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The Children's Oncology Group (COG) uses Clinical Group (CG) and modified Tumor Node Metastasis (TNM) stage to classify rhabdomyosarcoma (RMS). CG is based on surgicopathologic findings and is determined after the completion of initial surgical procedure(s) but prior to chemotherapy and/or radiation therapy. The modified TNM stage is based on clinical and radiographic findings and is assigned prior to any treatment. These systems have evolved over several decades. We review the history, evolution, and rationale behind the current CG and modified TNM classification systems used by COG for RMS. Data from the seven most recently completed and reported frontline COG trials (D9602, D9802, D9803, ARST0331, ARST0431, ARST0531, ARST08P1) were analyzed, and confirm that CG and modified TNM stage remain relevant and useful for predicting prognosis in RMS. We propose updates based on recent data and discuss factors warranting future study to further optimize these classification systems.

Background Esophageal cancer (EC) is one of the aggressive gastrointestinal malignancies. It has been reported that microRNAs (miRNAs) play key roles during the tumorigenesis of EC. To identify novel potential targets for EC, differential expressed miRNAs (DEG) between EC and adjacent normal tissues were analyzed with bioinformatics tool. Methods The differential expression of miRNAs between EC and adjacent normal tissues was analyzed. CCK-8 and Ki67 staining were used to detect the cell proliferation. Flow cytometry was performed to test the cell apoptosis. The correlation between miR-7-5p and KLF4 was detected by dual-luciferase report assay. Gene and protein expression in EC cells or in tissues were measured by qRT-PCR and Western blot, respectively. Cell migration and invasion were detected with transwell assay. Xenograft mice model was established to investigate the role of miR-7-5p in EC tumorigenesis in vivo. Results MiR-7-5p was found to be negatively correlated with the survival rate of patient with EC. In addition, downregulation of miR-7-5p significantly inhibited the growth and invasion of EC cells. Meanwhile, miR-7-5p directly targeted KLF4 in EC cells. Moreover, downregulation of miR-7-5p inhibited the tumorigenesis of EC via inactivating MAPK signaling pathway in vivo. Conclusion Downregulation of miR-7-5p notably suppressed the progression of EC via targeting KLF4. Thus, miR-7-5p might serve as a new target for the treatment of EC.

11504 Background: Pazopanib is a multi-targeted tyrosine kinase inhibitor (TKI) with activity in advanced soft tissue sarcoma. ARST1321 was a phase II study designed to compare the near complete pathologic response rate (≥ 90% necrosis) following preoperative chemoradiation +/- pazopanib in children and adults with intermediate/high risk chemotherapy-sensitive body wall/extremity NRSTS. Enrollment was stopped early following a predetermined interim analysis that found the rate of near complete pathologic response to be significantly greater with the addition of pazopanib. As a planned secondary analysis of the study, we now report the outcome data for this cohort. Methods: ARST1321 was a jointly designed COG and NRG Oncology study open to enrollment July 2014-October 2018. Eligible adult (≥18 years) and pediatric (< 18 years) patients with newly-diagnosed unresected body wall/extremity NRSTS were enrolled into the Chemotherapy Cohort (> 5 cm, FNCLCC grade 2/3, protocol-designated chemotherapy-sensitive histology). Following a dose-finding phase, patients were randomized to receive (Regimen A) or not receive (Regimen B) pazopanib (< 18 years: 350 mg/m2/day; ≥ 18 years: 600 mg/day) in combination with ifosfamide (7.5 gm/m2/cycle) and doxorubicin (75 mg/m2/cycle) + 45 Gy preoperative RT followed by primary resection at week 13, then further chemotherapy to week 25. Results: Eighty-five eligible patients were enrolled in the Chemotherapy Cohort and randomized to receive or not receive pazopanib. Median age 22.1 years (range: 5.7-64.2 years); 30 patients < 18 years. Most common histologies were synovial sarcoma (n = 42) and undifferentiated pleomorphic sarcoma (n = 19). As of December 31, 2021, at a median survivor follow-up of 3.3 years (range: 0.1 – 5.8 years), the 3-year event-free survival (EFS) for all patients in the intent-to-treat analysis was 52.5% (95% CI: 34.8%-70.2%) for Regimen A and 50.6% (32%-69.2%) for Regimen B (p = 0.8677); 3-year overall survival (OS) was 75.7% (59.7%-91.7%) for Regimen A and 65.4% (48.1%-82.7%) for Regimen B (p = 0.1919). Conclusions: Although the rate of near complete pathologic response was significantly greater with the addition of pazopanib to preoperative chemoradiation in children and adults with intermediate/high risk body wall/extremity NRSTS, outcomes were not statistically significantly different between the two regimens. Pathologic response could be a TKI-related phenomenon and may not be a good surrogate marker of outcome in future studies. Clinical trial information: NCT02180867.

