Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy

Radiotherapy is a well-established treatment for cancer. However, the existence of radioresistant cells is one of the major obstacles in radiotherapy. In order to understand the mechanism of cellular radioresistance and develop more effective radiotherapy, we have established clinically relevant radioresistant (CRR) cell lines, which continue to proliferate under daily exposure to 2 Gray (Gy) of X-rays for >30 days. X-ray irradiation significantly induced autophagic cells in parental cells, which was exiguous in CRR cells, suggesting that autophagic cell death is involved in cellular radiosensitivity. An autophagy inducer, rapamycin sensitized CRR cells to the level of parental cells and suppressed cell growth. An autophagy inhibitor, 3-methyladenine induced radioresistance of parental cells. Furthermore, inhibition of autophagy by knockdown of Beclin-1 made parental cells radioresistant to acute radiation. These suggest that the suppression of autophagic cell death but not apoptosis is mainly involved in cellular radioresistance. Therefore, the enhancement of autophagy may have a considerable impact on the treatment of radioresistant tumor.     

Genome-scale CRISPR-Cas9 knockout screening in nasopharyngeal carcinoma for radiosensitive and radioresistant genes

BackgroundGenome-scale CRISPR-Cas9 knockout screening may provide new insights into the mechanism underlying clinical radioresistance in nasopharyngeal carcinoma (NPC), which is remain largely unknown. Our objective was to screen the functional genes associated with radiosensitivity and radioresistance in NPC, laying a foundation for further research on its functional mechanismand.MethodsCRISPR-Cas9 library lentivirus screening in radiation-treated NPC cells was combined with second-generation sequence technology to identify functional genes, which were further validated in radioresistant NPC cells and patient tissues.ResultsEleven radiosensitive and radioresistant genes were screened. Among these genes, the expression of FBLN5, FAM3C, MUS81, and DNAJC17 were significantly lower and TOMM20, CDKN2AIP, SNX22, and SP1 were higher in the radioresistant NPC cells (C666-1R, 5-8FR) (p < 0.05). CALD1 was highly expressed in C666-1R. Furthermore, we found knockout of FBLN5, FAM3C, MUS81 and DNAJC17 promoted the proliferation of NPC cells, while CDKN2AIP and SP1 had the opposed results (p < 0.05). This result was verified in NPC patient tissues. Meanwhile, KEGG analysis showed that the Fanconi anemia pathway and the TGF-β signaling pathway possibly contributed to radiosensitivity or radioresistance in NPC.ConclusionsNine genes involved in the radiosensitivity or radioresistance of NPC: four genes for radiosensitivity (FBLN5, FAM3C, MUS81, and DNAJC17), two genes for radioresistance (CDKN2AIP, SP1), two potential radioresistant genes (TOMM20, SNX22), and a potential radiosensitive gene (CALD1). Genome-scale CRISPR-Cas9 knockout screening for radiosensitive and radioresistant genes in NPC may provide new insights into the mechanisms underlying clinical radioresistance to improve the efficacy of radiotherapy for NPC.     

Incidence,mortality, and survival trends of soft tissue and bone sarcoma in Switzerland between 1996 and 2015

