In vivo Molecular Imaging and Radionuclide (131I) Therapy of Human Nasopharyngeal Carcinoma Cells Transfected with a Lentivirus Expressing Sodium Iodide Symporter

Introduction

Despite recent improvements in the survival rates for nasopharyngeal carcinoma (NPC), novel treatment strategies are required to improve distant metastasis-free survival. The sodium iodine symporter (NIS) gene has been applied for in vivo imaging and cancer therapy. In this study, we examined the potential of NIS gene therapy as a therapeutic approach in NPC by performing non-invasive imaging using ¹²⁵I and ¹³¹I therapy in vivo.

Methods

We constructed a lentiviral vector expressing NIS and enhanced green fluorescent protein (EGFP) under the control of the human elongation factor-1α (EF1α) promoter, and stably transfected the vector into CNE-2Z NPC cells to create CNE-2Z-NIS cells. CNE-2Z and CNE-2Z-NIS tumor xenografts were established in nude mice; ¹²⁵I uptake, accumulation and efflux were measured using micro-SPECT/CT imaging; the therapeutic effects of treatment with ¹³¹I were assessed over 25 days by measuring tumor volume and immunohistochemical staining of the excised tumors.

Results

qPCR, immunofluorescence and Western blotting confirmed that CNE-2Z-NIS cells expressed high levels of NIS mRNA and protein. CNE-2Z-NIS cells and xenografts took up and accumulated significantly more ¹²⁵I than CNE-2Z cells and xenografts. In vitro, ¹³¹I significantly reduced the clonogenic survival of CNE-2Z-NIS cells. In vivo, ¹³¹I effectively inhibited the growth of CNE-2Z-NIS xenografts. At the end of ¹³¹I therapy, CNE-2Z-NIS xenograft tumor cells expressed higher levels of NIS and caspase-3 and lower levels of Ki-67.

Conclusion

Lentiviruses effectively delivered and mediated long-lasting expression of NIS in CNE-2Z cells which enabled uptake and accumulation of radioisotopes and provided a significant therapeutic effect in an in vivo model of NPC. NIS-mediated radioiodine treatment merits further investigation as a potentially effective, low toxicity therapeutic strategy for NPC.     

Theranostic Studies of Human Sodium Iodide Symporter Imaging and Therapy Using 188Re: A Human Glioma Study in Mice

Objective

To investigate the role of ¹⁸⁸Re in human sodium iodide symporter (hNIS) theranostic gene-mediated human glioma imaging and therapy in model mice.

Methods

The human glioma cell line U87 was transfected with recombinant lentivirus encoding the hNIS gene under the control of cytomegalovirus promoter (U87-hNIS). The uptake and efflux of ¹⁸⁸Re were determined after incubating the cells with ¹⁸⁸Re. ¹⁸⁸Re uptake experiments in the presence of various concentrations of sodium perchlorate were carried out. In vitro cell killing tests with ¹⁸⁸Re were performed. U87-hNIS mediated ¹⁸⁸Re distribution, imaging and therapy in nude mice were also tested.

Results

U87-hNIS cell line was successfully established. The uptake of ¹⁸⁸Re in U87-hNIS cells increased up to 26-fold compared to control cells, but was released rapidly with a half-life of approximately 4 minutes. Sodium perchlorate reduced hNIS-mediated ¹⁸⁸Re uptake to levels of control cell lines. U87-hNIS cells were selectively killed following exposure to ¹⁸⁸Re, with a survival of 21.4%, while control cells had a survival of 92.1%. Unlike in vitro studies, U87-hNIS tumor showed a markedly increased ¹⁸⁸Re retention even 48 hours after ¹⁸⁸Re injection. In the therapy study, there was a significant difference in tumor size between U87-hNIS mice (317±67 mm³) and control mice (861±153 mm³) treated with ¹⁸⁸Re for 4 weeks (P<0.01).

Conclusion

The results indicate that inserting the hNIS gene into U87 cells is sufficient to induce specific ¹⁸⁸Re uptake, which has a cell killing effect both in vitro and in vivo. Moreover, our study, based on the function of hNIS as a theranostic gene allowing noninvasive imaging of hNIS expression by ¹⁸⁸Re scintigraphy, provides detailed characterization of in vivo vector biodistribution and level, localization, essential prerequisites for precise planning and monitoring of clinical gene therapy that aims to individualize gene therapy concept.     

Quantitative Apparent Diffusion Coefficient Measurements Obtained by 3-Tesla MRI Are Correlated with Biomarkers of Bladder Cancer Proliferative Activity

Purpose

To investigate the association between Apparent Diffusion Coefficient (ADC) values and cell cycle and proliferative biomarkers (p53, p21, Ki67,) in order to establish its potential role as a noninvasive biomarker for prediction of cell cycle, proliferative activity and biological aggressiveness in bladder cancer.

