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Abstract 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.

Abstract 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.

The physiological process of iodine uptake in the thyroid is used for 131I treatment of thyroid diseases. Children are more sensitive to radiation compared to adults and may react differently to 131I exposure. The aims of this study were to evaluate the effects on thyroid protein expression in young and adult rats one year after 131I injection and identify potential biomarkers related to 131I exposure, absorbed dose, and age. Twelve Sprague Dawley rats (young and adults) were i.v. injected with 50 kBq or 500 kBq 131I and killed twelve months later. Twelve untreated rats were used as age-matched controls. Quantitative proteomics, statistical analysis, and evaluation of biological effects were performed. The effects of irradiation were most prominent in young rats. Protein biomarker candidates were proposed related to age, absorbed dose, thyroid function, and cancer, and a panel was proposed for 131I exposure. In conclusion, the proteome of rat thyroid was differentially regulated twelve months after low-intermediate dose exposure to 131I in both young and adult rats. Several biomarker candidates are proposed for 131I exposure, age, and many of them are known to be related to thyroid function or thyroid cancer. Further research on human samples is needed for validation. Data are avaiable via ProteomeXchange with identifier PXD024786.

Abstract Mutation analysis is typically performed at the DNA level since most technical approaches are developed for DNA analysis. However, some applications, like transcriptional mutagenesis, RNA editing and gene expression analysis, require RNA analysis. Here, we combine reverse transcription and digital DNA sequencing to enable low error digital RNA sequencing. We evaluate yield, reproducibility, dynamic range and error correction rate for seven different reverse transcription conditions using multiplexed assays. The yield, reproducibility and error rate vary substantially between the specific conditions, where the yield differs 9.9-fold between the best and worst performing condition. Next, we show that error rates similar to DNA sequencing can be achieved for RNA using appropriate reverse transcription conditions, enabling detection of mutant allele frequencies

Abstract 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.

Abstract Background The tumor microenvironment clearly influences cancer progressing properties but less is known about how individual cancer microenvironments potentially moderate cancer treatment effects. By cultivating and treating cancer cell lines in patient-derived scaffolds (PDS), the impact of specific characteristics of individual cancer microenvironments can be incorporated in human-like growth modelling and cancer drug treatment testing. Methods PDSs from 78 biobanked primary breast cancer samples with known patient outcomes, were prepared and repopulated with donor breast cancer cell lines, followed by treatment with 5-fluorouracil or doxorubicin after cellular adaption to the various microenvironments. Cancer cell responses to the treatments were monitored by RNA-analyses, highlighting changes in gene sets representative for crucial tumor biological processes such as proliferation, cancer stem cell features, differentiation and epithelial-to-mesenchymal transition. Results The chemotherapy treatments induced distinct gene expression patterns in adapted cancer cells with clusters of similar treatment responses depending on the patient-derived cancer microenvironment used as growth substrate. The doxorubicin treatment displayed a favorable gene signature among surviving cancer cells with low proliferation (MKI67) and pluripotency features (NANOG, POU5F1), in comparison to 5-fluorouracil showing low proliferation but increased pluripotency. Specific gene changes monitored post-treatment were also significantly correlated with clinical data, including histological grade (NANOG), lymph node metastasis (SLUG) and disease-free patient survival (CD44). Conclusions This laboratory-based treatment study using patient-derived scaffolds repopulated with cancer cell lines, clearly illustrates that the human cancer microenvironment influences chemotherapy responses. The differences in treatment responses defined by scaffold-cultures have potential prognostic and treatment predictive values.

Recombinant α1-microglobulin (A1M) is proposed as a protector during 177Lu-octreotate treatment of neuroendocrine tumors, which is currently limited by bone marrow and renal toxicity. Co-administration of 177Lu-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 177Lu-octreotate and/or A1M administration. Mice were injected with 177Lu-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 177Lu 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 177Lu-octreotate+A1M group. Altogether, a tissue-dependent proteomic response followed exposure to 177Lu-octreotate alone or together with A1M. Combining 177Lu-octreotate with A1M did not inhibit the radiation-induced protein expression early after exposure, and late effects should be further studied.