BackgroundResearch on soft-tissue sarcoma (STS) and bone sarcoma (BS) is increasingly in the focus of physicians and pharmaceutical companies. Expanding knowledge has improved the management of sarcoma and possibly survival. Here we provide the first population-based data on time trends of incidence, mortality, and survival of STS and BS diagnosed in Switzerland between 1996 and 2015.MethodsWe performed a retrospective registry study with data from the National Institute for Cancer Epidemiology and Registration (NICER) database in Switzerland between 1996 and 2015.ResultsWe identified 5384 STS patients and 940 BS patients. The three most common STS subtypes were undifferentiated/unclassified sarcoma (22.3%), liposarcoma (20.6%) and leiomyosarcoma (20.6%). Chondrosarcoma, osteosarcoma and Ewing sarcoma represented 40.4%, 27.0% and 15.2% of the BS group, respectively. The age-standardized incidence and mortality rates in 2011–2015 were 4.43 and 1.42 per 100,000 person-years for STS, and 0.91 and 0.42 for BS. Age-standardized incidence of STS in males was significantly higher during 1996–2000 than during 2001–2015; however, mortality rates did not change significantly over time. Five-year relative survival (RS) for STS improved significantly from 56.4% (95%CI 52.9–59.7 for 1996–2001) to 61.6% (95%CI 58.6–64.4 for 2011–2015) (p = 0.025). No improvement in 5-year RS for BS could be observed (RS 1996–2000: 69.6%, 95%CI 61.2–76.6; RS 2011–2015: 73.1%, 95%CI 66.6–78.6; p = 0.479).ConclusionIncidence rates of STS and BS have been stable since 2001. The longer RS in STS can be attributed to advances in sarcoma patient management.     

Micronuclei and apoptosis in glioma and neuroblastoma cell lines and role of other lesions in the reconstruction of cellular radiosensitivity

It is now well established that micronuclei frequency does not always rank cell lines according to radiosensitivity. There is, however, a growing interest in reconstructing cellular radiosensitivity (measured by colony assay) from concurrent micronucleus and apoptosis data. Using a variety of radiosensitive and radioresistant cell lines, we have derived a missing parameter –P _oe , the probability of cell death by other events such as small deletions, chromosome aberrations, late apoptosis and necrosis which are undetectable by micronucleus and apoptosis assays performed at a single time point. In the radioresistant glioma cell lines G120, G60, G28, G44 and G62 (SF2 ≥0.59), a characteristic threshold dose exists above which cell loss due to undetectable lesions occurs. In the radiosensitive SK-N-SH and KELLY cell lines (SF2 ≤0.43), the P _oe parameter is positive at very low doses, reaches a maximum and declines at higher doses. In the radiation resistant G28 cells, P _oe was found to be below zero for doses up to 6 Gy. In the G62, G44 and G120 cell lines, the threshold doses to induce P _oe events were 0.87, 3.04 and 3.85 Gy, respectively. Cell death by undetectable lesions is a cell-specific and time-dependent variable. Micronucleus and apoptosis assays performed concurrently and at a specific time point miss cell death due to other events and this may be the reason why reconstruction of cellular radiosensitivity from micronucleus and apoptosis data fails. Received: 8 March 2001 / Accepted: 1 September 2001     

Incidence of soft tissue sarcoma in Taiwan: A nationwide population-based study (2007–2013)

BackgroundAsian studies on soft tissue sarcoma (STS) incidence, irrespective of the primary site, are scant.MethodsSTS data were acquired from the population-based 2007–2013 Taiwan Cancer Registry of the Health and Welfare Data Science Center, Taiwan. Histological subtype-, site-, sex-, and age-specific STS incidence rates were analyzed according to the 2013 classification of the World Health Organization.ResultsIn total, 11,393 patients with an age-standardized incidence rate (ASIR) of 5.62 (95% confidence interval, 5.51–5.73) per 100,000 person-years were identified. Overall, a male predominance (sex-standardized incidence rate ratio, 1.2) was noted, and the rate increased with age, peaking at >75 years. Approximately 30% of STSs occurred in connective, subcutaneous, and other soft tissues and 70% in other sites. In addition to connective, subcutaneous, and other soft tissues, the three most common primary sites were the stomach (15.9%), skin (14.3%), and small intestines (10.5%). Gastrointestinal stromal tumor was the most common subtype (29.2%; ASIR, 1.55/100,000 person-years), followed by liposarcoma (11.5%; ASIR, 0.63/100,000 person-years) and leiomyosarcoma (9.7%; ASIR, 0.53/100,000 person-years). Compared with relevant data from Western countries, the incidence rate of angiosarcomas was higher than that in other regions, whereas the incidence rates of leiomyosarcoma and Kaposi sarcoma were lower than those in other regions.ConclusionSTS incidence varied by histological subtype, sex, age, and primary site in an Asian population. Our results suggested regional and racial discrepancies in the incidence rates of certain STS subtypes.     