Materials and Methods

Patients with bladder cancer who underwent 3,0 Tesla DW-MRI of the bladder before TUR-B or radical cystectomy were eligible for this prospective IRB-approved study. Histological specimen were immunohistochemically stained for the following markers: p53, p21 and ki67. Two board-certified uropathologists reviewed the specimens blinded to DW-MRI results. Histological grade and T-stage were classified according to the WHO 2004 and the 2009 TNM classification, respectively. Nonparametric univariate and multivariate statistics including correlation, logistic regression and ROC analysis were applied.

Results

Muscle invasive bladder cancer was histologically confirmed in 10 out of 41 patients. All examined tissue biomarkers were significantly correlated with ADC values (p<0.05, respectively). Based on multivariate analysis, p53 and ADC are both independent prognostic factors for muscle invasiveness of bladder cancer (>/ = T2). (p = 0.013 and p = 0.018).

Conclusion

ADC values are associated with cell cycle and proliferative biomarkers and do thereby reflect invasive and proliferative potential in bladder cancer. ADC and p53 are both independent prognostic factors for muscle invasiveness in bladder cancer.     

Differentiation of Central Lung Cancer from Atelectasis: Comparison of Diffusion-Weighted MRI with PET/CT

Objective

Prospectively assess the performance of diffusion-weighted magnetic resonance imaging (DW-MRI) for differentiation of central lung cancer from atelectasis.

Materials and Methods

38 consecutive lung cancer patients (26 males, 12 females; age range: 28–71 years; mean age: 49 years) who were referred for thoracic MR imaging examinations were enrolled. MR examinations were performed using a 1.5-T clinical scanner and scanning sequences of T1WI, T2WI, and DWI. Cancers and atelectasis were measured by mapping of the apparent diffusion coefficients (ADCs) obtained with a b-value of 500 s/mm².

Results

PET/CT and DW-MR allowed differentiation of tumor and atelectasis in all 38 cases, but T2WI did not allow differentiation in 9 cases. Comparison of conventional T2WI and DW-MRI indicated a higher contrast noise ratio of the central lung carcinoma than the atelectasis by DW-MRI. ADC maps indicated significantly lower mean ADC in the central lung carcinoma than in the atelectasis (1.83±0.58 vs. 2.90±0.26 mm²/s, p<0.0001). ADC values of small cell lung carcinoma were significantly greater than those from squamous cell carcinoma and adenocarcinoma (p<0.0001 for both).

Conclusions

DW-MR imaging provides valuable information not obtained by conventional MR and may be useful for differentiation of central lung carcinoma from atelectasis. Future developments may allow DW-MR imaging to be used as an alternative to PET-CT in imaging of patients with lung cancer.     

Non-Gaussian Analysis of Diffusion Weighted Imaging in Head and Neck at 3T: A Pilot Study in Patients with Nasopharyngeal Carcinoma

Purpose

To technically investigate the non-Gaussian diffusion of head and neck diffusion weighted imaging (DWI) at 3 Tesla and compare advanced non-Gaussian diffusion models, including diffusion kurtosis imaging (DKI), stretched-exponential model (SEM), intravoxel incoherent motion (IVIM) and statistical model in the patients with nasopharyngeal carcinoma (NPC).

Materials and Methods

After ethics approval was granted, 16 patients with NPC were examined using DWI performed at 3T employing an extended b-value range from 0 to 1500 s/mm². DWI signals were fitted to the mono-exponential and non-Gaussian diffusion models on primary tumor, metastatic node, spinal cord and muscle. Non-Gaussian parameter maps were generated and compared to apparent diffusion coefficient (ADC) maps in NPC.

Results

Diffusion in NPC exhibited non-Gaussian behavior at the extended b-value range. Non-Gaussian models achieved significantly better fitting of DWI signal than the mono-exponential model. Non-Gaussian diffusion coefficients were substantially different from mono-exponential ADC both in magnitude and histogram distribution.

Conclusion

Non-Gaussian diffusivity in head and neck tissues and NPC lesions could be assessed by using non-Gaussian diffusion models. Non-Gaussian DWI analysis may reveal additional tissue properties beyond ADC and holds potentials to be used as a complementary tool for NPC characterization.     

Interobserver Reproducibility of Diffusion-Weighted MRI in Monitoring Tumor Response to Neoadjuvant Therapy in Esophageal Cancer

Objective

To investigate the reproducibility of diffusion-weighted magnetic resonance imaging (DW-MRI) in assessing tumor response early in the course of neoadjuvant chemoradiotherapy in patients with operable esophageal cancer.

