Single-cell transcriptome analysis reveals periodontal ligament fibroblast heterogeneity with distinct IL-1ß and RANKL expression in periodontitis.

Periodontitis (PD) is an inflammatory disease with alveolar bone destruction by osteoclasts (OCs). In PD, both inflammation and OC activation are significantly influenced by periodontal ligament fibroblasts (PDL-Fib). Yet, whether PDL-Fib has heterogeneity and whether distinct PDL-Fib subsets have specific functions have not been investigated. In this study, we discovered the complexity of PDL-Fib in PD, utilizing single-cell RNA sequencing data from human PD patients. We identified distinct subpopulations of PDL-Fib: one expressing interleukin-1 beta (IL-1ß) and another expressing the receptor activator of nuclear factor-kappa B ligand (RANKL), both crucial in OC differentiation and bone resorption. In periodontal tissues of mice with PD, active IL-1ß, cleaved caspase 1, and nucleotide-binding oligomerization domain-like receptor 3 (NLPR3) were significantly elevated, implicating the NLRP3 inflammasome in IL-1ß production. Upon stimulation of PDL-Fib with LPS from Porphyromonas gingivalis (pg), the most well-characterized periodontal bacteria, a more rapid increase in IL-1ß, followed by RANKL induction, was observed. IL-1ß and tumor necrosis factor alpha (TNF-α), another LPS-responsive cytokine, effectively increased RANKL in PDL-Fib, suggesting an indirect effect of pgLPS through IL-1ß and TNF-α on RANKL induction. Immunohistological analyses of mouse periodontal tissues also showed markedly elevated levels of IL-1ß and RANKL upon PD induction and displayed separate locations of IL-1ß-expressing PDL-Fib and RANKL-expressing PDL-Fib in PD. The heterogenic feature of fibroblasts expressing IL-1ß and RANKL was also mirrored in our combined cross-tissue single-cell RNA sequencing datasets analysis. In summary, our study elucidates the heterogeneity of PDL-Fib, highlighting distinct functional groups for producing RANKL and IL-1ß, which collectively promote OC generation and bone destruction in PD.

PINK1 restrains periodontitis-induced bone loss by preventing osteoclast mitophagy impairment.

The oral colonization of periodontal pathogens onto gingival tissues establishes hypoxic microenvironment, often disrupting periodontal homeostasis in conjunction with oxidative stress. The association between reactive oxygen species (ROS) and osteolytic periodontitis have been suggested by recent studies. PTEN-induced kinase 1 (PINK1), a mitochondrial serine/threonine kinase, is an essential protein for mitochondrial quality control as it protects cells from oxidative stress by promoting degradation of damaged mitochondria through mitophagy. However, the pathophysiological roles of PINK1 in osteoclast-mediated bone loss have not been explored. Here we aimed to determine whether PINK1 plays a role in the regulation of osteoclastogenesis and alveolar bone resorption associated with periodontitis. C57BL/6 wild type (WT) and Pink1 knockout (KO) mice were subjected to ligature-induced periodontitis (LIP), and alveolar bones were evaluated by µCT-analysis and tartrate-resistant acid phosphatase (TRAP) staining. The µCT-analysis showed that bone volume fraction and travecular thickness were lower in Pink1 KO compared to WT mice. The number of TRAP-positive osteoclasts was markedly increased in the periodontal tissues of Pink1 KO mice with LIP. The genetic silencing or deletion of Pink1 promoted excessive osteoclast differentiation and bone resorption in vitro, as respectively indicated by TRAP staining and resorption pits on dentin slices. PINK1 deficiency led to mitochondrial instabilities as indicated by confocal microscopy of mitochondrial ROS, mitochondrial oxygen consumption rate (OCR) analysis, and transmission electron microscopy (TEM). Consequently, a significant increase in Ca2+-nuclear factor of activated T cells 1 (NFATc1) signaling was also found. On the other hand, restoration of mitophagy and autophagy by spermidine (SPD) treatment and the resolution of oxidative stress by N-acetyl-l-cysteine (NAC) treatment protected PINK1 deficiency-induced excessive generation of osteoclasts. Taken together, our findings demonstrate that PINK1 is essential for maintaining mitochondrial homeostasis during osteoclast differentiation. Therefore, targeting PINK1 may provide a novel therapeutic strategy for severe periodontitis with fulminant osteolysis.

