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Background: Patients with high-risk neuroblastoma (NB) have a 5-year event-free survival of less than 50 %, and novel and improved treatment options are needed. Radiolabeled somatostatin analogs (SSTAs) could be a treatment option. The aims of this work were to compare the biodistribution and the therapeutic effects of 177Lu-octreotate and 177Lu-octreotide in mice bearing the human CLB-BAR NB cell line, and to evaluate their regulatory effects on apoptosis-related genes. Methods: The biodistribution of 177Lu-octreotide in mice bearing CLB-BAR tumors was studied at 1, 24, and 168 h after administration, and the absorbed dose was estimated to tumor and normal tissues. Further, animals were administered different amounts of 177Lu-octreotate or 177Lu-octreotide. Tumor volume was measured over time and compared to a control group given saline. RNA was extracted from tumors, and the expression of 84 selected genes involved in apoptosis was quantified with qPCR. Results: The activity concentration was generally lower in most tissues for 177Lu-octreotide compared to 177Lu-octreotate. Mean absorbed dose per administered activity to tumor after injection of 1.5 MBq and 15 MBq was 0.74 and 0.03 Gy/MBq for 177Lu-octreotide and 2.9 and 0.45 Gy/MBq for 177Lu-octreotate, respectively. 177Lu-octreotide treatment resulted in statistically significant differences compared to controls. Fractionated administration led to a higher survival fraction than after a single administration. The pro-apoptotic genes TNSFS8, TNSFS10, and TRADD were regulated after administration with 177Lu-octreotate. Treatment with 177Lu-octreotide yielded regulation of the pro-apoptotic genes CASP5 and TRADD, and of the anti-apoptotic gene IL10 as well as the apoptosis-related gene TNF. Conclusion: 177Lu-octreotide gave somewhat better anti-tumor effects than 177Lu-octreotate. The similar effect observed in the treated groups with 177Lu-octreotate suggests saturation of the somatostatin receptors. Pronounced anti-tumor effects following fractionated administration merited receptor saturation as an explanation. The gene expression analyses suggest apoptosis activation through the extrinsic pathway for both radiopharmaceuticals.

Wild European perch (Perca fluviatilis) is one of the most important freshwater fish species, in Sweden, due to its widespread and his value for recreational fishing. Little it is known regarding the biodistribution of naturally occurring radionuclides such as 238U, 234U, 226Ra, 210Po in perch. Therefore, in this study, perches from five lakes located in different counties in Sweden were collected to investigate the biodistribution of 238U, 234U, 226Ra, 210Po and 137Cs in organs and tissues of perch as well as their radiological impact. The results showed that uranium radionuclides ranged between 0.1 and 6 Bq/kg with an average value of 1.1 ± 1.5 Bq/kg. 226Ra varied from 0.4 to 8 Bq/kg with a mean concentration of 1.7 ± 1.9 Bq/kg. The ranged of 210Po was 0.5 – 250 Bq/kg, with an average value of 24 ± 52 Bq/kg. On the other hand, the highest activity concentration of 137Cs, 151 ± 1 Bq/kg, was detected in muscle samples of perch from Redsjösjön lake. For uranium radionuclides and 226Ra uptake from water is the main source whereas for 210Po and 137Cs the uptake is controlled by the perch diet. Regarding naturally occurring radionuclides, the perch tended to accumulated uranium radionuclides in fins, gills, and skin; 226Ra in bones, fins and skin and 210Po in the organs linked to digestive system. Finally, in case of consumption, it is advised the consumption of skinned fillets of perch due to the higher bioaccumulation of the radionuclides investigated in the skin and scales.

Background: In phenylketonuria (PKU), weaning is considered more challenging when compared to feeding healthy infants. The primary aim of weaning is to gradually replace natural protein from breast milk or standard infant formula with solids containing equivalent phenylalanine (Phe). In addition, a Phe-free second stage L-amino acid supplement is usually recommended from around 6 months to replace Phe-free infant formula. Our aim was to assess different weaning approaches used by health professionals across Europe. Methods: A cross sectional questionnaire (survey monkey®) composed of 31 multiple and single choice questions was sent to European colleagues caring for inherited metabolic disorders (IMD). Centres were grouped into geographical regions for analysis. Results: Weaning started at 17–26 weeks in 85% (n = 81/95) of centres, >26 weeks in 12% (n = 11/95) and 26 weeks. First solids were mainly low Phe vegetables (59%, n = 56/95) and fruit (34%, n = 32/95).A Phe exchange system to allocate dietary Phe was used by 52% (n = 49/95) of centres predominantly from Northern and Southern Europe and 48% (n = 46/95) calculated most Phe containing food sources (all centres in Eastern Europe and the majority from Germany and Austria). Some centres used a combination of both methods.A second stage Phe-free L-amino acid supplement containing a higher protein equivalent was introduced by 41% (n = 39/95) of centres at infant age 26–36 weeks (mainly from Germany, Austria, Northern and Eastern Europe) and 37% (n = 35/95) at infant age > 1y mainly from Southern Europe. 53% (n = 50/95) of centres recommended a second stage Phe-free L-amino acid supplement in a spoonable or semi-solid form. Conclusions: Weaning strategies vary throughout European PKU centres. There is evidence to suggest that different infant weaning strategies may influence longer term adherence to the PKU diet or acceptance of Phe-free L-amino acid supplements; rendering prospective long-term studies important. It is essential to identify an effective weaning strategy that reduces caregiver burden but is associated with acceptable dietary adherence and optimal infant feeding development. Keywords: Weaning, Infant, Phenylketonuria, Phenylalanine, Phe-free infant formula

Background: In infants with phenylketonuria (PKU), dietary management is based on lowering and titrating phenylalanine (Phe) intake from breast milk or standard infant formula in combination with a Phe-free infant formula in order to maintain blood Phe levels within target range. Professionals use different methods to feed infants with PKU and our survey aimed to document practices across Europe. Methods: We sent a cross sectional, survey monkey® questionnaire to European health professionals working in IMD. It contained 31 open and multiple-choice questions. The results were analysed according to different geographical regions. Results: Ninety-five centres from 21 countries responded. Over 60% of centres commenced diet in infants by age 10 days, with 58% of centres implementing newborn screening by day 3 post birth. At diagnosis, infant hospital admission occurred in 61% of metabolic centres, mainly in Eastern, Western and Southern Europe. Breastfeeding fell sharply following diagnosis with only 30% of women still breast feeding at 6 months.53% of centres gave pre-measured Phe-free infant formula before each breast feed and 23% alternated breast feeds with Phe-free infant formula. With standard infant formula feeds, measured amounts were followed by Phe-free infant formula to satiety in 37% of centres (n = 35/95), whereas 44% (n = 42/95) advised mixing both formulas together. Weaning commenced between 17 and 26 weeks in 85% centres, ≥26 weeks in 12% and

In Sweden, around 20 % of the potable water comes from groundwater sources and about one million people drink water from their private wells. In areas with moderate or high abundance of naturally occurring radionuclides in the bedrock, the groundwater could be enhanced with radio and chemically toxic elements such as uranium, radium, lead and polonium. Therefore, this study aims to carry out a comprehensive analysis of the behaviour and radiological impact of naturally occurring radionuclides in well water. For that, a well water survey was done in 2020 along Sweden, focusing the study on the determination of 210 Po, 210 Pb, 226 Ra, 228 Ra, 238 U and 234 U by alpha spectrometry. The results of the present study showed that the naturally occurring radionuclides distributions in the well water investigated were highly variable. In addition, the radionuclides with the highest contribution to the indicative dose were 210 Pb >  228 Ra >  210 Po >  226 Ra >  234 U >  238 U. On the other hand, the filtration and aeration purification treatment could help to remediate the radiological impact. However, the dose assessment results point out the need to include well water in the framework of the current regulations of requirements for the protection of the health of the public regarding radioactive substances in water intended for human consumption, to guarantee the quality of water from the radiological point of view., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Computational pharmacogenomics can potentially identify new indications for already approved drugs and pinpoint compounds with similar mechanism-of-action. Here, we used an integrated drug repositioning approach based on transcriptomics data and structure-based virtual screening to identify compounds with gene signatures similar to three known proteasome inhibitors (PIs; bortezomib, MG-132, and MLN-2238). In vitro validation of candidate compounds was then performed to assess proteasomal proteolytic activity, accumulation of ubiquitinated proteins, cell viability, and drug-induced expression in A375 melanoma and MCF7 breast cancer cells. Using this approach, we identified six compounds with PI properties ((-)-kinetin-riboside, manumycin-A, puromycin dihydrochloride, resistomycin, tegaserod maleate, and thapsigargin). Although the docking scores pinpointed their ability to bind to the β5 subunit, our in vitro study revealed that these compounds inhibited the β1, β2, and β5 catalytic sites to some extent. As shown with bortezomib, only manumycin-A, puromycin dihydrochloride, and tegaserod maleate resulted in excessive accumulation of ubiquitinated proteins and elevated HMOX1 expression. Taken together, our integrated drug repositioning approach and subsequent in vitro validation studies identified six compounds demonstrating properties similar to proteasome inhibitors., (© 2024. The Author(s).)