Abstract Recombinant α1-microglobulin (A1M) is a proposed radioprotector during 177Lu-octreotate therapy of neuroendocrine tumors (NETs). To ensure a maintained therapeutic effect, we previously demonstrated that A1M does not affect the 177Lu-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 177Lu-octreotate with and without A1M or A1M alone. Human GOT1 tumor-bearing mice received 30 MBq 177Lu-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 177Lu-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 177Lu-octreotate in GOT1 tumors.

Abstract 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.

Abstract 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.

IntroductionOvarian cancer (OC) is the leading cause of gynecological cancer-related death. Of the main OC histologic subtypes, invasive mucinous carcinomas (MC) account for only 3% of OC cases and are frequently associated with favorable prognosis. Nevertheless, MCs differ greatly from the other OC histotypes in clinical, pathological, and biological behavior. However, the origin and molecular pathogenesis of MC are not yet fully understood. Therefore, identification of novel diagnostic markers could potentially facilitate early diagnosis of OC, particularly the MC histotype, thereby leading to the development of histotype-specific treatment regimens and improved survival rates.MethodsIn the present study, Trefoil factor gene family members (TFF1, TFF2 and TFF3) were identified as MC histotype-specific biomarkers using RNA sequencing (RNA-seq) data for 95 stage I-II OCs. The diagnostic value of TFF1, TFF2 and TFF3 was then evaluated by immunohistochemistry on 206 stage I-II OCs stratified by histotype (high-grade serous carcinoma [HGSC], endometrioid carcinoma [EC], clear cell carcinoma [CCC], and MC).ResultsWe showed significantly elevated intracytoplasmic protein expression levels for TFF1, TFF2 and TFF3 in MC samples, thereby revealing an association between expression of Trefoil factor gene family members and the MC histotype. Taken together, these findings suggest that the TFF proteins may play a pivotal role in tumor initiation and progression for the MC histotype.ConclusionTaken together, these findings suggest that the TFF proteins may play a pivotal role in tumor initiation and progression for the MC histotype. Moreover, these novel histotype-specific diagnostic biomarkers may not only improve patient stratification of early-stage ovarian carcinomas but may also be candidates for the development of molecular targeted therapies.

Abstract Background The BIRC5 gene encodes for the Survivin protein, which is a member of the inhibitor of apoptosis family. Survivin is found in humans during fetal development, but generally not in adult cells thereafter. Previous studies have shown that Survivin is abundant in most cancer cells, thereby making it a promising target for anti-cancer drugs and a potential prognostic tool. Methods To assess genetic alterations and mutations in the BIRC5 gene as well as BIRC5 co-expression with other genes, genomic and transcriptomic data were downloaded via cBioPortal for approximately 9000 samples from The Cancer Genome Atlas (TCGA) representing 33 different cancer types and 11 pan-cancer organ systems, and validated using the ICGC Data Portal and COSMIC. TCGA BIRC5 RNA sequencing data from 33 different cancer types and matching normal tissue samples for 16 cancer types were downloaded from Broad GDAC Firehose and validated using breast cancer microarray data from our previous work and data sets from the GENT2 web-based tool. Survival data were analyzed with multivariable Cox proportional hazards regression analysis and validated using KM plotter for breast-, ovarian-, lung- and gastric cancer. Results Although genetic alterations in BIRC5 were not common in cancer, BIRC5 expression was significantly higher in cancer tissue compared to normal tissue in the 16 different cancer types. For 14/33 cancer types, higher BIRC5 expression was linked to worse overall survival (OS, 4/14 after adjusting for both age and tumor grade and 10/14 after adjusting only for age). Interestingly, higher BIRC5 expression was associated with better OS in lung squamous cell carcinoma and ovarian serous cystadenocarcinoma. Higher BIRC5 expression was also linked to shorter progressive-free interval (PFI) for 14/33 cancer types (4/14 after adjusting for both age and tumor grade and 10/14 after adjusting only for age). External validation showed that high BIRC5 expression was significantly associated with worse OS for breast-, lung-, and gastric cancer. Conclusions Our findings suggest that BIRC5 overexpression is associated with the initiation and progression of several cancer types, and thereby a promising prognostic biomarker.