Adaptive radiotherapy in patients receiving neoadjuvant radiation for soft tissue sarcoma

AimThe aim of this study is to evaluate tumor volume changes during preoperative radiotherapy and to assess the role of adaptive radiation.BackgroundContemporary neoadjuvant radiotherapy utilizes image guidance for precise treatment delivery. Moreover, it may depict changes in tumor size and shape.Materials and methodsBetween 2016 and 2018, 23 patients aged ≥18 years with soft tissue sarcoma were treated with neoadjuvant radiation followed by surgical resection. The tumor volumes (cc) were measured using the Pinnacle planning system prior to starting radiotherapy and during treatment, the changes in volume and absolute differences were estimated. Moreover, patient's position on the machine was evaluated to assess setup offsets. The triggers for plan adaptation were >1 cm expansion or unacceptable setup offsets.ResultsThe mean tumors volume at presentation was 810 cc (range, 55–4000). At last cone beam CT the tumor volume had changed in 14 patients (61%); it was stable in nine patients (39%). Disease regression was documented in eight patients (35%), with median shrinkage of −20.5% (range, −2 to −29%), while tumor progression was observed in six cases (26%), the median change was 12.5% (range, +10 to +25%).Adaptive radiation was required in four patients (17%). For the remaining 19 cases (83%), the dose distribution was adequate to cover target volumes.ConclusionsChange in soft tissue sarcoma volume during radiation is not uncommon. Image guidance should be used to reduce setup errors and to detect differences in tumor volume. Image guidance and adaptive radiation are paramount to ensure optimal radiation delivery.     

Knock-down of glutaminase 2 expression decreases glutathione,NADH, and sensitizes cervical cancer to ionizing radiation

Phosphate-activated mitochondrial glutaminase (GLS2) is suggested to be linked with elevated glutamine metabolism. It plays an important role in catalyzing the hydrolysis of glutamine to glutamate. The present study was to investigate the potent effect of GLS2 on radioresistance of cervical carcinoma. GLS2 was examined in 144 cases of human cervical cancer specimens (58 radioresistant specimens, 86 radiosensitive specimens) and 15 adjacent normal cervical specimens with immunohistochemistry. HeLa cells were treated with a cumulative dose of 50 Gy X-rays, over 6 months, yielding the resistant sub-line HeLaR. The expressions of GLS2 were measured by Western blot. Radioresistance was tested by colony survival assay. Apoptosis was determined by flow cytometry. The levels of glutathione (GSH), reactive oxygen species (ROS), NAD⁺/NADH ratio and NADP⁺/NADPH ratio were detected by quantization assay kit. Xenografts were used to confirm the effect of GLS2 on radioresistance in vivo. The expressions of GLS2 were significantly enhanced in tumor tissues of radioresistant patients compared with that in radiosensitive patients. In vitro, the radioresistant cell line HeLaR exhibited significantly increased GLS2 levels than its parental cell line HeLa. GLS2 silenced radioresistant cell HeLaR shows substantially enhanced radiosensitivity with lower colony survival and higher apoptosis in response to radiation. In vivo, xenografts with GLS2 silenced HeLaR were more sensitive to radiation. At the molecular level, knock-down of GLS2 increased the intracellular ROS levels of HeLaR exposed to irradiation by decreasing the productions of antioxidant GSH, NADH and NADPH. GLS2 may have an important role in radioresistance in cervical cancer patients.     

Expeditious and Stereoselective Synthesis of α-Guanosine

Abstract

The preparation of the unnatural nucleoside α-guanosine (1) has been achieved from readily available, literature precursors in ca. 6–9% overall yield. The key step, construction of the α-anomeric bond between the purine and the sugar, was accomplished by S_N2 displacement of protected β-chlororibose derivative 2 with 2-amino-6-chloropurine. The optimal conditions for this reaction involved cesium carbonate in N-methylpyrrolidinone (α/β ratio: 7.7:1).     