Methods

Eleven male patients (mean age 54.8 years) with newly diagnosed esophageal cancer underwent DW-MRI before and 10 days after start of chemoradiotherapy. Reproducibility of apparent diffusion coefficient (ADC) measurements by manual (freehand) and semi-automated volumetric methods was assessed.

Results

Interobserver reproducibility for the assessment of mean tumor ADC by the manual measurement method was good, with an ICC of 0.69 (95% CI, 0.36 to 0.85; P = 0.001). Interobserver reproducibility for the assessment of mean tumor ADC by the semi-automated volumetric measurement method was very good, with an ICC of 0.96 (95% CI, 0.91 to 0.98; P<0.001).

Conclusion

Semi-automated volumetric ADC measurements have higher reproducibility than manual ADC measurements in assessing tumor response to chemoradiotherapy in patients with esophageal adenocarcinoma.     

Imaging characteristics, tissue distribution, and spread of a novel oncolytic vaccinia virus carrying the human sodium iodide symporter

Introduction

Oncolytic viruses show promise for treating cancer. However, to assess therapy and potential toxicity, a noninvasive imaging modality is needed. This study aims to determine the in vivo biodistribution, and imaging and timing characteristics of a vaccinia virus, GLV-1h153, encoding the human sodium iodide symporter (hNIS.

Methods

GLV-1h153 was modified from GLV-1h68 to encode the hNIS gene. Timing of cellular uptake of radioiodide ¹³¹I in human pancreatic carcinoma cells PANC-1 was assessed using radiouptake assays. Viral biodistribution was determined in nude mice bearing PANC-1 xenografts, and infection in tumors confirmed histologically and optically via Green Fluorescent Protein (GFP) and bioluminescence. Timing characteristics of enhanced radiouptake in xenografts were assessed via ¹²⁴I-positron emission tomography (PET). Detection of systemic administration of virus was investigated with both ¹²⁴I-PET and 99m-technecium gamma-scintigraphy.

Results

GLV-1h153 successfully facilitated time-dependent intracellular uptake of ¹³¹I in PANC-1 cells with a maximum uptake at 24 hours postinfection (P<0.05). In vivo, biodistribution profiles revealed persistence of virus in tumors 5 weeks postinjection at 10⁹ plaque-forming unit (PFU)/gm tissue, with the virus mainly cleared from all other major organs. Tumor infection by GLV-1h153 was confirmed via optical imaging and histology. GLV-1h153 facilitated imaging virus replication in tumors via PET even at 8 hours post radiotracer injection, with a mean %ID/gm of 3.82±0.46 (P<0.05) 2 days after intratumoral administration of virus, confirmed via tissue radiouptake assays. One week post systemic administration, GLV-1h153-infected tumors were detected via ¹²⁴I-PET and 99m-technecium-scintigraphy.

Conclusion

GLV-1h153 is a promising oncolytic agent against pancreatic cancer with a promising biosafety profile. GLV-1h153 facilitated time-dependent hNIS-specific radiouptake in pancreatic cancer cells, facilitating detection by PET with both intratumoral and systemic administration. Therefore, GLV-1h153 is a promising candidate for the noninvasive imaging of virotherapy and warrants further study into longterm monitoring of virotherapy and potential radiocombination therapies with this treatment and imaging modality.     

Optimization of Multimodal Imaging of Mesenchymal Stem Cells Using the Human Sodium Iodide Symporter for PET and Cerenkov Luminescence Imaging

Purpose

The use of stably integrated reporter gene imaging provides a manner to monitor the in vivo fate of engrafted cells over time in a non-invasive manner. Here, we optimized multimodal imaging (small-animal PET, Cerenkov luminescence imaging (CLI) and bioluminescence imaging (BLI)) of mesenchymal stem cells (MSCs), by means of the human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) as reporters.

Methods

First, two multicistronic lentiviral vectors (LV) were generated for multimodal imaging: BLI, ¹²⁴I PET/SPECT and CLI. Expression of the imaging reporter genes was validated in vitro using ^99mTcO₄ ⁻ radioligand uptake experiments and BLI. Uptake kinetics, specificity and tracer elution were determined as well as the effect of the transduction process on the cell''s differentiation capacity. MSCs expressing the LV were injected intravenously or subcutaneously and imaged using small-animal PET, CLI and BLI.

Results

The expression of both imaging reporter genes was functional and specific. An elution of ^99mTcO₄ ⁻ from the cells was observed, with 31% retention after 3 h. After labeling cells with ¹²⁴I in vitro, a significantly higher CLI signal was noted in hNIS expressing murine MSCs. Furthermore, it was possible to visualize cells injected intravenously using BLI or subcutaneously in mice, using ¹²⁴I small-animal PET, CLI and BLI.

Conclusions

This study identifies hNIS as a suitable reporter gene for molecular imaging with PET and CLI, as confirmed with BLI through the expression of Fluc. It supports the potential for a wider application of hNIS reporter gene imaging and future clinical applications.     