Repurposing of FDA-approved drugs is a quick and cost-effective alternative to de novo drug development. Here, we identify genes involved in bortezomib sensitivity, predict cancer types that may benefit from treatment with bortezomib, and evaluate the mechanism-of-action of bortezomib in breast cancer (BT-474 and ZR-75-30), melanoma (A-375), and glioblastoma (A-172) cells in vitro. Cancer cell lines derived from cancers of the blood, kidney, nervous system, and skin were found to be significantly more sensitive to bortezomib than other organ systems. The in vitro studies confirmed that although bortezomib effectively inhibited the β5 catalytic site in all four cell lines, cell cycle arrest was only induced in G2/M phase and apoptosis in A-375 and A-172 after 24h. The genomic and transcriptomic analyses identified 33 genes (e.g. ALDH18A1, ATAD2) associated with bortezomib resistance. Taken together, we identified biomarkers predictive of bortezomib sensitivity and cancer types that might benefit from treatment with bortezomib., (© 2024. The Author(s).)

Recombinant α 1 -microglobulin (A1M) is proposed as a protector during 177 Lu-octreotate treatment of neuroendocrine tumors, which is currently limited by bone marrow and renal toxicity. Co-administration of 177 Lu-octreotate and A1M could result in a more effective treatment by protecting healthy tissue, but the radioprotective action of A1M is not fully understood. The aim of this study was to examine the proteomic response of kidneys and bone marrow early after 177 Lu-octreotate and/or A1M administration. Mice were injected with 177 Lu-octreotate and/or A1M, while control mice received saline or A1M vehicle solution. Bone marrow, kidney medulla, and kidney cortex were sampled after 24 h or 7 d. The differential protein expression was analyzed with tandem mass spectrometry. The dosimetric estimation was based on 177 Lu activity in the kidney. PHLDA3 was the most prominent radiation-responsive protein in kidney tissue. In general, no statistically significant difference in the expression of radiation-related proteins was observed between the irradiated groups. Most canonical pathways were identified in bone marrow from the 177 Lu-octreotate+A1M group. Altogether, a tissue-dependent proteomic response followed exposure to 177 Lu-octreotate alone or together with A1M. Combining 177 Lu-octreotate with A1M did not inhibit the radiation-induced protein expression early after exposure, and late effects should be further studied.

Triple-negative breast cancer (TNBC) is associated with poor prognosis and limited treatment options due to the lack of important receptors (estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor receptor 2 [HER2]) used for targeted therapy. However, high-throughput in vitro drug screening of cell lines is a powerful tool for identifying effective drugs for a disease. Here, we determine the intrinsic chemosensitivity of TNBC cell lines to proteasome inhibitors (PIs), thereby identifying potentially potent 2-drug combinations for TNBC. Eight TNBC cell lines (BT-549, CAL-148, HCC1806, HCC38, HCC70, MDA-MB-436, MDA-MB-453, and MDA-MB-468) and two controls (MCF-10A and MCF-7) were first exposed to 18 drugs (11 PIs and 7 clinically relevant chemotherapeutic agents) as monotherapy, followed by prediction of potent 2-drug combinations using the IDACombo pipeline. The synergistic effects of the 2-drug combinations were evaluated with SynergyFinder in four TNBC cell lines (CAL-148, HCC1806, HCC38, and MDA-MB-468) and three controls (BT-474, MCF-7, and T47D) in vitro, followed by further evaluation of tumor regression in zebrafish tumor models established using HCC1806 and MCF-7 cells. Monotherapy identified nine effective drugs (bortezomib, carfilzomib, cisplatin, delanzomib, docetaxel, epoxomicin, MLN-2238, MLN-9708, and nedaplatin) across all cell lines. PIs (e.g., bortezomib, delanzomib, and epoxomicin) were highly potent drugs in TNBC cells, of which bortezomib and delanzomib inhibited the chymotrypsin-like activity of the 20 S proteasome by 100% at 10 µM. Moreover, several potent 2-drug combinations (e.g., bortezomib+nedaplatin and epoxomicin+epirubicin) that killed virtually 100% of cells were also identified. Although HCC1806- and MCF-7-derived xenografts treated with bortezomib+nedaplatin and carboplatin+paclitaxel were smaller, HCC1806 cells frequently metastasized to the trunk region. Taken together, we show that PIs used in combination with platinum agents or topoisomerase inhibitors exhibit increased efficiency with almost 100% inhibition in TNBC cell lines, indicating that PIs are therefore promising compounds to use as combination therapy for TNBC., (© 2024. The Author(s).)

177 Lu-DOTATATE peptide receptor radionuclide therapy (PRRT) is used clinically to treat metastasized or unresectable neuroendocrine tumors (NETs). Although 177 Lu-DOTATATE is mostly well tolerated in patients, bone marrow suppression and long-term renal toxicity are still side effects that should be considered. Amino acids are often used to minimize renal radiotoxicity, however, they are associated with nausea and vomiting in patients. α 1 -microglobulin (A1M) is an antioxidant with heme- and radical-scavenging abilities. A recombinant form (rA1M) has previously been shown to be renoprotective in preclinical models, including in PRRT-induced kidney damage. Here, we further investigated rA1M's renal protective effect in a mouse 177 Lu-DOTATATE model in terms of administration route and dosing regimen and as a combined therapy with amino acids (Vamin). Moreover, we investigated the protective effect of rA1M on peripheral blood and bone marrow cells, as well as circulatory biomarkers. Intravenous (i.v.) administration of rA1M reduced albuminuria levels and circulatory levels of the oxidative stress-related protein fibroblast growth factor-21 (FGF-21). Dual injections of rA1M (i.e., at 0 and 24 h post- 177 Lu-DOTATATE administration) preserved bone marrow cellularity and peripheral blood reticulocytes. Administration of Vamin, alone or in combination with rA1M, did not show any protection of bone marrow cellularity or peripheral reticulocytes. In conclusion, this study suggests that rA1M, administered i.v. for two consecutive days in conjunction with 177 Lu-DOTATATE, may reduce hematopoietic and kidney toxicity during PRRT with 177 Lu-DOTATATE.

Recombinant α 1 -microglobulin (A1M) is a proposed radioprotector during 177 Lu-octreotate therapy of neuroendocrine tumors (NETs). To ensure a maintained therapeutic effect, we previously demonstrated that A1M does not affect the 177 Lu-octreotate induced decrease in GOT1 tumor volume. However, the underlying biological events of these findings are still unknown. The aim of this work was to examine the regulation of apoptosis-related genes in GOT1 tumors short-time after i.v. administration of 177 Lu-octreotate with and without A1M or A1M alone. Human GOT1 tumor-bearing mice received 30 MBq 177 Lu-octreotate or 5 mg/kg A1M or co-treatment with both. Animals were sacrificed after 1 or 7 days. Gene expression analysis of apoptosis-related genes in GOT1 tissue was performed with RT-PCR. In general, similar expression patterns of pro- and anti-apoptotic genes were found after 177 Lu-octreotate exposure with or without co-administration of A1M. The highest regulated genes in both irradiated groups compared to untreated controls were FAS and TNFSFRS10B. Administration of A1M alone only resulted in significantly regulated genes after 7 days. Co-administration of A1M did not negatively affect the transcriptional apoptotic response of 177 Lu-octreotate in GOT1 tumors., (© 2023. The Author(s).)

Background: The human proteasome gene family (PSM) consists of 49 genes that play a crucial role in cancer proteostasis. However, little is known about the effect of PSM gene expression and genetic alterations on clinical outcome in different cancer forms., Methods: Here, we performed a comprehensive pan-cancer analysis of genetic alterations in PSM genes and the subsequent prognostic value of PSM expression using data from The Cancer Genome Atlas (TCGA) containing over 10,000 samples representing up to 33 different cancer types. External validation was performed using a breast cancer cohort and KM plotter with four cancer types., Results: The PSM genetic alteration frequency was high in certain cancer types (e.g. 67%; esophageal adenocarcinoma), with DNA amplification being most common. Compared with normal tissue, most PSM genes were predominantly overexpressed in cancer. Survival analysis also established a relationship with PSM gene expression and adverse clinical outcome, where PSMA1 and PSMD11 expression were linked to more unfavorable prognosis in ≥ 30% of cancer types for both overall survival (OS) and relapse-free interval (PFI). Interestingly, PSMB5 gene expression was associated with OS (36%) and PFI (27%), and OS for PSMD2 (42%), especially when overexpressed., Conclusion: These findings indicate that several PSM genes may potentially be prognostic biomarkers and novel therapeutic targets for different cancer forms., (© 2022. The Author(s).)

