Comprehensive genomic variation profiling of cervical intraepithelial neoplasia and cervical cancer identifies potential targets for cervical cancer early warning.

BACKGROUND: To better understand the pathogenesis of cervical cancer (CC), we systematically analysed the genomic variation and human papillomavirus (HPV) integration profiles of cervical intraepithelial neoplasia (CIN) and CC. METHODS: We performed whole-genome sequencing or whole-exome sequencing of 102 tumour-normal pairs and human papillomavirus probe capture sequencing of 45 CCs, 44 CIN samples and 25 normal cervical samples, and constructed strict integrated workflow of genomic analysis. RESULTS: Mutational analysis identified eight significantly mutated genes in CC including four genes (FAT1, MLL3, MLL2 and FADD), which have not previously been reported in CC. Targetable alterations were identified in 55.9% of patients. In addition, HPV integration breakpoints occurred in 97.8% of the CC samples, 70.5% of the CIN samples and 42.8% of the normal cervical samples with HPV infection. Integrations of high-risk HPV strains in CCs, including HPV16, 18, 33 and 58, also occurred in the CIN samples. Moreover, gene mutations were detected in 52% of the CIN specimens, and 54.8% of these mutations occurred in genes that also mutated in CCs. CONCLUSION: Our results lay the foundation for a deep understanding of the molecular mechanisms and finding new diagnostic and therapeutic targets of CC.

[Nonparametric method of estimating survival functions containing right-censored and interval-censored data].

Missing data represent a general problem in many scientific fields, especially in medical survival analysis. Dealing with censored data, interpolation method is one of important methods. However, most of the interpolation methods replace the censored data with the exact data, which will distort the real distribution of the censored data and reduce the probability of the real data falling into the interpolation data. In order to solve this problem, we in this paper propose a nonparametric method of estimating the survival function of right-censored and interval-censored data and compare its performance to SC (self-consistent) algorithm. Comparing to the average interpolation and the nearest neighbor interpolation method, the proposed method in this paper replaces the right-censored data with the interval-censored data, and greatly improves the probability of the real data falling into imputation interval. Then it bases on the empirical distribution theory to estimate the survival function of right-censored and interval-censored data. The results of numerical examples and a real breast cancer data set demonstrated that the proposed method had higher accuracy and better robustness for the different proportion of the censored data. This paper provides a good method to compare the clinical treatments performance with estimation of the survival data of the patients. This pro vides some help to the medical survival data analysis.

Excess multi-cause mortality linked to influenza virus infection in China, 2012-2021: a population-based study.

Objectives: The aim of this study is to estimate the excess mortality burden of influenza virus infection in China from 2012 to 2021, with a concurrent analysis of its associated disease manifestations. Methods: Laboratory surveillance data on influenza, relevant population demographics, and mortality records, including cause of death data in China, spanning the years 2012 to 2021, were incorporated into a comprehensive analysis. A negative binomial regression model was utilized to calculate the excess mortality rate associated with influenza, taking into consideration factors such as year, subtype, and cause of death. Results: There was no evidence to indicate a correlation between malignant neoplasms and any subtype of influenza, despite the examination of the effect of influenza on the mortality burden of eight diseases. A total of 327,520 samples testing positive for influenza virus were isolated between 2012 and 2021, with a significant decrease in the positivity rate observed during the periods of 2012-2013 and 2019-2020. China experienced an average annual influenza-associated excess deaths of 201721.78 and an average annual excess mortality rate of 14.53 per 100,000 people during the research period. Among the causes of mortality that were examined, respiratory and circulatory diseases (R&C) accounted for the most significant proportion (58.50%). Fatalities attributed to respiratory and circulatory diseases exhibited discernible temporal patterns, whereas deaths attributable to other causes were dispersed over the course of the year. Conclusion: Theoretically, the contribution of these disease types to excess influenza-related fatalities can serve as a foundation for early warning and targeted influenza surveillance. Additionally, it is possible to assess the costs of prevention and control measures and the public health repercussions of epidemics with greater precision.

