Discriminating metastasised from non-metastasised seminoma based on transcriptional changes in primary tumours using NGS.

BACKGROUND: We aimed to better discriminate (occult) metastasised from non-metastasised seminoma based on transcriptional changes of small RNAs in the primary tumour. METHODS: Total RNAs including small RNAs were isolated from five testicular tumours of each, lymphogenic, occult and non-metastasised patients. Next-generation sequencing (SOLID, Life Technologies) was used to examine transcriptional changes. Small RNAs showing ⩾50 reads and a significant ⩾2-fold difference using non-metastasised tumours as the reference group were examined in univariate logistic regression analysis and combinations of two small RNAs were further examined using support vector machines. RESULTS: On average, 1.3 × 10(7), 1.4 × 10(7) and 1.7 × 10(7) small RNA reads were detectable in non-metastasised, occult and lymphogenic metastasised seminoma, respectively, of which 30-32% remained after trimming. Between 59 and 68% represented annotated reads and between 8.6 and 11% were annotated small RNA tags. Of them, 137 small RNAs showed>50 reads and a two-fold difference to the reference. In univariate analysis, 32-38 small RNAs significantly discriminated lymphogenic/occult from non-metastasised seminoma, and among these different comparisons, it were the same small RNAs in 51-88%. Many combinations of two of these small RNAs allowed a complete discrimination of metastasised from non-metastasised seminoma irrespective of the metastasis subtype. CONCLUSIONS: Metastasised and non-metastasised seminoma can be completely discriminated with a combination of two small RNAs.

Prediction of metastatic status in non-seminomatous testicular cancer.

PURPOSE: To examine the significance of 90 biomarkers for predicting metastatic status in non-seminomatous germ cell tumors (NSGCT). By predicting metastatic status, it may be possible to eliminate unnecessary therapeutic or diagnostic efforts. MATERIALS AND METHODS: We investigated 552 males who were diagnosed with non-metastatic (n = 273) and metastatic (n = 279) NSGCT between 2000 and 2011. The sample included cancers of different histologies: embryonal cell carcinoma (n = 131), teratoma (n = 55), and mixed histology (n = 366). We collected and analyzed more than 90 parameters via logistic regression: demographic characteristics, medical history, histopathological parameters, and levels of tumor markers and hormones. RESULTS: Testis histology (p = 0.004), clinical symptoms (p = 0.0005), tumor length (p = 0.005), infiltration of the rete testis (p = 0.008), invasion of lymphatic (pL1) and blood vessels (pV1) (p < 0.0001), and levels of enzymes such as LDH, ßHCG, AFP, and FSH (p values as small as <0.0001) were associated with metastatic status. With one model, we identified 14 out of 76 (18.4 %) metastatic NSGCT cases with 93-100 % certainty (positive predictive value) at 99 % specificity by the peripheral blood levels of LDH (day of operation) in combination with FSH measurements (1 day after operation). A second model included pV, tumor length, and FSH (1 day after operation). It identified 25 out of 90 (27.8 %) non-metastatic NSGCT with approximately 90 % certainty (negative predictive value) at 94-98 % sensitivity. CONCLUSIONS: No single parameter was able to discriminate metastatic from non-metastatic NSGCT, but combinations of parameters in two predictive models accurately identified the metastatic status in 23 % of the cases in our sample.

Iodine-131 dose-dependent gene expression: alterations in both normal and tumour thyroid tissues of post-Chernobyl thyroid cancers.

