Structural basis for SARS-CoV-2 neutralizing antibodies with novel binding epitopes.

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) threatens global public health and economy unprecedentedly, requiring accelerating development of prophylactic and therapeutic interventions. Molecular understanding of neutralizing antibodies (NAbs) would greatly help advance the development of monoclonal antibody (mAb) therapy, as well as the design of next generation recombinant vaccines. Here, we applied H2L2 transgenic mice encoding the human immunoglobulin variable regions, together with a state-of-the-art antibody discovery platform to immunize and isolate NAbs. From a large panel of isolated antibodies, 25 antibodies showed potent neutralizing activities at sub-nanomolar levels by engaging the spike receptor-binding domain (RBD). Importantly, one human NAb, termed PR1077, from the H2L2 platform and 2 humanized NAb, including PR953 and PR961, were further characterized and subjected for subsequent structural analysis. High-resolution X-ray crystallography structures unveiled novel epitopes on the receptor-binding motif (RBM) for PR1077 and PR953, which directly compete with human angiotensin-converting enzyme 2 (hACE2) for binding, and a novel non-blocking epitope on the neighboring site near RBM for PR961. Moreover, we further tested the antiviral efficiency of PR1077 in the Ad5-hACE2 transduction mouse model of COVID-19. A single injection provided potent protection against SARS-CoV-2 infection in either prophylactic or treatment groups. Taken together, these results shed light on the development of mAb-related therapeutic interventions for COVID-19.

Screening of Botanical Drugs against Lassa Virus Entry.

Lassa virus (LASV) belongs to the Old World Mammarenavirus genus (family Arenaviridae). At present, there are no approved drugs or vaccines specific for LASV. In this study, high-throughput screening of a botanical drug library was performed against LASV entry using a pseudotype virus bearing the LASV envelope glycoprotein complex (GPC). Two hit compounds, bergamottin and casticin, were identified as micromolar range inhibitors of LASV entry. A mechanistic study revealed that casticin inhibited LASV entry by blocking low pH-induced membrane fusion. Analysis of adaptive mutants demonstrated that the F446L mutation, located in the transmembrane domain of GP2, conferred resistance to casticin. Furthermore, casticin antiviral activity extends to the New World (NW) pathogenic mammarenaviruses, and mutation of the conserved F446 also conferred resistance to casticin in these viruses. Unlike casticin, bergamottin showed little effect on LASV GPC-mediated membrane fusion, instead inhibiting LASV entry by blocking endocytic trafficking. Notably, both compounds showed inhibitory effects on authentic lymphocytic choriomeningitis virus. Our study shows that both casticin and bergamottin are candidates for LASV therapy and that the conserved F446 in LASV GPC is important in drug resistance in mammarenaviruses.IMPORTANCE: Currently, there is no approved therapy to treat Lassa fever (LASF). Our goal was to identify potential candidate molecules for LASF therapy. Herein, we screened a botanical drug library and identified two compounds, casticin and bergamottin, that inhibited LASV entry via different mechanisms.

Crystal Structure of African Swine Fever Virus pS273R Protease and Implications for Inhibitor Design.

African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs that is responsible for serious economic and production losses. It is caused by the African swine fever virus (ASFV), a large and complex icosahedral DNA virus of the Asfarviridae family. Currently, there is no effective treatment or approved vaccine against the ASFV. pS273R, a specific SUMO-1 cysteine protease, catalyzes the maturation of the pp220 and pp62 polyprotein precursors into core-shell proteins. Here, we present the crystal structure of the ASFV pS273R protease at a resolution of 2.3 Å. The overall structure of the pS273R protease is represented by two domains named the "core domain" and the N-terminal "arm domain." The "arm domain" contains the residues from M1 to N83, and the "core domain" contains the residues from N84 to A273. A structure analysis reveals that the "core domain" shares a high degree of structural similarity with chlamydial deubiquitinating enzyme, sentrin-specific protease, and adenovirus protease, while the "arm domain" is unique to ASFV. Further, experiments indicated that the "arm domain" plays an important role in maintaining the enzyme activity of ASFV pS273R. Moreover, based on the structural information of pS273R, we designed and synthesized several peptidomimetic aldehyde compounds at a submolar 50% inhibitory concentration, which paves the way for the design of inhibitors to target this severe pathogen.IMPORTANCE African swine fever virus, a large and complex icosahedral DNA virus, causes a deadly infection in domestic pigs. In addition to Africa and Europe, countries in Asia, including China, Vietnam, and Mongolia, were negatively affected by the hazards posed by ASFV outbreaks in 2018 and 2019, at which time more than 30 million pigs were culled. Until now, there has been no vaccine for protection against ASFV infection or effective treatments to cure ASF. Here, we solved the high-resolution crystal structure of the ASFV pS273R protease. The pS273R protease has a two-domain structure that distinguishes it from other members of the SUMO protease family, while the unique "arm domain" has been proven to be essential for its hydrolytic activity. Moreover, the peptidomimetic aldehyde compounds designed to target the substrate binding pocket exert prominent inhibitory effects and can thus be used in a potential lead for anti-ASFV drug development.