Characterization of CagI in the cag pathogenicity island of Helicobacter pylori.

Helicobacter pylori is a highly successful human-specific gastric pathogen that infects up to 50% of the world's population. Virulent H. pylori isolates harbor the cytotoxin-associated genes pathogenicity island (cag-PAI), which encodes a type IV secretion system that translocates bacterial effector (e.g., CagA oncoprotein) molecules into host cells. Although some cag-PAI genes are shown to be required for CagA delivery or localization, the majority have no known function. In the current study, the authors performed a cell components fractionation assay and showed that CagI, one of the cag-PAI proteins located in the bacterial membrane, was not translocated into host cells. The homologous recombination method then was used to construct the isogenic mutant of H. pylori cagI, and the translocation assay was performed. The results showed that the isogenic mutant of H. pylori NCTC 11637 cagI could cause a reduction in the degree of CagA translocation. Overall, the results suggested that CagI might be an accessory component of the CagA secretion system not translocated into host cells and that it is located in the bacterial membrane.

Hedgehog signaling plays a conserved role in inhibiting fat formation.

Hedgehog (Hh) signals regulate invertebrate and vertebrate development, yet the role of the cascade in adipose development was undefined. To analyze a potential function, we turned to Drosophila and mammalian models. Fat-body-specific transgenic activation of Hh signaling inhibits fly fat formation. Conversely, fat-body-specific Hh blockade stimulated fly fat formation. In mammalian models, sufficiency and necessity tests showed that Hh signaling also inhibits mammalian adipogenesis. Hh signals elicit this function early in adipogenesis, upstream of PPARgamma, potentially diverting preadipocytes as well as multipotent mesenchymal prescursors away from adipogenesis and toward osteogenesis. Hh may elicit these effects by inducing the expression of antiadipogenic transcription factors such as Gata2. These data support the notion that Hh signaling plays a conserved role, from invertebrates to vertebrates, in inhibiting fat formation and highlighting the potential of the Hh pathway as a therapeutic target for osteoporosis, lipodystrophy, diabetes, and obesity.

Murine orthotopic model for the assessment of chemoradiotherapeutic interventions in rectal cancer.

A murine orthotopic model for the study of colon cancer has been described earlier. However, for the study of rectal cancer, three issues remain: (i) the relative sensitivity of the implanted tumors to ionizing radiation (IR); (ii) the location of the tumor for the delivery of external beam IR; and (iii) the assessment of a given modality over time before necropsy. In this protocol, we have modified an orthotopic model for colon cancer described earlier for the specific assessment of chemoradiation in rectal cancer by (i) cecal transplantation of tumors with a known response to IR; (ii) securing the tumor to the lateral abdominal wall with a permanent suture for the administration of IR; and (iii) transfection of cells with luciferase before tumor implantation for the assessment of the chemoradiotherapeutic interventions over time by bioluminescence imaging before the end on the study. This technique allows targeted delivery of IR in an intraperitoneal tumor. Imaging throughout the course of the treatment is possible such that the timing of chemoradiation can be determined and permits comparison between groups before the end of the treatment. This model represents a modified technique that allows the assessment of chemoradiotherapeutic interventions in rectal cancer.

Radiosensitization of HT-29 cells and xenografts by the nitric oxide donor DETANONOate.