Tubo-Ovarian Abscess (with/without Pseudotumor Area) Mimicking Ovarian Malignancy: Role of Diffusion-Weighted MR Imaging with Apparent Diffusion Coefficient Values

Objective

To assess the added value of diffusion-weighted magnetic resonance imaging (DWI) with apparent diffusion coefficient (ADC) values compared to MRI, for characterizing the tubo-ovarian abscesses (TOA) mimicking ovarian malignancy.

Materials and Methods

Patients with TOA (or ovarian abscess alone; n = 34) or ovarian malignancy (n = 35) who underwent DWI and MRI were retrospectively reviewed. The signal intensity of cystic and solid component of TOAs and ovarian malignant tumors on DWI and the corresponding ADC values were evaluated, as well as clinical characteristics, morphological features, MRI findings were comparatively analyzed. Receiver operating characteristic (ROC) curve analysis based on logistic regression was applied to identify different imaging characteristics between the two patient groups and assess the predictive value of combination diagnosis with area under the curve (AUC) analysis.

Results

The mean ADC value of the cystic component in TOA was significantly lower than in malignant tumors (1.04 ± 0 .41 × 10⁻³ mm²/s vs. 2.42 ± 0.38 × 10⁻³ mm²/s; p < 0.001). The mean ADC value of the enhanced solid component in 26 TOAs was 1.43 ± 0.16×10⁻³mm²/s, and 46.2% (12 TOAs; pseudotumor areas) showed significantly higher signal intensity on DW-MRI than in ovarian malignancy (mean ADC value 1.44 ± 0.20×10⁻³ mm²/s vs.1.18 ± 0.36 × 10⁻³ mm²/s; p = 0.043). The combination diagnosis of ADC value and dilated tubal structure achieved the best AUC of 0.996. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of MRI vs. DWI with ADC values for predicting TOA were 47.1%, 91.4%, 84.2%, 64%, and 69.6% vs. 100%, 97.1%, 97.1%, 100%, and 98.6%, respectively.

Conclusions

DW-MRI is superior to MRI in the assessment of TOA mimicking ovarian malignancy, and the ADC values aid in discriminating the pseudotumor area of TOA from the solid portion of ovarian malignancy.     

Prognostic Factors and Survival in Patients with Radiation-Related Second Malignant Neoplasms Following Radiotherapy for Nasopharyngeal Carcinoma

Purpose

To analyze the clinicopathological characteristics, treatment modalities, and potential prognostic factors of radiation-related second malignant neoplasms (SMNs) in a large group of nasopharyngeal carcinoma (NPC) cases.

Methods and Materials

Institutional electronic medical records of 39,118 patients with NPC treated by definitive radiotherapy between February 1964 and December 2003 were reviewed. A total of 247 patients with confirmed SMN attributable to radiotherapy were included.

Results

Median latency between radiotherapy for NPC and the diagnosis of SMN was 9.5 years (range, 3.1–36.8 years). Squamous cell carcinoma was the most common histologic type, followed by fibrosarcoma and adenocarcinoma. Median progression-free survival and overall survival (OS) of the 235 patients who underwent treatment were 17.3 months and 28.5 months, respectively. The 5-year OS rates were 42.9%, 23.7%, and 0% for the surgery, radiotherapy, and chemotherapy groups, respectively. The independent prognostic factors associated with survival were sex, histologic type, and treatment modality in both the early stage subgroup and the advanced stage subgroup of SMN.

Conclusions

Sex, histologic type, and treatment modality were the significant prognostic factors for SMN. Complete resection offers the best chance for long-term survival. In select patients with locally advanced and unresectable SMN, reirradiation should be strongly considered as a curative option.     

Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

Background

Nasopharyngeal carcinoma (NPC) is known for its high metastatic potential and locoregional recurrence, although the molecular alterations that are driving NPC metastasis remain unclear at this time. This study aimed to examine the expression of fibulin-5 in NPC, correlate the results with clinicopathological variables and survival, and to investigate the role of fibulin-5 in human NPC cell lines.

Material and Methods

Standard semi-quantitative-RT-PCR, quantitative-RT-PCR, immunoblotting, and immunohistochemistry were used to investigate the mRNA and protein expression profiles of fibulin-5 in normal and NPC tissues. Immunohistochemistry of fibulin-5 was correlated with clinicopathological characteristics by univariate analyses. NPC cells overexpressing fibulin-5 or fibulin-5-siRNA cells were generated by stable transfection to characterize the molecular mechanisms of fibulin-5-elicited cell growth and metastasis.

