. In this study, we attempted to clear this mechanism using X-ray resistant cancer cells. X-ray resistant cells produce high amounts of mitochondria-derived ROS, which enhanced nuclear translocation of NF-κB, resulting in increased NO production. Moreover, the expression of PEPT1 that imports 5-aminolevulinic acid, the precursor of photosensitizers, was upregulated in X-ray resistant cancer cells. This was accompanied by an increase in intracellular 5-aminolevulinic acid-derived porphyrin accumulation, resulting in enhancement of PDT-induced cytotoxicity. Therefore, effective accumulation of photosensitizers induced by ROS and NO may achieve PDT after radiation therapy and PDT could be a promising treatment for radioresistant cancer cells.

A risk-based subgroup analysis of the effect of adjuvant S-1 in estrogen receptor-positive, HER2-negative early breast cancer.

PURPOSE: The Phase III POTENT trial demonstrated the efficacy of adding S-1 to adjuvant endocrine therapy for estrogen receptor-positive, HER2-negative early breast cancer. We investigated the efficacy of S-1 across different recurrence risk subgroups. METHODS: This was a post-hoc exploratory analysis of the POTENT trial. Patients in the endocrine-therapy-only arm were divided into three groups based on composite risk values calculated from multiple prognostic factors. The effects of S-1 were estimated using the Cox model in each risk group. The treatment effects of S-1 in patients meeting the eligibility criteria of the monarchE trial were also estimated. RESULTS: A total of 1,897 patients were divided into three groups: group 1 (≤ lower quartile of the composite values) (N = 677), group 2 (interquartile range) (N = 767), and group 3 (> upper quartile) (N = 453). The addition of S-1 to endocrine therapy resulted in 49% (HR: 0.51, 95% CI: 0.33-0.78) and 29% (HR: 0.71, 95% CI 0.49-1.02) reductions in invasive disease-free survival (iDFS) events in groups 2 and 3, respectively. We could not identify any benefit from the addition of S-1 in group 1. The addition of S-1 showed an improvement in iDFS in patients with one to three positive nodes meeting the monarchE cohort 1 criteria (N = 290) (HR: 0.47, 95% CI: 0.29-0.74). CONCLUSIONS: The benefit of adding adjuvant S-1 was particularly marked in group 2. Further investigations are warranted to explore the optimal usage of adjuvant S-1.

Long-Term Fluorescent Tissue Marking Using Tissue-Adhesive Porphyrin with Polycations Consisting of Quaternary Ammonium Salt Groups.

Localization of tumors during laparoscopic surgery is generally performed by locally injecting India ink into the submucosal layer of the gastrointestinal tract using endoscopy. However, the location of the tumor is obscured because of the black-stained surgical field and the blurring caused by India ink. To solve this problem, in this study, we developed a tissue-adhesive porphyrin with polycations consisting of quaternary ammonium salt groups. To evaluate the ability of tissue-adhesive porphyrin in vivo, low-molecular-weight hematoporphyrin and tissue-adhesive porphyrin were injected into the anterior wall of the exposed stomach in rats. Local injection of low-molecular-weight hematoporphyrin into the anterior wall of the stomach was not visible even after 1 day because of its rapid diffusion. In contrast, the red fluorescence of the tissue-adhesive porphyrin was visible even after 7 days due to the electrostatic interactions between the positively-charged moieties of the polycation in the tissue-adhesive porphyrin and the negatively-charged molecules in the tissue. In addition, intraperitoneal injection of tissue-adhesive porphyrin in rats did not cause adverse effects such as weight loss, hepatic or renal dysfunction, or organ adhesion in the abdominal cavity. These results indicate that tissue-adhesive porphyrin is a promising fluorescent tissue-marking agent.

Hydrocoel morphogenesis forming the pentaradial body plan in a sea cucumber, Apostichopus japonicus.