BACKGROUND: Mechanisms of radioresistance in rectal cancer remain unclear. OBJECTIVES: To determine mechanisms of radioresistance in rectal cancer cells and to assess the role of the nitric oxide donor DETANONOate as a radiosensitizing agent. METHODS: Survival was determined by clonogenic assays, apoptosis by PARP-1 cleavage, and phenotypic differences by Western blot analysis. SCID mice bearing HT-29 xenografts were treated with ionizing radiation (IR) [2.0 Gy x 5], DETANONOate [0.4 mg/kg i.p.], or combination treatment. RESULTS: Colorectal cancer HT-29-p53-null cells were resistant and HCT-116-p53 wild-type cells sensitive to IR, which correlated with cleaved PARP-1. Increased levels of p21 occurred in HCT-116 cells, while Bcl-2 and survivin were elevated in HT-29 cells. Radiosensitization was achieved with a substantial elevation of cleaved PARP-1 in DETANONOate-HT-29-treated versus control cells, which was accompanied by elevation of p21, p27, and BAX, and a concomitant decrease in Bcl-2. SCID mice bearing HT-29 xenografts demonstrated a 37.6%, 51.1%, and 70.1% inhibition in tumor growth in mice receiving IR, DETANONOate, and combination treatment versus control, respectively. CONCLUSIONS: Radioresistant HT-29 cells are p53-null and have substantially decreased levels of p21. DETANONOate radiosensitized HT-29 cells in vitro and in vivo by an additive effect in apoptosis.

A prospective study on peptide mapping of human fatigue saliva markers based on magnetic beads.

In order to explore convenient and stable fatigue markers, we studied various high-molecular-weight peptide fragments under fatigue state and non-fatigue state in the saliva using time of flight mass spectrometry. The saliva samples were collected from 10 healthy volunteers that were in the condition of fatigue and non-fatigue, respectively. Moreover, the time of flight mass spectrometry was conducted using two kinds of sample treatment methods, the magnetic beads enrichment (MB) and direct detection of stock solution. This was followed by modeling via the mass spectra of MB and supernatant (stock solution) directly collected after centrifugation. Both MB and direct sampling produced good spectrograms between 1,000 and 15,000 Da, while some peaks were lost in the enrichment. The spectrograms in the early and late period were different in each individual. Due to the limited sample size, 20 early and 20 late spectrograms were used for modeling analysis. Three different peptides were identified in the stock solution samples that can be detected in both fatigue and non-fatigue groups. The cross validity of MB model was 92.06%, while that of the stock solution model was 95.49%. The results showed that there were different peaks within the molecular weight of 2,000-15,000 Da, which provided a scientific basis for further realization of the convenient fatigue detection method based on the biosensor technique, with important theoretical and practical significance.

Targeted next-generation sequencing of dedifferentiated chondrosarcoma in the skull base reveals combined TP53 and PTEN mutations with increased proliferation index, an implication for pathogenesis.

Dedifferentiated chondrosarcoma (DDCS) is a rare disease with a dismal prognosis. DDCS consists of two morphologically distinct components: the cartilaginous and noncartilaginous components. Whether the two components originate from the same progenitor cells has been controversial. Recurrent DDCS commonly displays increased proliferation compared with the primary tumor. However, there is no conclusive explanation for this mechanism. In this paper, we present two DDCSs in the sellar region. Patient 1 exclusively exhibited a noncartilaginous component with a TP53 frameshift mutation in the pathological specimens from the first surgery. The tumor recurred after radiation therapy with an exceedingly increased proliferation index. Targeted next-generation sequencing (NGS) revealed the presence of both a TP53 mutation and a PTEN deletion in the cartilaginous and the noncartilaginous components of the recurrent tumor. Fluorescence in situ hybridization and immunostaining confirmed reduced DNA copy number and protein levels of the PTEN gene as a result of the PTEN deletion. Patient 2 exhibited both cartilaginous and noncartilaginous components in the surgical specimens. Targeted NGS of cells from both components showed neither TP53 nor PTEN mutations, making Patient 2 a naïve TP53 and PTEN control for comparison. In conclusion, additional PTEN loss in the background of the TP53 mutation could be the cause of increased proliferation capacity in the recurrent tumor.

Inhibition of DNA double-strand break repair by the dual PI3K/mTOR inhibitor NVP-BEZ235 as a strategy for radiosensitization of glioblastoma.