Abstract 131I 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 131I 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 131I exposure. Twelve young (5 weeks) and twelve adult Sprague Dawley rats (17 weeks) were i.v. injected with 50 kBq 131I (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 131I 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.

Abstract 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.

Background: The understanding of per- and polyfluoroalkyl substances (PFAS) health effects is rapidly advancing among critical populations. Therefore, the objective of this study was to assess PFAS serum levels among Lebanese pregnant women, cord serum and human milk levels, their determinants, and effects on newborn anthropometry. Methods: We measured concentrations of six PFAS (PFHpA, PFOA, PFHxS, PFOS, PFNA and PFDA) using liquid chromatography MS/MS for 419 participants, of which 269 had sociodemographic, anthropometric, environmental and dietary information. Results: The percentage of detection for PFHpA, PFOA, PFHxS and PFOS was 36.3–37.7%. PFOA and PFOS levels (95th percentile) were higher than HBM-I and HBM-II values. While PFAS were not detected in cord serum, five compounds were detected in human milk. Multivariate regression showed that fish/shellfish consumption, vicinity to illegal incineration and higher educational level were associated with an almost twice higher risk of elevated PFHpA, PFOA, PFHxS and PFOS serum levels. Higher PFAS levels in human milk were observed with higher eggs and dairy products consumption, in addition to tap water (preliminary findings). Higher PFHpA was significantly associated with lower newborn weight-for-length Z-score at birth. Conclusions: Findings establish the need for further studies, and urgent action to reduce exposure among subgroups with higher PFAS levels.

We present a young male patient with breast cancer having several risk factors likely acting in consort: irradiation of the breast for gynecomastia in adolescence and a life-long administration of phenothiazine for schizophrenia from the age of 16 years, with elevated serum prolactin level resulting in breast cancer development 24 years after irradiation.

Abstract Background The food frequency questionnaire (FFQ) is the most frequently used method to assess dietary intake in epidemiological studies evaluating diet-disease association. The objective of this study was to validate a FFQ for use among Lebanese adults by evaluating various facets of validity and reproducibility. Methods The quantitative 164-items FFQ was validated against the average of six 24-h dietary recalls (DRs) in a sample of 238 Lebanese adults. Reproducibility of the FFQ was assessed by administering it twice within 1 month’ time interval. Results Positive statistically significant Pearson correlations were observed in most macro and micronutrients between the FFQ and the six 24-h DRs, ranging from 0.16 to 0.65, with two thirds of the correlation coefficients exceeding 0.3. Energy, gender, and age-adjusted statistically significant Pearson correlation coefficients ranged from 0.14 to 0.64, with two thirds of the coefficients exceeding 0.2. Intakes from the FFQ were mostly higher than those of the 24-h DRs. Mean percent difference between nutrient intakes from both dietary methods decreased remarkably after using energy-adjusted mean intakes. Values were acceptable to good for all macronutrients and several micronutrients. Cross-classification analysis revealed that around 64.3 to 83.9% of participants were classified into the same and adjacent quartile whereas grossly misclassified proportions ranged from 3.7 to 12.2%. Weighted kappa values ranged from 0.02 to 0.36 with most of them exceeding 0.2. In indirect validity analysis, key nutrient mean intakes estimated from the six 24-h DRs were significantly positively associated with tertiles of food groups derived from the FFQ. Bland Altman plots showed that the majority of data points fell within the limits of agreement (LOA) for all nutrients. As for reproducibility analysis, ICC values were all statistically significant ranging from 0.645 to 0.959 and Bland Altman plots confirmed these results. Conclusions Based on various aspects of validity and reproducibility, and an extensive range of statistical tests, the present FFQ developed for a Lebanese community is an acceptable tool for dietary assessment and is useful for evaluating diet-disease associations in future studies.

