SLFN11 is a BRCA Independent Biomarker for the Response to Platinum-Based Chemotherapy in High-Grade Serous Ovarian Cancer and Clear Cell Ovarian Carcinoma.

BRCA1/2 mutations are robust biomarkers for platinum-based chemotherapy in epithelial ovarian cancers. However, BRCA1/2 mutations in clear cell ovarian carcinoma (CCC) are less frequent compared with high-grade serous ovarian cancer (HGSC). The discovery of biomarkers that can be applied to CCC is an unmet need in chemotherapy. Schlafen 11 (SLFN11) has attracted attention as a novel sensitizer for DNA-damaging agents including platinum. In this study, we investigated the utility of SLFN11 in HGSC and CCC for platinum-based chemotherapy. SLFN11 expression was analyzed retrospectively by IHC across 326 ovarian cancer samples. The clinicopathologic significance of SLFN11 expression was analyzed across 57 advanced HGSC as a discovery set, 96 advanced HGSC as a validation set, and 57 advanced CCC cases, all of whom received platinum-based chemotherapy. BRCA1/2 mutation was analyzed using targeted-gene sequencing. In the HGSC cohort, the SLFN11-positive and BRCA mutation group showed significantly longer whereas the SLFN11-negative and BRCA wild-type group showed significantly shorter progression-free survival and overall survival. Moreover, SLFN11-positive HGSC shrunk significantly better than SLFN11-negative HGSC after neoadjuvant chemotherapy. Comparable results were obtained with CCC but without consideration of BRCA1/2 mutation due to a small population. Multivariate analysis identified SLFN11 as an independent factor for better survival in HGSC and CCC. The SLFN11-dependent sensitivity to platinum and PARP inhibitors were validated with genetically modified non-HGSC ovarian cancer cell lines. Our study reveals that SLFN11 predicts platinum sensitivity in HGSC and CCC independently of BRCA1/2 mutation status, indicating that SLFN11 assessment can guide treatment selection in HGSC and CCC.

Temporal changes in magnetic resonance imaging appearance of adult granulosa cell tumor.

Granulosa cell tumors (GCTs) can have a wide variety of appearances on magnetic resonance imaging (MRI), ranging from entirely solid to multilocular cystic, suggesting that GCTs undergo remarkable morphological changes during growth. These temporal changes in MRI appearance of individual GCTs have not been documented. A 54-year-old asymptomatic postmenopausal woman was referred to our department for a small ovarian mass. This 3-cm solid mass showed high intensity on diffusion-weighted MRI and low intensity on apparent diffusion coefficient mapping. Close clinical follow-up was recommended, but she did not return to our hospital until the age of 63, when she was referred for a large ovarian tumor. MRI showed a 15-cm multilocular cyst containing a solid component with hemorrhaging. Postoperative diagnosis was adult GCT (AGCT). These temporal changes demonstrate a possible reason why GCTs can have such a wide range of MRI appearance. This knowledge might promote accurate preoperative diagnosis of AGCTs.

Diagnostic performance of 68Ga-DOTATOC PET/CT in tumor-induced osteomalacia.

OBJECTIVE: Tumor-induced osteomalacia (TIO) is caused by typically small tumors that secrete fibroblast growth factor 23 (FGF23). As tumor resection is the only effective treatment for TIO, it is important to detect the culprit tumor. We aimed to assess the utility of 68Gallium-DOTA-D-Phe(1)-Tyr(3)-octreotide (68Ga-DOTATOC) PET/CT in TIO and the correlation between biochemical parameters and the PET/CT results. METHODS: Thirty-five patients with clinically suspected TIO who had undergone 68Ga-DOTATOC PET/CT were retrospectively analyzed. 68Ga-DOTATOC PET/CT results were compared with biochemical parameters and the final diagnosis, including histopathology. RESULTS: 68Ga-DOTATOC PET/CT detected focal uptake consistent with TIO in 21/35 patients, one of which was considered false positive. In 16 patients, the cause of osteomalacia was confirmed histologically as phosphaturic mesenchymal tumor (n = 15) or fibrous dysplasia (n = 1). The other four patients were judged clinically as true positive by subsequent MRI and the clinical course. Overall, the detection rate of 68Ga-DOTATOC PET/CT was 57% (20/35). Median tumor maximum standardized uptake value (SUVmax) was 6.9 (range 1.5-37.7). There was no significant difference in serum intact FGF23 level between DOTATOC-positive and DOTATOC-negative cases, and no significant correlation was observed between intact FGF23 level and tumor SUVmax. CONCLUSIONS: 68Ga-DOTATOC PET/CT was clinically useful in detecting culprit tumors and subsequent patient management in TIO.