BACKGROUND: A strong, consistent association between childhood irradiation and subsequent thyroid cancer provides an excellent model for studying radiation carcinogenesis. METHODS: We evaluated gene expression in 63 paired RNA specimens from frozen normal and tumour thyroid tissues with individual iodine-131 (I-131) doses (0.008-8.6 Gy, no unirradiated controls) received from Chernobyl fallout during childhood (Ukrainian-American cohort). Approximately half of these randomly selected samples (32 tumour/normal tissue RNA specimens) were hybridised on 64 whole-genome microarrays (Agilent, 4 × 44 K). Associations between I-131 dose and gene expression were assessed separately in normal and tumour tissues using Kruskal-Wallis and linear trend tests. Of 155 genes significantly associated with I-131 after Bonferroni correction and with ≥2-fold increase per dose category, we selected 95 genes. On the remaining 31 RNA samples these genes were used for validation purposes using qRT-PCR. RESULTS: Expression of eight genes (ABCC3, C1orf9, C6orf62, FGFR1OP2, HEY2, NDOR1, STAT3, and UCP3) in normal tissue and six genes (ANKRD46, CD47, HNRNPH1, NDOR1, SCEL, and SERPINA1) in tumour tissue was significantly associated with I-131. PANTHER/DAVID pathway analyses demonstrated significant over-representation of genes coding for nucleic acid binding in normal and tumour tissues, and for p53, EGF, and FGF signalling pathways in tumour tissue. CONCLUSION: The multistep process of radiation carcinogenesis begins in histologically normal thyroid tissue and may involve dose-dependent gene expression changes.

The search for biomarkers of metastatic seminoma.

PURPOSE: We screened 90 potential parameters as biomarkers of metastatic seminoma to facilitate detection and eliminate unnecessary therapeutic or diagnostic efforts. MATERIALS AND METHODS: A total of 527 men with pure seminoma (diagnosed 2000 to 2011) were followed during therapy. More than 90 demographic/anamnestic (eg age, height, weight) histopathological parameters (testicular/tumor size, testicular intraepithelial neoplasia) and levels of tumor markers (eg α-fetoprotein, ß-human chorionic gonadotropin, lactate dehydrogenase) in peripheral blood and testicular vein were collected for analysis via logistic regression. Previously described risk factors (tumors larger than 4 cm, infiltration of rete testis) were assessed separately. RESULTS: Established parameters such as tumor length (p = 0.0003), involvement of lymphatic (p <0.0001) or vascular channels (p = 0.0009), extent of primary tumor (p <0.0001) and infiltration of the tunica albuginea (p = 0.02) as well as new biomarkers such as absence of testicular intraepithelial neoplasia in tumor bearing testis (p = 0.03), testicular volume (p = 0.04) and tumor volume (p = 0.02) showed a significant association with metastatic disease. This association was also true of lactate dehydrogenase, human chorionic gonadotropin and α-fetoprotein (p <0.0001 at maximum). However, the discriminatory capacity of these biomarkers (concordance or ROC area) did not exceed 65% when examined alone or in combination, and higher values (up to 80%) were detected for enzyme levels. A subset of metastatic seminoma (2% to 27%) was detectable with high accuracy (positive predictive value 92% to 100%) based on enzyme measurements (p <0.0006). CONCLUSIONS: New biomarkers of metastatic seminoma were identified and previously described risk factors were validated. Further prospective studies of these novel parameters are warranted to verify our findings and to explore a potential use for detecting occult metastases.

[Cutaneous damage after acute exposure to ionizing radiation: decisive for the prognosis of radiation accident victims]. / Hautschäden nach akuter Strahlenexposition mit ionisierender Strahlung: Entscheidend für die Prognose von Strahlenunfallopfern.

The cutaneous radiation syndrome includes all deterministic effects on the skin and visible parts of the mucosa from ionizing radiation. The Intensity and duration of radiation-induced skin symptoms depend on the kind and quality of ionizing radiation. The aim of this study was the investigation of the importance of the time of the development of radiation induced-skin effects on the prognosis of radiation accident victims. Clinical data about radiation accident victims from the database SEARCH were used. 211 cases with good documentation regarding radiation-induced skin effects were selected. From these 211 patients, 166 survived the acute phase of the acute radiation syndrome, while 45 died during the acute phase. Among those patients who did not survive the acute phase, 82.2 % showed their first documented radiation-induced skin symptoms during the first 3 days after radiation exposure. Of those patients whose first documented radiation-induced skin symptoms appeared on or after day four, 94.2 % survived the acute phase. The time to the occurrence of the first radiation-induced skin effects is diagnostically significant. The skin plays an important role in the clinical course of radiation syndromes and in the development of radiation-induced multi-organ failure. In a retrospective data analysis like this, the quality of data might be a limitation.