Molecular biomarkers of ionizing radiation (IR) exposure are a promising new tool in various disciplines: they can give necessary information for adaptive treatment planning in cancer radiotherapy, enable risk projection for radiation-induced survivorship diseases, or facilitate triage and intervention in radiation hazard events. However, radiation biomarker discovery has not yet resolved the most basic features of personalized medicine: age and sex. To overcome this critical bias in biomarker identification, we quantitated age and sex effects and assessed their relevance in the radiation response across the blood proteome. We used high-throughput mass spectrometry on blood plasma collected 24 h after 0.5 Gy total body irradiation (15 MV nominal photon energy) from male and female C57BL/6 N mice at juvenile (7-weeks-old) or adult (18-weeks-old) age. We also assessed sex and strain effects using juvenile male and female BALB/c nude mice. We showed that age and sex created significant effects in the proteomic response regarding both extent and functional quality of IR-induced responses. Furthermore, we found that age and sex effects appeared non-linear and were often end-point specific. Overall, age contributed more to differences in the proteomic response than sex, most notably in immune responses, oxidative stress, and apoptotic cell death. Interestingly, sex effects were pronounced for DNA damage and repair pathways and associated cellular outcome (pro-survival vs. pro-apoptotic). Only one protein (AHSP) was identified as a potential general biomarker candidate across age and sex, while GMNN, REG3B, and SNCA indicated some response similarity across age. This low yield advocated that unisex or uniage biomarker screening approaches are not feasible. In conclusion, age- and sex-specific screening approaches should be implemented as standard protocol to ensure robustness and diagnostic power of biomarker candidates. Bias-free molecular biomarkers are a necessary progression towards personalized medicine and integral for advanced adaptive cancer radiotherapy and risk assessment., (© 2022. The Author(s).)

Northern Sweden has been the object of intense metal mining in the last decades producing several water-filled open-pits, or pit lakes. Most of these pit lakes have been limed to maintain a good water quality and to prevent generation of acidic water that could leach the exposed rocks and release metals into water. The aim of this work was to examine the concentration of stable elements and naturally occurring radionuclides in water and sediment samples from pit lakes originating from non-uranium mining activities in Northern Sweden. Surface water and surface sediments were collected from 27 pit lakes in Northern Sweden. Water quality parameters, concentration of stable elements and radionuclides were measured by a water probe, ICP-MS and XRF, and alpha and gamma spectrometry, respectively. Furthermore, a multivariate statistical analysis (PCA) was performed on the water samples and sediments. In general, the quality of the surface water was good, but some lakes had low pH values (2.5-5.7), and high concentrations of Fe (up to 200 mg/L) and other metals (e.g. Zn, Cu). When relating the metal concentrations in sediments in pit lakes with the concentration found in natural lakes, some sites had relatively high levels of Cu, As, Cr and Pb. The activity concentration of 210Po, and U and Th isotopes in water and sediment samples were at environmental levels, as was the ambient dose equivalent rate at these sites (range 0.08-0.14 μSv/h)., Competing Interests: The authors have declared that no competing interests exist.

131 I is used clinically for therapy, and may be released during nuclear accidents. After the Chernobyl accident papillary thyroid carcinoma incidence increased in children, but not adults. The aims of this study were to compare 131 I irradiation-dependent differences in RNA and protein expression in the thyroid and plasma of young and adult rats, and identify potential age-dependent biomarkers for 131 I exposure. Twelve young (5 weeks) and twelve adult Sprague Dawley rats (17 weeks) were i.v. injected with 50 kBq 131 I (absorbed dose to thyroid = 0.1 Gy), and sixteen unexposed age-matched rats were used as controls. The rats were killed 3-9 months after administration. Microarray analysis was performed using RNA from thyroid samples, while LC-MS/MS analysis was performed on proteins extracted from thyroid tissue and plasma. Canonical pathways, biological functions and upstream regulators were analysed for the identified transcripts and proteins. Distinct age-dependent differences in gene and protein expression were observed. Novel biomarkers for thyroid 131 I exposure were identified: (PTH), age-dependent dose response (CA1, FTL1, PVALB (youngsters) and HSPB6 (adults)), thyroid function (Vegfb (adults)). Further validation using clinical samples are needed to explore the role of the identified biomarkers., (© 2022. The Author(s).)

Radionuclide treatment of patients with neuroendocrine tumors has advanced in the last decades with favorable results using 177 Lu-octreotate. However, the gap between the high cure rate in animal studies vs. patient studies indicates a potential to increase the curation of patients. The aim of this study was to investigate the tumor response for different fractionation schemes with 177 Lu-octreotate. BALB/c mice bearing a human small-intestine neuroendocrine GOT1 tumor were either mock treated with saline or injected intravenously with a total of 30-120 MBq of 177 Lu-octreotate: 1 × 30, 2 × 15, 1 × 60, 2 × 30, 1 × 120, 2 × 60, or 3 × 40 MBq. The tumor volume was measured twice per week until the end of the experiment. The mean tumor volume for mice that received 2 × 15 = 30 and 1 × 30 MBq 177 Lu-octreotate was reduced by 61% and 52%, respectively. The mean tumor volume was reduced by 91% and 44% for mice that received 2 × 30 = 60 and 1 × 60 MBq 177 Lu-octreotate, respectively. After 120 MBq 177 Lu-octreotate, given as 1-3 fractions, the mean tumor volume was reduced by 91-97%. Multiple fractions resulted in delayed regrowth and prolonged overall survival by 20-25% for the 120 MBq groups and by 45% for lower total activities, relative to one fraction. The results indicate that fractionation and hyperfractionation of 177 Lu-octreotate are beneficial for tumor reduction and prolongs the time to regrowth.

Background: Neuroblastoma (NB) is one of the most frequently diagnosed tumors in infants. NB is a neuroendocrine tumor type with various characteristics and features, and with diverse outcome. The most malignant NBs have a 5-year survival rate of only 40-50%, indicating the need for novel and improved treatment options. 177 Lu-octreotate is routinely administered for treatment of neuroendocrine tumors overexpressing somatostatin receptors (SSTR). The aim of this study was to examine the biodistribution of 177 Lu-octreotate in mice bearing aggressive human NB cell lines, in order to evaluate the potential usefulness of 177 Lu-octreotate for treatment of NB., Methods: BALB/c nude mice bearing CLB-BAR, CLB-GE or IMR-32 tumor xenografts (n = 5-7/group) were i.v. injected with 0.15 MBq, 1.5 MBq or 15 MBq 177 Lu-octreotate and sacrificed 1 h, 24 h, 48 h and 168 h after administration. The radioactivity concentration was determined for collected tissue samples, tumor-to-normal-tissue activity concentration ratios (T/N) and mean absorbed dose for each tissue were calculated. Immunohistochemical (IHC) staining for SSTR1-5, and Ki67 were carried out for tumor xenografts from the three cell lines., Results: High 177 Lu concentration levels and T/N values were observed in all NB tumors, with the highest for CLB-GE tumor xenografts (72%IA/g 24 h p.i.; 1.5 MBq 177 Lu-octreotate). The mean absorbed dose to the tumor was 6.8 Gy, 54 Gy and 29 Gy for CLB-BAR, CLB-GE and IMR-32, respectively, p.i. of 15 MBq 177 Lu-octreotate. Receptor saturation was clearly observed in CLB-BAR, resulting in higher concentration levels in the tumor when lower activity levels where administered. IHC staining demonstrated highest expression of SSTR2 in CLB-GE, followed by CLB-BAR and IMR-32., Conclusion: T/N values for all three human NB tumor xenograft types investigated were high relative to previously investigated neuroendocrine tumor types. The results indicate a clear potential of 177 Lu-octreotate as a therapeutic alternative for metastatic NB., (© 2021. The Author(s).)