Abstract Background Ovarian cancer is the main cause of gynecological cancer-associated death. However, 5-year survival rates differ dramatically between the five main ovarian carcinoma histotypes. Therefore, we need to have a better understanding of the mechanisms that promote histotype-specific ovarian carcinogenesis and identify novel prognostic biomarkers. Methods Here, we evaluated the prognostic role of 29 genes for early-stage (I and II) ovarian carcinomas (n = 206) using immunohistochemistry (IHC). Results We provide evidence of aberrant protein expression patterns for Collagen type III alpha 1 chain (COL3A1), G protein-coupled receptor 158 (GPR158) and PITH domain containing 1 (PITHD1). Kaplan-Meier survival analysis revealed that COL3A1 expression was associated with shorter overall survival in the four major histotypes of epithelial ovarian carcinoma patients (P value = 0.026, HR = 2.99 (95% CI 1.089–8.19)). Furthermore, GPR158 and PITHD1 were shown to be histotype-specific prognostic biomarkers, with elevated GPR158 expression patterns in mucinous ovarian carcinoma patients with unfavorable overall survival (P value = 0.00043, HR = 6.13 (95% CI 1.98–18.98)), and an association with lower PITHD1 protein expression and unfavorable overall and disease-specific survival in clear-cell ovarian carcinoma patients (P value = 0.012, HR = 0.22 (95% CI 0.058–0.80); P value = 0.003, HR = 0.17 (95% CI 0.043–0.64)). Conclusions The novel biomarkers identified here may improve prognostication at the time of diagnosis and may assist in the development of future individualized therapeutic strategies for ovarian carcinoma patients.

Abstract Background 177Lu-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 177Lu-octreotate 24 h before the main injection of 177Lu-octreotate resulted in higher 177Lu 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 177Lu-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 177Lu-octreotate followed by a second injection of 10 MBq of 177Lu-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

Abstract Background Current prognostic markers cannot adequately predict the clinical outcome of breast cancer patients. Therefore, additional biomarkers need to be included in routine immune panels. FOXA1 was a significant predictor of favorable outcome in primary breast cancer, while Nestin expression is preferentially found in triple-negative tumors with increased rate of nodal metastases, and reduced survival. No studies have investigated the prognostic value of FOXA1 and Nestin expression in breast cancer metastases. Methods Breast cancer metastases (n = 164) from various anatomical sites were retrospectively analyzed by immunohistochemistry for FOXA1, Nestin and GATA3 expression. Cox regression analysis assessed the prognostic value of FOXA1 and Nestin expression. Results In breast cancer metastases, FOXA1 expression was associated with Nestin-negativity, GATA3-positivity, ER-positivity, HER2-positivity and non-triple-negative status (P

Early-stage (I and II) ovarian carcinoma patients generally have good prognosis. Yet, some patients die earlier than expected. Thus, it is important to stratify early-stage patients into risk groups to identify those in need of more aggressive treatment regimens. The prognostic value of 29 histotype-specific biomarkers identified using RNA sequencing was evaluated for early-stage clear-cell (CCC), endometrioid (EC) and mucinous (MC) ovarian carcinomas (n = 112) using immunohistochemistry on tissue microarrays. Biomarkers with prognostic significance were further evaluated in an external ovarian carcinoma data set using the web-based Kaplan-Meier plotter tool. Here, we provide evidence of aberrant protein expression patterns and prognostic significance of 17 novel histotype-specific prognostic biomarkers [10 for CCC (ARPC2, CCT5, GNB1, KCTD10, NUP155, RPL13A, RPL37, SETD3, SMYD2, TRIO), three for EC (CECR1, KIF26B, PIK3CA), and four for MC (CHEK1, FOXM1, KIF23, PARPBP)], suggesting biological heterogeneity within the histotypes. Combined predictive models comprising the protein expression status of the validated CCC, EC and MC biomarkers together with established clinical markers (age, stage, CA125, ploidy) improved the predictive power in comparison with models containing established clinical markers alone, further strengthening the importance of the biomarkers in ovarian carcinoma. Further, even improved predictive powers were demonstrated when combining these models with our previously identified prognostic biomarkers PITHD1 (CCC) and GPR158 (MC). Moreover, the proteins demonstrated improved risk prediction of CCC-, EC-, and MC-associated ovarian carcinoma survival. The novel histotype-specific prognostic biomarkers may not only improve prognostication and patient stratification of early-stage ovarian carcinomas, but may also guide future clinical therapy decisions.