Improvement of biodistribution profile of a radiogallium-labeled, αvß6 integrin-targeting peptide probe by incorporation of negatively charged amino acids.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. Since αvß6 integrin has been reported as a promising target for PDAC diagnosis, we previously developed H-Cys(mal-NOTA-67Ga)-(Gly)6-A20FMDV2-NH2 ([67Ga]CG6) as an αvß6 integrin-targeting probe. Although [67Ga]CG6 specifically binds to αvß6 integrin-positive xenografts, the uptake of [67Ga]CG6 in the organs surrounding the pancreas, such as the liver and spleen, was comparable to that in the αvß6 integrin-positive xenografts. We hypothesized that the undesirable accumulation of [67Ga]CG6 in those organs was caused by the positive charges of [67Ga]CG6 (+ 3). In this study, we aimed to decrease [67Ga]CG6 uptake in the liver and spleen by reducing the electric charges of the probe. METHODS: We synthesized H-Cys(mal-NOTA-67Ga)-(Asp)6-A20FMDV2-NH2 ([67Ga]CD6) and evaluated its affinity to αvß6 integrin via in vitro competitive binding assay. Isoelectric points of the probes were determined by electrophoresis. Biodistribution study, autoradiography, and immunostaining for ß6 integrin were conducted using αvß6 integrin-positive and negative tumor-bearing mice. RESULTS: In vitro competitive binding assay showed that the alteration of the linker had a negligible impact on the affinity of [67Ga]CG6 to αvß6 integrin. The results of electrophoresis revealed that [67Ga]CG6 was positively charged whereas [67Ga]CD6 was negatively charged. In the biodistribution study, the uptake of [67Ga]CD6 in the αvß6 integrin-positive xenografts was significantly higher than that in the αvß6 integrin-negative ones at 60 and 120 min. The uptake of [67Ga]CD6 in the liver and spleen was more than two-fold lower than that of [67Ga]CG6 at both time points. In the immunohistochemistry study, the radioactivity accumulated areas in the autoradiogram of the αvß6 integrin-positive xenograft roughly coincided with ß6 integrin-expressing areas. CONCLUSION: We have successfully reduced the nonspecific uptake in the liver and spleen by altering the linker amino acid from G6 to D6. [67Ga]CD6 overcame the drawbacks of [67Ga]CG6 in its biodistribution.