Breast cancer (BC) patients are frequently at risk of developing other malignancies following treatment. Although studies have been conducted to elucidate the etiology of multiple primary malignancies (MPM) after a BC diagnosis, few studies have investigated other previously diagnosed primary malignancies (OPPM) before BC. Here, genome-wide profiling was used to identify potential driver DNA copy number alterations and somatic mutations that promote the development of MPMs. To compare the genomic profiles for two primary tumors (BC and OPPM) from the same patient, tumor pairs from 26 young women (≤50 years) diagnosed with one or more primary malignancies before breast cancer were analyzed. Malignant melanoma was the most frequent OPPM, followed by gynecologic- and hematologic malignancies. However, significantly more genetic alterations were detected in BC compared to the OPPM. BC also showed more genetic similarity as a group than the tumor pairs. Clonality testing showed that genetic alterations on chromosomes 1, 3, 16, and 19 were concordant in both tumors in 13 patients. TP53 mutations were also found to be prevalent in BC, MM, and HM. Although all samples were classified as genetically unstable, chromothripsis-like patterns were primarily observed in BC. Taken together, few recurrent genetic alterations were identified in both tumor pairs that can explain the development of MPMs in the same patient. However, larger studies are warranted to further investigate key driver mutations associated with MPMs., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Radioiodide (131I) is commonly used to treat thyroid cancer and hyperthyroidis.131I released during nuclear accidents, have resulted in increased incidence of thyroid cancer in children. Therefore, a better understanding of underlying cellular mechanisms behind 131I exposure is of great clinical and radiation protection interest. The aim of this work was to study the long-term dose-related effects of 131I exposure in thyroid tissue and plasma in young rats and identify potential biomarkers., Materials and Methods: Male Sprague Dawley rats (5-week-old) were i.v. injected with 0.5, 5.0, 50 or 500 kBq 131I (Dthyroid ca 1-1000 mGy), and killed after nine months at which time the thyroid and blood samples were collected. Gene expression microarray analysis (thyroid samples) and LC-MS/MS analysis (thyroid and plasma samples) were performed to assess differential gene and protein expression profiles in treated and corresponding untreated control samples. Bioinformatics analyses were performed using the DAVID functional annotation tool and Ingenuity Pathway Analysis (IPA). The gene expression microarray data and LC-MS/MS data were validated using qRT-PCR and ELISA, respectively., Results: Nine 131I exposure-related candidate biomarkers (transcripts: Afp and RT1-Bb, and proteins: ARF3, DLD, IKBKB, NONO, RAB6A, RPN2, and SLC25A5) were identified in thyroid tissue. Two dose-related protein candidate biomarkers were identified in thyroid (APRT and LDHA) and two in plasma (DSG4 and TGM3). Candidate biomarkers for thyroid function included the ACADL and SORBS2 (all activities), TPO and TG proteins (low activities). 131I exposure was shown to have a profound effect on metabolism, immune system, apoptosis and cell death. Furthermore, several signalling pathways essential for normal cellular function (actin cytoskeleton signalling, HGF signalling, NRF2-mediated oxidative stress, integrin signalling, calcium signalling) were also significantly regulated., Conclusion: Exposure-related and dose-related effects on gene and protein expression generated few expression patterns useful as biomarkers for thyroid function and cancer., Competing Interests: The authors have declared that no competing interests exist.

A pit lake arises as a consequence of anthropogenic activities in opencast mining areas. These water bodies may be enriched in hazardous stable contaminants and/or in naturally occurring radionuclides depending on the local geological conditions. Mining legacy in Sweden produced hundreds of these pit lakes and most of them are used for recreational purposes in the southern part of the country. In this paper, one pit lake was selected for having enhanced levels of natural radionuclides. Physico-chemical parameters (temperature, pH, oxidation-reduction potential, dissolved oxygen and depth), elemental composition (via Inductive Coupled Plasma Mass Spectrometry) and radiometric characterization (via alpha spectrometry of 226 Ra, 210 Po and 210 Pb) were carried along the depth of a 60 m depth pit lake, with the main aim to describe how natural radionuclides and elements behaves with depth in a non-uraniferous pit lake. Based on observed changes in physico-chemical parameters, a thermocline and a chemocline region were identified at around 10 and 30 m depth respectively. Concerning radionuclides, 226 Ra ranged from 75 ± 3 up to 360 ± 12 mBq/kg while 210 Po ranged from 11 ± 1 up to 71 ± 3 mBq/kg. 210 Pb distribution with depth was also determined via secular equilibrium with 210 Po after 2 years and also stable Pb was measured. Disequilibrium 226 Ra- 210 Pb was found and the residence time of 210 Pb in the water column was assessed. Additionally, different vertical distributions between 210 Pb and Pb were found which points out different sources for different lead isotopes in the water body., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Recent data suggest that the transcription factor Zfp148 represses activation of the tumor suppressor p53 in mice and that therapeutic targeting of the human orthologue ZNF148 could activate the p53 pathway without causing detrimental side effects. We have previously shown that Zfp148 deficiency promotes p53-dependent proliferation arrest of mouse embryonic fibroblasts (MEFs), but the underlying mechanism is not clear. Here, we showed that Zfp148 deficiency downregulated cell cycle genes in MEFs in a p53-dependent manner. Proliferation arrest of Zfp148-deficient cells required increased expression of ARF, a potent activator of the p53 pathway. Chromatin immunoprecipitation showed that Zfp148 bound to the ARF promoter, suggesting that Zfp148 represses ARF transcription. However, Zfp148 preferentially bound to promoters of other transcription factors, indicating that deletion of Zfp148 may have pleiotropic effects that activate ARF and p53 indirectly. In line with this, we found no evidence of genetic interaction between TP53 and ZNF148 in CRISPR and siRNA screen data from hundreds of human cancer cell lines. We conclude that Zfp148 deficiency, by increasing ARF transcription, downregulates cell cycle genes and cell proliferation in a p53-dependent manner. However, the lack of genetic interaction between ZNF148 and TP53 in human cancer cells suggests that therapeutic targeting of ZNF148 may not increase p53 activity in humans.

Natural radioactivity in the environment is a field gaining more attention in last decades. This work is focused on the study of natural radioactivity complemented with elementary characterization at former non-uraniferous mining areas in Sweden. This aim is addressed through the study of mining lakes, called pit lakes, which are water bodies generated after opencast mining. Environmental matrices (water, sediments and rocks) from 32 Swedish pit lakes, commonly used for recreational purposes were radiometrically characterized via alpha ( 238 U, 234 U, 232 Th, 230 Th, 210 Po isotopes) and gamma spectrometry ( 238 U and 232 Th series radionuclides). Additionally, ambient dose rate equivalent in the immediate surrounding of each pit lake was quantified. Physico-chemical parameters (pH, specific conductivity, dissolved oxygen, oxidation-reduction potential) and elemental composition (major and trace elements by ICP-MS) were analysed in water samples and elementary composition of sediments/rocks was measured by XRF and SEM-EDX in some specific cases. A non-negligible number of pit lakes (26%) with enhanced U levels in water was found. At some sites, rocks contained up to 4% of U in areas with high degree of interaction with local population. Concerning the elementary perspective, another popular site (due to its turquoise water) was found to have elevated dissolved heavy metal levels. Results obtained in this work prove that measurement of natural radioactivity is another component that should be included in routine analysis of characterization in mining areas, especially if restauration of post-mining sites is intended for human recreational.

Since patients with medullary thyroid cancer (MTC) often have metastatic disease at the time of diagnosis, the development of efficient systemic treatment options for MTC is important. Vandetanib and cabozantinib are two tyrosine kinase inhibitors (TKIs) that were recently approved by FDA and EMA for systemic treatment of metastatic MTC. Additionally, since MTC is of a neuroendocrine tumour type, treatment with radiolabelled somatostatin analogues (e.g. 177Lu-octreotate) is a valid option for patients with MTC. The aim of this study was to investigate the potentially increased therapeutic effect of combining radiation therapy with these TKIs for treatment of MTC in a mouse model. Nude mice carrying patient-derived MTC tumours (GOT2) were treated with external beam radiotherapy (EBRT) and/or one of the two TKIs vandetanib or cabozantinib. The tumour volume was determined and compared with that of mock-treated controls. The treatment doses were chosen to give a moderate effect as monotherapy to be able to detect any increased therapeutic effect from the combination therapy. At the end of follow-up, tumours were processed for immunohistochemical (IHC) analyses. The animals in the combination therapy groups showed the largest reduction in tumour volume and the longest time to tumour progression. Two weeks after start of treatment, the tumour volume for these mice was reduced by about 70-75% compared with controls. Furthermore, also EBRT and TKI monotherapy resulted in a clear anti-tumour effect with a reduced tumour growth compared with controls. The results show that an increased therapeutic effect could be achieved when irradiation is combined with TKIs for treatment of MTC. Future studies should evaluate the potential of using 177Lu-octreotate therapy in combination with TKIs in patients., Competing Interests: The authors have declared that no competing interests exist.

Cancer drug development has been riddled with high attrition rates, in part, due to poor reproducibility of preclinical models for drug discovery. Poor experimental design and lack of scientific transparency may cause experimental biases that in turn affect data quality, robustness and reproducibility. Here, we pinpoint sources of experimental variability in conventional 2D cell-based cancer drug screens to determine the effect of confounders on cell viability for MCF7 and HCC38 breast cancer cell lines treated with platinum agents (cisplatin and carboplatin) and a proteasome inhibitor (bortezomib). Variance component analysis demonstrated that variations in cell viability were primarily associated with the choice of pharmaceutical drug and cell line, and less likely to be due to the type of growth medium or assay incubation time. Furthermore, careful consideration should be given to different methods of storing diluted pharmaceutical drugs and use of DMSO controls due to the potential risk of evaporation and the subsequent effect on dose-response curves. Optimization of experimental parameters not only improved data quality substantially but also resulted in reproducible results for bortezomib- and cisplatin-treated HCC38, MCF7, MCF-10A, and MDA-MB-436 cells. Taken together, these findings indicate that replicability (the same analyst re-performs the same experiment multiple times) and reproducibility (different analysts perform the same experiment using different experimental conditions) for cell-based drug screens can be improved by identifying potential confounders and subsequent optimization of experimental parameters for each cell line.