BackgroundRadioiodide (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 methodsMale 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.ResultsNine 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.ConclusionExposure-related and dose-related effects on gene and protein expression generated few expression patterns useful as biomarkers for thyroid function and cancer.

Abstract 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.

The number of patients with multiple primary malignancies has been increasing steadily in recent years. In the present study, we describe a unique case of an 81-year-old woman with 5 metachronous and synchronous primary malignant neoplasms. The patient was first diagnosed with an endometrium adenocarcinoma in 1997 and a colon adenocarcinoma in 2002. Eleven years after her colon surgery, in 2013, the patient presented with 3 other primary malignancies within a 4-month time span: an invasive malignant melanoma on the lower leg, an invasive mucinous breast carcinoma in the right breast, and a pleomorphic spindle cell sarcoma on the left upper arm. Subsequent routine medical checkups in 2013–2017 revealed no metastases of the primary malignancies. The patient mentioned a familial aggregation of malignant tumors, including 2 sisters with breast cancer and a brother with lung cancer. Interestingly, next-generation sequencing analysis of the patient’s blood sample detected no mutations in the BRCA1, BRCA2, TP53, PTEN, CDH1, PALB2, RAD51C, RAD51D, MLH1, MSH2, MSH6, PMS2, EPCAM, APC, MUTYH, STK11, BMPR1A, SMAD4, PTEN, POLE, POLD1, GREM1, and GALNT12 genes. Therefore, whole genome sequencing is warranted to identify cancer-related genetic alterations in this patient with quintuple primary malignancies.

Breast cancer (BC) histological and molecular classifications significantly improved the treatment strategy and prognosis. Inhibitor of apoptosis BIRC5/survivin is often overexpressed in cancers, however, indications of its importance in BC are inconsistent. We integrate BIRC5 protein and mRNA measures with clinical associates and long-term outcome in three independent cohorts Protein levels of BIRC5 were measured in primary lysates of 845 patients of the West Swedish BC cohort (VGR-BC) and linked to 5- and 27-years survival. The results were externally validated in transcriptomic data from METABRIC and SCAN-B cohorts. Survival analysis showed that high levels of BIRC5 were consistently associated with a poor probability of 5-year overall survival. High BIRC5 in VGR-BC contributed negatively to the disease-specific survival at 5 and 27 years. Subsets with different status by ER (estrogen receptor) expression and presence of nodal metastasis supported independent association of high BIRC5 with poor prognosis in all cohorts. In METABRIC and SCAN-B cohorts, high levels of BIRC5 mRNA were associated with the basal-like and luminal B molecular BC subtypes and with increasing histologic grade. BIRC5 is a sensitive survival marker that acts independent of ER and nodal status, and its levels need to be considered when making treatment decisions.

Abstract Background 177Lu-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 177Lu-octreotate therapy. The aim of this study was to determine the therapeutic effect of combination therapy using 177Lu-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 177Lu-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 177Lu-octreotate monotherapy and combination therapy. Time to progression was prolonged in the combination therapy group compared with 177Lu-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 177Lu-octreotate monotherapy and combination therapy. Conclusions A comparative analysis of the different treatment groups showed that combination therapy using sonidegib and 177Lu-octreotate could be beneficial to patients with neuroendocrine tumors. Gene expression analysis revealed a functional interaction between sonidegib and 177Lu-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.