Unique expression and critical role of metallothionein 3 in the control of osteoclastogenesis and osteoporosis.

Bone homeostasis is maintained by an intricate balance between osteoclasts and osteoblasts, which becomes disturbed in osteoporosis. Metallothioneins (MTs) are major contributors in cellular zinc regulation. However, the role of MTs in bone cell regulation has remained unexplored. Single-cell RNA sequencing analysis discovered that, unlike the expression of other MT members, the expression of MT3 was unique to osteoclasts among various macrophage populations and was highly upregulated during osteoclast differentiation. This unique MT3 upregulation was validated experimentally and supported by ATAC sequencing data analyses. Downregulation of MT3 by gene knockdown or knockout resulted in excessive osteoclastogenesis and exacerbated bone loss in ovariectomy-induced osteoporosis. Transcriptome sequencing of MT3 knockdown osteoclasts and gene set enrichment analysis indicated that the oxidative stress and redox pathways were enriched, which was verified by MT3-dependent regulation of reactive oxygen species (ROS). In addition, MT3 deficiency increased the transcriptional activity of SP1 in a manner dependent on intracellular zinc levels. This MT3-zinc-SP1 axis was crucial for the control of osteoclasts, as zinc chelation and SP1 knockdown abrogated the promotion of SP1 activity and osteoclastogenesis by MT3 deletion. Moreover, SP1 bound to the NFATc1 promoter, and overexpression of an inactive SP1 mutant negated the effects of MT3 deletion on NFATc1 and osteoclastogenesis. In conclusion, MT3 plays a pivotal role in controlling osteoclastogenesis and bone metabolism via dual axes involving ROS and SP1. The present study demonstrated that MT3 elevation is a potential therapeutic strategy for osteolytic bone disorders, and it established for the first time that MT3 is a crucial bone mass regulator.

Extracellular vesicles from periodontal pathogens regulate hepatic steatosis via Toll-like receptor 2 and plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is associated with nonalcoholic fatty liver disease (NAFLD) by lipid accumulation in the liver. In this study, we showed that extracellular vesicles (EVs) from the periodontal pathogens Filifactor alocis and Porphyromonas gingivalis induced steatosis by inducing PAI-1 in the liver and serum of mice fed a low-fat diet. PAI-1 induction was not observed in TLR2-/- mice. When tested using HEK-Blue hTLR2 cells, human TLR2 reporter cells, the TLR2-activating ability of serum from NAFLD patients (n = 100) was significantly higher than that of serum from healthy subjects (n = 100). Correlation analysis confirmed that PAI-1 levels were positively correlated with the TLR2-activating ability of serum from NAFLD patients and healthy subjects. Amphiphilic molecules in EVs were involved in PAI-1 induction. Our data demonstrate that the TLR2/PAI-1 axis is important for hepatic steatosis by EVs of periodontal pathogens.

PARK2 Induces Osteoclastogenesis through Activation of the NF-κB Pathway.

Osteoclast generation from monocyte/macrophage lineage precursor cells needs to be tightly regulated to maintain bone homeostasis and is frequently over-activated in inflammatory conditions. PARK2, a protein associated with Parkinson's disease, plays an important role in mitophagy via its ubiquitin ligase function. In this study, we investigated whether PARK2 is involved in osteoclastogenesis. PARK2 expression was found to be increased during the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. PARK2 gene silencing with siRNA significantly reduced osteoclastogenesis induced by RANKL, LPS (lipopolysaccharide), TNFα (tumor necrosis factor α), and IL-1ß (interleukin-1ß). On the other hand, overexpression of PARK2 promoted osteoclastogenesis. This regulation of osteoclastogenesis by PARK2 was mediated by IKK (inhibitory κB kinase) and NF-κB activation while MAPK (mitogen-activated protein kinases) activation was not involved. Additionally, administration of PARK2 siRNA significantly reduced osteoclastogenesis and bone loss in an in vivo model of inflammatory bone erosion. Taken together, this study establishes a novel role for PARK2 as a positive regulator in osteoclast differentiation and inflammatory bone destruction.