Screening and Identification of Lassa Virus Entry Inhibitors from an FDA-Approved Drug Library.

Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae) and causes severe hemorrhagic fever in humans. At present, there are no Food and Drug Administration (FDA)-approved drugs or vaccines specific for LASV. Here, high-throughput screening of an FDA-approved drug library was performed against LASV entry by using pseudotype virus bearing LASV envelope glycoprotein (GPC). Two hit compounds, lacidipine and phenothrin, were identified as LASV entry inhibitors in the micromolar range. A mechanistic study revealed that both compounds inhibited LASV entry by blocking low-pH-induced membrane fusion. Accordingly, lacidipine showed virucidal effects on the pseudotype virus of LASV. Adaptive mutant analyses demonstrated that replacement of T40, located in the ectodomain of the stable-signal peptide (SSP), with lysine (K) conferred LASV resistance to lacidipine. Furthermore, lacidipine showed antiviral activity against LASV, the closely related Mopeia virus (MOPV), and the New World arenavirus Guanarito virus (GTOV). Drug-resistant variants indicated that V36M in the ectodomain of the SSP mutant and V436A in the transmembrane domain of the GP2 mutant conferred GTOV resistance to lacidipine, suggesting the interface between SSP and GP2 is the target of lacidipine. This study shows that lacidipine is a candidate for LASV therapy, reinforcing the notion that the SSP-GP2 interface provides an entry-targeted platform for arenavirus inhibitor design.IMPORTANCE Currently, there is no approved therapy to treat Lassa fever; therefore, repurposing of approved drugs will accelerate the development of a therapeutic stratagem. In this study, we screened an FDA-approved library of drugs and identified two compounds, lacidipine and phenothrin, which inhibited Lassa virus entry by blocking low-pH-induced membrane fusion. Additionally, both compounds extended their inhibition against the entry of Guanarito virus, and the viral targets were identified as the SSP-GP2 interface.

Comparison of 3D and 2D gamma passing rate criteria for detection sensitivity to IMRT delivery errors.

This study compared three-dimensional (3D) and two-dimensional (2D) percentage gamma passing rates (%GPs) for detection sensitivity to IMRT delivery errors and investigated the correlation between two kinds of %GP. Eleven prostate IMRT cases were selected, and errors in multileaf collimator (MLC) bank sag, MLC leaf traveling, and machine output were simulated by recalculating the dose distributions in patients. 2D doses were extracted from the 3D doses at the isocenter position. The 3D and 2D %GPs with different gamma criteria were then obtained by comparing the recalculated and original doses in specific regions of interest (ROI), such as the whole body, the planning target volume (PTV), the bladder, and the rectum. The sensitivities to simulated errors of the two types of %GP were compared, and the correlation between the 2D and 3D %GPs for different ROIs were analyzed. For the whole-body evaluation, both the 2D and 3D %GPs with the 3%/3 mm criterion were above 90% for all tested MLC errors and for MU deviations up to 4%, and the 3D %GP was higher than the 2D %GP. In organ-specific evaluations, the PTV-specific 2D and 3D %GP gradients were -4.70% and -5.14% per millimeter of the MLC traveling error, and -17.79% and -20.50% per percentage of MU error, respectively. However, a stricter criterion (2%/1 mm) was needed to detect the tested MLC sag error. The Pearson correlation analysis showed a significant strong correlation (r > 0.8 and P < 0.001) between the 2D and 3D %GPs in the whole body and PTV-specific gamma evaluations. The whole-body %GP with the 3%/3 mm criterion was inadequate to detect the tested MLC and MU errors, and a stricter criterion may be needed. The PTV-specific gamma evaluation helped to improve the sensitivity of the error detection, especially using the 3D GP%.