Development and characterization of a 68Ga-labeled A20FMDV2 peptide probe for the PET imaging of αvß6 integrin-positive pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is known to be one of the most lethal cancers. Since the majority of patients are diagnosed at an advanced stage, development of a detection method for PDAC at an earlier stage of disease progression is strongly desirable. Integrin αVß6 is a promising target for early PDAC detection because its expression increases during precancerous changes. The present study aimed to develop an imaging probe for positron emission tomography (PET) which targets αVß6 integrin-positive PDAC. We selected A20FMDV2 peptide, which binds specifically to αvß6 integrin, as a probe scaffold, and 68Ga as a radioisotope. A20FMDV2 peptide has not been previously labeled with 68Ga. A cysteine residue was introduced to the N-terminus of the probe at a site-specific conjugation of maleimide-NOTA (mal-NOTA) chelate. Different numbers of glycine residues were also introduced between cysteine and the A20FMDV2 sequence as a spacer in order to reduce the steric hindrance of the mal-NOTA on the binding probe to αVß6 integrin. In vitro, the competitive binding assay revealed that probes containing a 6-glycine linker ([natGa]CG6 and [natGa]Ac-CG6) showed high affinity to αVß6 integrin. Both probes could be labeled by 67/68Ga with high radiochemical yield (>50%) and purity (>98%). On biodistribution analysis, [67Ga]Ac-CG6 showed higher tumor accumulation, faster blood clearance, and lower accumulation in the surrounding organs of pancreas than did [67Ga]CG6. The αVß6 integrin-positive xenografts were clearly visualized by PET imaging with [68Ga]Ac-CG6. The intratumoral distribution of [68Ga]Ac-CG6 coincided with the αVß6 integrin-positive regions detected by immunohistochemistry. Thus, [68Ga]Ac-CG6 is a useful peptide probe for the imaging of αVß6 integrin in PDAC.

Cullin-4B E3 ubiquitin ligase mediates Apaf-1 ubiquitination to regulate caspase-9 activity.

Apoptotic protease-activating factor 1 (Apaf-1) is a component of apoptosome, which regulates caspase-9 activity. In addition to apoptosis, Apaf-1 plays critical roles in the intra-S-phase checkpoint; therefore, impaired expression of Apaf-1 has been demonstrated in chemotherapy-resistant malignant melanoma and nuclear translocation of Apaf-1 has represented a favorable prognosis of patients with non-small cell lung cancer. In contrast, increased levels of Apaf-1 protein are observed in the brain in Huntington's disease. The regulation of Apaf-1 protein is not yet fully understood. In this study, we show that etoposide triggers the interaction of Apaf-1 with Cullin-4B, resulting in enhanced Apaf-1 ubiquitination. Ubiquitinated Apaf-1, which was degraded in healthy cells, binds p62 and forms aggregates in the cytosol. This complex of ubiquitinated Apaf-1 and p62 induces caspase-9 activation following MG132 treatment of HEK293T cells that stably express bcl-xl. These results show that ubiquitinated Apaf-1 may activate caspase-9 under conditions of proteasome impairment.

Endovascular trophoblast expresses CD59 to evade complement-dependent cytotoxicity.

In the human placenta, extravillous trophoblasts (EVTs) invade maternal decidual tissues (interstitial trophoblasts) and maternal spiral arteries (endovascular trophoblasts). Although endovascular trophoblasts are directly exposed to maternal blood containing complement components, they are not eliminated by complement-dependent cytotoxicity (CDC). In this study, we investigated the expression and possible function of CD59, one of the membrane-bound complement regulators, in EVTs. Immunohistochemistry of early embryo implantation sites revealed that CD59 was hardly expressed on interstitial trophoblasts, whereas it was intensely expressed on endovascular trophoblasts. Using the human EVT-like cell line Swan71, we established CD59-silencing Swan71 cells (Sw_CD59sh) and non-silencing control Swan71 cells (Sw_CTRsh). In vitro cell apoptosis assay showed that Sw_CD59sh cells were significantly more susceptible to CDC as compared to Sw_CTRsh. Our results suggest that CD59 confers some protection against maternal complement attack to the endovascular trophoblasts.

Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro.

The endometrium extracellular matrix (ECM) is essential for embryo implantation. Versican, a large chondroitin sulfate proteoglycan that binds hyaluronan and forms large ECM aggregates, can influence fundamental physiological phenomena, such as cell proliferation, adhesion and migration. The present study investigated the possible role of versican in human embryo implantation. Versican V1 expression and secretion in human endometrial epithelial cells (EECs) was most prominent in the mid-secretory phase. Versican expression in EECs significantly increased after treatment with estrogen and progesterone, but not by estrogen alone. We also established versican V1-overexpressing Ishikawa (endometrial cancer cell line) cells (ISKW-V1), versican V3-overexpressing (ISKW-V3) and control GFP-overexpressing (ISKW-GFP) Ishikawa cells. By the in vitro implantation model, the attachment ratio of BeWo (choriocarcinoma cell line) spheroids to the monolayer of ISKW-V1, but not of ISKW-V3, was found significantly enhanced compared with attachment to the ISKW-GFP monolayer. The conditioned medium derived from ISKW-V1 (V1-CM) also promoted the attachment of BeWo spheroids to the ISKW monolayer. However, this attachment-promoting effect was abolished when V1-CM was pretreated with chondroitinase ABC, which degrades chondroitin sulfate. Therefore, out of the ECM components, versican V1 may facilitate human embryo implantation.