Echinoderms constitute an animal phylum characterized by the pentaradial body plan. During the development from bilateral larvae to pentaradial adults, the formation of the multiple of five hydrocoel lobes, i.e., the buddings from the mesodermal coelom, is the firstly emerging pentameral character. The developmental mechanism underlying the hydrocoel-lobe formation should be revealed to understand the evolutionary process of this unique and highly derived body plan of echinoderms, although the morphogenetic mechanisms of hydrocoel lobes are largely uninvestigated. In this study, using the sea cucumber Apostichopus japonicus, in which hydrocoel is easily observable, the developmental process of hydrocoel lobes was described in detail, focusing on cell proliferation and rearrangement. Cell proliferation was not specifically distributed in the growing tips of the hydrocoel lobes, and inhibition of cell proliferation did not affect lobe formation. During lobe formation, the epithelium of the hydrocoel lobes was firstly thickened and then transformed into a simple epithelium, suggesting that tissue expansion via tissue remodeling contributes to the hydrocoel-lobe formation.

Systemic chemotherapy combined with anti-epidermal growth factor receptor antibody therapy for RAS wild-type appendiceal signet-ring cell carcinoma: a series of three cases.

The effect of anti-epidermal growth factor receptor (EGFR) antibody-containing chemotherapy on appendiceal signet-ring cell carcinoma (SRCC) remains unknown. Herein, we report three patients, diagnosed as having synchronous metastases, who underwent this treatment for unresectable appendiceal SRCC with RAS wild type. Cases 1, 2, and 3 received FOLFOX with panitumumab, FOLFOX with cetuximab, and FOLFIRI with cetuximab, respectively, and their progression-free survival were 6.2, 7.2, and 18.7 months, respectively. The subsequent anti-vascular endothelial growth factor antibody-containing therapy was ineffective, and their overall survival was 8.2, 11.4, and 22.9 months, respectively. The anti-EGFR antibody-containing chemotherapy showed moderate efficacy for appendiceal SRCC. Further studies including molecular analysis should be needed.

Reemployment of Kupffer's vesicle cells into axial and paraxial mesoderm via transdifferentiation.

Kupffer's vesicle (KV) in the teleost embryo is a fluid-filled vesicle surrounded by a layer of epithelial cells with rotating primary cilia. KV transiently acts as the left-right organizer and degenerates after the establishment of left-right asymmetric gene expression. Previous labelling experiments in zebrafish embryos indicated that descendants of KV-epithelial cells are incorporated into mesodermal tissues after the collapse of KV. However, the overall picture of their differentiation potency had been unclear due to the lack of suitable genetic tools and molecular analyses. In the present study, we established a novel zebrafish transgenic line with a promoter of dand5, in which all KV-epithelial cells and their descendants are specifically labelled until the larval stage. We found that KV-epithelial cells undergo epithelial-mesenchymal transition upon KV collapse and infiltrate into adjacent mesodermal progenitors, the presomitic mesoderm and chordoneural hinge. Once incorporated, the descendants of KV-epithelial cells expressed distinct mesodermal differentiation markers and contributed to the mature populations such as the axial muscles and notochordal sheath through normal developmental process. These results indicate that differentiated KV-epithelial cells possess unique plasticity in that they are reemployed into mesodermal lineages through transdifferentiation after they complete their initial role in KV.

Quantitative analyses reveal extracellular dynamics of Wnt ligands in Xenopus embryos.

The mechanism of intercellular transport of Wnt ligands is still a matter of debate. To better understand this issue, we examined the distribution and dynamics of Wnt8 in Xenopus embryos. While Venus-tagged Wnt8 was found on the surfaces of cells close to Wnt-producing cells, we also detected its dispersal over distances of 15 cell diameters. A combination of fluorescence correlation spectroscopy and quantitative imaging suggested that only a small proportion of Wnt8 ligands diffuses freely, whereas most Wnt8 molecules are bound to cell surfaces. Fluorescence decay after photoconversion showed that Wnt8 ligands bound on cell surfaces decrease exponentially, suggesting a dynamic exchange of bound forms of Wnt ligands. Mathematical modeling based on this exchange recapitulates a graded distribution of bound, but not free, Wnt ligands. Based on these results, we propose that Wnt distribution in tissues is controlled by a dynamic exchange of its abundant bound and rare free populations.