Cryptotanshinone enhances the effect of Arsenic trioxide in treating liver cancer cell by inducing apoptosis through downregulating phosphorylated- STAT3 in vitro and in vivo.

BACKGROUND: Arsenic trioxide (ATO) is approved for treating terminal-stage liver cancer in China. Cryptotanshinone (CT), a STAT3 inhibitor, has exhibited certain anti-tumor potency; however, the use of CT enhanced ATO for treating liver cancer has not been reported. Here we try to elucidate how CT could enhance the efficacy of ATO for treating liver cancer and its correlation to STAT3 in vitro and in vivo. METHODS: Cell viability of ATO combined with CT was assessed by 1MTT assay. Cell apoptosis induced by ATO combined with CT was detected by Annexin V/PI staining and apoptosis-related proteins were detected by western blotting. STAT3-related proteins were analysis by western blotting analysis and Immunofluorescence assays. Efficacy evaluation of ATO combined with CT on xenograft was carried in nude mice and related proteins were analysis by Immunohistochemistry assays. RESULTS: First we evaluated cell vitality, and our data indicated that the ATO combined with CT showed obvious growth inhibition of Bel-7404 cells compared to ATO or CT alone. Next we found that ATO combined with CT induced cell apoptosis in Bel-7404 cells and upregulated the activation of apoptosis-related proteins cleaved-caspase-3, cleaved-caspase-9, and cleaved-poly(ADP-ribose) polymerase in a time-dependent manner. Next, we found that ATO combined with CT not only inhibited the constitutive levels of phosphorylated-JAK2 and phosphorylated-STAT3Tyr705 but did so in a time-dependent manner. We also found that ATO combined with CT reversed the upregulated expression of phosphorylated-STAT3Tyr705 stimulated by interleukin-6 and downregulated STAT3 direct target genes and the anti-apoptotic proteins Bcl-2, XIAP, and survivin but obviously upregulated the promoting apoptosis proteins Bak,.In vivo studies showed that ATO combined with CT decreased tumor growth. Tumors from ATO combined with CT-treated mice showed decreased levels of phosphorylated-STAT3Tyr705 and the anti-apoptotic protein Bcl-2 but an increased level of pro-apoptotic protein Bax. CONCLUSIONS: Our study provides strong evidence that CT could enhance the efficacy of ATO in treating liver cancer both in vitro and in vivo. Downregulation of phosphorylated-STAT3 expression may play an important role in inducing apoptosis of Bel-7404 cells.

Dosimetric benefit to organs at risk following margin reductions in nasopharyngeal carcinoma treated with intensity-modulated radiation therapy.

INTRODUCTION: It is important to decrease the radiation exposure of normal tissue in intensity-modulated radiation therapy (IMRT). Minimizing planning target volume (PTV) margins with more precise target localization techniques can achieve this goal. This study aimed to quantify the extent to which organs at risk (OARs) are spared when using reduced margins in the treatment of nasopharyngeal carcinoma (NPC). METHODS: Two IMRT plans were regenerated for 40 patients with NPC based on two PTV margins, which were reduced or unchanged following cone beam computed tomography online correction. The reduced-margin plan was optimized based on maximal dose reduction to OARs without compromising target coverage. Dosimetric comparisons were evaluated in terms of target coverage and OAR sparing. RESULTS: Improvements in target coverage occurred with margin reduction, and significant improvements in dosimetric parameters were observed for all OARs (P < 0.05) except for the right optic nerve, chiasm, and lens. Doses to OARs decreased at a rate of 1.5% to 7.7%. Sparing of the left parotid and right parotid, where the mean dose (Dmean) decreased at a rate of 7.1% and 7.7%, respectively, was greater than the sparing of other OARs. CONCLUSIONS: Significant improvements in OAR sparing were observed with margin reduction, in addition to improvement in target coverage. The parotids benefited most from the online imaging-guided approach.

