Transcriptional response of kidney tissue after 177Lu-octreotate administration in mice

AbstractIntroductionThe kidneys are one of the main dose limiting organs in 177Lu-octreotate therapy of neuroendocrine tumors. Therefore, biomarkers for radiation damage would be of great importance in this type of therapy. The purpose of this study was to investigate the absorbed dose dependency on early transcriptional changes in the kidneys from 177Lu-octreotate exposure.MethodsFemale Balb/c nude mice were i.v. injected with 1.3, 3.6, 14, 45 or 140MBq 177Lu-octreotate. The animals were killed 24h after injection followed by excision of the kidneys. The absorbed dose to the kidneys ranged between 0.13 and 13Gy. Total RNA was extracted from separated renal tissue samples, and applied to Illumina MouseRef-8 Whole-Genome Expression Beadchips to identify regulated transcripts after irradiation. Nexus Expression 2.0 and Gene Ontology terms were used for data processing and to determine affected biological processes.ResultsDistinct transcriptional responses were observed following 177Lu-octreotate administration. A higher number of differentially expressed transcripts were observed in the kidney medulla (480) compared to cortex (281). In addition, 39 transcripts were regulated at all absorbed dose levels in the medulla, compared to 32 in the cortex. Three biological processes in the cortex and five in the medulla were also shared by all absorbed dose levels. Strong association to metabolism was found among the affected processes in both tissues. Furthermore, an association with cellular and developmental processes was prominent in kidney medulla, while transport and immune response were prominent in kidney cortex.ConclusionSpecific biological and dose-dependent responses were observed in both tissues. The number of affected transcripts and biological processes revealed distinct response differences between the absorbed doses delivered to the tissues

Clinical relevance of breast cancer-related genes as potential biomarkers for oral squamous cell carcinoma

Background: Squamous cell carcinoma of the oral cavity (OSCC) is a common cancer form with relatively low 5-year survival rates, due partially to late detection and lack of complementary molecular markers as targets for treatment. Molecular profiling of head and neck cancer has revealed biological similarities with basal-like breast and lung carcinoma. Recently, we showed that 16 genes were consistently altered in invasive breast tumors displaying varying degrees of aggressiveness. Methods: To extend our findings from breast cancer to another cancer type with similar characteristics, we performed an integrative analysis of transcriptomic and proteomic data to evaluate the prognostic significance of the 16 putative breast cancer-related biomarkers in OSCC using independent microarray datasets and immunohistochemistry. Predictive models for disease-specific (DSS) and/or overall survival (OS) were calculated for each marker using Cox proportional hazards models. Results: We found that CBX2, SCUBE2, and STK32B protein expression were associated with important clinicopathological features for OSCC (peritumoral inflammatory infiltration, metastatic spread to the cervical lymph nodes, and tumor size). Consequently, SCUBE2 and STK32B are involved in the hedgehog signaling pathway which plays a pivotal role in metastasis and angiogenesis in cancer. In addition, CNTNAP2 and S100A8 protein expression were correlated with DSS and OS, respectively. Conclusions: Taken together, these candidates and the hedgehog signaling pathway may be putative targets for drug development and clinical management of OSCC patients

Up-regulation of cell cycle arrest protein BTG2 correlates with increased overall survival in breast cancer, as detected by immunohistochemistry using tissue microarray