Adjuvant S-1 plus endocrine therapy for oestrogen receptor-positive, HER2-negative, primary breast cancer: a multicentre, open-label, randomised, controlled, phase 3 trial.

BACKGROUND: Oral fluoropyrimidines, such as S-1, have been shown to have a role in controlling disease progression in metastatic breast cancer. We examined adjuvant treatment with S-1 in patients with oestrogen receptor (ER)-positive and HER2-negative primary breast cancer. METHODS: We did a multicentre, open-label, randomised, controlled, phase 3 trial in 139 sites (137 hospitals and two clinics). Eligible patients were women aged 20-75 years with histologically diagnosed stage I to IIIB invasive breast cancer (intermediate to high risk of recurrence). Patients were temporarily registered at participating institutions and biopsy or surgical samples were collected and sent for central pathological assessment. Patients received 5 years of standard adjuvant endocrine therapy (selective oestrogen receptor modulators with or without ovarian suppression and aromatase inhibitors) with or without 1 year of S-1. Oral S-1 80-120 mg/day was administered twice a day for 14 days with 7 days off. Randomisation (1:1) using the minimisation method was done with six stratification factors (age, axillary lymph node metastasis at surgery or sentinel lymph node biopsy, preoperative or postoperative (neoadjuvant or adjuvant) chemotherapy, preoperative endocrine therapy, proportion of ER-positive cells, and study site). The primary endpoint was invasive disease-free survival, in the full analysis set (all randomly assigned patients, excluding those with significant protocol deviations). The safety analysis set consisted of all patients who received at least one dose of study treatment. Here, we report the results from the interim analysis at the data cutoff date Jan 31, 2019. This trial is registered with Japan Registry of Clinical Trials, jRCTs051180057, and the University hospital Medical Information Network, UMIN000003969. FINDINGS: Between Feb 1, 2012, and Feb 1, 2016, 1930 patients were enrolled in the full analysis set, 957 (50%) received endocrine therapy plus S-1 and 973 (50%) received endocrine therapy alone. Median follow-up was 52·2 months (IQR 42·1-58·9). 155 (16%) patients in the endocrine therapy alone group and in 101 (11%) patients in the endocrine therapy plus S-1 group had invasive disease-free survival events (hazard ratio 0·63, 95% CI 0·49-0·81, p=0·0003). As the primary endpoint was met at interim analysis, the trial was terminated early. The most common grade 3 or worse adverse events were decreased neutrophil count (72 [8%] of 954 patients in the endocrine therapy plus S-1 group vs seven [1%] of 970 patients in the endocrine therapy alone group), diarrhoea (18 [2%] vs none), decreased white blood cells (15 [2%] vs two [<1%]), and fatigue (six [<1%] vs none). Serious adverse events were reported in nine (1%) of 970 patients in the endocrine therapy alone group and 25 (3%) of 954 patients in the endocrine therapy plus S-1 group. There was one (<1%) possible treatment-related death in the endocrine therapy plus S-1 group due to suspected pulmonary artery thrombosis. INTERPRETATION: These data suggest that this combination of S-1 with endocrine therapy could be a potential treatment option for this intermediate and high-risk group of patients with ER-positive, HER2-negative primary breast cancer. FUNDING: Public Health Research Foundation (Japan), Taiho Pharmaceutical.

A Durable Response after the Discontinuation of Nivolumab in an Advanced Gastric Cancer Patient.

A durable response after the discontinuation of immune checkpoint-inhibitor therapy has previously been reported in several cancers. We herein describe a patient with gastric cancer who maintained a durable response after the discontinuation of nivolumab. A 65-year-old man was treated with nivolumab as a sixth-line therapy for recurrent gastric cancer. After four cycles of nivolumab therapy, he showed a partial response. But the treatment was discontinued when two immune-related adverse events occurred after six cycles. Disease regression was sustained for approximately 2 years, without the re-administration of nivolumab. The characteristics leading to such responses are unclear, and further studies are warranted in this regard.