Discovery and characterization of potent pan-variant SARS-CoV-2 neutralizing antibodies from individuals with Omicron breakthrough infection.

The SARS-CoV-2 Omicron variant evades most currently approved neutralizing antibodies (nAbs) and caused drastic decrease of plasma neutralizing activity elicited by vaccination or prior infection, urging the need for the development of pan-variant antivirals. Breakthrough infection induces a hybrid immunological response with potentially broad, potent and durable protection against variants, therefore, convalescent plasma from breakthrough infection may provide a broadened repertoire for identifying elite nAbs. We performed single-cell RNA sequencing (scRNA-seq) and BCR sequencing (scBCR-seq) of B cells from BA.1 breakthrough-infected patients who received 2 or 3 previous doses of inactivated vaccine. Elite nAbs, mainly derived from the IGHV2-5 and IGHV3-66/53 germlines, showed potent neutralizing activity across Wuhan-Hu-1, Delta, Omicron sublineages BA.1 and BA.2 at picomolar NT50 values. Cryo-EM analysis revealed diverse modes of spike recognition and guides the design of cocktail therapy. A single injection of paired antibodies cocktail provided potent protection in the K18-hACE2 transgenic female mouse model of SARS-CoV-2 infection.

Impact on xerostomia for nasopharyngeal carcinoma patients treated with superficial parotid lobe-sparing intensity-modulated radiation therapy (SPLS-IMRT): A prospective phase II randomized controlled study.

PURPOSE: To compare the incidence of xerostomia in nasopharyngeal carcinoma (NPC) patients treated with superficial parotid lobe-sparing intensity-modulated radiation therapy (SPLS-IMRT) and conventional IMRT (C-IMRT). METHODS: Patients with histologically confirmed NPC who met the eligibility criteria were randomly assigned to receive either SPLS-IMRT or C-IMRT. The primary endpoint was the incidence of xerostomia at 12 months post-IMRT. The secondary endpoints included the xerostomia questionnaire (XQ) score, unstimulated salivary flow rate (USFR), stimulated salivary flow rate (SSFR), and survival outcomes. RESULTS: Ninety patients were enrolled. Eighty-two patients were included for xerostomia analysis (42 in the SPLS-IMRT group and 40 in the C-IMRT group). At 12 months post-IMRT, the incidence of xerostomia in the SPLS-IMRT group was significantly lower than that in the C-IMRT group (83.4% vs 95.0%; P = 0.007), especially the grade 3 xerostomia (0% vs 12.5%; P < 0.001). The median change in XQ score was similar between the two groups (11.9 points vs 14.1 points; P = 0.194). There was a significantly higher median fractional USFR (0.67 vs 0.35; P = 0.024) and SSFR (0.66 vs 0.32; P = 0.021) in the SPLS-IMRT group than the C-IMRT group. The 3-year LRRFS, DMFS, and OS in the SPLS-IMRT and C-IMRT groups were 92.5% vs 90.9%, 83.8% vs 81.7%, and 88.9% vs 88.2% (all P > 0.05). CONCLUSION: SPLS-IMRT significantly reduced the incidence of xerostomia at 12 months post-IMRT in NPC by recovering parotid gland function earlier than C-IMRT, without compromising survivals. Phase III clinical trials are warranted. (ClinicalTrials.gov, number NCT05020067).

Multivariate NTCP Model of Hypothyroidism After Intensity-Modulated Radiotherapy for Nasopharyngeal Carcinoma.