This study aimed to investigate the function of long non-coding RNA FOXD2 adjacent opposite strand RNA 1 (lncRNA FOXD2-AS1) during the progression of esophagus cancer (EC) and explore its underlying molecular mechanisms. The level of FOXD2-AS1 in EC tissues and paracancerous tissues was detected by using RT-qPCR; ROC curve was used to evaluate the diagnostic value of FOXD2-AS1 for EC. In addition, CCK8 assay and immunofluorescence staining assay were used to detect the proliferation of Eca-109 and TE-1 cells. To investigate the function of FOXD2-AS1 on cell apoptosis and cell cycle, flow cytometry was performed. To detect the invasion ability of EC cells, transwell invasion assay was performed. Starbase3.0 and Targetscan were used to predict the target genes of FOXD2-AS1 and miR-145-5p, and protein expressions were detected with western blot. We found FOXD2-AS1 was significantly upregulated in EC tissues compared with adjacent normal tissues, which was positively correlated with clinicopathological parameters of patients with EC. Downregulation of FOXD2-AS1 inhibited the proliferation and invasion by inducing apoptosis of EC cells. Moreover, FOXD2-AS1 may regulate the expression of CDK6 by targeting miR-145-3p. Meanwhile, silencing of FOXD2-AS1 caused G1 phase arrest of EC cells by reducing the expression of CDK6. In conclusion, silening FOXD2-AS1 significantly inhibited the proliferation and invasion of EC cells by regulating the miR-145-5p/CDK6 axis. Therefore, FOXD2-AS1 might be used as diagnostic biomarker and therapeutic target for EC.

δ-tocotrienol (DT3), a member of vitamin E family, has been shown to have potent radio-protective effect. However, its application as a radioprotectant has limited, at least in part, by its short plasma elimination half-life and low bioavailability. In an effort to increase the metabolic stability of DT3, a deuterium substituted DT3 derivative, d(6)-DT3, was designed and synthesized. d(6)-DT3 showed improved in vitro and in vivo metabolic stability compared to DT3. The unexpected lower potency of d(6)-DT3 in inducing granulocyte-colony stimulating factor (G-CSF) production in mouse revealed that the metabolite(s) of DT3 might play a major role in inducing G-CSF induction.

Background: Non-small cell lung carcinoma (NSCLC) is a common malignant tumor with poor prognosis. CircRNA-100876 has been considered to be involved in NSCLC. However, the mechanism by which circRNA_100876 mediated the progression of NSCLC remains unclear. Methods: CCK8 assay and immunofluorescence were used to detect cell proliferation. Flow cytometry and transwell assay were performed to analyze cell apoptosis, migration and invasion, respectively. Verification of possible target for circRNA_100876 and related miR-636 were done using luciferase assay. In addition, western blot was performed to detect the protein expressions in NSCLC cells. Results: Silencing of circRNA_100876 notably inhibited the proliferation of NSCLC cells. Moreover, downregulation of circRNA_100876 significantly induce the apoptosis of NSCLC cells via mediation of apoptosis-related proteins. In addition, silencing of circRNA_100876 significantly inhibited migration and invasion of NSCLC cells. MiR-636 was the downstream target of circRNA_100876. Meanwhile, RET was the direct target of miR-636. Finally, circRNA_100876 shRNA2 notably suppressed the progression of NSCLC through PI3K/Akt signaling. Conclusion: CircRNA_100876 knockdown notably suppressed the progression of NSCLC through regulation of miR-636/RET axis, which may serve as a potential target for treatment of NSCLC.