PURPOSE: Inhibitors of the DNA damage response (DDR) have great potential for radiosensitization of numerous cancers, including glioblastomas, which are extremely radio- and chemoresistant brain tumors. Currently, there are no DNA double-strand break (DSB) repair inhibitors that have been successful in treating glioblastoma. Our laboratory previously demonstrated that the dual phosphoinositide 3-kinase/mTOR inhibitor NVP-BEZ235 can potently inhibit the two central DDR kinases, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated (ATM), in vitro. Here, we tested whether NVP-BEZ235 could also inhibit ATM and DNA-PKcs in tumors in vivo and assessed its potential as a radio- and chemosensitizer in preclinical mouse glioblastoma models. EXPERIMENTAL DESIGN: The radiosensitizing effect of NVP-BEZ235 was tested by following tumor growth in subcutaneous and orthotopic glioblastoma models. Tumors were generated using the radioresistant U87-vIII glioma cell line and GBM9 neurospheres in nude mice. These tumors were then treated with ionizing radiation and/or NVP-BEZ235 and analyzed for DNA-PKcs and ATM activation, DSB repair inhibition, and attenuation of growth. RESULTS: NVP-BEZ235 potently inhibited both DNA-PKcs and ATM kinases and attenuated the repair of ionizing radiation-induced DNA damage in tumors. This resulted in striking tumor radiosensitization, which extended the survival of brain tumor-bearing mice. Notably, tumors displayed a higher DSB-load when compared with normal brain tissue. NVP-BEZ235 also sensitized a subset of subcutaneous tumors to temozolomide, a drug routinely used concurrently with ionizing radiation for the treatment of glioblastoma. CONCLUSIONS: These results demonstrate that it may be possible to significantly improve glioblastoma therapy by combining ionizing radiation with potent and bioavailable DNA repair inhibitors such as NVP-BEZ235.

Role of p53, Bax, p21, and DNA-PKcs in radiation sensitivity of HCT-116 cells and xenografts.

BACKGROUND: Molecular factors that dictate tumor response to ionizing radiation in rectal cancer are not well described. METHODS: We investigated the contribution of p53, p21, Bax, and DNA-PKcs in response to ionizing radiation in an isogeneic colorectal cancer system in vitro and in vivo. RESULTS: HCT-116 DNA-PKcs(-/-) cells and xenografts were radiosensitive compared with wild-type (WT) HCT-116 cells. HCT-116 p53(-/-) cells and tumor xenografts displayed a radioresistant phenotype. Separately, p21 or Bax deficiency was associated with a radiosensitive phenotype in vitro and in vivo. In vivo, Bax deficiency led to increased tumor necrosis and decreased microvessel density. In vitro, HCT-116 Bax(-/-) cells had decreased levels of vascular endothelial growth factor. HCT-116 WT cells had a more radioresistant phenotype after pancaspase inhibition, but pancaspase inhibition did not alter radiosensitivity in HCT-116 Bax(-/-) cells subjected to ionizing radiation. There was no difference in cell growth in HCT-116 WT cells subjected to transient apoptosis-inducing factor (AIF) inhibition; however, HCT-116 Bax(-/-) cells treated with AIF siRNA followed by ionizing radiation had a significant survival advantage compared with control-treated cells, implicating AIF in the radiosensitivity of Bax(-/-) cells. CONCLUSION: These data might be used along with other markers to predict response to radiation in patients with rectal cancer.

In vitro and in vivo radiosensitization of colorectal cancer HT-29 cells by the smac mimetic JP-1201.