Odor preference and olfactory memory are impaired in Olfaxin-deficient mice.

Olfaxin, which is a BNIP2 and Cdc42GAP homology (BCH) domain-containing protein, is predominantly expressed in mitral and tufted (M/T) cells in the olfactory bulb (OB). Olfaxin and Caytaxin, which share 56.3% amino acid identity, are similar in their glutamatergic terminal localization, kidney-type glutaminase (KGA) interaction, and caspase-3 substrate. Although the deletion of Caytaxin protein causes human Cayman ataxia and ataxia in the mutant mouse, the function of Olfaxin is largely unknown. In this study, we generated Prune2 gene mutant mice (Prune2Ex16-/-; knock out [KO] mice) using the CRISPR/Cas9 system, during which the exon 16 containing start codon of Olfaxin mRNA was deleted. Exon 16 has 80 nucleotides and is contained in four of five Prune2 isoforms, including PRUNE2, BMCC1, BNIPXL, and Olfaxin/BMCC1s. The levels of Olfaxin mRNA and Olfaxin protein in the OB and piriform cortex of KO mice significantly decreased. Although Prune2 mRNA also significantly decreased in the spinal cord, the gross anatomy of the spinal cord and dorsal root ganglion (DRG) was intact. Further, disturbance of the sensory and motor system was not observed in KO mice. Therefore, in the current study, we examined the role of Olfaxin in the olfactory system where PRUNE2, BMCC1, and BNIPXL are scarcely expressed. Odor preference was impaired in KO mice using opposite-sex urinary scents as well as a non-social odor stimulus (almond). Results of the odor-aversion test demonstrated that odor-associative learning was disrupted in KO mice. Moreover, the NMDAR2A/NMDAR2B subunits switch in the piriform cortex was not observed in KO mice. These results indicated that Olfaxin may play a critical role in odor preference and olfactory memory.

Associations among apolipoproteins, oxidized high-density lipoprotein and cardiovascular events in patients on hemodialysis.

Apolipoproteins are associated with survival among patients on hemodialysis (HD), but these associations might be influenced by dysfunctional (oxidized) high-density lipoprotein (HDL). We assessed associations among apolipoproteins and oxidized HDL, mortality and cardiovascular disease (CVD) events in patients on HD. This prospective observational study examined 412 patients on prevalent HD. Blood samples were obtained before dialysis at baseline to measure lipids, apolipoproteins, oxidized LDL, oxidized HDL, high-sensitivity C-reactive protein (hs-CRP) and interleukin (IL)-6 at baseline, and HDL-C and hs-CRP were measured 12 months later. Patients were then prospectively followed-up (mean, 40 months) and all-cause mortality and composite CVD events were analyzed. Associations between variables at baseline and clinical outcome were assessed by Cox proportional hazards modeling (n = 412) and Cox hazards modeling with a time-varying covariate with HDL-C and hs-CRP (n = 369). Quartiles of apolipoproteins and oxidized HDL were not associated with all-cause mortality. However, Cox proportional hazards models with quartiles of each variable adjusted for confounders and hs-CRP or IL-6 identified apolipoprotein (apo)B-to-apoA-I ratio (apoB/apoA-I) and oxidized HDL, but not apoA-I or apoA-II, as independent risk factors for composite CVD events. These associations were confirmed by Cox proportional hazards modeling with time-varying covariates for hs-CRP. ApoB/apoA-I was independently associated with composite CVD events in 1-standard deviation (SD) increase-of-variables models adjusted for the confounders, oxidized HDL and hs-CRP. However, these associations disappeared from the model adjusted with IL-6 instead of hs-CRP, and oxidized HDL and IL-6 were independently associated with composite CVD events. Findings resembled those from Cox proportional hazards modeling using time-varying covariates with HDL-C adjusted with IL-6. In conclusion, both oxidized HDL and apoB/apoA-I might be associated with CVD events in patients on prevalent HD, while associations of apoB/apoA-I with CVD events differed between models of apoB/apoA-I quartiles and 1-SD increases, and were influenced by IL-6.