Accumulation of DSBs in gamma-H2AX domains fuel chromosomal aberrations.

DNA double strand breaks (DSBs) pose a severe hazard to the genome as erroneous rejoining of DSBs can lead to mutation and cancer. Here, we have investigated the correlation between X irradiation-induced gamma-H2AX foci, theoretically induced DSBs, and the minimal number of mis-rejoined DNA breaks (MNB) in irradiated lymphocytes obtained from two healthy humans by painting of the whole chromosome complement by spectral karyotyping. There were less gamma-H2AX foci/dose than theoretically expected, while misrepair, as expressed by MNB/gamma-H2AX focus, was similar at 0.5 and 1Gy but 3.6-fold up at 3Gy. Hence, our results suggest that X-ray-induced gamma-H2AX foci in G0 lymphocyte nuclei contain more than one DSB and that the increasing number of DSBs per gamma-H2AX repair factory lead to an increased rate of misrepair.

A radiation-induced gene signature distinguishes post-Chernobyl from sporadic papillary thyroid cancers.

We investigated selected gene targets to differentiate radiation-induced papillary thyroid cancers (PTCs) from other etiologies. Total RNA was isolated from 11 post-Chernobyl PTCs and 41 sporadic PTCs characterized by a more aggressive tumor type and lacking a radiation exposure history. RNA from 10 tumor samples from both groups was pooled and hybridized separately on a whole genome microarray for screening. Then 92 selected gene targets were examined quantitatively on each tumor sample using an RTQ-PCR-based low-density array (LDA). Screening for more than fivefold differences in gene expression between the groups by microarray detected 646 up-regulated and 677 down-regulated genes. Categorization of these genes revealed a significant (P < 0.0006) over-representation of the number of up-regulated genes coding for oxidoreductases, G-proteins and growth factors, while the number of genes coding for immunoglobulin appeared to be significantly down-regulated. With the LDA, seven genes (SFRP1, MMP1, ESM1, KRTAP2-1, COL13A1, BAALC and PAGE1) made a complete differentiation between the groups possible. Gene expression patterns known to be associated with a more aggressive tumor type in older patients appeared to be more pronounced in post-Chernobyl PTC, thus underlining the known aggressiveness of radiation-induced PTC. Seven genes were found that completely distinguished post-Chernobyl (PTC) from sporadic PTC.

Rapid Prediction of Hematologic Acute Radiation Syndrome in Radiation Injury Patients Using Peripheral Blood Cell Counts.

Rapid clinical triage of radiation injury patients is essential for determining appropriate diagnostic and therapeutic interventions. We examined the utility of blood cell counts (BCCs) in the first three days postirradiation to predict clinical outcome, specifically for hematologic acute radiation syndrome (HARS). We analyzed BCC test samples from radiation accident victims (n = 135) along with their clinical outcome HARS severity scores (H1-4) using the System for Evaluation and Archiving of Radiation Accidents based on Case Histories (SEARCH) database. Data from nonirradiated individuals (H0, n = 132) were collected from an outpatient facility. We created binary categories for severity scores, i.e., 1 (H0 vs. H1-4), 2 (H0-1 vs. H2-4) and 3 (H0-2 vs. H3-4), to assess the discrimination ability of BCCs using unconditional logistic regression analysis. The test sample contained 454 BCCs from 267 individuals. We validated the discrimination ability on a second independent group comprised of 275 BCCs from 252 individuals originating from SEARCH (HARS 1-4), an outpatient facility (H0) and hospitals (e.g., leukemia patients, H4). Individuals with a score of H0 were easily separated from exposed individuals based on developing lymphopenia and granulocytosis. The separation of H0 and H1-4 became more prominent with increasing hematologic severity scores and time. On day 1, lymphocyte counts were most predictive for discriminating binary categories, followed by granulocytes and thrombocytes. For days 2 and 3, an almost complete separation was achieved when BCCs from different days were combined, supporting the measurement of sequential BCC. We found an almost complete discrimination of H0 vs. irradiated individuals during model validation (negative predictive value, NPV > 94%) for all three days, while the correct prediction of exposed individuals increased from day 1 (positive predictive value, PPV 78-89%) to day 3 (PPV > 90%). The models were unable to provide predictions for 10.9% of the test samples, because the PPVs or NPVs did not reach a 95% likelihood defined as the lower limit for a prediction. We developed a prediction model spreadsheet to provide early and prompt diagnostic predictions and therapeutic recommendations including identification of the worried well, requirement of hospitalization or development of severe hematopoietic syndrome. These results improve the provisional classification of HARS. For the final diagnosis, further procedures (sequential diagnosis, retrospective dosimetry, clinical follow-up, etc.) must be taken into account. Clinical outcome of radiation injury patients can be rapidly predicted within the first three days postirradiation using peripheral BCC.