Patients with medullary thyroid cancer (MTC) are often diagnosed with spread tumour disease and the development of better systemic treatment options for these patients is important. Treatment with the radiolabelled somatostatin analogue 177Lu-octreotate is already a promising option but can be optimised. For example, combination treatment with another substance could increase the effect on tumour tissue. Gemcitabine is a nucleoside analogue that has been shown to sensitise tumour cells to radiation. The aim of this study was to investigate potentially additive or synergistic effects of combining radiation with gemcitabine for treatment of MTC. Nude mice transplanted with patient-derived MTC tumours (GOT2) were divided into groups and treated with radiation and/or gemcitabine. Radiation treatment was given as 177Lu-octreotate or external beam radiotherapy (EBRT). The volume of treated and untreated tumours was followed. The absorbed dose and amount of gemcitabine were chosen to give moderate tumour volume reduction when given as monotherapy to enable detection of increased effects from combination treatment. After follow-up, the mice were killed and tumours were immunohistochemically (IHC) analysed. Overall, the animals that received a combination of EBRT and gemcitabine showed the largest reduction in tumour volume. Monotherapy with EBRT or gemcitabine also resulted in a clear detrimental effect on tumour volume, while the animals that received 177Lu-octreotate monotherapy showed similar response as the untreated animals. The GOT2 tumour was confirmed in the IHC analyses by markers for MTC. The IHC analyses also revealed that the proliferative activity of tumour cells was similar in all tumours, but indicated that fibrotic tissue was more common after EBRT and/or gemcitabine treatment. The results indicate that an additive, or even synergistic, effect may be achieved by combining radiation with gemcitabine for treatment of MTC. Future studies should be performed to evaluate the full potential of combining 177Lu-octreotate with gemcitabine in patients., Competing Interests: The authors have declared that no competing interests exist.

177Lu-octreotate is an FDA-approved radionuclide therapy for patients with gastroenteropancreatic neuroendocrine tumours (NETs) expressing somatostatin receptors. The 177Lu-octreotate therapy has shown promising results in clinical trials by prolonging progression-free survival, but complete responses are still uncommon. The aim of this study was to improve the 177Lu-octreotate therapy by means of combination therapy. To identify radiosensitising inhibitors, two cell lines, GOT1 and P-STS, derived from small intestinal neuroendocrine tumours (SINETs), were screened with 1,224 inhibitors alone or in combination with external radiation. The screening revealed that inhibitors of Hsp90 can potentiate the tumour cell-killing effect of radiation in a synergistic fashion (GOT1; false discovery rate <3.2×10-11). The potential for Hsp90 inhibitor ganetespib to enhance the anti-tumour effect of 177Lu-octreotate in an in vivo setting was studied in the somatostatin receptor-expressing GOT1 xenograft model. The combination led to a larger decrease in tumour volume relative to monotherapies and the tumour-reducing effect was shown to be synergistic. Using patient-derived tumour cells from eight metastatic SINETs, we could show that ganetespib enhanced the effect of 177Lu-octreotate therapy for all investigated patient tumours. Levels of Hsp90 protein expression were evaluated in 767 SINETs from 379 patients. We found that Hsp90 expression was upregulated in tumour cells relative to tumour stroma in the vast majority of SINETs. We conclude that Hsp90 inhibitors enhance the tumour-killing effect of 177Lu-octreotate therapy synergistically in SINET tumour models and suggest that this potentially promising combination should be further evaluated.

Background: 177 Lu-octreotate is used for therapy of somatostatin receptor expressing neuroendocrine tumors with promising results, although complete tumor remission is rarely seen. Previous studies on nude mice bearing the human small intestine neuroendocrine tumor, GOT1, have shown that a priming injection of 177 Lu-octreotate 24 h before the main injection of 177 Lu-octreotate resulted in higher 177 Lu concentration in tumor, resulting in increased absorbed dose, volume reduction, and time to regrowth. To our knowledge, the cellular effects of a priming treatment schedule have not yet been studied. The aim of this study was to identify transcriptional changes contributing to the enhanced therapeutic response of GOT1 tumors in nude mice to 177 Lu-octreotate therapy with priming, compared with non-curative monotherapy., Results: RNA microarray analysis was performed on tumor samples from GOT1-bearing BALB/c nude mice treated with a 5 MBq priming injection of 177 Lu-octreotate followed by a second injection of 10 MBq of 177 Lu-octreotate after 24 h and killed after 1, 3, 7, and 41 days after the last injection. Administered activity amounts were chosen to be non-curative, in order to facilitate the study of tumor regression and regrowth. Differentially regulated transcripts (RNA samples from treated vs. untreated animals) were identified (change ≥ 1.5-fold; adjusted p value < 0.01) using Nexus Expression 3.0. Analysis of the biological effects of transcriptional regulation was performed using the Gene Ontology database and Ingenuity Pathway Analysis. Transcriptional analysis of the tumors revealed two stages of pathway regulation for the priming schedule (up to 1 week and around 1 month) which differed distinctly from cellular responses observed after monotherapy. Induction of cell cycle arrest and apoptotic pathways (intrinsic and extrinsic) was found at early time points after treatment start, while downregulation of pro-proliferative genes were found at a late time point., Conclusions: The present study indicates increased cellular stress responses in the tumors treated with a priming treatment schedule compared with those seen after conventional 177 Lu-octreotate monotherapy, resulting in a more profound initiation of cell cycle arrest followed by apoptosis, as well as effects on PI3K/AKT-signaling and unfolded protein response.

Background: Molecular classification of tumour clonality is currently not evaluated in multiple invasive breast carcinomas, despite evidence suggesting common clonal origins. There is no consensus about which type of data (e.g. copy number, mutation, histology) and especially which statistical method is most suitable to distinguish clonal recurrences from independent primary tumours., Methods: Thirty-seven invasive breast tumour pairs were stratified according to laterality and time interval between the diagnoses of the two tumours. In a multi-omics approach, tumour clonality was analysed by integrating clinical characteristics (n = 37), DNA copy number (n = 37), DNA methylation (n = 8), gene expression microarray (n = 7), RNA sequencing (n = 3), and SNP genotyping data (n = 3). Different statistical methods, e.g. the diagnostic similarity index (SI), were used to classify the tumours as clonally related recurrences or independent primary tumours., Results: The SI and hierarchical clustering showed similar tendencies and the highest concordance with the other methods. Concordant evidence for tumour clonality was found in 46% (17/37) of patients. Notably, no association was found between the current clinical guidelines and molecular tumour features., Conclusions: A more accurate classification of clonal relatedness between multiple breast tumours may help to mitigate treatment failure and relapse by integrating tumour-associated molecular features, clinical parameters, and statistical methods. Guidelines need to be defined with exact thresholds to standardise clonality testing in a routine diagnostic setting.

High-throughput gene expression analysis is increasingly used in radiation research for discovery of damage-related or absorbed dose-dependent biomarkers. In tissue samples, cell type-specific responses can be masked in expression data due to mixed cell populations which can preclude biomarker discovery. In this study, we deconvolved microarray data from thyroid tissue in order to assess possible bias from mixed cell type data. Transcript expression data [GSE66303] from mouse thyroid that received 5.9 Gy from 131I over 24 h (or 0 Gy from mock treatment) were deconvolved by cell frequency of follicular cells and C-cells using csSAM and R and processed with Nexus Expression. Literature-based signature genes were used to assess the relative impact from ionizing radiation (IR) or thyroid hormones (TH). Regulation of cellular functions was inferred by enriched biological processes according to Gene Ontology terms. We found that deconvolution increased the detection rate of significantly regulated transcripts including the biomarker candidate family of kallikrein transcripts. Detection of IR-associated and TH-responding signature genes was also increased in deconvolved data, while the dominating trend of TH-responding genes was reproduced. Importantly, responses in biological processes for DNA integrity, gene expression integrity, and cellular stress were not detected in convoluted data-which was in disagreement with expected dose-response relationships-but upon deconvolution in follicular cells and C-cells. In conclusion, previously reported trends of 131I-induced transcriptional responses in thyroid were reproduced with deconvolved data and usually with a higher detection rate. Deconvolution also resolved an issue with detecting damage and stress responses in enriched data, and may reduce false negatives in other contexts as well. These findings indicate that deconvolution can optimize microarray data analysis of heterogeneous sample material for biomarker screening or other clinical applications., Competing Interests: The authors have declared that no competing interests exist.