Effects of extracellular vesicles derived from oral bacteria on osteoclast differentiation and activation.

Dysbiosis of the oral microbiota plays an important role in the progression of periodontitis, which is characterized by chronic inflammation and alveolar bone loss, and associated with systemic diseases. Bacterial extracellular vesicles (EVs) contain various bioactive molecules and show diverse effects on host environments depending on the bacterial species. Recently, we reported that EVs derived from Filifactor alocis, a Gram-positive periodontal pathogen, had osteoclastogenic activity. In the present study, we analysed the osteoclastogenic potency and immunostimulatory activity of EVs derived from the Gram-negative periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia, the oral commensal bacterium Streptococcus oralis, and the gut probiotic strain Lactobacillus reuteri. Bacterial EVs were purified by density gradient ultracentrifugation using OptiPrep (iodixanol) reagent. EVs from P. gingivalis, T. forsythia, and S. oralis increased osteoclast differentiation and osteoclstogenic cytokine expression in osteoclast precursors, whereas EVs from L. reuteri did not. EVs from P. gingivalis, T. forsythia, and S. oralis preferentially activated Toll-like receptor 2 (TLR2) rather than TLR4 or TLR9, and induced osteoclastogenesis mainly through TLR2. The osteoclastogenic effects of EVs from P. gingivalis and T. forsythia were reduced by both lipoprotein lipase and polymyxin B, an inhibitor of lipopolysaccharide (LPS), while the osteoclastogenic effects of EVs from S. oralis were reduced by lipoprotein lipase alone. These results demonstrate that EVs from periodontal pathogens and oral commensal have osteoclastogenic activity through TLR2 activation by lipoproteins and/or LPS.

20-hydroxyecdysone (20E) signaling regulates amnioserosa morphogenesis during Drosophila dorsal closure: EcR modulates gene expression in a complex with the AP-1 subunit, Jun.

Steroid hormones influence diverse biological processes throughout the animal life cycle, including metabolism, stress resistance, reproduction, and lifespan. In insects, the steroid hormone, 20-hydroxyecdysone (20E), is the central hormone regulator of molting and metamorphosis, and plays roles in tissue morphogenesis. For example, amnioserosa contraction, which is a major driving force in Drosophila dorsal closure (DC), is defective in embryos mutant for 20E biosynthesis. Here, we show that 20E signaling modulates the transcription of several DC participants in the amnioserosa and other dorsal tissues during late embryonic development, including zipper, which encodes for non-muscle myosin. Canonical ecdysone signaling typically involves the binding of Ecdysone receptor (EcR) and Ultraspiracle heterodimers to ecdysone-response elements (EcREs) within the promoters of responsive genes to drive expression. During DC, however, we provide evidence that 20E signaling instead acts in parallel to the JNK cascade via a direct interaction between EcR and the AP-1 transcription factor subunit, Jun, which together binds to genomic regions containing AP-1 binding sites but no EcREs to control gene expression. Our work demonstrates a novel mode of action for 20E signaling in Drosophila that likely functions beyond DC, and may provide further insights into mammalian steroid hormone receptor interactions with AP-1.

Upfront radiosurgery plus targeted agents followed by active brain control using radiosurgery delays neurological death in non-small cell lung cancer with brain metastasis.