Results

Our results demonstrated that fibulin-5 overexpression in NPC specimens and significantly correlated with advanced tumor metastasis indicating a poor 5-year overall survival. Fibulin-5 was mainly expressed in the nucleus in human NPC specimens and cell lines. Functionally, fibulin-5 overexpression yielded fast growth in NPC cells. In addition, fibulin-5 promotes cell metastasis in NPC cells through increased FLJ10540 and phosphor-AKT activity. In contrast, siRNA depletion of fibulin-5 suppressed FLJ10540 expression and phosphor-AKT activity. Suppression of either fibulin-5 or FLJ10540 can cause significant inhibition with regards to cell motility in NPC cells. Finally, immunohistochemical analysis of human aggressive NPC specimens showed a significant and positive correlation between fibulin-5 and FLJ10540 expression.

Conclusion

Higher fibulin-5 expression is not only an important indicator of poor survival, but also contributes to the development of new therapeutic strategies in the FLJ10540/AKT pathway for NPC treatment.     

Quantitative Immunohistochemical Analysis Reveals Association between Sodium Iodide Symporter and Estrogen Receptor Expression in Breast Cancer

Background

Human sodium iodide symporter (hNIS) gene over-expression is under active consideration worldwide as an alternative target molecule for breast cancer (BC) diagnosis and targeted radio-iodine treatment. However, the field demands better stratified analysis of endogenous hNIS expression across major BC subtypes. Therefore, we have analyzed subtype-specific variation of hNIS overexpression in breast tumor tissue samples by immunohistochemistry (IHC) and also report the development of a homogeneous, quantitative analysis method of digital IHC images.

Methods

hNIS expression was analyzed from 108 BC tissue samples by IHC. Sub-cellular localization of hNIS protein was analyzed by dual immunofluorescence (IF) staining method using hNIS and HER2 antibodies. An ImageJ based two-step digital analysis method was developed and applied for the bias-free analysis of the images.

Results

Staining of the tumor samples show 70% cases are hNIS positive indicating high incidence of hNIS positive cases in BC. More importantly, a subtype specific analysis done for the first time shows that hNIS expression is overly dominated in estrogen receptor (ER) positive cases than the receptor negative cases. Further, 56% of the ER+ve, PgR+ve, HER2-ve and 36% of ER+ve, PgR+ve, HER2+ve cases show highest intensity staining equivalent to the thyroid tissue. A significant positive correlation is also observed between hNIS and estrogen receptor expression (p = 0.0033, CI = 95%) suggesting hNIS mediated targeted radio-iodine therapy procedures may benefit both ER+ve, PgR+ve, HER2–ve as well as HER2+ve cases. Further, in a few cases, hNIS and HER2 protein localization is demonstrated by overlapping membrane co-expression. ImageJ based image analysis method shows over 70% match with manual pathological scoring method.

Conclusion

The study indicates a positive link between hNIS and ER expression in BC. The quantitative IHC image analysis method reported here will further help in patient stratification and potentially benefit global clinical assessment where hNIS mediated targeted ¹³¹I radio-ablative therapy is aimed.     

Glycosylation of Sodium/Iodide Symporter (NIS) Regulates Its Membrane Translocation and Radioiodine Uptake

Purpose

Human sodium/iodide symporter (hNIS) protein is a membrane glycoprotein that transports iodide ions into thyroid cells. The function of this membrane protein is closely regulated by post-translational glycosylation. In this study, we measured glycosylation-mediated changes in subcellular location of hNIS and its function of iodine uptake.

Methods

HeLa cells were stably transfected with hNIS/tdTomato fusion gene in order to monitor the expression of hNIS. Cellular localization of hNIS was visualized by confocal microscopy of the red fluorescence of tdTomato. The expression of hNIS was evaluated by RT-PCR and immunoblot analysis. Functional activity of hNIS was estimated by radioiodine uptake. Cyclic AMP (cAMP) and tunicamycin were used to stimulate and inhibit glycosylation, respectively. In vivo images were obtained using a Maestro fluorescence imaging system.

Results

cAMP-mediated Glycosylation of NIS resulted in increased expression of hNIS, stimulating membrane translocation, and enhanced radioiodine uptake. In contrast, inhibition of glycosylation by treatment with tunicamycin dramatically reduced membrane translocation of intracellular hNIS, resulting in reduced radioiodine uptake. In addition, our hNIS/tdTomato fusion reporter successfully visualized cAMP-induced hNIS expression in xenografted tumors from mouse model.

Conclusions

These findings clearly reveal that the membrane localization of hNIS and its function of iodine uptake are glycosylation-dependent, as our results highlight enhancement of NIS expression and glycosylation with subsequent membrane localization after cAMP treatment. Therefore, enhancing functional NIS by the increasing level of glycosylation may be suggested as a promising therapeutic strategy for cancer patients who show refractory response to conventional radioiodine treatment.     