We attempt to dissect the pathology of non-small cell lung cancer (NSCLC) patients at different stages and discover the novel candidate genes. Microarray data (GSE21933) were retrieved from the Gene Expression Omnibus database. The differential expression profiles of lung tumor tissues during different stages were analyzed. The significantly altered functions and pathways were assessed and the key nodes in a protein-protein interaction (PPI) network were screened out. Then, the coexpression gene pairs and tumor-related genes were assessed. RT-PCR analysis was performed to validate the candidate gene, natural killer-tumor recognition sequence (NKTR). The number of differentially expressed genes (DEGs) for stage IB, IIB, IIIA and IV tumors were 499, 602, 592 and 457, respectively. Most of the DEGs were NSCLC-related genes identified through literature research. A few genes were commonly downregulated in all the 4 stages of tumors, such as CNTN6 and LBX2. The DEGs in early‑stage tumors were closely related with the negative regulation of signal transduction, the apoptosis pathway and the p53 signaling pathway. DEGs in late-stage tumors were significantly enriched in transcription, response to organic substances and synapse regulation-related biological processes. A total of 16 genes (including NKTR) made up the significant coexpression network. NKTR was a key node in the PPI network and was significantly upregulated in lung cancer cells. The mechanism of NSCLC progression in different tumor stages may be different. NKTR may be the target candidate for NSCLC prevention and treatment.

The thermodynamics of the interaction between Ca(2+) and calmodulin (CaM) was examined using isothermal titration calorimetry (ITC). The chemical denaturation of calmodulin was monitored spectroscopically to determine the stability of Ca(2+)-free (apo) and Ca(2+)-loaded (holo) CaMs. We explored the conformational and structural dynamics of CaM using amide hydrogen-deuterium (H-D) exchange coupled with Fourier transform infrared (FT-IR) spectroscopy. The results of H-D exchange and FT-IR suggest that CaM activation by Ca(2+) binding involves significant conformational changes. The results have also revealed that while the overall conformation of holo-CaM is more stable than that of the apo-CaM, some part of its α-helix structures, most likely the EF-hand domain region, has more solvent exposure, thus, has a faster H-D exchange rate than that of the apo-CaM. The ITC method provides a new strategy for obtaining site-specific Ca(2+) binding properties and a better estimation of the cooperativity and conformational change contributions of coupled EF-hand proteins.

The cAMP receptor protein (CRP) requires cAMP for an allosteric change and regulates more than 150 genes in Escherichia coli. In this study, the modular half of cAMP receptor protein was used to investigate the allosteric signal transmission pathway induced by cAMP binding. The activation of CRP upon cAMP binding is indicated to be realignment of the two subunits within the CRP dimer. The interaction of loop 3 and Phe136 do not involve in signal transmission.

Circular dichroism (CD) is a spectroscopic technique widely used for estimating protein secondary structures in aqueous solution, but its accuracy has been doubted in recent work. In the present paper, the contents of nine globular proteins with known secondary structures were determined by CD spectroscopy and Fourier transform infrared spectroscopy (FTIR) in aqueous solution. A large deviation was found between the CD spectra and X-ray data, even when the experimental conditions were optimized. The content determined by FTIR was in good agreement with the X-ray crystallography data. Therefore, CD spectra are not recommended for directly calculating the content of a protein's secondary structure.

The X-ray crystal structure of the cAMP-liganded D138L mutant of Escherichia coli catabolite gene activator protein (CAP) was determined at a resolution of 1.66 A. This high resolution crystal structure reveals four cAMP binding sites in the homodimer. Two anti conformations of cAMPs ( anti -cAMP) locate between the β-barrel and the C-helix of each subunit; two syn conformations of cAMPs ( syn -cAMP) bind on the surface of the C-terminal domain. With two syn -cAMP molecules bound, the D138L CAP is highly symmetrical with both subunits assuming a “closed” conformation. These differences make the hinge region of the mutant more flexible. Protease susceptibility measurements indicate that D138L is more susceptible to proteases than that of wild type (WT) CAP. The results of protein dynamic experiments (H/D exchange measurements) indicate that the structure of D138L mutant is more dynamic than that of WT CAP, which may impact the recognition of specific DNA sequences.

Circular dichroism (CD) is a spectroscopic technique widely used for estimating protein secondary structures in aqueous solution, but its accuracy has been doubted in recent work. In the present paper, the contents of nine globular proteins with known secondary structures were determined by CD spectroscopy and Fourier transform infrared spectroscopy (FTIR) in aqueous solution. A large deviation was found between the CD spectra and X-ray data, even when the experimental conditions were optimized. The content determined by FTIR was in good agreement with the X-ray crystallography data. Therefore, CD spectra are not recommended for directly calculating the content of a protein’s secondary structure.