Genomic instability contributes to the neoplastic phenotype by deregulating key cancer-related genes, which in turn can have a detrimental effect on patient outcome. DNA amplification of the 8p11-p12 genomic region has clinical and biological implications in multiple malignancies, including breast carcinoma where the amplicon has been associated with tumor progression and poor prognosis. However, oncogenes driving increased cancer-related death and recurrent genetic features associated with the 8p11-p12 amplicon remain to be identified. In this study, DNA copy number and transcriptome profiling data for 229 primary invasive breast carcinomas (corresponding to 185 patients) were evaluated in conjunction with clinicopathological features to identify putative oncogenes in 8p11-p12 amplified samples. Illumina paired-end whole transcriptome sequencing and whole-genome SNP genotyping were subsequently performed on 23 samples showing high-level regional 8p11-p12 amplification to characterize recurrent genetic variants (SNPs and indels), expressed gene fusions, gene expression profiles and allelic imbalances. We now show previously undescribed chromothripsis-like patterns spanning the 8p11-p12 genomic region and allele-specific DNA amplification events. In addition, recurrent amplification-specific genetic features were identified, including genetic variants in the HIST1H1E and UQCRHL genes and fusion transcripts containing MALAT1 non-coding RNA, which is known to be a prognostic indicator for breast cancer and stimulated by estrogen. In summary, these findings highlight novel candidate targets for improved treatment of 8p11-p12 amplified breast carcinomas., Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

Magnetic resonance (MR) methods enable noninvasive, regional tumor therapy response assessment, but associations between MR parameters, underlying biology, and therapeutic effects must be investigated. The aim of this study was to investigate response assessment efficacy and biological associations of MR parameters in a neuroendocrine tumor (NET) model subjected to radionuclide treatment. Twenty-one mice with NETs received 177 Lu-octreotate at day 0. MR experiments (day -1, 1, 3, 8, and 13) included T2-weighted, dynamic contrast-enhanced (DCE) and diffusion-weighted imaging (DWI) and relaxation measurements (T1/T2*). Tumor tissue was analyzed using proteomics. MR-derived parameters were evaluated for each examination day and for different radial distances from the tumor center. Response assessment efficacy and biological associations were evaluated using feature selection and protein expression correlations, respectively. Reduced tumor growth rate or shrinkage was observed until day 8, followed by reestablished growth in most tumors. The most important MR parameter for response prediction was DCE-MRI-derived pretreatment signal enhancement ratio (SER) at 40% to 60% radial distance, where it correlated significantly also with centrally sampled protein CCD89 (association: DNA damage and repair, proliferation, cell cycle arrest). The second most important was changed diffusion (D) between day -1 and day 3, at 60% to 80% radial distance, where it correlated significantly also with peripherally sampled protein CATA (association: oxidative stress, proliferation, cell cycle arrest, apoptotic cell death). Important information regarding tumor biology in response to radionuclide therapy is reflected in several MR parameters, SER and D in particular. The spatial and temporal information provided by MR methods increases the sensitivity for tumor therapy response., (Copyright © 2017 Oncoinvent AS. Published by Elsevier Inc. All rights reserved.)

Background: Patients with somatostatin receptor-expressing neuroendocrine tumours can be treated with intravenously administered 177 Lu-octreotate. Few patients are cured with the present protocol due to the current dose limitation of normal organs at risk, such as the kidneys. By locally administering 177 Lu-octreotate to the liver for the purpose of treating liver metastases, a substantially reduced absorbed dose to organs at risk could be achieved. The development of such a technique requires the capability of measuring the 177 Lu activity concentration in tissues in vivo. The aim of this study was to evaluate different performance parameters of two commercially available intraoperative gamma detectors in order to investigate whether intraoperative gamma detector measurements could be used to determine 177 Lu activity concentration in vivo., Results: Measurements were made using different sources containing 177 Lu. Response linearity, sensitivity, spatial resolution and its depth dependence, organ thickness dependence of the measured count rate and tumour detectability were assessed for two intraoperative gamma detectors. The two detectors (a scintillation and a semiconductor detector) showed differences in technical performance. For example, the sensitivity was higher for the scintillation detector, while the spatial resolution was better for the semiconductor detector. Regarding organ thickness dependence and tumour detectability, similar results were obtained for both detectors, and even relatively small simulated tumours of low tumour-to-background activity concentration ratios could be detected., Conclusions: Acceptable results were obtained for both detectors, although the semiconductor detector proved more advantageous for our purpose. The measurements demonstrated factors that must be corrected for, such as organ thickness or dead-time effects. Altogether, intraoperative gamma detector measurements could be used to determine 177 Lu activity concentration in vivo.

Background: 177 Lu-[DOTA 0 , Tyr 3 ]-octreotate ( 177 Lu-octreotate) is used for treatment of patients with somatostatin receptor (SSTR) expressing neuroendocrine tumors. However, complete tumor remission is rarely seen, and optimization of treatment protocols is needed. In vitro studies have shown that irradiation can up-regulate the expression of SSTR1, 2 and 5, and increase 177 Lu-octreotate uptake. The aim of the present study was to examine the anti-tumor effect of a 177 Lu-octreotate priming dose followed 24 h later by a second injection of 177 Lu-octreotate compared to a single administration of 177 Lu-octreotate, performed on the human small intestine neuroendocrine tumor cell line, GOT1, transplanted to nude mice., Results: Priming resulted in a 1.9 times higher mean absorbed dose to the tumor tissue per administered activity, together with a reduced mean absorbed dose for kidneys. Priming gave the best overall anti-tumor effects. Magnetic resonance imaging showed no statistically significant difference in tumor response between treatment with and without priming. Gene expression analysis demonstrated effects on cell cycle regulation. Biological processes associated with apoptotic cell death were highly affected in the biodistribution and dosimetry study, via differential regulation of, e.g., APOE, BAX, CDKN1A, and GADD45A., Conclusions: Priming had the best overall anti-tumor effects and also resulted in an increased therapeutic window. Results indicate that potential biomarkers for tumor regrowth may be found in the p53 or JNK signaling pathways. Priming administration is an interesting optimization strategy for 177 Lu-octreotate therapy of neuroendocrine tumors, and further studies should be performed to determine the mechanisms responsible for the reported effects.

Background: Gene expression profiling has made considerable contributions to our understanding of cancer biology and clinical care. This study describes a novel gene expression signature for breast cancer-specific survival that was validated using external datasets. Methods: Gene expression signatures for invasive breast carcinomas (mainly luminal B subtype) corresponding to 136 patients were analyzed using Cox regression, and the effect of each gene on disease-specific survival (DSS) was estimated. Iterative Bayesian model averaging was applied on multivariable Cox regression models resulting in an 18-marker panel, which was validated using three external validation datasets. The 18 genes were analyzed for common pathways and functions using the Ingenuity Pathway Analysis software. This study complied with the REMARK criteria. Results: The 18-gene multivariable model showed a high predictive power for DSS in the training and validation cohort and a clear stratification between high- and low-risk patients. The differentially expressed genes were predominantly involved in biological processes such as cell cycle, DNA replication, recombination, and repair. Furthermore, the majority of the 18 genes were found to play a pivotal role in cancer. Conclusions: Our findings demonstrated that the 18 molecular markers were strong predictors of breast cancer-specific mortality. The stable time-dependent area under the ROC curve function (AUC(t)) and high C-indices in the training and validation cohorts were further improved by fitting a combined model consisting of the 18-marker panel and established clinical markers. Impact: Our work supports the applicability of this 18-marker panel to improve clinical outcome prediction for breast cancer patients. Cancer Epidemiol Biomarkers Prev; 26(11); 1619-28. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: 177 Lu-octreotate can be used to treat somatostatin receptor expressing neuroendocrine tumors. It is highly effective in animal models, but clinical studies have so far only demonstrated low cure rates. Hedgehog inhibitors have shown therapeutic effect as monotherapy in neuroendocrine tumor model systems and might be one option to enhance the efficacy of 177 Lu-octreotate therapy. The aim of this study was to determine the therapeutic effect of combination therapy using 177 Lu-octreotate and the Hedgehog signaling pathway inhibitor sonidegib., Methods: GOT1-bearing BALB/c nude mice were treated with either sonidegib (80 mg/kg twice a week via oral gavage), a single injection of 30 MBq 177 Lu-octreotate i.v., or a combination of both. Untreated animals served as controls. Tumor size was measured twice-weekly using calipers. The animals were killed 41 d after injection followed by excision of the tumors. Total RNA was extracted from each tumor sample and then subjected to gene expression analysis. Gene expression patterns were compared with those of untreated controls using Nexus Expression 3.0, IPA and Gene Ontology terms. Western blot was carried out on total protein extracted from the tumor samples to analyze activation-states of the Hh and PI3K/AKT/mTOR pathways., Results: Sonidegib monotherapy resulted in inhibition of tumor growth, while a significant reduction in mean tumor volume was observed after 177 Lu-octreotate monotherapy and combination therapy. Time to progression was prolonged in the combination therapy group compared with 177 Lu-octreotate monotherapy. Gene expression analysis revealed a more pronounced response following combination therapy compared with both monotherapies, regarding the number of regulated genes and biological processes. Several cancer-related signaling pathways (i.e. Wnt/β-catenin, PI3K/AKT/mTOR, G-protein coupled receptor, and Notch) were affected by the combination therapy, but not by either monotherapy. Protein expression analysis revealed an activation of the Hh- and PI3K/AKT/mTOR pathways in tumors exposed to 177 Lu-octreotate monotherapy and combination therapy., Conclusions: A comparative analysis of the different treatment groups showed that combination therapy using sonidegib and 177 Lu-octreotate could be beneficial to patients with neuroendocrine tumors. Gene expression analysis revealed a functional interaction between sonidegib and 177 Lu-octreotate, i.e. several cancer-related signaling pathways were modulated that were not affected by either monotherapy. Protein expression analysis indicated a possible PI3K/AKT/mTOR-dependent activation of the Hh pathway, independent of SMO.