Influences of TP53 expression on cellular radiation response and its relevance to diagnostic biodosimetry for mission environmental monitoring.

TP53 is a transcriptional activator and regulates genomic instability and cellular responses to DNA damage in response to ionising radiation. The molecular mechanism behind p53-mediated responses, such as, apoptosis and genomic instability remains unclear. An in vitro model of biological effects to irradiation was established. In order to elucidate the functional role of TP53 under different stress-reaction pathways and identify possible biological indicators, p53 was stably transfected into HL-60 cells, which provides a p53 minus background. Significantly enhanced radiosensitivity and growth suppression were observed. G(2) accumulation was obtained. Radiation-induced apoptosis of HL-60 cells was significantly inhibited by TP53, indicating that, in the event of DNA damage, TP53 is able to prevent cell death of HL-60 leukaemia cells by sustaining an arrest of the cell cycle at G(2) phase. Further evidence will be presented to identify specific radiation-targeted genes or signals as possible biomarkers for early diagnosis of radiation damage as well as mission environmental monitoring.

Human testicular mast cells contain tryptase: increased mast cell number and altered distribution in the testes of infertile men.

OBJECTIVE: To determine whether human testicular mast cells contain the potent fibroblast growth factor tryptase and to examine changes in mast cell morphology and intratesticular distribution in testes with normal spermatogenesis versus abnormal spermatogenesis. DESIGN: Retrospective evaluation of testicular biopsies with the use of immunohistochemistry, morphometry, and electron microscopy. SETTING: University research and clinical institutes. PATIENT(S): Infertile men (total of 24) with severe hypospermatogenesis, germ cell arrest syndrome, or Sertoli cell only syndrome, and men without pathologies. INTERVENTION(S): Diagnostic testicular biopsy. MAIN OUTCOME MEASURE(S): Location, number, and distribution of testicular mast cells. RESULT(S): All groups showed tryptase-positive mast cells. In specimens with normal spermatogenesis, mast cells were round and located mainly in the interstitial spaces close to Leydig cells. In germ cell arrest syndrome, a 2-fold increase was evident, and in Sertoli cell only syndrome, a >3-fold increase of tryptase-immunoreactive mast cells became evident. Moreover, there was a statistically significant shift of the cells from the interstitium to the tubular walls in Sertoli cell only syndrome and germ cell arrest syndrome. Mast cells in specimens of Sertoli cell only syndrome and germ cell arrest syndrome were heterogeneous, with rounded or elongated shapes and signs of degranulation. The thickness of the tubular walls was doubled in specimens of germ cell arrest syndrome and Sertoli cell only syndrome in comparison with normal specimens, and this increase was positively correlated with the number of mast cells in these patients. CONCLUSION(S): Our results suggest that mast cell products, including the potent fibroblast growth factor tryptase, are involved in the thickening of the tubular wall and other changes in infertile testes.

Ionizing radiation induces up-regulation of functional beta1-integrin in human lung tumour cell lines in vitro.