The role of radiosurgery has become further accentuated in the era of targeted agents (TA). Thus, the neurologic outcome of radiosurgery in brain metastasis (BM) of non-small cell lung cancer (NSCLC) was reviewed. We analyzed 135 patients with BM of NSCLC who were administered Cyberknife radiosurgery (CKRS) as either initial or salvage therapy. We evaluated local failure (LF), intracranial failure (IF), and neurological death (ND) due to BM. Primary outcome was neurological death-free survival (NDFS). Median follow-up was 16.2 months. Median CKRS dose of 22 Gy was administered to median 2 targets per patient. Among 99 deaths, 14 (14%) were ND. Upfront treatment for BM included CKRS alone in 85 patients (63%), CKRS + TA in 26 patients (19%), and WBRT in 24 patients (18%). No patients or tumor related factors were associated with ND. However, the type of upfront treatment for BM was significantly associated with ND [HR 0.07 (95% CI 0.01-0.57) for CKRS + TA, HR 0.56 (95% CI 0.19-1.68) for CKRS alone] compared with the WBRT group (P = 0.01). The 2-year NDFS rates for the CKRS + TA, CRKS alone, and WBRT groups were 94%, 87%, and 78%, respectively (P = 0.03). Upfront CKRS showed significantly higher 2-year LF-free survival rate (P < 0.01). IF rate was insignificantly lower in the WBRT group compared with CKRS group (P = 0.38). Upfront CKRS + TA was associated with the best neurological outcome with high NDFS. Active brain control by early delivery of radiosurgery could achieve better neurological outcome in NSCLC with BM.

Association of concurrent hepatitis B surface antigen and antibody to hepatitis B surface antigen with hepatocellular carcinoma in chronic hepatitis B virus infection.

Antibody to hepatitis B surface antigen (HBsAg) (anti-HBs) can exist in patients with chronic hepatitis B virus (HBV) infection. To date, little is known about the association of concurrent HBsAg and anti-HBs (concurrent HBsAg/ anti-HBs) with hepatocellular carcinoma (HCC). The aim of this study was to investigate the clinical relevance of concurrent HBsAg/anti-HBs with preS deletion mutations and HCC in chronic HBV infection. A total of 755 patients with chronic HBV infection were included consecutively at a tertiary center. Logistic regression analysis was used to identify risk factors for HCC, and serum HBV DNA was amplified, followed by direct sequencing to detect preS deletions. The prevalence of concurrent HBsAg/anti-HBs was 6.4% (48/755) and all HBVs tested were genotype C. HCC occurred more frequently in the concurrent HBsAg/anti-HBs group than in the HBsAg only group [22.9% (11/48) vs. 7.9% (56/707), P = 0.002]. In multivariate analyses, age >40 years [odds ratio (OR), 14.712; 95% confidence interval (CI), 4.365-49.579; P < 0.001], male gender (OR 2.431; 95% CI, 1.226-4.820; P = 0.011), decompensated cirrhosis (OR, 3.642; 95% CI, 1.788-7.421; P < 0.001) and concurrent HBsAg/anti-HBs (OR, 4.336; 95% CI, 1.956-9.613; P < 0.001) were associated independently with HCC. In molecular analysis, preS deletion mutations were more frequent in the concurrent HBsAg/anti-HBs and HCC groups than in the HBsAg without HCC group (42.3% and 32.5% vs. 11.3%; P = 0.002 and 0.012, respectively). In conclusion, concurrent HBsAg/anti-HBs is associated with preS deletion mutations and may be one of the risk factors for HCC in chronic HBV infection with genotype C.

Prognostic values of alpha-fetoprotein and protein induced by vitamin K absence or antagonist-II in hepatitis B virus-related hepatocellular carcinoma: a prospective study.

BACKGROUND/AIM: Alpha-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist-II (PIVKA-II) are most widely used tumor markers in detecting hepatocellular carcinoma (HCC). Recently, there have been some studies about them as prognostic markers in hepatitis C virus-associated HCC. However, prognostic values of AFP and PIVKA-II remain clarified in hepatitis B virus (HBV)-associated HCC. This study was aimed to evaluate the prognostic values of AFP and PIVKA-II in HBV-associated HCC. METHODS: Patients (n=126) were divided into 4 groups according to median levels of AFP and PIVKA-II (L; low/low, A; high/low, P; low/high, H; high/high) at diagnosis. Clinical characteristics and survival were compared among the groups, and Cox regression analysis was performed to find independent factors for survival. RESULTS: Baseline host and viral factors were not significantly different among the 4 groups. High PIVKA-II groups (P and H) had more aggressive tumor characteristics (larger size of tumors, higher number of tumors, frequent portal vein thrombosis, P<0.05) and much shorter median survival time than low PIVKA-II groups (L and A) (P<0.05). In multivariate analysis, high PIVKA-II level was an independent predictor for survival (risk ration: 2.377, 95% confidence interval: 1.359-4.157, P=0.002) together with Child-Pugh score, advanced TMN stages, and treatment modality. Even after excluding 33 patients who had Child-Pugh class C and advanced tumor stages (tumor-nodes-metastasis stage III-IV) at diagnosis, high PIVKA-II level was still an independent predictor for survival (risk ration: 4.258, 95% confidence interval: 2.418-8.445, P<0.001). CONCLUSIONS: Serum PIVKA-II level, not serum AFP, was a valuable independent prognostic factor in HBV-related HCC.