Safety and effectiveness of a novel neuroprotectant, KUS121, in patients with non-arteritic central retinal artery occlusion: An open-label, non-randomized, first-in-humans, phase 1/2 trial.

Kyoto University Substance (KUS) 121, an ATPase inhibitor of valosin-containing protein, is a novel neuroprotectant. We tested the safety and effectiveness of KUS121 in patients with acute central retinal artery occlusion (CRAO). We conducted an investigator-initiated, first-in-humans, phase 1/2 clinical trial. Nine patients with non-arteritic CRAO symptoms lasting for 4-48 h were enrolled. These patients received daily intravitreal injections of KUS121 for 3 days: 25 µg (low-dose) in the first three patients and 50 µg (high-dose) in the next six patients. The primary endpoint was the safety of the drug. As a secondary endpoint, pharmacokinetics was evaluated. Other key secondary endpoints were changes in best-corrected visual acuity (BCVA), measured using the Early Treatment Diabetic Retinopathy Study chart, visual field scores, and retinal sensitivities between baseline and week 12; and decimal BCVA at week 12. Administration of KUS121 did not result in serious adverse events. All nine patients (100%) showed significant improvement of BCVA. Average readable letter counts, visual field scores, and retinal sensitivities also improved. Decimal BCVA at week 12 was better than 0.1 in four patients (44%) and equal to or better than 0.05 in seven patients (78%). This first-in-humans clinical trial provides support for the safety and efficacy of intravitreal KUS121 injection. To substantiate the safety and effectiveness for patients with acute CRAO, further larger scale clinical studies will be needed.

Induced pluripotent stem cell-based Drug Repurposing for Amyotrophic lateral sclerosis Medicine (iDReAM) study: protocol for a phase I dose escalation study of bosutinib for amyotrophic lateral sclerosis patients.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a progressive and severe neurodegenerative disease caused by motor neuron death. There have as yet been no fundamental curative medicines, and the development of a medicine for ALS is urgently required. Induced pluripotent stem cell (iPSC)-based drug repurposing identified an Src/c-Abl inhibitor, bosutinib, as a candidate molecular targeted therapy for ALS. The objectives of this study are to evaluate the safety and tolerability of bosutinib for the treatment of patients with ALS and to explore the efficacy of bosutinib on ALS. This study is the first clinical trial of administered bosutinib for patients with ALS. METHODS AND ANALYSIS: An open-label, multicentre phase I dose escalation study has been designed. The study consists of a 12-week observation period, a 1-week transitional period, a 12-week study treatment period and a 4-week follow-up period. After completion of the transitional period, subjects whose total ALS Functional Rating Scale-Revised (ALSFRS-R) score decreased by 1-3 points during the 12-week observation period receive bosutinib for 12 weeks. Three to six patients with ALS are enrolled in each of the four bosutinib dose levels (100, 200, 300 or 400 mg/day) to evaluate the safety and tolerability under a 3+3 dose escalation study design. Dose escalation and maximum tolerated dose are determined by the safety assessment committee comprising oncologists/haematologists and neurologists based on the incidence of dose-limiting toxicity in the first 4 weeks of the treatment at each dose level. A recommended phase II dose is determined by the safety assessment committee on completion of the 12-week study treatment in all subjects at all dose levels. The efficacy of bosutinib is also evaluated exploratorily using ALS clinical scores and biomarkers. ETHICS AND DISSEMINATION: This study received full ethical approval from the institutional review board of each participating site. The findings of the study will be disseminated in peer-reviewed journals and at scientific conferences. TRIAL REGISTRATION NUMBER: UMIN000036295; Pre-results, JMA-IIA00419; Pre-results.

A therapeutic angiogenesis of sustained release of basic fibroblast growth factor using biodegradable gelatin hydrogel sheets in a canine chronic myocardial infarction model.