PURPOSE: Cell-matrix interactions are in part mediated through the beta1-integrin pathway regulating cell survival, proliferation, adhesion and migration. This study was performed to elucidate alterations of expression of the beta1-integrin and its co-localized protein kinase, integrin-linked kinase (ILK), after exposure to ionizing radiation in two lung carcinoma cell lines in the presence or absence of different beta1-integrin-dependent matrix proteins. MATERIALS AND METHODS: Exponentially growing A549 and SKMES1 cells grown on fibronectin, laminin, BSA or plastic were exposed to 2 Gy or 6 Gy. Besides colony formation assays (0.5-8 Gy) and immediate plating experiments, flow cytometry (for beta1-integrin) and immunoblotting (for beta1-integrin and ILK) were carried out to analyze the protein expression. The localization of both proteins plus filamentous (f-) actin was further examined by immunofluorescence staining and laser confocal scanning microscopy. Functionality of the beta1 receptor subunit after irradiation was investigated in adhesion assays. RESULTS: A549 and SKMES1 cells grown on fibronectin or laminin demonstrated a significant increase in cell survival after irradiation compared to cells grown on BSA or plastic. Immediate plating of cells after irradiation on fibronectin did not show an improved survival. Flow cytometric and Western blot data showed a dose- and matrix-dependent induction of beta1-integrin and ILK expression after irradiation within 48 h. Adhesion to fibronectin or laminin compared to BSA or plastic was increased by 10-fold after irradiation, demonstrating these specific cell surface receptors to be functional. The staining of beta1-integrin and ILK in A549 cells confirmed the radiation-induced up-regulation of both proteins. Additionally, beta1-integrin and ILK co-localized with accumulated actin fibers at the cytoplasmic face of the cell membrane at confined areas. CONCLUSIONS: Ionizing radiation strongly induced the expression of functional beta1-integrin and ILK in the two lung cancer cell lines, A549 and SKMES1, dependent on different matrices used. Additionally, the subcellular localization of both proteins was altered by irradiation, and the individual cellular radiosensitivity was reduced in the presence of an extracellular matrix. On the one hand, this may result in aggravated therapeutic approaches and on the other hand, cells could adhere more strongly in their environment by the increase in functional surface receptor density preventing metastasis. Concerning intravascular located tumour cells, beta1-integrin up-regulation might enable these cells to adhere to the endothelium, which represents a prerequisite for metastatic disease. Identification of such mechanisms will provide considerable insights into the understanding of tumorigenicity and metastatic phenotypes, possibly leading to new, optimized radiochemotherapeutic regimens.

Protein kinase inhibitors modulate time-dependent effects of UV and ionizing irradiation on ICAM-1 expression on human hepatoma cells.

PURPOSE: To investigate modulation of the expression of the adhesion protein ICAM-1 by UV and ionizing irradiation. MATERIALS AND METHODS: HepG2 hepatoma cells were irradiated in vitro with UVB (20 mJ cm(-2)) or X-rays (5 Gy), respectively. Gene expression of ICAM-1 after irradiation was quantified by RT-PCR. Cell surface density of ICAM-1 was determined by flow cytometry. Protein or lipid kinase inhibitors were used to clarify radiation-induced transduction pathways that control ICAM-1 expression. Immuno-electron microscopy and dot-blot analysis were used to examine localization of ICAM-1. RESULTS: The study showed time-dependent effects of ionizing and UV irradiation on ICAM-1 expression of HepG2 cells. After an immediate transient decrease of ICAM-1 cell surface expression within minutes to hours, ICAM-1 expression increased up to 1.35-fold over normal level at 48 h post-irradiation. Irradiation caused ICAM-1 to become internalized into lysosomes. Additionally, ICAM-1 together with parts of the cell were pinched off. Finally, ICAM-1 levels were down- and up-regulated by decreased or increased gene expression. The early decrease of ICAM-1 expression could be blocked by a potent PKC inhibitor (BisX), whereas the increase of ICAM-1 after 24 h was prevented by addition of the p38 MAP kinase inhibitor SB 203580. CONCLUSION: The data suggest that ICAM-1 expression is modulated by UV, as well as ionizing radiation, in a time-dependent way involving PKC and p38 MAP kinase pathways.

Laboratory intercomparison on the γ-H2AX foci assay.