Intraoperative electron radiation therapy after salvage surgery in gynecological cancers and retroperitoneal sarcomas: outcomes and adverse effects

BackgroundSalvage surgery is considered an option for isolated recurrences of retroperitoneal and pelvic tumors, in patients who have undergone previous radiotherapy. In order to increase local control intra operative electron radiation therapy (IOERT) can be used in these patients to administer additional radiation dose. We evaluated the outcomes and adverse effects in patients with retroperitoneal sarcoma and gynecologic tumors after salvage surgery and IOERT.Materials and methodsTwenty patients were retrospectively analyzed. Twenty-three IOERT treatments were performed after surgery. Six (30%) were sarcoma and 14 (70%) were gynecological carcinoma. Administered dose depended on previous dose received with external beam radiotherapy (EBRT) and proximity to critical structures. The toxicities were scored using the Common Terminology Criteria for Adverse Events version 4.0.ResultsThe median age of the patients was 51 years (range 34–70). After a median follow-up of 32 months (range 1–68), in the sarcoma group the local control rate was 66.6%; while in the gynecological group the local control rate was 64.3%. In relation to late toxicity, one patient had a Grade 2 vesicovaginal fistula, and one patient presented Grade 4 enterocolitis and enteric intestinal fistula.ConclusionsIOERT could have a role in the treatment of retroperitoneal sarcomas in primary tumors after EBRT, as it may suggest a benefit in local control or recurrences after surgical resection in those at high risk of microscopic residual disease. The addition of IOERT to salvage resection for isolated recurrence of gynecologic cancers suggest favorable local control in cases with concern for residual microscopic disease.     

A population-based study of soft tissue sarcoma incidence and survival in Australia: An analysis of 26,970 cases

BackgroundSoft tissue sarcomas (STS) are rare, often fatal tumors, but little is known of the epidemiology and survival in the Australian population. This study aims to provide the first epidemiological analysis of incidence and survival rates of STS in the Australian population.MethodsA retrospective population-based observational study was conducted between 1982 and 2009 of all patients with a diagnosis of STS using the Australian Institute of Health and Welfare (AIHW) Australian Cancer Database. Incidence rates per 100,000; incidence rate ratios, age-standardized incidence rates, prevalence and incidence rates of subtypes of STS, median, one-year and 5-year survival rates were examined.ResultsA total of 26,970 patients were identified. Between 1982 and 2009 STS incidence rates significantly increased from 3.99 [95% CI 3.68–4.32] to 6.12 [95% CI 5.80–6.46] per 100,000 Australian population, with a peak incident rate ratio (IRR) of 1.59 [95% CI 1.51–1.69] (p < 0.0001) in 2001. Median age at diagnosis increased from 58 to 63 years. Incidence rates were stable across all 10-year age cohorts, except for people aged over 70 where it increased. Overall, age-standardized incidence rates increased from 4.70 [95% CI 4.42–5.00] in 1982 to 5.87 [95% CI 5.63–6.11] per 100 000 Australians in 2009. Leiomyosarcoma (20.43%), malignant fibrous histiocytoma (16.14%), and soft tissue tumors/sarcomas, not otherwise specified (10.18%) were the most common STS subtypes. Median survival from diagnosis increased from 5.80 years [95% CI 5.06–6.54] in 1985–1989 cohort to 8.18 years [95% CI 7.54–8.81] in the 2000–2004 cohort (log-rank test p < 0.0001).ConclusionThe incidence of STS is increasing in Australia, most noticeably in those aged over 70 years, with a small but statistically significant increase in overall survival rates.     

Correlation Between Beta- and Alpha-Adrenergic Receptor Concentrations in Human Placenta

Abstract

α₂- and β-adrenergic receptors in human placental membranes have been investigated using the radioligands [³H]-RX 821002 and [³H]-dihydroalprenolol, respectively. The specific binding of the α₂-adrenoceptor antagonist RX 821002 confirms the presence of an α₂-adrenoceptor in the human placenta, which has been characterized previously with [³H]-rauwolscine. The major finding presented here is a correlation between the α₂- and β-adrenergic receptor concentrations (r=0.765) in the human placenta at term. It is suggested that the α₂/β adrenoceptor balance may play an important role in regulation of the vascular bed of the placenta. Determination of the α₂/β ratio may help towards an understanding of the contractility of the placental vascular muscles.     