Humans are exposed to 131I in medical diagnostics and treatment but also from nuclear accidents, and better knowledge of the molecular response in thyroid is needed. The aim of the study was to examine the transcriptional response in thyroid tissue 24 h after 131I administration in rats. The exposure levels were chosen to simulate both the clinical situation and the case of nuclear fallout. Thirty-six male rats were i.v. injected with 0-4700 kBq 131I, and killed at 24 h after injection (Dthyroid = 0.0058-3.0 Gy). Total RNA was extracted from individual thyroid tissue samples and mRNA levels were determined using oligonucleotide microarray technique. Differentially expressed transcripts were determined using Nexus Expression 3.0. Hierarchical clustering was performed in the R statistical computing environment. Pathway analysis was performed using the Ingenuity Pathway Analysis tool and the Gene Ontology database. T4 and TSH plasma concentrations were measured using ELISA. Totally, 429 differentially regulated transcripts were identified. Downregulation of thyroid hormone biosynthesis associated genes (e.g. thyroglobulin, thyroid peroxidase, the sodium-iodine symporter) was identified in some groups, and an impact on thyroid function was supported by the pathway analysis. Recurring downregulation of Dbp and Slc47a2 was found. Dbp exhibited a pattern with monotonous reduction of downregulation with absorbed dose at 0.0058-0.22 Gy. T4 plasma levels were increased and decreased in rats whose thyroids were exposed to 0.057 and 0.22 Gy, respectively. Different amounts of injected 131I gave distinct transcriptional responses in the rat thyroid. Transcriptional response related to thyroid function and changes in T4 plasma levels were found already at very low absorbed doses to thyroid.

Targeted α-therapy is a promising treatment option for various types of malignant tumors. Radiolabeled cancer-seeking agents, however, undergo degradation, resulting in a certain percentage of free radionuclide in the body. The radiohalogen 211 At accumulates in various tissues, with specifically high uptake in the thyroid. When normal thyroid function is disturbed because of ionizing radiation (IR) exposure, deleterious effects can occur in tissues that depend on thyroid hormone (TH) regulation for normal physiologic function. However, knowledge of systemic effects is still rudimentary. We previously reported similarities in transcriptomic regulation between the thyroid and other tissues despite large differences in absorbed dose from 211 At. Here, we present supportive evidence on systemic effects after 211 At administration., Methods: Expression microarray data from the kidney cortex and medulla, liver, lungs, and spleen were used from previous studies in which mice were intravenously injected with 0.064-42 kBq of 211 At and killed after 24 h or injected with 1.7 kBq of 211 At and killed after 1, 6, or 168 h. Controls were mock-treated and killed after 24 h. Literature-based gene signatures were used to evaluate the relative impact from IR- or TH-induced regulation. Thyroid- and TH-associated upstream regulators as well as thyroid-related diseases and functions were generated using functional analysis software., Results: Responses in IR- or TH-associated gene signatures were tissue-specific and varied over time, and the relative impact of each gene signature differed between the investigated tissues. The liver showed a clear dominance of TH-responding genes. In the kidney cortex, kidney medulla, and lungs, the TH-associated signature was detected to at least an extent similar to the IR-associated signature. The spleen was the single tissue showing regulation of only IR-associated signature genes. Various thyroid-associated diseases and functions were inferred from the data: L-triiodothyronine, TH, TH receptor, and triiodothyronine (reverse) were inferred as upstream regulators with differences in incidence and strength of regulation depending on tissue type., Conclusion: These findings indicate that transcriptional regulation in various nonthyroid tissues was-in part-induced by thyroid (hormone)-dependent signaling. Consideration of the systemic context between tissues could contribute to normal tissue risk assessment and planning of remedial measures., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

Neuroendocrine tumors (NETs) can be treated by peptide receptor radionuclide therapy using radiolabeled somatostatin analogs. However, the efficacy of such treatment is low and needs to be optimized. Our study evaluated the potential radiosensitizing effects of inhibition of nicotineamide phosphoribosyltransferase on 177 Lu-DOTATATE treatment in a NET model., Methods: Nude mice xenografted with the human NET cell line GOT1 were treated with semiefficient doses of 177 Lu-DOTATATE (7.5 MBq, intravenously) or the nicotineamide phosphoribosyltransferase inhibitor GMX1778 (100 mg/kg/wk, orally)., Results: Median time to tumor progression (tumor volume larger than at day 0) was 3 d for controls, 7 d for single-dose GMX1778, 28 d for single-dose 177 Lu-DOTATATE, 35 d for 3 weekly doses of GMX1778, and 98 d for combined treatment with 177 Lu-DOTATATE and GMX1778 × 1. After 177 Lu-DOTATATE and 3 weekly doses of GMX1778, none of the tumors progressed within 120 d., Conclusion: GMX1778 enhances the efficacy of 177 Lu-DOTATATE treatment and induces a prolonged antitumor response., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

Non-targeted effects can induce responses in tissues that have not been exposed to ionizing radiation. Despite their relevance for risk assessment, few studies have investigated these effects in vivo. In particular, these effects have not been studied in context with thyroid exposure, which can occur e.g. during irradiation of head and neck tumors. To determine the similarity between in-field and out-of-field responses in normal tissue, we used a partial body irradiation setup with female mice where the thyroid region, the thorax and abdomen, or all three regions were irradiated. After 24 h, transcriptional regulation in the kidney cortex, kidney medulla, liver, lungs, spleen, and thyroid was analyzed using microarray technology. Thyroid irradiation resulted in transcriptional regulation in the kidney medulla and liver that resembled regulation upon direct exposure of these tissues regarding both strength of response and associated biological function. The kidney cortex showed fewer similarities between the setups, while the lungs and spleen showed little similarity between in-field and out-of-field responses. Interestingly, effects were generally not found to be additive. Future studies are needed to identify the molecular mechanisms that mediate these systemic effects, so that they may be used as targets to minimize detrimental side effects in radiotherapy.

Background: (131)I and (211)At are used in nuclear medicine and accumulate in the thyroid gland and may impact normal thyroid function. The aim of this study was to determine transcriptional profile variations, assess the impact on cellular activity, and identify genes with biomarker properties in thyroid tissue after (131)I and (211)At administration in mice., Methods: To further investigate thyroid tissue transcriptional responses to (131)I and (211)At administration, we generated a new transcriptional dataset that includes re-evaluated raw intensity values from our previous (131)I and (211)At studies. Differential transcriptional profiles were identified by comparing treated and mock-treated samples using Nexus Expression 3.0 software. Further data analysis was performed using R/Bioconductor and IPA., Results: A total of 1144 genes were regulated. Hierarchical clustering subdivided the groups into two clusters containing the lowest and highest absorbed dose levels, respectively, and revealed similar transcriptional regulation patterns for many kallikrein-related genes. Twenty-seven of the 1144 genes were recurrently regulated after (131)I and (211)At exposure and divided into six clusters. Several signalling pathways were affected, including calcium, integrin-linked kinase, and thyroid cancer signalling, and the peroxisomal proliferator-activated receptor network., Conclusions: Substantial changes in transcriptional regulation were shown in (131)I and (211)At-treated samples, and 27 genes were identified as potential biomarkers for (131)I and (211)At exposure. Clustering revealed distinct differences between transcriptional profiles of both similar and different exposures, demonstrating the necessity for better understanding of radiation-induced effects on cellular activity. Additionally, ionizing radiation-induced changes in kallikrein gene expression and identified canonical pathways should be further assessed.