The focus of the study is an intercomparison of laboratories' dose-assessment performances using the γ-H2AX foci assay as a diagnostic triage tool for rapid individual radiation dose assessment. Homogenously X-irradiated (240 kVp, 1 Gy/min) blood samples for establishing calibration data (0.25-4 Gy) as well as blinded test samples (0.1-6.4 Gy) were incubated at 37°C for 2 and 24 h (repair time) and sent to the participants. The foci assay was performed according to protocols individually established in participating laboratories and therefore varied. The time taken to report dose estimates was documented for each laboratory. Additional information concerning laboratory organization/characteristics as well as assay performance was collected. The mean absolute difference (MAD) of estimated doses relative to the actual doses was calculated and radiation doses were merged into four triage categories reflecting clinical relevance to calculate accuracy, sensitivity and specificity. First γ-H2AX based dose estimates were reported 7 h after sample receipt. Estimates were similarly accurate for 2 and 24 h repair times, providing scope for its use in the early phase of a radiation exposure incident. Equal accuracy was achieved by scoring 20, 30, 40 or 50 cells per sample. However, MAD values of 0.5-0.7 Gy and 1.3-1.7 Gy divided the data sets into two groups, driven mainly by the considerable differences in foci yields between calibration and blind samples. Foci yields also varied dramatically between laboratories, highlighting reproducibility issues as an important caveat of the foci assay. Nonetheless, foci counts could distinguish high- and low-dose samples in all data sets and binary dose categories of clinical significance could be discriminated with satisfactory accuracy (mean 84%, ±0.03 SEM). Overall, the results suggest that the γ-H2AX assay is a useful tool for rapidly screening individuals for significant exposures that occurred up to at least 24 h earlier, and may help to prioritize cytogenetic dosimetry follow-up.

Laboratory intercomparison of gene expression assays.

The possibility of a large-scale acute radiation exposure necessitates the development of new methods that could provide rapid individual dose estimates with high sample throughput. The focus of the study was an intercomparison of laboratories' dose-assessment performances using gene expression assays. Lithium-heparinized whole blood from one healthy donor was irradiated (240 kVp, 1 Gy/min) immediately after venipuncture at approximately 37°C using single X-ray doses. Blood samples to establish calibration curves (0.25-4 Gy) as well as 10 blinded test samples (0.1-6.4 Gy) were incubated for 24 h at 37°C supplemented with an equal volume of medium and 10% fetal calf serum. For quantitative reverse transcription polymerase chain reaction (qRT-PCR), samples were lysed, stored at -20°C and shipped on ice. For the Chemical Ligation Dependent Probe Amplification methodology (CLPA), aliquots were incubated in 2 ml CLPA reaction buffer (DxTerity), mixed and shipped at room temperature. Assays were run in each laboratory according to locally established protocols. The mean absolute difference (MAD) of estimated doses relative to the true doses (in Gy) was calculated. We also merged doses into binary categories reflecting aspects of clinical/diagnostic relevance and examined accuracy, sensitivity and specificity. The earliest reported time on dose estimates was <8 h. The standard deviation of technical replicate measurements in 75% of all measurements was below 11%. MAD values of 0.3-0.5 Gy and 0.8-1.3 Gy divided the laboratories contributions into two groups. These fourfold differences in accuracy could be primarily explained by unexpected variances of the housekeeping gene (P = 0.0008) and performance differences in processing of calibration and blinded test samples by half of the contributing laboratories. Reported gene expression dose estimates aggregated into binary categories in general showed an accuracies and sensitivities of 93-100% and 76-100% for the groups, with low MAD and high MAD, respectively. In conclusion, gene expression-based dose estimates were reported quickly, and for laboratories with MAD between 0.3-0.5 Gy binary dose categories of clinical significance could be discriminated with an accuracy and sensitivity comparable to established cytogenetic assays.

Laboratory intercomparison of the dicentric chromosome analysis assay.

The study design and obtained results represent an intercomparison of various laboratories performing dose assessment using the dicentric chromosome analysis (DCA) as a diagnostic triage tool for individual radiation dose assessment. Homogenously X-irradiated (240 kVp, 1 Gy/min) blood samples for establishing calibration data (0.25-5 Gy) as well as blind samples (0.1-6.4 Gy) were sent to the participants. DCA was performed according to established protocols. The time taken to report dose estimates was documented for each laboratory. Additional information concerning laboratory organization/characteristics as well as assay performance was collected. The mean absolute difference (MAD) was calculated and radiation doses were merged into four triage categories reflecting clinical aspects to calculate accuracy, sensitivity and specificity. The earliest report time was 2.4 days after sample arrival. DCA dose estimates were reported with high and comparable accuracy, with MAD values ranging between 0.16-0.5 Gy for both manual and automated scoring. No significant differences were found for dose estimates based either on 20, 30, 40 or 50 cells, suggesting that the scored number of cells can be reduced from 50 to 20 without loss of precision of triage dose estimates, at least for homogenous exposure scenarios. Triage categories of clinical significance could be discriminated efficiently using both scoring procedures.