This study investigated the safety and efficacy of a sustained release of basic fibroblast growth factor (bFGF) with biodegradable gelatin hydrogel sheets as therapeutic angiogenesis in canine chronic myocardial infarction (MI) models. Canine chronic MI model was induced by ligating the left anterior descending coronary artery and its diagonal branches. At 4 week post-induction, we applied either saline (Control group, n = 5) or 200 µg of bFGF (Treatment group, n = 6) soaked gelatin hydrogel sheets on the ischemic area of the left ventricular (LV) wall. At 6 weeks after the procedure, we evaluated the efficacy by echocardiography and immunohistochemical study. There were no procedure-related adverse events or deaths. The serum bFGF level was under detectable levels in all animals at any sampling points. In terms of efficacy, echocardiographic evaluation demonstrated that fractional shortening was significantly improved in the treatment group. In addition, immunohistochemical study showed that the capillary density in the border zone of the MI area, as well as the MI area, significantly increased in the treatment group. Therapeutic angiogenesis by bFGF using biodegradable gelatin hydrogel sheets was safe, increased the capillary density, and improved LV function in canine chronic MI models.

Combined effects of neoadjuvant letrozole and zoledronic acid on γδT cells in postmenopausal women with early-stage breast cancer.

INTRODUCTION: Adjuvant bisphosphonates lead to better prognosis in postmenopausal breast cancer. However, the association between clinical outcomes and immune modulation by them is still unclear. METHODS: In this prospective, open-label phase II study, postmenopausal women with estrogen receptor-positive and human epidermal growth factor receptor 2-negative early-stage breast cancer received neoadjuvant letrozole (LET) for one month, followed by treatment with a single dose of zoledronic acid. The patients underwent an additional 5 months of treatment with LET prior to surgery. The primary endpoint was the tumor objective response rate (ORR) determined by diameter via MRI. The association between the ORR and γδT cell frequencies was assessed as a secondary endpoint. RESULTS: Out of sixty patients, 55 patients were evaluable for response by MRI. The ORR for LET with zoledronic acid was 38.2% (21/55), which was comparable to that of historical controls (45%). A decrease in the frequency of the Vδ2 T cell subset was observed throughout treatment, and Vδ2 T cells were activated for 6 months. In planned subgroup analyses, patients with low frequencies of Vδ2 T cells prior to zoledronic acid infusion experienced a favorable tumor response compared to those with high frequencies (59.3% [16/27] vs 17.9% [5/28], p = .002). There were no serious adverse events with this treatment regimen. CONCLUSION: These results showed that neoadjuvant LET with zoledronic acid could not achieve overall effect for local tumor response. However, patients with a low frequency of γδ T cells would benefit from the treatment including zoledronic acid. (UMIN 000008701).

High variability of expression profiles of homeologous genes for Wnt, Hh, Notch, and Hippo signaling pathways in Xenopus laevis.

Cell signaling pathways, such as Wnt, Hedgehog (Hh), Notch, and Hippo, are essential for embryogenesis, organogenesis, and tissue homeostasis. In this study, we analyzed 415 genes involved in these pathways in the allotetraploid frog, Xenopus laevis. Most genes are retained in two subgenomes called L and S (193 homeologous gene pairs and 29 singletons). This conservation rate of homeologs is much higher than that of all genes in the X. laevis genome (86.9% vs 60.2%). Among singletons, 24 genes are retained in the L subgenome, a rate similar to the average for all genes (82.8% vs 74.6%). In addition, as general components of signal transduction, we also analyzed 32 heparan sulfate proteoglycan (HSPG)-related genes and eight TLE/Groucho transcriptional corepressors-related genes. In these gene sets, all homeologous pairs have been retained. Transcriptome analysis using RNA-seq data from developmental stages and adult tissues demonstrated that most homeologous pairs of signaling components have variable expression patterns, in contrast to the conservative expression profiles of homeologs for transcription factors. Our results indicate that homeologous gene pairs for cell signaling regulation have tended to become subfunctionalized after allotetraploidization. Diversification of signaling pathways by subfunctionalization of homeologs may enhance environmental adaptability. These results provide insights into the evolution of signaling pathways after polyploidization.

Patterns of clinical practice for sentinel lymph node biopsy in women with node-negative breast cancer: the results of a national survey in Japan.