Stereotactic body radiation therapy with or without transarterial chemoembolization for patients with primary hepatocellular carcinoma: preliminary analysis.

BACKGROUND: The objectives of this retrospective study was to evaluate the efficacy of stereotactic body radiation therapy (SBRT) for small non-resectable hepatocellular carcinoma (HCC) and SBRT combined with transarterial chemoembolization (TACE) for advanced HCC with portal vein tumor thrombosis (PVTT). METHODS: Thirty one patients with HCC who were treated with SBRT were used for the study. We studied 32 HCC lesions, where 23 lesions (22 patients) were treated targeting small non-resectable primary HCC, and 9 lesions (9 patients) targeting PVTT using the Cyberknife. All the 9 patients targeting PVTT received TACE for the advanced HCC. Tumor volume was 3.6-57.3 cc (median, 25.2 cc) and SBRT dose was 30-39 Gy (median, 36 Gy) in 3 fractions for consecutive days for 70-85% of the planned target volume. RESULTS: The median follow up was 10.5 months. The overall response rate was 71.9% [small HCC: 82.6% (19/23), advanced HCC with PVTT: 44.4% (4/9)], with the complete and partial response rates of 31.3% [small HCC: 26.1% (6/23), advanced HCC with PVTT: 11.1% (1/9)], and 50.0% [small HCC: 56.5% (13/23), advanced HCC with PVTT: 33.3% (3/9)], respectively. The median survival period of small HCC and advanced HCC with PVTT patients was 12 months and 8 months, respectively. No patient experienced Grade 4 toxicity. CONCLUSION: SBRT for small HCC and SBRT combined with TACE for advanced HCC with PVTT showed feasible treatment modalities with minimal side effects in selected patients with primary HCC.

Polymorphisms of interleukin-1 and interleukin-2 genes in patients with gastric cancer in Korea.

BACKGROUND AND AIM: Interleukin (IL)-1 gene polymorphism has been reported to be associated with the increment of gastric cancer (GC) and the decrement of duodenal ulcers (DU). In addition, IL-2 is known to induce Helicobacter pylori (H. pylori)-associated gastric atrophy, but it is not known whether IL-2 gene polymorphism increases the risk of GC (GC) or peptic ulcer diseases. Therefore, we compared the genotypes of IL-1B, IL-1RN, and IL-2 gene polymorphisms with risk of gastric ulcers (GU), GC, and DU in Korean patients. METHODS: In total, 116 GU, 122 GC, and 104 DU patients were included consecutively and compared with 100 healthy controls. Polymorphisms of the IL-1B-511/-31 gene, the penta-allelic variable number of tandem repeats of the IL-1RN gene, and the IL-2-330 gene were analyzed by polymerase chain reaction with restriction fragment length polymorphism or confronting two-pair primers methods. RESULTS: The age-sex-adjusted odds ratios (OR) for the IL-1B-511 T genotype relative to the C/C genotype (OR = 0.82, 95% confidence interval [CI] 0.41-1.65), IL-1RN*2 genotype relative to the L/L genotype (OR = 0.85, 95% CI 0.41-1.78), and IL-2-330 T genotype relative to the G/G genotype (OR = 1.94, 95% CI 0.76-4.96) were not increased in GC. There was also no significant difference in the genotypes of these cytokine polymorphisms between the study group (GU or DU) and control group. In addition, genotypic frequency was not associated with H. pylori positivity and histological type of GC. CONCLUSION: IL-1B-511, IL-1RN, and IL-2 genetic polymorphisms were not important contributors to the pathogenesis of GU, GC, and DU in Korean patients.

Stereotactic radiosurgery for brain metastasis in non-small cell lung cancer.