OBJECTIVE: To evaluate the incidence of hypothyroidism in patients with nasopharyngeal carcinoma after intensity-modulated radiotherapy (IMRT), analyze its correlation with multiple influencing factors such as thyroid exposure dose, thyroid volume, and gender, and construct a multivariate-based normal tissue complication probability (NTCP) model for the occurrence of hypothyroidism after IMRT. MATERIALS AND METHODS: The thyroid hormone levels of patients at different points in time before and after radiotherapy were tested, and statistics on the incidence of hypothyroidism after treatment were obtained. The dose-volume data of patients' thyroids were converted into EQD2 equivalent dose values. The correlation between hypothyroidism after radiotherapy and thyroid exposure dose, thyroid volume, gender, and other factors was analyzed, and an NTCP model was constructed. RESULTS: A total of 69 patients with nasopharyngeal carcinoma were enrolled in this study. Twelve months after radiotherapy, a total of 24 patients (34.8%) developed hypothyroidism. Univariate analysis and multivariate analysis revealed that the average thyroid dose and thyroid volume are the most important factors affecting hypothyroidism after radiotherapy. The NTCP model constructed based on the average dose and thyroid volume has a good degree of fit. CONCLUSION: The volume and average dose of the thyroid gland are the key factors affecting the occurrence of hypothyroidism in patients with nasopharyngeal carcinoma after radiotherapy. The NTCP model constructed based on multivariate construction suggests that reducing the average dose of the thyroid to the greatest extent is an effective way to protect thyroid functions.

Characterizing the Lassa Virus Envelope Glycoprotein Membrane Proximal External Region for Its Role in Fusogenicity.

The membrane-proximal external region (MPER) of Lassa virus (LASV) glycoprotein complex (GPC) is critical in modulating its functionality. Till now, the high-resolution structure of the intact GPC, including MPER is not available. In this study, we used alanine substitution to scan all 16 residues located in LASV MPER. Western blotting and quantification fusion assay showed that the residues located at the C terminus of the HR2 (M414 and L415) and N terminus of the MPER (K417 and Y419) are critical for GPC-mediated membrane fusion function. Furthermore, cell surface biotinylation experiments revealed that M414A, K417A and Y419A expressed similar levels as WT, whereas L415A mutant led to a reduction of mature GPC on the cell surface. Moreover, substitution of these residues with the similar residue such as M414L, L415I, K417R and Y419F would partly compensate the loss of the fusion activity caused by the alanine mutant in these sites. Results from this study showed that several key residues in the MPER region are indispensable to promote the conformational changes that drive fusion events and shed light on the structure analysis of LASV GPC and anti-LASV therapeutics.

A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes.

COVID-19 pandemic caused by SARS-CoV-2 constitutes a global public health crisis with enormous economic consequences. Monoclonal antibodies against SARS-CoV-2 can provide an important treatment option to fight COVID-19, especially for the most vulnerable populations. In this work, potent antibodies binding to SARS-CoV-2 Spike protein were identified from COVID-19 convalescent patients. Among them, P4A1 interacts directly with and covers majority of the Receptor Binding Motif of the Spike Receptor-Binding Domain, shown by high-resolution complex structure analysis. We further demonstrate the binding and neutralizing activities of P4A1 against wild type and mutant Spike proteins or pseudoviruses. P4A1 was subsequently engineered to reduce the potential risk for Antibody-Dependent Enhancement of infection and to extend its half-life. The engineered antibody exhibits an optimized pharmacokinetic and safety profile, and it results in complete viral clearance in a rhesus monkey model of COVID-19 following a single injection. These data suggest its potential against SARS-CoV-2 related diseases.

Efficacy and safety of primary surgery with postoperative radiotherapy in head and neck mucosal melanoma: a single-arm Phase II study.