Gemcitabine-mediated radiosensitization of human soft tissue sarcoma

Background/Purpose

Local and systemic control of soft tissue sarcoma (STS) remains a clinical challenge, particularly for retroperitoneal, deep truncal, or advanced extremity disease. 2′,2′-Difluoro-2′-deoxycytidine (gemcitabine) is a potent radiosensitizer in many tumor types, but it has not been studied in human STS. The purpose of this study was to determine the radiosensitizing potential of gemcitabine in preclinical models of human STS.

Materials and Methods

The in vitro radiosensitizing activity of gemcitabine was assessed with clonogenic survival assay on three human STS cell lines: SK-LMS-1 (leiomyosarcoma), SW-872 (liposarcoma), and HT-1080 (fibrosarcoma). Cell cycle distribution was determined using dual-channel flow cytometry. The in vivo radiosensitizing activity of gemcitabine was assessed with subcutaneous SK-LMS-1 nude mice xenografts. Tumor-bearing mice were treated with concurrent weekly gemcitabine and fractionated daily radiotherapy (RT) (2 Gy daily) for 3 weeks (a total dose of 30 Gy).

Results

The 50% inhibitory concentration (IC₅₀) of gemcitabine for the human STS cell lines ranged from 10 to 1000 nM. Significant in vitro radiosensitization was demonstrated in all three human STS cell lines using gemcitabine concentrations at and below the IC₅₀. Maximal radiosensitization was associated with accumulation of cells in early S-phase. SK-LMS-1 xenografts displayed significant tumor growth delay with combined gemcitabine and RT compared to either treatment alone. Treatment related toxicity was greatest in the gemcitabine plus RT arm, but remained at an acceptable level.

Conclusions

Gemcitabine is a potent radiosensitizer in preclinical models of human STS. Clinical trials combining gemcitabine and RT in human STS are warranted.     

Lithuanian cohort of Chernobyl cleanup workers: Cancer incidence follow-up 1986–2012

BackgroundCancer risks following radiation exposure in adulthood after Chernobyl are less studied compared to those after exposure in childhood. We aimed to evaluate cancer risk in the Lithuanian cohort of Chernobyl cleanup workers 26 years after their exposure in Chernobyl.MethodsStudy population (6707 men) was followed for cancer incidence upon return from Chernobyl till the end of 2012 by linkage procedure with the Lithuanian Cancer Registry and for migration and death – with Central Population Registry. The site-specific cancer risk in the cohort was estimated by calculating the standardised incidence ratio (SIR) with 95 % confidence interval (CI).ResultsA total of 596 cancer cases was observed in the cohort, against 584 expected (SIR 1.02; 95 % CI 0.94, 1.11). Only incidence of mouth and pharynx cancers was increased compared to the expected (SIR 1.41; 95 % CI 1.07, 1.86). Nevertheless, an increased risk of thyroid cancer was observed among cleanup workers who were younger than 30 years when entering the Chernobyl zone (SIR 2.90; 95 % CI 1.09, 7.72), whose radiation dose was above 100 milisievert (mSv) (SIR 3.13; 95 % CI 1.30, 7.52) and who had shorter duration of stay (SIR 2.30; 95 % CI 1.03, 5.13).ConclusionsOur findings are consistent with those observed in other cohorts of workers, namely, the increased risk of cancer sites related to behavioural factors. The increased risk of thyroid cancer among cleanup workers who were younger than 30 years when entering Chernobyl and whose radiation dose was above 100 mSv cannot exclude the association with the radiation exposure in Chernobyl.     