PURPOSE: Stereotactic radiosurgery (SRS) has been introduced for small-sized single and oligo-metastases in the brain. The aim of this study is to assess treatment outcome, efficacy, and prognostic variables associated with survival and intracranial recurrence. MATERIALS AND METHODS: This study retrospectively reviewed 123 targets in 64 patients with non-small cell lung cancer (NSCLC) treated with SRS between January 2006 and December 2012. Treatment responses were evaluated using magnetic resonance imaging. Overall survival (OS) and intracranial progression-free survival (IPFS) were determined. RESULTS: The median follow-up was 13.9 months. The median OS and IPFS were 14.1 and 8.9 months, respectively. Fifty-seven patients died during the follow-up period. The 5-year local control rate was achieved in 85% of 108 evaluated targets. The 1- and 2-year OS rates were 55% and 28%, respectively. On univariate analysis, primary disease control (p < 0.001), the Eastern Cooperative Oncology Group (ECOG) performance status (0-1 vs. 2; p = 0.002), recursive partitioning analysis class (1 vs. 2; p = 0.001), and age (<65 vs. ≥65 years; p = 0.036) were significant predictive factors for OS. Primary disease control (p = 0.041) and ECOG status (p = 0.017) were the significant prognostic factors for IPFS. Four patients experienced radiation necrosis. CONCLUSION: SRS is a safe and effective local treatment for brain metastases in patients with NSCLC. Uncontrolled primary lung disease and ECOG status were significant predictors of OS and intracranial failure. SRS might be a tailored treatment option along with careful follow-up of the intracranial and primary lung disease status.

Stereotactic body radiotherapy for prostate cancer: a preliminary report.

AIMS: We report the results of a retrospective study of stereotactic body radiotherapy (SBRT) using a Cyberknife for prostate cancer. METHODS: In all 29 patients were treated with hypofractionated SBRT using a Cyberknife at median 36 Gy in five fractions. All the patients were treated with a radical aim. Prostate-specific antigen (PSA) was evaluated at baseline and after radiotherapy. Acute (≤3 months) and late (>3 months) urinary and rectal toxicities were recorded according to the CTCAE version 4.0. RESULTS: The median duration of follow up was 41 months. PSA values decreased in a time-dependent way. The median PSA nadir was 0.329 ng/mL, achieved after a median of 23 months' follow up. Two patients had a PSA failure according to the definition of nadir + 2 ng/mL. Eight patients (28%) had a benign PSA bounce at median 9 months after radiotherapy. CTCAE Grade 2 and 3 late urinary toxicities were reported in 3 and 3%, respectively. One patient had exacerbated urinary symptoms and received an operation. There were no severe late rectal toxicities. CONCLUSIONS: The preliminary findings of our study suggest SBRT is feasible for prostate cancer treatment. Further studies with more patients and longer follow-up duration are required.

Predictable factors of histologic discrepancy of gastric cancer between the endoscopic forceps biopsy and endoscopic treatment specimen.

BACKGROUND/AIMS: Recently, variable gastrointestinal track tumors including early stage malignancies are treated by endoscopic procedure. However, the discrepancy of histologic diagnosis may sometimes exist between the pretreatment forceps biopsy results and those of post treatment specimen. So the prediction of malignant lesion is important in the aspect of treatment selection. In this study, we investigated the predictable factors of the histologic discrepancy through the clinical, endoscopic features of the lesion diagnosed as adenocarcinoma in the post-endoscopic treatment specimen after the adenoma was diagnosed by the endoscopic forceps biopsy. METHODS: From March 2005 to April 2009, 129 gastric tumor lesions (129 patients) which were not diagnosed as malignancy and treated with endoscopic procedure were enrolled retrospectively. We compared the pretreatment endoscopic forceps biopsy results and post-treatment specimen biopsy results, then, analyzed the tumor characteristics. RESULTS: Twenty-one cases (16.3%) were diagnosed as malignancy after endoscopic treatment. Especially, discrepancy occurred more frequently in depressed lesions than in flat or elevated lesions (41.7% vs. 13.7%, p=0.012), and in lesions diagnosed as high grade adenomas than low or moderate grade adenomas (33.3% vs. 11.1%. p=0.004). CONCLUSIONS: In cases of depressed type lesions in the pretreatment endoscopy or those diagnosed as high grade adenoma in the pretreatment forceps biopsy, we should consider combined malignant lesion. Therefore, treatment modalities ensuring accurate diagnosis and potentially curative resection, should be carefully selected and performed in cases which have these features.