Imaging of Treatment Response to the Combination of Carboplatin and Paclitaxel in Human Ovarian Cancer Xenograft Tumors in Mice Using FDG and FLT PET

Introduction

A combination of carboplatin and paclitaxel is often used as first line chemotherapy for treatment of ovarian cancer. Therefore the use of imaging biomarkers early after initiation of treatment to determine treatment sensitivity would be valuable in order to identify responders from non-responders. In this study we describe the non-invasive PET imaging of glucose uptake and cell proliferation using 2-deoxy-2-[¹⁸F]fluoro-D-glucose (FDG) and 3’-deoxy-3’-[¹⁸F]fluorothymidine (FLT) for early assessment of treatment response in a pre-clinical mouse model of human ovarian cancer treated with carboplatin and paclitaxel.

Methods

In vivo uptake of FLT and FDG in human ovarian cancer xenografts in mice (A2780) was determined before treatment with carboplatin and paclitaxel (CaP) and repeatedday 1, 4 and 8 after treatment start. Tracer uptake was quantified using small animal PET/CT. Tracer uptake was compared with gene expression of Ki67, TK1, GLUT1, HK1 and HK2.

Results

Tumors in the CaP group was significantly smaller than in the control group (p=0.03) on day 8. On day 4 FDG SUVmax ratio was significantly lower in the CaP group compared to the control group (105±4% vs 138±9%; p=0.002) and on day 8 the FDG SUVmax ratio was lower in the CaP compared to the control group (125±13% vs 167±13%; p=0.05). On day 1 the uptake of FLT SUVmax ratio was 89±9% in the CaP group and 109±6% in the control group; however the difference was not statistically significant (p=0.08).

Conclusions

Our data suggest that both FDG and FLT PET may be used for the assessment of anti-tumor effects of a combination of carboplatin and paclitaxel in the treatment of ovarian cancer. FLT provides an early and transient signal and FDG a later and more prolonged response. This underscores the importance of optimal timing between treatment and FLT or FDG imaging since treatment response may otherwise be overlooked.     

Genome-Wide Analyses of Radioresistance-Associated miRNA Expression Profile in Nasopharyngeal Carcinoma Using Next Generation Deep Sequencing

Background

Rapidly growing evidence suggests that microRNAs (miRNAs) are involved in a wide range of cancer malignant behaviours including radioresistance. Therefore, the present study was designed to investigate miRNA expression patterns associated with radioresistance in NPC.

Methods

The differential expression profiles of miRNAs and mRNAs associated with NPC radioresistance were constructed. The predicted target mRNAs of miRNAs and their enriched signaling pathways were analyzed via biological informatical algorithms. Finally, partial miRNAs and pathways-correlated target mRNAs were validated in two NPC radioreisitant cell models.

Results

50 known and 9 novel miRNAs with significant difference were identified, and their target mRNAs were narrowed down to 53 nasopharyngeal-/NPC-specific mRNAs. Subsequent KEGG analyses demonstrated that the 53 mRNAs were enriched in 37 signaling pathways. Further qRT-PCR assays confirmed 3 down-regulated miRNAs (miR-324-3p, miR-93-3p and miR-4501), 3 up-regulated miRNAs (miR-371a-5p, miR-34c-5p and miR-1323) and 2 novel miRNAs. Additionally, corresponding alterations of pathways-correlated target mRNAs were observed including 5 up-regulated mRNAs (ICAM1, WNT2B, MYC, HLA-F and TGF-β1) and 3 down-regulated mRNAs (CDH1, PTENP1 and HSP90AA1).

Conclusions

Our study provides an overview of miRNA expression profile and the interactions between miRNA and their target mRNAs, which will deepen our understanding of the important roles of miRNAs in NPC radioresistance.     

Monitoring Early Response to Anti-Angiogenic Therapy: Diffusion-Weighted Magnetic Resonance Imaging and Volume Measurements in Colon Carcinoma Xenografts

Objectives

To evaluate the use of diffusion-weighted MRI (DW-MRI) and volume measurements for early monitoring of antiangiogenic therapy in an experimental tumor model.

Materials and Methods

23 athymic nude rats, bearing human colon carcinoma xenografts (HT-29) were examined before and after 6 days of treatment with regorafenib (n = 12) or placebo (n = 11) in a clinical 3-Tesla MRI. For DW-MRI, a single-shot EPI sequence with 9 b-values (10–800 s/mm²) was used. The apparent diffusion coefficient (ADC) was calculated voxelwise and its median value over a region of interest, covering the entire tumor, was defined as the tumor ADC. Tumor volume was determined using T2-weighted images. ADC and volume changes between first and second measurement were evaluated as classifiers by a receiver-operator-characteristic (ROC) analysis individually and combined using Fisher''s linear discriminant analysis (FLDA).