Laboratory intercomparison of the cytokinesis-block micronucleus assay.

The focus of the study is an intercomparison of laboratories' dose-assessment performances using the cytokinesis-block micronucleus (CBMN) assay as a diagnostic triage tool for individual radiation dose assessment. Homogenously X-irradiated (240 kVp, 1 Gy/min) blood samples for establishing calibration data (0.25-5 Gy) as well as blind samples (0.1-6.4 Gy) were sent to the participants. The CBMN assay was performed according to protocols individually established and varying among participating laboratories. The time taken to report dose estimates was documented for each laboratory. Additional information concerning laboratory organization/characteristics as well as assay performance was collected. The mean absolute difference (MAD) was calculated and radiation doses were merged into four triage categories reflecting clinical aspects to calculate accuracy, sensitivity and specificity. The earliest report time was 4 days after sample arrival. The CBMN dose estimates were reported with high accuracy (MAD values of 0.20-0.50 Gy at doses below 6.4 Gy for both manual and automated scoring procedures), but showed a limitation of the assay at the dose point of 6.4 Gy, which resulted in a clear dose underestimation in all cases. The MAD values (without 6.4 Gy) differed significantly (P = 0.03) between manual (0.25 Gy, SEM = 0.06, n = 4) or automated scoring procedures (0.37 Gy, SEM = 0.08, n = 5), but lowest MAD were equal (0.2 Gy) for both scoring procedures. Likewise, both scoring procedures led to the same allocation of dose estimates to triage categories of clinical significance (about 83% accuracy and up to 100% specificity).

Comparison of established and emerging biodosimetry assays.

Rapid biodosimetry tools are required to assist with triage in the case of a large-scale radiation incident. Here, we aimed to determine the dose-assessment accuracy of the well-established dicentric chromosome assay (DCA) and cytokinesis-block micronucleus assay (CBMN) in comparison to the emerging γ-H2AX foci and gene expression assays for triage mode biodosimetry and radiation injury assessment. Coded blood samples exposed to 10 X-ray doses (240 kVp, 1 Gy/min) of up to 6.4 Gy were sent to participants for dose estimation. Report times were documented for each laboratory and assay. The mean absolute difference (MAD) of estimated doses relative to the true doses was calculated. We also merged doses into binary dose categories of clinical relevance and examined accuracy, sensitivity and specificity of the assays. Dose estimates were reported by the first laboratories within 0.3-0.4 days of receipt of samples for the γ-H2AX and gene expression assays compared to 2.4 and 4 days for the DCA and CBMN assays, respectively. Irrespective of the assay we found a 2.5-4-fold variation of interlaboratory accuracy per assay and lowest MAD values for the DCA assay (0.16 Gy) followed by CBMN (0.34 Gy), gene expression (0.34 Gy) and γ-H2AX (0.45 Gy) foci assay. Binary categories of dose estimates could be discriminated with equal efficiency for all assays, but at doses ≥1.5 Gy a 10% decrease in efficiency was observed for the foci assay, which was still comparable to the CBMN assay. In conclusion, the DCA has been confirmed as the gold standard biodosimetry method, but in situations where speed and throughput are more important than ultimate accuracy, the emerging rapid molecular assays have the potential to become useful triage tools.

Gene expression comparisons performed for biodosimetry purposes on in vitro peripheral blood cellular subsets and irradiated individuals.