Long-term outcome and toxicity of hypofractionated stereotactic body radiotherapy as a boost treatment for head and neck cancer: the importance of boost volume assessment.

BACKGROUND: The aim of this study was to report the long-term clinical outcomes of patients who received stereotactic body radiotherapy (SBRT) as a boost treatment for head and neck cancer. MATERIALS AND METHODS: Between March 2004 and July 2007, 26 patients with locally advanced, medically inoperable head and neck cancer or gross residual tumors in close proximity to critical structures following head and neck surgery were treated with SBRT as a boost treatment. All patients were initially treated with standard external beam radiotherapy (EBRT). SBRT boost was prescribed to the median 80% isodose line with a median dose of 21 (range 10-25) Gy in 2-5 (median, 5) fractions. RESULTS: The median follow-up after SBRT was 56 (range 27.6 - 80.2) months. The distribution of treatment sites in 26 patients was as follows: the nasopharynx, including the base of the skull in 10 (38.5%); nasal cavity or paranasal sinus in 8 (30.8%); periorbit in 4 (15.4%); tongue in 3 (11.5%); and oropharyngeal wall in 1 (3.8%). The median EBRT dose before SBRT was 50.4 Gy (range 39.6 - 70.2). The major response rate was 100% with 21 (80.8%) complete responses (CR). Severe (grade ≥ 3) late toxicities developed in 9 (34.6%) patients, and SBRT boost volume was a significant parameter predicting severe late complication. CONCLUSIONS: The present study demonstrates that a modern SBRT boost is a highly efficient tool for local tumor control. However, we observed a high frequency of serious late complications. More optimized dose fractionation schedule and patient selection are required to achieve excellent local control without significant late morbidities in head and neck boost treatment.

Stereotactic body radiotherapy for patients with unresectable primary hepatocellular carcinoma: dose-volumetric parameters predicting the hepatic complication.

PURPOSE: To identify the parameters that predict hepatic toxicity and deterioration of hepatic function. MATERIALS AND METHODS: A total of 47 patients with small unresectable primary hepatocellular carcinoma received hypofractionated stereotactic body radiotherapy (SBRT) using the CyberKnife. Of those, 36 patients received no other local treatments that could influence hepatic toxicity at least for 3 months after the completion of SBRT. The gross tumor volume (GTV) was 18.3 ± 15.9 cm(3) (range, 3.0-81.3 cm(3)), and the total dose administered was 30-39 Gy (median, 36 Gy). To assess the deterioration of hepatic function, we evaluated the presence or absence of the progression of Child-Pugh class (CP class). To identify the parameters of predicting the radiation-induced hepatic toxicity and deterioration of the hepatic function, several clinical and dose-volumetric parameters were evaluated. RESULTS: Of 36 patients, 12 (33%) developed Grade 2 or higher hepatic toxicity and 4 (11%) developed progression of CP class. The multivariate analysis showed that the only significant parameter associated with the progression of CP class was the total liver volume receiving a dose less than 18 Gy (<18 Gy). CONCLUSIONS: The progression of CP class after SBRT limits other additional local treatments and also reflects the deterioration of hepatic function. Therefore, it would be important to note that the presence or absence of the progression of CP class is a dose-limiting factor. The total liver volume receiving <18 Gy should be greater than 800 cm(3) to reduce the risk of the deterioration of hepatic function.

Fractionated stereotactic radiotherapy as reirradiation for locally recurrent head and neck cancer.