One size does not fit all: Factors associated with increased frequency of radiation overexposure alerts based on fixed-alert thresholds

ObjectivesQuantify the expected rate of CT radiation dose alerts for three body regions using accepted radiation dose benchmarks and assess key determinants of alert frequency.MethodsThis IRB-approved retrospective cohort study evaluated consecutive CT examinations performed between July and December 2013 within an academic medical system. CTDI_vol x-ray tube output metrics were compared to the body-region-specific benchmark levels, Achievable Doses (AD), Diagnostic Reference Levels (DRL), and Dose Notification Values (DNV). A logistic regression model for the simulated alerts was fit as a function of the independent predictors: scanner, body region, gender, weight, and age.ResultsFor 17,000 exams, the proportion of events triggering alerts increased with patient weight. Significant covariates were scanner, body region, patient weight and patient age (all p < 0.0001). Odds of alert generation for the AD, DRL, and DNV benchmarks increased by 7.6%, 6.6% and 2.9% per kilogram, respectively, and by 0.8%, 1.1% and −2.7% per year of age (all p < 0.0001). Compared to the most highly optimized scanner, odds of alert generation varied by a factor of 595 for AD, 1126 for DRL, and 13 for DNV.ConclusionAlert frequency was significantly correlated with weight, age, body region and scanner. Controllable factors include scanner functionality and associated protocol optimization. Patient factors driving alert frequency are predominantly weight, and to a lesser degree, age. Size-agnostic fixed dose thresholds can frequently produce false positive alerts in appropriately performed exams of large patients, while missing opportunities to identify outlier scans of higher-than-expected dose in small patients.     

Immediate and long-term effects of radiation on the immune system of specific-pathogen-free mice

Studies on the immediate and long-term effects of radiation on the immune system of specific-pathogen-free mice are summarized in this paper. There was a striking difference in the radiation response of lymphocyte subsets; B cells consist of a fairly radiosensitive homogeneous population, whereas T cells consist of a large percentage (greater than 90 per cent) of radiosensitive and a small percentage (less than 10 per cent) of extremely radioresistant subpopulations. Ly 1+ and Ly 2+ lymphocytes appear equally radiosensitive, although the percentage of radioresistant cells was slightly larger for the former (approximately 5.5 per cent) than the latter (approximately 2.5 per cent). There was a significant strain difference in the radiosensitivity of immune-response potential in mice; immunocompetent cells of C3H mice were more radioresistant than those of BALB/c, C57BL/6, and B10.BR mice. Studies on the long-term effect of radiation on immune system in mice indicated no evidence for accelerated ageing of the immunologic functions when radiation exposure was given to young adults. Preliminary results on the enhancing effect of low dose radiation on cytotoxic T cell response in vitro are also discussed.     

Influence of 6 MV and 20 MV X-radiation dose rate on in vitro survive of the K-562 cell line

BackgroundAnalysis of the survival rate of cells after irradiation with a specified dose of X-radiation might be one of the basic foundations for assessment of biological implications of ionizing radiation. Investigation of the influence of X-radiation dose rate on cells was carried out in vitro using the SF2 test.AimThe aim of this study was to investigate the influence of X-radiation dose rate on the surviving fraction of the K-562 cell line for two photon energies of 6 MV and 20 MV.Materials/MethodsTo measure the cells' reaction to X-radiation of variable dose rate human leukaemic K-562 cells were used. In order to fulfil the main aim of the study, the cell line was subjected to irradiation at two different dose rates. Total dose applied at once was 2 Gy. A quantitative evaluation of cell survival rate was carried out at every step of the experiment using a clonogenic assay.ResultsHigh dose rate at the energy of 6 MV decreased the percentage of surviving cells to 23%, while lower dose rate decreased it only to 36%. A similar effect is observed at the energy of 20MV-namely at the higher dose rate the percentage of surviving cells is 18%, whereas at the lower one it is only 34%.ConclusionsThe experiment has shown that when using a lower dose rate, the biological effect of ionizing radiation is less pronounced. However, at a higher dose rate higher radiosensitivity of cells is observed.