Background: Circadian variation of gene expression is often neglected when ionizing radiation-induced effects are studied, whether in animal models or in cell culture. This study characterized diurnal variation of genome-wide transcriptional regulation and responses of potential biomarkers and signature genes in normal mouse tissues at 24 h after i.v. administration of (131)I., Methods: Female BALB/c nude mice were i.v. injected with 90 kBq (131)I at 9:00 a.m., 12:00 p.m., or 3:00 p.m. and killed after 24 h (n = 4/group). Paired control groups were mock-treated (n = 3-4/group). The kidneys, liver, lungs, spleen, and thyroid were excised, snap-frozen, and stored at -80 °C until extraction of total RNA. RNA microarray technology was used for genome-wide expression analysis. Enriched biological processes were categorized after cellular function. Signature genes for ionizing radiation and thyroid hormone-induced responses were taken from the literature. Absorbed dose was estimated using the Medical Internal Radiation Dose (MIRD) formalism., Results: The thyroid received an absorbed dose of 5.9 Gy and non-thyroid tissues received 0.75-2.2 mGy over 24 h. A distinct peak in the total number of significantly regulated transcripts was observed at 9:00 a.m. in the thyroid, but 3 h later in the kidney cortex, kidney medulla, and liver. Transcriptional regulation in the lungs and spleen was marginal. Associated cellular functions generally varied in quality and response strength between morning, noon, and afternoon. In the thyroid, 25 genes were significantly regulated at all investigated times of day, and 24 thereof showed a distinct pattern of pronounced down-regulation at 9:00 a.m. and comparatively weak up-regulation at later times. Eleven of these genes belonged to the species-specific kallikrein subfamily Klk1b. Responses in signature genes for thyroid hormone-induced responses were more frequent than for ionizing radiation, and trends persisted irrespective of time of day., Conclusion: Diurnal variation of genome-wide transcriptional responses to 90 kBq (131)I was demonstrated for the thyroid, kidney cortex and medulla, and liver, whereas variation was only marginal in the lungs and spleen. Overall, significant detection of potential biomarkers and signature genes was validated at each time of day, although direction of regulation and fold-change differed between morning, noon, and afternoon. These findings suggest that circadian rhythm should be considered in radiation research and that biological and analytical endpoints should be validated for circadian robustness.

Unlabelled: The kidneys are one of the main dose-limiting organs in peptide receptor radionuclide therapy and due to large inter-individual variations in renal toxicity, biomarkers are urgently needed in order to optimize therapy and reduce renal tissue damage. The aim of this study was to investigate the transcriptional, functional, and morphological effects on renal tissue after 177Lu-octreotate administration in normal mice, and to identify biomarkers for radiation induced renal toxicity., Methods: C57BL/6N mice were i.v. injected with 0, 30, 60, 90, 120, or 150 MBq 177Lu-octreotate (0, 16, 29, 40, 48, and 54 Gy to the kidneys). At 4, 8, and 12 months after administration, radiation-induced effects were evaluated in relation to (a) global transcriptional variations in kidney tissues, (b) morphological changes in the kidneys, (c) changes in white and red blood cell count as well as blood levels of urea, and (d) changes in renal function using 99mTc-DTPA/99mTc-DMSA scintigraphy., Results: In general, the highest number of differentially regulated transcripts was observed at 12 months after administration. The Cdkn1a, C3, Dbp, Lcn2, and Per2 genes displayed a distinct dose-dependent regulation, with increased expression level with increasing absorbed dose. Ifng, Tnf, and Il1B were identified as primary up-stream regulators of the recurrently regulated transcripts. Furthermore, previously proposed biomarkers for kidney injury and radiation damage were also observed. The functional investigation revealed reduced excretion of 99mTc-DTPA after 150 MBq, an increased uptake of 99mTc-DMSA at all dose levels compared with the controls, and markedly increased urea level in blood after 150 MBq at 12 months., Conclusion: Distinct dose-response relationships were found for several of the regulated transcripts. The Cdkn1a, Dbp, Lcn2, and Per2 genes are proposed as biomarkers for 177Lu-octreotate exposure of kidney. Correlations to functional and morphological effects further confirm applicability of these genes as markers of radiation damage in kidney tissue.

Background: 211At-labeled radiopharmaceuticals are potentially useful for tumor therapy. However, a limitation has been the preferential accumulation of released 211At in the thyroid gland, which is a critical organ for such therapy. The aim of this study was to determine the effect of absorbed dose, dose-rate, and time after 211At exposure on genome-wide transcriptional expression in mouse thyroid gland., Methods: BALB/c mice were i.v. injected with 1.7, 7.5 or 100 kBq 211At. Animals injected with 1.7 kBq were killed after 1, 6, or 168 h with mean thyroid absorbed doses of 0.023, 0.32, and 1.8 Gy, respectively. Animals injected with 7.5 and 100 kBq were killed after 6 and 1 h, respectively; mean thyroid absorbed dose was 1.4 Gy. Total RNA was extracted from pooled thyroids and the Illumina RNA microarray platform was used to determine mRNA levels. Differentially expressed transcripts and enriched GO terms were determined with adjusted p-value <0.01 and fold change >1.5, and p-value <0.05, respectively., Results: In total, 1232 differentially expressed transcripts were detected after 211At administration, demonstrating a profound effect on gene regulation. The number of regulated transcripts increased with higher initial dose-rate/absorbed dose at 1 or 6 h. However, the number of regulated transcripts decreased with mean absorbed dose/time after 1.7 kBq 211At administration. Furthermore, similar regulation profiles were seen for groups administered 1.7 kBq. Interestingly, few previously proposed radiation responsive genes were detected in the present study. Regulation of immunological processes were prevalent at 1, 6, and 168 h after 1.7 kBq administration (0.023, 0.32, 1.8 Gy).

Background: In cancer radiotherapy, knowledge of normal tissue responses and toxicity risks is essential in order to deliver the highest possible absorbed dose to the tumor while maintaining normal tissue exposure at non-critical levels. However, few studies have investigated normal tissue responses in vivo after (211)At administration. In order to identify molecular biomarkers of ionizing radiation exposure, we investigated genome-wide transcriptional responses to (very) low mean absorbed doses from (211)At in normal mouse tissues., Methods: Female BALB/c nude mice were intravenously injected with 1.7 kBq (211)At and killed after 1 h, 6 h, or 7 days or injected with 105 or 7.5 kBq and killed after 1 and 6 h, respectively. Controls were mock-treated. Total RNA was extracted from tissue samples of kidney cortex and medulla, liver, lungs, and spleen and subjected to microarray analysis. Enriched biological processes were categorized after cellular function based on Gene Ontology terms., Results: Responses were tissue-specific with regard to the number of significantly regulated transcripts and associated cellular function. Dose rate effects on transcript regulation were observed with both direct and inverse trends. In several tissues, Angptl4, Per1 and Per2, and Tsc22d3 showed consistent transcript regulation at all exposure conditions., Conclusions: This study demonstrated tissue-specific transcriptional responses and distinct dose rate effects after (211)At administration. Transcript regulation of individual genes, as well as cellular responses inferred from enriched transcript data, may serve as biomarkers in vivo. These findings expand the knowledge base on normal tissue responses and may help to evaluate and limit side effects of radionuclide therapy.

Background: The kidneys are regarded as one of the main dose-limiting organs in the treatment of neuroendocrine tumours with (177)Lu-[DOTA(0), Tyr(3)]-octreotate ((177)Lu-octreotate), despite the successful use of kidney uptake blocking agents such as lysine and arginine. To avoid renal toxicity but still give each patient as high amount of (177)Lu-octreotate as possible, there is a need for methods/biomarkers that indicate renal injury in an early stage of the treatment. The aim of this study was to investigate the potential of using urinary retinol binding protein 4 (RBP4) and carbamoylated haemoglobin (Hb) in blood as biomarkers of nephrotoxic effects on adult mice after (177)Lu-octreotate treatment., Methods: Adult BALB/c nude mice were injected with 60 MBq or 120 MBq of (177)Lu-octreotate or with saline (control). Urine was collected before injection and concentrations of urinary RBP4 and creatinine were determined 14 to 90 days after injection Blood samples were collected after 90 days, and carbamoylated N-terminal valine in Hb, formed from urea, was measured as valine hydantoin (VH) after detachment from Hb., Results: The RBP4 values increased with administered activity and time. For the 60 and 120 MBq groups, statistically significantly higher RBP4 levels (p <0.05) were found at day 60 and 90 compared to baseline, also at day 30 for 120 MBq group. For VH, the mean values were similar for the 60 MBq and control groups, while a small increase was observed for the 120 MBq group; but there were no statistically significant differences between any of the groups (p >0.05). No morphological changes in the kidney tissue were found., Conclusions: Urinary RBP4 is a promising new biomarker for radiation-induced renal toxicity. For the conditions used in this experiment, carbamoylated Hb (from urea) measured as VH may not be a sufficiently sensitive biomarker to be used for renal toxicity., Trial Registration: ID 326-2008.