PURPOSE: Sentinel lymph node (SLN) biopsy is now accepted as the standard of care for axillary staging in women with node-negative breast cancer. Currently, dye, radioisotope (RI), and fluorescence indocyanine green (fICG) are tracers available. Importance of these three tracers has been recognized for SLN biopsy but the trend for SLN mapping has not been reported. Aim of this national wide survey was to evaluate practice patterns of SLN biopsy in Japan. METHODS: This survey was conducted to examine the clinical practice of SLN biopsy in centers where one or more Japanese Breast Cancer Society (JBCS) board-certified surgeons practice breast cancer care. Their responses were recorded from 1 to 30 Oct 2014 and received by mail or fax in Japan. The questionnaire included three items: the number of breast cancer patients treated per year, the number of SLN biopsy procedures in a single year, and the methods for SLN detection. RESULTS: A total of 412 responses excluding the 63 centers that do not perform the surgery were analyzed. Out of them, 206 (50 %) centers had a gamma probe, 118 (29 %) had an NIR fluorescence imaging system, and both were available at 49 (12 %) of the centers. Neither RI nor fICG was available in 137 (33 %). The dye method was preferentially used in private hospitals. In 412 centers, a total of 36,221 patients underwent SLN biopsy per year and 23,038 (64 %) received radioactive tracer. fICG was co-applied with RI in 83 and 13 % of patients, respectively. Single mapping with RI alone was used in only 4 % of patients. The non-radioactive method was used for routine SLN biopsy in 13,183 (36 %) patients [8533 (24 %) for dye alone and 4650 (12 %) for fICG alone]. CONCLUSIONS: A radioactive tracer was used in 64 % of women with early breast cancer for SLN biopsy while approximately 24 % received dye alone, which was especially prevalent in PHs. The fICG was used in only 12 % as a non-radioactive method but incentive package for fICG by national health insurance plan could increase the number of NIR imaging systems and improve the sensitivity for SLN biopsy in Japan.

Sentinel lymph node biopsy using indocyanine green fluorescence in early-stage breast cancer: a meta-analysis.

Sentinel lymph node (SLN) biopsy using indocyanine green (ICG) fluorescence is safe and has a high detection rate for SLNs. However, the results of this novel technique are heterogeneous. The objective of this meta-analysis was to evaluate the diagnostic performance of the ICG fluorescence method compared with the standard radioisotope (RI) method. All eligible studies were identified from 2005 through 2015. A proportion meta-analysis was performed using a fixed effects and/or random effects model based on the study heterogeneity. A total of 12 studies met the inclusion criteria and included 1736 women. There was no significant difference between ICG fluorescence and RI for SLN detection using either the fixed effects model [odds ratio (OR) 1.29, 95% confidence interval (CI) 0.87-1.90] or the random effects model (OR 1.32, 95% CI 0.54-3.18). There were seven studies reporting the detection rate for tumor-positive SLN. The ICG fluorescence method was significantly better than the RI method in the fixed effects model (OR 1.87, 95% CI 1.00-3.49) for staging axilla. However, there was no difference in the random effects model (OR 1.90, 95% CI 0.74-4.86). There was study outcome heterogeneity for the detection of SLN but not for tumor-positive SLN. There was no publication bias observed in the studies included. The ICG fluorescence method has valid diagnostic performance for SLN detection and shows a trend toward better axilla staging compared with the RI method. ICG fluorescence is a useful alternative to RI for SLN biopsy.

Transcutaneous oxygen pressure as a surrogate index of lower limb amputation.