We examined the benefit of gene expression analysis on peripheral blood cellular subsets of different radiosensitivity to elucidate their utility as biodosimeters for estimation of dose in irradiated individuals. Peripheral mononucleated cells were isolated from 18 healthy volunteers employing density separation in a CPT-NH tube. Peripheral mononucleated cells were cultured in RPMI 1640 medium containing 10% autologous serum and were irradiated with 0.1-1 Gy (240 kV, 13 mA, X rays at 1 Gy/min). A low-dose study was performed with isolated peripheral mononucleated cells from one healthy donor in three independent experiments. Peripheral mononucleated cells were irradiated at 0 (sham), 1, 2.5 and 5 cGy (70 kV, 13 mA X rays at 1 cGy/min) and gene expression was measured 24 and 48 h after irradiation. After irradiation, CD4(+) or CD8(+) cells were isolated by magnetic beads in independent experiments. RNA from lymphocyte subsets and peripheral mononucleated cells was isolated after 24 and 48 h and converted into cDNA. Gene expression of GADD45, CDKN1A, DDB2, PCNA, BAX and ATF3 were determined using RTQ-PCR. Data were analyzed employing linear and logistic regression analysis. The same examinations were performed in 5 individuals either diagnosed using CT scans (up to 4.3 cGy) or by administering (F-18)-fluoro-2-deoxy-d-glucose (F-18 FDG, 0.6 cGy). Methodological, intra- and inter-individual variability in 90-95% of measurements did not exceed the introduced twofold change over sham-irradiated control values in peripheral mononucleated cells and CD4(+) cells, and therefore no false positive results were observed. Dose reconstruction in peripheral mononucleated cells in opposite to CD4(+) lymphocytes required fewer genes and appeared more efficient (R-square = 84.8% compared to 51.8%). In vitro samples exposed to 10 cGy could be completely discriminated from sham-irradiated samples without individual pre-exposure controls, which coincided with our preliminary in vivo results. However, in vitro differential gene expression was measured relative to control values and did not differ significantly at 24 and 48 h after irradiation in contrast to our preliminary in vivo data. In addition, below 5 cGy in vitro data did not show reproducible significant changes in gene expression, which was opposite to our preliminary in vivo data. Therefore a twofold change in gene expression over control sufficiently controls for different sources of variance, and measuring gene expression in peripheral mononucleated cell for biological dosimetry purposes appears superior over measurements in lymphocyte subsets. The increased gene expression measured after low absorbed doses in vivo and in vitro might indicate a particular applicability of this method for a low-level radiation scenario in the absence of individual pre-exposure controls. However, the constant gene expression values measured up to 48 h in our in vitro model at doses >10 cGy, and the absence of reproducible and statistically significant gene expression changes below 5 cGy contrast to the preliminary in vivo results performed at similar doses. Therefore, measurements with our in vitro models should be interpreted cautiously.

Acute high-dose X-radiation-induced genomic changes in A549 cells.

Accidents with ionizing radiation often involve single, acute high-dose exposures that can lead to acute radiation syndrome and late effects such as carcinogenesis. To study such effects at the cellular level, we investigated acute ionizing radiation-induced chromosomal aberrations in A549 adenocarcinoma cells at the genome-wide level by exposing the cells to an acute dose of 6 Gy 240 kV X rays. One sham-irradiated clone and four surviving irradiated clones were recovered by minimal dilution and further expanded and analyzed by chromosome painting and tiling-path array CGH, with the nonirradiated clone 0 serving as the control. Acute X-ray exposure induced specific translocations and changes in modal chromosome number in the four irradiated clones. Array CGH disclosed unique and recurrent genomic changes, predominantly losses, and revealed that the fragile sites FRA3B and FRA16D were preferential regions of genomic alterations in all irradiated clones, which is likely related to radioresistant S-phase progression and genomic stress. Furthermore, clone 4 displayed an increased radiosensitivity at doses >5 Gy. Pairwise comparisons of the gene expression patterns of all irradiated clones to the sham-irradiated clone 0 revealed an enrichment of the Gene Ontology term "M Phase" (P = 6.2 × 10(-7)) in the set of differentially expressed genes of clone 4 but not in those of clones 1-3. Ionizing radiation-induced genomic changes and fragile site expression highlight the capacity of a single acute radiation exposure to affect the genome of exposed cells by inflicting genomic stress.