Results

All ADCs and volumes are stated as median±standard deviation. Tumor ADC increased significantly in the therapy group (0.76±0.09×10⁻³ mm²/s to 0.90±0.12×10⁻³ mm²/s; p<0.001), with significantly higher changes of tumor ADC than in the control group (0.10±0.11×10⁻³ mm²/s vs. 0.03±0.09×10⁻³ mm²/s; p = 0.027). Tumor volume increased significantly in both groups (therapy: 347.8±449.1 to 405.3±823.6 mm³; p = 0.034; control: 219.7±79.5 to 443.7±141.5 mm³; p<0.001), however, the therapy group showed significantly reduced tumor growth (33.30±47.30% vs. 96.43±31.66%; p<0.001). Area under the curve and accuracy of the ADC-based ROC analysis were 0.773 and 78.3%; and for the volume change 0.886 and 82.6%. The FLDA approach yielded an AUC of 0.985 and an accuracy of 95.7%.

Conclusions

Regorafenib therapy significantly increased tumor ADC after 6 days of treatment and also significantly reduced tumor growth. However, ROC analyses using each parameter individually revealed a lack of accuracy in discriminating between therapy and control group. The combination of both parameters using FLDA substantially improved diagnostic accuracy, thus highlighting the potential of multi-parameter MRI as an imaging biomarker for non-invasive early tumor therapy monitoring.     

Periaortic Brown Adipose Tissue as a Major Determinant of [18F]-Fluorodeoxyglucose Vascular Uptake in Atherosclerosis-Prone,ApoE?/? Mice

Background

[¹⁸F]-fluorodeoxyglucose (FDG) has been suggested for the clinical and experimental imaging of inflammatory atherosclerotic lesions. Significant FDG uptake in brown adipose tissue (BAT) has been observed both in humans and mice. The objective of the present study was to investigate the influence of periaortic BAT on apolipoprotein E-deficient (apoE^−/−) mouse atherosclerotic lesion imaging with FDG.

Methods

ApoE^−/− mice (36±2 weeks-old) were injected with FDG (12±2 MBq). Control animals (Group A, n = 7) were injected conscious and kept awake at room temperature (24°C) throughout the accumulation period. In order to minimize tracer activity in periaortic BAT, Group B (n = 7) and C (n = 6) animals were injected under anaesthesia at 37°C and Group C animals were additionally pre-treated with propranolol. PET/CT acquisitions were performed prior to animal euthanasia and ex vivo analysis of FDG biodistribution.

Results

Autoradiographic imaging indicated higher FDG uptake in atherosclerotic lesions than in the normal aortic wall (all groups, P<0.05) and the blood (all groups, P<0.01) which correlated with macrophage infiltration (R = 0.47; P<0.001). However, periaortic BAT uptake was either significantly higher (Group A, P<0.05) or similar (Group B and C, P = NS) to that observed in atherosclerotic lesions and was shown to correlate with in vivo quantified aortic FDG activity.

Conclusion

Periaortic BAT FDG uptake was identified as a confounding factor while using FDG for the non-invasive imaging of mouse atherosclerotic lesions.     

Predicting Prostate Biopsy Outcomes: A Preliminary Investigation on Screening with Ultrahigh B-Value Diffusion-Weighted Imaging as an Innovative Diagnostic Biomarker

Background

Routine screening of prostate specific antigen (PSA) is no longer recommended because of a high rate of over-diagnosis of prostate cancer (PCa).

Objective

To evaluate the efficacy of diffusion-weighted magnetic resonance imaging (DW-MRI) for PCa detection, and to explore the clinical utility of ultrahigh b-value DW-MRI in predicting prostate biopsy outcomes.

Methodology

73 male patients were selected for the study. They underwent 3T MRI using T2WI conventional DW-MRI with b-value 1000 s/mm², and ultrahigh b-value DW-MRI with b-values of 2000 s/mm² and 3000 s/mm². Two radiologists evaluated individual prostate gland images on a 5-point rating scale using PI-RADS, for the purpose of region-specific comparisons among modalities. Sensitivity, specificity, accuracy, positive predictive value (PPV), negative predictive value (NPV) and likelihood ratios (LR) were investigated for each MRI modality. The area under the receiver operating characteristic (ROC) curve (AUC) was also calculated.

Results

Results showed the improved diagnostic value of ultrahigh b-value DWI-MRI for detection of PCa when compared to other b values and conventional MRI protocols. Sensitivity values for 3000 s/mm² in both peripheral zone (PZ) and transition zone (TZ) were significantly higher than those observed with conventional DW-MRI—Specificity values for 3000 s/mm² in the TZ were significantly higher than other b-value images, whereas specificity values using 3000 s/mm² in the PZ were not significantly higher than 2000 s/mm² images. PPV and NPV between 3000 s/mm² and the other three modalities were significantly higher for both PZ and TZ images. The PLRs and NLRs of b-value 3000 s/mm² DW-MRI in the PZ and TZ were also recorded. ROC analysis showed greater AUCs for the b value 3000 s/mm² DWI than for the other three modalities.