BACKGROUND: There still remains no well-established treatment strategy for head and neck mucosal melanoma (HNMM). We aim to evaluate the effectiveness and safety of primary surgery with postoperative radiotherapy for this disease. PATIENTS AND METHODS: A single-arm, Phase II clinical trial was conducted at Sun Yat-Sen University Cancer Center. Patients with nonmetastatic, histologically proven HNMM were prospectively enrolled. Patients received primary surgery followed by intensity-modulated radiotherapy with an equivalent dose at 2 Gy per fraction of 65-70 Gy to CTV1 (high-risk regions including tumor bed) and 50-55 Gy to CTV2 (low-risk regions). Additional use of adjuvant chemotherapy (AC) depended on consultation from a multidisciplinary team. This trial is registered with ClinicalTrials.gov, number NCT03138642. RESULTS: A total of 33 patients were enrolled and analyzed between July 2010 and November 2016. There were 18 (54.5%) patients with T3 disease and 15 (45.5%) patients with T4a disease. The median age at diagnosis was 58 years (range 27-83 years), and 61% of the cohort were males. The overall median follow-up duration was 25.3 months (range 5.3-67.1 months). The 3-year overall survival (OS), local relapse-free survival (LRFS), regional relapse-free survival (RRFS), and distant metastasis-free survival (DMFS) rates were 44.4, 91.7, 78.1, and 41.7%, respectively. Patients with T4a disease showed significantly inferior OS (P=0.049) and DMFS (P=0.040) than those with T3 disease. Prophylactic neck radiation (PNR) was nearly associated with superior RRFS (P=0.078). However, there was no significant difference in OS, LRFS, RRFS, and DMFS for patients treated with or without AC (P>0.05 for all). Toxicities were generally mild to moderate. CONCLUSION: Primary surgery with postoperative radiotherapy yielded excellent local control and acceptable toxicity profile for HNMM. Nevertheless, high rates of distant metastases resulted in limited survival.

Temporal lobe injury patterns following intensity modulated radiotherapy in a large cohort of nasopharyngeal carcinoma patients.

OBJECTIVES: To analyze the correlation between dose-volume-histograms (DVHs) with three patterns (edema, enhancement, and necrosis) of temporal lobe injury (TLI) in patients receiving intensity modulated radiation therapy (IMRT) for nasopharyngeal carcinoma (NPC) and to determine optimal thresholds to predict the incidence of each TLI pattern, with particular emphasis on the relationship between edema volume and the risk of enhancement and necrosis. MATERIALS AND METHODS: A cohort of 4186 NPC patients treated with IMRT was retrospectively reviewed with TLI presenting in 188 patients. The atlases of complication incidence (ACI) for each pattern were constructed using DVH curves of temporal lobes. Optimal threshold for predicting incidence of each pattern was determined using the point closest to top-left of the plot. The accuracy of using edema volume to predict enhancement and necrosis incidence was evaluated via area under curve (AUC) of receiver operator characteristics (ROC). RESULTS: All DVH parameters, Dmean, Dmax, D0.25cc, D0.5cc, D1cc, D3cc, D6cc, V20Gy, V30Gy, V40Gy, V50Gy, V60Gy, and V70Gy, except Dmin showed statistically significant differences between subgroups of each pattern (p < 0.05). For predicting incidence of each pattern, optimal DVH thresholds over the range of D0.25-D1cc, Dmean and V20-V70 were derived. The optimal thresholds of edema volume for predicting enhancement were 0.96 and 2.2cc and for predicting necrosis were 0.94 and 11.5cc. CONCLUSION: Optimal DVH thresholds were generated for limiting risk of each injury pattern. Edema volume was a strong predictor for risk of enhancement and necrosis, which could potentially be reduced by lowering edema volume below threshold.

Diterpenoid Tanshinones, the extract from Danshen (Radix Salviae Miltiorrhizae) induced apoptosis in nine human cancer cell lines.