BACKGROUND: The response to neoadjuvant chemoradiation in rectal cancer is variable and unpredictable. Resistance to chemoradiation has been directly correlated with the levels of the inhibitors of apoptosis (IAPs) in several malignancies. Because smac-DIABLO is a pro-apoptotic gene product that directly inhibits the activity of the IAPs, molecules with similar activity might radiosensitize rectal tumors with phenotypes that express high levels of IAPs. This study was undertaken to assess the radiosensitizing properties of the smac mimetic JP-1201 in radioresistant HT-29 colorectal cancer cells in vitro and established xenografts in SCID mice. METHODS: Survival was determined by clonogenic assays. PARP-1, caspase-8 cleavage, and IAP levels were assessed by Western blot analysis. SCID mice bearing HT-29 xenografts were treated with ionizing radiation: 2.0 Gy x 5; (n = 6), JP-1201 (5.0 mg/Kg i.p., n = 5) or combination treatment (n = 7) and compared to control (n = 8). DNA repair mechanisms were interrogated by gammaH2AX positive foci. RESULTS: Pretreatment of HT-29 cells with JP-1201 (5.0 microM) prior to ionizing radiation (IR) significantly decreased the survival of these cells. SCID mice bearing HT-29 xenografts demonstrated no difference in tumor load in the group receiving exclusively JP-1201 versus control. At the end of the treatment (day 40), a 46% reduction of tumor load was observed in the IR+JP-1201-treated group compared to the IR-only treated group. Radiosensitization was achieved with a substantial elevation of cleaved PARP-1 in JP-1201- treated HT-29 cells versus control cells with a concomitant decrease of XIAP, but not of survivin or cIAP1/2. JP-1201-treated HT-29 cells had a reduced ability to repair double-stranded DNA breaks (DSBs). CONCLUSION: The smac mimetic JP-1201 decreased the survival of HT-29 cells and tumor growth by an additive effect in apoptosis and a reduction in the level of XIAP and an impairment of DNA repair mechanisms. The pathways leading to this response need to be further investigated.

Tissue microarray constructs to predict a response to chemoradiation in rectal cancer.

PURPOSE: To identify, using tissue microarray (TMA), an immunohistochemical panel predictive of response to ionizing radiation (IR) in rectal cancer. METHODS: TMA constructs were prepared from archived stage II/III rectal tumors and matching adjacent mucosa (n=38) from patients treated with pre-operative chemoradiation. Immunohistochemistry (IHC) was performed for MIB, Cyclin E, p21, p27, p53, survivin, Bcl-2, and BAX. Immunoreactivity along with clinical variables was subjected to univariate and forward stepwise logistic regression analyses. RESULTS: Pathological complete response (pCR) was 23.9%. The number of positive lymph nodes obtained in the resected specimen was associated with pCR. Immunoreactivity for MIB (Sn 15%, Sp 65%, OR 0.33), p53 (Sn 3%, Sp 84%, OR 0.16), Bcl-2 (Sn 11%, Sp 74%, OR 0.35), and BAX (Sn 92%, Sp 80%, OR 46) was associated with pathological response (all p's<0.001). Forward stepwise logistic regression analysis demonstrated that MIB was an independent predictor of a response to chemoradiation (p=0.001). CONCLUSIONS: A combined panel of mediators of apoptosis alone or combined with clinical factors is a feasible approach that can be applied to rectal tumor biopsies to predict a response to chemoradiation. The most sensitive factor was BAX; while MIB independently predicted a response to chemoradiation.

Mechanisms of resistance to ionizing radiation in rectal cancer.

While patients with breast cancers are not subjected to the adverse side effects of tamoxifen or trastuzumab if their tumors are negative for estrogen, progesterone or Her-2/Neu, neoadjuvant ionizing radiation with concurrent chemotherapeutic agents is administered almost universally to patients with stage II/III rectal cancers. There is, however, a tremendously wide range of response to this preoperative modality from complete pathological response to continuous tumor growth in patients receiving the same form of treatment. The specific phenotype of the tumor plays a major role in rendering tumor cells survival advantage to the cytotoxic effects of chemoradiation. Pathways such as proliferation, cell cycle, apoptosis and hypoxia have been investigated under a variety of conditions in preirradiated tissues and postirradiated tumors. This article reviews the current evidence available to identify a molecular profile predictive of the best response to ionizing radiation.