Conclusions

DW-MRI with a b-value of 3000 s/mm² was found to be the most accurate and reliable MRI modality for PCa tumor detection and localization, particularly for TZ lesion discrimination. It may be stated that the b-value of 3000 s/mm² is a novel, improved diagnostic biomarker with greater predictive accuracy for PCa prior to biopsy.     

High b Value (2,000 s/mm2) Diffusion-Weighted Magnetic Resonance Imaging in Prostate Cancer at 3 Tesla: Comparison with 1,000 s/mm2 for Tumor Conspicuity and Discrimination of Aggressiveness

Objective

The objective of our study was to investigate tumor conspicuity and the discrimination potential for tumor aggressiveness on diffusion-weighted magnetic resonance imaging (DW-MRI) with high b value at 3-T.

Materials and Methods

The institutional review board approved this study and waived the requirement for informed consent. A total of 50 patients with prostate cancer (69 cancer foci; 48 in the PZ, 20 in the TZ, and one in whole prostate) who underwent multiparametric prostate MRI including DW-MRI (b values: 0, 1000 s/mm² and 0, 2000 s/mm²) on a 3-T system were included. Lesion conspicuity score (LCS) using visual assessment (1 = invisible for surrounding normal site; 2 = slightly high intensity; 3 = moderately high; and 4 = very high) and tumor-normal signal intensity ratio (TNR) were assessed, and apparent diffusion coefficient (ADC, ×10⁻³ mm²/s) of the tumor regions and normal regions were measured.

Results

Mean LCS and TNR at 0, 2000 s/mm² was significantly higher than those at 0, 1000 s/mm² (p<0.001 for both). In addition, ADC at both 0, 1000 and 0, 2000 s/mm² was found to distinguish intermediate or high risk cancer with Gleason score ≥7 from low risk cancer with Gleason score ≤6 (p<0.001 for both). Furthermore, ADC of tumor regions correlated with Gleason score at both 0, 1000 s/mm² (ρ = −0.602; p<0.001) and 0, 2000 s/mm² (ρ = −0.645; p<0.001).

Conclusions

For tumor conspicuity and characterization of prostate cancer on DW-MRI of 3-T MRI, b = 0, 2000 s/mm² is more useful than b = 0, 1000 s/mm².     

An Experimental Study on 131I-CHIBA-1001: A Radioligand for α7 Nicotinic Acetylcholine Receptors

Objective

The α7 nicotinic acetylcholine receptors (nAChRs) play a vital role in the pathophysiology of neuropsychiatric diseases such as Alzheimer’s disease and depression. However, there is currently no suitable positron emission tomography (PET) or Single-Photon Emission Computed Tomography (SPECT) radioligands for imaging α7 nAChRs in brain. Here our aim is to radiosynthesize a novel SPECT radioligand ¹³¹I-CHIBA-1001 for whole body biodistribution study and in vivo imaging of α7 nAChRs in brain.

Method

¹³¹I-CHIBA-1001 was radiosynthesized by chloramine-T method. Different conditions of reaction time and temperature were tested to get a better radiolabeling yield. Radiolabeling yield and radiochemical purities of ¹³¹I-CHIBA-1001 were analyzed by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) system. Whole body biodistribution study was performed at different time points post injection of ¹³¹I-CHIBA-1001 in KM mice. Monkey subject was used for in vivo SPECT imaging in brain.

Result

The radiolabeling yield of ¹³¹I-CHIBA-1001 reached 96% within 1.5∼2.0 h at 90∼95°C. The radiochemical purity reached more than 99% after HPLC purification. ¹³¹I-CHIBA-1001 was highly stable in saline and fresh human serum in room temperature and 37°C separately. The biodistribution data of brain at 15, 30, and 60 min were 11.05±1.04%ID/g, 8.8±0.04%ID/g and 6.28±1.13%ID/g, respectively. In experimental SPECT imaging, the distribution of radioactivity in the brain regions was paralleled with the distribution of α7 nAChRs in the monkey brain. Moreover, in the blocking SPECT imaging study, the selective α7 nAChR agonist SSR180711 blocked the radioactive uptake in the brain successfully.

Conclusion

The CHIBA-1001 can be successfully radiolabeled with ¹³¹I using the chloramine-T method. ¹³¹I-CHIBA-1001 can successfully accumulate in the monkey brain and image the α7 acetylcholine receptors. ¹³¹I-CHIBA-1001 can be a candidate for imagingα7 acetylcholine receptors, which will be of great value for the diagnosis and treatment of mental diseases.