OBJECTIVE: To identify the active anti-tumor constituents in the extract from Danshen (Radix Salviae Miltiorrhizae) and investigate the mechanisms underlying the actions. METHODS: First, we introduced a two-step counter- current chromatography to extract the therapeutically active diterpenoid, tanshinone from Danshen (Radix Salviae Miltiorrhizae). The cholecystokinin (CCK-8) method was used to evaluate the inhibitory effect of diterpenoid tanshinone in liver cancer QGY-7703, lung cancer PC9, lung cancer A549, gastric cancer MKN-45, gastric cancer HGC-27, colon cancer HCT116, myeloma cellU266/ RPMI8226, and human breast cancer MCF-7 in vitro. Fluorescence staining was used to observe the cytotoxicity ofditerpenoid tanshinone on PC9 cells. The Western blot was used to detect apoptosis- related protein poly ADP-ribose polymerase (PARP), cysteinyl aspartate specific proteinase3/9 (caspase3/9), and cleaved-cysteinyl aspartate specific proteinase3/9 (cleaved-caspase3/9). The endoplasmic reticulum stress-related activating transcription factor 4 (ATF4), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), and phosphorylated jun amino-terminal kinase (p-JNK), and caspase- 12 were also analyzed using the Western blot. RESULTS: Diterpenoid tanshinone inhibited the nine human tumor cell lines, with an IC50 of 4.37-29 µg/mL, with the PC9 and MCF-7 displaying the lowest values. Fluorescence staining showed a lethal effect of diterpenoid tanshinone on PC9 cells. The Western blot showed that the expression of caspase3/9 protein and ATF-4 protein decreased gradually. However, the PARP, cleaved-caspase 3/9 and the expression of p-eIF2 α, P-JNK, and caspase- 12 increased gradually, in a dose-dependent fashion. CONCLUSION: We successfully introduced a two-step counter-current chromatography method to extract diterpenoid tanshinone, and demonstrated its antitumor activity. Diterpenoid tanshinone can induce apoptosis in nine human cancer cell lines.

Reduction of clinical target volume in patients with lateralized cancer of the nasopharynx and without contralateral lymph node metastasis receiving intensity-modulated radiotherapy.

BACKGROUND: This study investigated lymph node distribution in patients with lateralized cancer of the nasopharynx to identify areas suitable for clinical target volume (CTV) reduction. METHODS: A total of 1680 patients with nasopharyngeal carcinoma (NPC) whose tumor involvement was assessed by MRI were reviewed retrospectively. RESULTS: One hundred and twelve patients (7%) had a lateralized primary lesion. Of these, 9 patients (8%) had contralateral lymph nodes including 4 (4%) who had contralateral cervical lymph nodes (CLNs). The rates of contralateral level III/Va metastasis were <1% (1 of 104) and 12.5% (1 of 8) in patients without and with contralateral retropharynx/level II involvement, respectively. No known risk factors were significantly associated with contralateral lymph node metastasis. CONCLUSION: Reduced cervical CTV coverage, including the contralateral level II, is feasible in patients with lateralized primary NPC, which may help to better protect the cervical OAR, including the thyroid, larynx, and esophagus. © 2015 Wiley Periodicals, Inc. Head Neck 38: E468-E472, 2016.

Multi-subject atlas-based auto-segmentation reduces interobserver variation and improves dosimetric parameter consistency for organs at risk in nasopharyngeal carcinoma: A multi-institution clinical study.

BACKGROUND AND PURPOSE: To assess whether consensus guideline-based atlas-based auto-segmentation (ABAS) reduces interobserver variation and improves dosimetric parameter consistency for organs at risk (OARs) in nasopharyngeal carcinoma (NPC). MATERIALS AND METHODS: Eight radiation oncologists from 8 institutes contoured 20 OARs on planning CT images of 16 patients via manual contouring and manually-edited ABAS contouring. Interobserver variation [volume coefficient of variation (CV), Dice similarity coefficient (DSC), three-dimensional isocenter difference (3D-ICD)] and dosimetric parameters were compared between the two methods of contouring for each OAR. RESULTS: Interobserver variation was significant for all OARs in manual contouring, resulting in significant dosimetric parameter variation (P<0.05). Edited ABAS significantly improved multiple metrics and reduced dosimetric parameter variation for most OARs; brainstem, spinal cord, cochleae, temporomandibular joint (TMJ), larynx and pharyngeal constrictor muscle (PCM) obtained most benefit (range of mean DSC, volume CV and main ICD values was 0.36-0.83, 12.1-84.3%, 2.2-5.0mm for manual contouring and 0.42-0.86, 7.2-70.6%, 1.2-3.5mm for edited ABAS contouring, respectively; range of dose CV reduction: 1.0-3.0%). CONCLUSION: Substantial objective interobserver differences occur during manual contouring, resulting in significant dosimetric parameter variation. Edited ABAS reduced interobserver variation and improved dosimetric parameter consistency, particularly for brainstem, spinal cord, cochleae, TMJ, larynx and PCM.