Abstract Background The International Physical Actvity Questionnaire (IPAQ) is a validated tool for physical activity assessment used in many countries however no Arabic version of the long-form of this questionnaire exists to this date. Hence, the aim of this study was to cross-culturally adapt and validate an Arabic version of the long International Physical Activity Questionnaire (AIPAQ) equivalent to the French version (F-IPAQ) in a Lebanese population. Methods The guidelines for cross-cultural adaptation provided by the World Health Organization and the International Physical Activity Questionnaire committee were followed. One hundred fifty-nine students and staff members from Saint Joseph University of Beirut were randomly recruited to participate in the study. Items of the A-IPAQ were compared to those from the F-IPAQ for concurrent validity using Spearman’s correlation coefficient. Content validity of the questionnaire was assessed using factor analysis for the A-IPAQ’s items. The physical activity indicators derived from the A-IPAQ were compared with the body mass index (BMI) of the participants for construct validity. The instrument was also evaluated for internal consistency reliability using Cronbach’s alpha and Intraclass Correlation Coefficient (ICC). Finally, thirty-one participants were asked to complete the A-IPAQ on two occasions three weeks apart to examine its test–retest reliability. Bland-Altman analyses were performed to evaluate the extent of agreement between the two versions of the questionnaire and its repeated administrations. Results A high correlation was observed between answers of the F-IPAQ and those of the A-IPAQ, with Spearman’s correlation coefficients ranging from 0.91 to 1.00 (p

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.

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.

UnlabelledThe 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.MethodsC57BL/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.ResultsIn 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.ConclusionDistinct 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.

Background211At-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.MethodsBALB/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 1.5, and p-value ResultsIn 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).

AimThe aim of this study was to investigate the variation of the miRNA expression levels in normal renal cortical tissue after 177Lu-octreotate administration, a radiopharmaceutical used for treatment of neuroendocrine cancers.MethodsFemale BALB/c nude mice were i.v. injected with 1.3, 3.6, 14, 45, or 140 MBq 177Lu-octreotate, while control animals received saline. The animals were killed at 24 h after injection and total RNA, including miRNA, was extracted from the renal cortical tissue and hybridized to the Mouse miRNA Oligo chip 4plex to identify differentially regulated miRNAs between exposed and control samples.ResultsIn total, 57 specific miRNAs were differentially regulated in the exposed renal cortical tissues with 1, 29, 21, 27, and 31 miRNAs identified per dose-level (0.13, 0.34, 1.3, 4.3, and 13 Gy, respectively). No miRNAs were commonly regulated at all dose levels. miR-194, miR-107, miR-3090, and miR-3077 were commonly regulated at 0.34, 1.3, 4.3, and 13 Gy. Strong effects on cellular mechanisms ranging from immune response to p53 signaling and cancer-related pathways were observed at the highest absorbed dose. Thirty-nine of the 57 differentially regulated miRNAs identified in the present study have previously been associated with response to ionizing radiation, indicating common radiation responsive pathways.ConclusionIn conclusion, the 177Lu-octreotate associated miRNA signatures were generally dose-specific, thereby illustrating transcriptional regulation of radiation responsive miRNAs. Taken together, these results imply the importance of miRNAs in early immunological responses in the kidneys following 177Lu-octreotate administration.

Nemo-like kinase (NLK), a proline-directed serine/threonine kinase regulated by phosphorylation, can be localized in the cytosol or in the nucleus. Whether the localization of NLK can affect cell survival or cell apoptosis is yet to be disclosed. In the present study we found that NLK was mainly localized in the nuclei of breast cancer cells, in contrast to a cytosolic localization in non-cancerous breast epithelial cells. The nuclear localization of NLK was mediated through direct interaction with Heat shock protein 27 (HSP27) which further protected cancer cells from apoptosis. The present study provides evidence of a novel mechanism by which HSP27 recognizes NLK in the breast cancer cells and prevents NLK-mediated cell apoptosis.