<p>Abstract</p> <p>Background</p> <p>Previous studies have shown that the <it>ADIPOR1</it>, <it>ADORA1</it>, <it>BTG2 </it>and <it>CD46 </it>genes differ significantly between long-term survivors of breast cancer and deceased patients, both in levels of gene expression and DNA copy numbers. The aim of this study was to characterize the expression of the corresponding proteins in breast carcinoma and to determine their correlation with clinical outcome.</p> <p>Methods</p> <p>Protein expression was evaluated using immunohistochemistry in an independent breast cancer cohort of 144 samples represented on tissue microarrays. Fisher's exact test was used to analyze the differences in protein expression between dead and alive patients. We used Cox-regression multivariate analysis to assess whether the new markers predict the survival status of the patients better than the currently used markers.</p> <p>Results</p> <p>BTG2 expression was demonstrated in a significantly lower proportion of samples from dead patients compared to alive patients, both in overall expression (<it>P </it>= 0.026) and cell membrane specific expression (<it>P </it>= 0.013), whereas neither ADIPOR1, ADORA1 nor CD46 showed differential expression in the two survival groups. Furthermore, a multivariate analysis showed that a model containing BTG2 expression in combination with HER2 and Ki67 expression along with patient age performed better than a model containing the currently used prognostic markers (tumour size, nodal status, HER2 expression, hormone receptor status, histological grade, and patient age). Interestingly, BTG2 has previously been described as a tumour suppressor gene involved in cell cycle arrest and p53 signalling.</p> <p>Conclusions</p> <p>We conclude that high-level BTG2 protein expression correlates with prolonged survival in patients with breast carcinoma.</p

High-risk breast cancer: From biology to personalized therapeutic strategies

Adjuvant treatment regimens for breast cancer are primarily based on patient- and tumor-related factors, e.g. patient menopausal status, tumor stage and histological grade, and the status of molecular tumor markers (HER2/neu and the estrogen receptor). Despite improvements in survival rates, about 20% of patients experience recurrence within five years of initial therapy. There is therefore a need to improve patient risk assessment and to personalize therapy according to a combination of patient-specific clinicopathological features and tumor characteristics. This doctoral thesis is a multidisciplinary effort between molecular biologists, clinicians, and pathologists to identify potential therapeutic targets for high-risk breast carcinoma. This work exploits common knowledge that the accumulation of deleterious genetic and epigenetic modulators contribute to breast cancer risk for recurrence and death by deregulating key cellular processes within a specific tumor. In the first work, we found that tumors from high-risk breast cancer patients were genetically instable, containing a 2-fold increase in genetic alterations, an overrepresentation of alterations on chromosomes 3, 18, and 20, and the recurrent deregulation of a 13-marker transcriptome signature associated with significantly shorter disease-specific survival rates (AZGP1, CBX2, DNALI1, LOC389033, NME5, PIP, S100A8, SCUBE2, SERPINA11, STC2, STK32B, SUSD3, and UBE2C). Second, subsequent validation of the 13-marker signature demonstrated the importance of not only performing external validation in independent breast cancer microarray datasets, but also to assess the biological and clinical relevance of individual markers at the protein level because of frequent poor mRNA-protein correlation. It was shown that breast cancer outcome prediction was improved significantly by combining a four-marker immunohistochemical panel (AZGP1, PIP, S100A8, UBE2C) together with established clinicopathological features. Third, we showed that several putative markers previously identified by us may not only be useful for breast cancer prognostication, but may also be clinically relevant in oral squamous cell carcinoma, a cancer form bearing biological similarities to breast carcinoma. Lastly, we found that the 8p11-p12 genomic region is a hotspot for DNA amplification in breast cancer, where the WHSC1L1 gene may be one of several genes located in region with oncogenic potential and a substantial contributor to the aggressive breast cancer phenotype. Taken together, these findings further emphasize the importance of complementing established clinicopathological features with tumor-specific molecular markers to improve breast cancer risk assessment and develop more individualized treatment regimens

Circadian rhythm influences genome-wide transcriptional responses to I-131 in a tissue-specific manner in mice

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 I-131. Methods: Female BALB/c nude mice were i.v. injected with 90 kBq I-131 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 degrees 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 I-131 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

Digital RNA sequencing using unique molecular identifiers enables ultrasensitive RNA mutation analysis

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 <0.1% at RNA level. We also detect mutations at both DNA and RNA levels in tumor tissue using a breast cancer panel. Finally, we demonstrate that digital RNA sequencing can be applied to liquid biopsies, analyzing cell-free gene transcripts. In conclusion, we demonstrate that digital RNA sequencing is suitable for ultrasensitive RNA mutation analysis, enabling several basic research and clinical applications

Potential Biomarkers for Radiation-Induced Renal Toxicity following 177Lu-Octreotate Administration in Mice.

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