INTRODUCTION: A large number of clinical trials of therapeutic angiogenesis in patients with critical limb ischemia have been conducted in recent years. However, limb amputation, which is used as a primary endpoint in such studies, is not often required in Japan, which can make it difficult to carry out related clinical trials. Transcutaneous oxygen pressure (TcPO2) is widely used to evaluate the severity of limb ischemia, to decide the level of amputation, and to predict wound healing after limb amputation. The aim of the present study was to elucidate whether TcPO2 can be a surrogate index of limb ischemia, and to define an appropriate cutoff value for wound healing after limb amputation using meta-analysis. EVIDENCE ACQUISITION: A computer search was performed to identify studies describing the association between TcPO2 and limb ischemic events. From these, studies focused on wound healing after limb amputation were combined and analyzed. EVIDENCE SYNTHESIS: Eleven studies were identified for inclusion in this analysis. The analysis demonstrated that TcPO2 20 mmHg was a valid cutoff value for limb amputation and TcPO2 30 mmHg would be an appropriate value for wound healing after limb amputation. CONCLUSIONS: TcPO2 of 20 and 30 mmHg were considered appropriate cutoff values for limb amputation and wound healing after amputation, respectively.

Safety and efficacy of sustained release of basic fibroblast growth factor using gelatin hydrogel in patients with critical limb ischemia.

As a form of therapeutic angiogenesis, we sought to investigate the safety and efficacy of a sustained-release system of basic fibroblast growth factor (bFGF) using biodegradable gelatin hydrogel in patients with critical limb ischemia (CLI). We conducted a phase I-IIa study that analyzed 10 CLI patients following a 200-µg intramuscular injection of bFGF-incorporated gelatin hydrogel microspheres into the ischemic limb. Primary endpoints were safety and transcutaneous oxygen pressure (TcO2) at 4 and 24 weeks after treatment. During the follow-up, there was no death or serious procedure-related adverse event. After 24 weeks, TcO2 (28.4 ± 8.4 vs. 46.2 ± 13.0 mmHg for pretreatment vs after 24 weeks, p < 0.01) showed significant improvement. Regarding secondary endpoints, the distance walked in 6 min (255 ± 105 vs. 318 ± 127 m, p = 0.02), the Rutherford classification (4.4 ± 0.5 vs. 3.1 ± 1.4, p = 0.02), the rest pain scale (1.7 ± 1.0 vs. 1.2 ± 1.3, p = 0.03), and the cyanotic scale (2.0 ± 1.1 vs. 0.9 ± 0.9, p < 0.01) also showed improvement. The blood levels of bFGF were within the normal range in all patients. A subanalysis of patients with arteriosclerosis obliterans (n = 7) or thromboangiitis obliterans (Buerger's disease) (n = 3) revealed that TcO2 had significantly improved in both subgroups. TcO2 did not differ between patients with or without chronic kidney disease. The sustained release of bFGF from biodegradable gelatin hydrogel may offer a safe and effective form of angiogenesis for patients with CLI.

Evaluation of the Clinical Utility of the ICG Fluorescence Method Compared with the Radioisotope Method for Sentinel Lymph Node Biopsy in Breast Cancer.

PURPOSE: This study compared the clinical utility of indocyanine green (ICG) fluorescence and radioisotope (RI) for sentinel lymph node (SLN) detection in breast cancer. METHODS: Women with node-negative breast cancer underwent SLN biopsy using ICG fluorescence and RI. The primary end point was the sensitivity of ICG fluorescence compared with RI in the patients with tumor-positive SLNs. Secondary end points included detection rates for SLN, the additive effect of ICG fluorescence to RI, signature of positive SLNs according to tier, and adverse events related to ICG administration. RESULTS: A total of 847 women with clinical node-negative breast cancer underwent SLN biopsy, and 821 patients were included in the per-protocol analysis. SLN mapping was performed using ICG fluorescence and RI. The overall detection of SLNs using ICG fluorescence was identical to RI (97.2 vs. 97.0 %, P = 0.88), and the combination of both methods achieved a significant improvement compared with RI alone (99.8 vs. 97.0 %, P < 0.001). The detection rate for tumor-positive SLN was 93.3 % for ICG fluorescence and 90.0 % for RI, and the sensitivity of the ICG fluorescence method was 95.7 % (95 % CI 91.3-98.3, P = 0.11). The additional use of ICG significantly improved positive SLN detection for RI (97.2 vs. 90.0 %, P < 0.001). There were no serious adverse events related to hypersensitivity to ICG. CONCLUSIONS: The ICG fluorescence method may be an acceptable alternative to SLN detection using RI in breast cancer.