PURPOSE: We report early preliminary experience with CyberKnife radiosurgery (RS) as salvage treatment for locally recurrent head and neck cancer (HNC). METHODS AND MATERIALS: Between March 2004 and August 2006, 36 patients (44 sites) were treated with CyberKnife RS as reirradiation for locally recurrent HNC. Treatment sites were as follows: nasopharynx (8), maxillary sinus (8), neck lymph nodes (8), skull base (7), nasal cavity (4), retropharyngeal lymph nodes (3), orbit (2), and others (4). Total doses administered were 18-40 Gy (median, 30 Gy) in 3 to 5 fractions to the 65%-85% isodose line for 3-5 consecutive days. Previous external radiation dose ranged from 39.6 to 134.4 Gy (median, 70.2 Gy). Gross tumor volume ranged from 0.2 to 114.9 cm(3) (median, 22.6 cm(3)). Median follow-up was 17.3 months. RESULTS: Thirty-five of 44 sites were evaluated for response. Fifteen (42.9%) sites achieved complete response, 13 sites (37.1%) achieved a partial response, 3 (8.6%) sites maintained stable disease, and 4 sites (11.4%) showed tumor progression. Grade III acute complications were noted in 13 patients. Late complications were observed in three patients (1 bone necrosis, 2 soft tissue necrosis) during follow-up. CONCLUSION: These preliminary results suggest that fractionated stereotactic radiosurgery is an effective treatment modality as a salvage treatment with good short-term local control. The early overall response rate is encouraging. However, more experience and a longer follow-up are necessary to determine the role of fractionated stereotactic radiosurgery as a salvage treatment of locally recurrent HNC and to define long-term complications.

Initial thrombocytopenia as a simple, valuable predictor for clinical manifestation in acute hepatitis A.

OBJECTIVE: Although acute hepatitis A (AH-A) is usually self-limited, the clinical manifestations can vary from mild to severe liver dysfunction. However, little is known about the simple, valuable predictors for clinical manifestation in AH-A. The objective of this study was to identify the simple clinical predictors for severe liver dysfunction and its clinical course. MATERIAL AND METHODS: A total of 162 IgM anti-hepatitis A virus (HAV) positive patients were enrolled in the study. Severe AH-A was defined as a prothrombin time <40% of control activity. Various liver-unrelated and liver-related parameters at presentation were compared separately between the severe AH-A group and the non-severe group. RESULTS: Mean age (+/-SD) was 27.5 (+/-7.1) years and the proportion of males was 54% (88/162). Fourteen patients (8.7%) experienced severe AH-A. Of the liver-unrelated parameters, leukopenia (<4000/microl), thrombocytopenia (<150,000/microl), and high serum C-reactive protein levels (>8 mg/l) at presentation were significant predictors for severe AH-A in a univariate analysis (p<0.05). On multivariate analysis, only thrombocytopenia was an independent predictor for severe AH-A (odds ratio (OR) 5.562, 95% confidence interval (CI) 1.153-26.834, p=0.033). Of the liver-related parameters, there were no independent predictors, as shown by multivariate analysis. The thrombocytopenia group (33%, 54/162) not only had a longer recovery time (28 days (range, 14-140) versus 37 days (20-128), p<0.001), but also more frequent complications (OR 4.632, 95% CI 1.886-11.372, p=0.001) than the non-thrombocytopenia group. CONCLUSIONS: Initial thrombocytopenia may be a simple, valuable predictor for severity and clinical course in AH-A.

Identification of parasite DNA in common bile duct stones by PCR and DNA sequencing.

We attempted to identify parasite DNA in the biliary stones of humans via PCR and DNA sequencing. Genomic DNA was isolated from each of 15 common bile duct (CBD) stones and 5 gallbladder (GB) stones. The patients who had the CBD stones suffered from cholangitis, and the patients with GB stones showed acute cholecystitis, respectively. The 28S and 18S rDNA genes were amplified successfully from 3 and/or 1 common bile duct stone samples, and then cloned and sequenced. The 28S and 18S rDNA sequences were highly conserved among isolates. Identity of the obtained 28S D1 rDNA with that of Clonorchis sinensis was higher than 97.6%, and identity of the 18S rDNA with that of other Ascarididae was 97.9%. Almost no intra-specific variations were detected in the 28S and 18S rDNA with the exception of a few nucleotide variations, i.e., substitution and deletion. These findings suggest that C. sinensis and Ascaris lumbricoides may be related with the biliary stone formation and development.