Investigation of Biodistribution and Speciation Changes of Orally Administered Dual Radiolabeled Complex, Bis(5-chloro-7-[131I]iodo-8-quinolinolato)[65Zn]zinc.

Many zinc (Zn) complexes have been developed as promising oral antidiabetic agents. In vitro assays using adipocytes have demonstrated that the coordination structures of Zn complexes affect the uptake of Zn into cells and have insulinomimetic activities, for which moderate stability of Zn complexes is vital. The complexation of Zn plays a major role improving its bioavailability. However, investigation of the speciation changes of Zn complexes after oral administration is lacking. A dual radiolabeling approach was applied in order to investigate the speciation of bis(5-chloro-7-iodo-8-quinolinolato)zinc complex [Zn(Cq)2], which exhibits the antidiabetic activity in diabetic mice. In the present study, 65Zn- and 131I-labeled [Zn(Cq)2] were synthesized, and their biodistribution were analyzed after an oral administration using both invasive conventional assays and noninvasive gamma-ray emission imaging (GREI), a novel nuclear medicine imaging modality that enables analysis of multiple radionuclides simultaneously. The GREI experiments visualized the behavior of 65Zn and [131I]Cq from the stomach to large intestine and through the small intestine; most of the administered Zn was transported together with clioquinol (5-chloro-7-iodo-8-quinolinol) (Cq). Higher accumulation of 65Zn for [Zn(Cq)2] than ZnCl2 suggests that the Zn associated with Cq was highly absorbed by the intestinal tract. In particular, the molar ratio of administered iodine to Zn decreased during the distribution processes, indicating the dissociation of most [Zn(Cq)2] complexes. In conclusion, the present study successfully evaluated the speciation changes of orally administered [Zn(Cq)2] using the dual radiolabeling method.

Noninvasive evaluation of nicotinic acetylcholine receptor availability in mouse brain using single-photon emission computed tomography with [(123)I]5IA.

INTRODUCTION: Nicotinic acetylcholine receptors (nAChRs) are of great interest because they are implicated in higher brain functions. Nuclear medical imaging is one of the useful techniques for noninvasive evaluation of physiological and pathological function in living subjects. Recent progress in nuclear medical imaging modalities enables the clear visualization of the organs of small rodents. Thus, translational research using nuclear medical imaging in transgenic mice has become possible and helps to elucidate human disease pathology. However, imaging of α4ß2 nAChRs in the mouse brain has not yet been performed. The purpose of this study was to assess the feasibility of single-photon emission computed tomography (SPECT) with 5-[(123)I]iodo-3-[2(S)-azetidinylmethoxy]pyridine ([(123)I]5IA) for evaluating α4ß2 nAChR availability in the mouse brain. METHODS: A 60-min dynamic SPECT imaging session of α4ß2 nAChRs in the mouse brain was performed. The regional distribution of radioactivity in the SPECT images was compared to the density of α4ß2 nAChRs measured in an identical mouse. Alteration of nAChR density in the brains of Tg2576 mice was also evaluated. RESULTS: The mouse brain was clearly visualized by [(123)I]5IA-SPECT and probe accumulation was significantly inhibited by pretreatment with (-)-nicotine. The regional distribution of radioactivity in SPECT images showed a significant positive correlation with α4ß2 nAChR density measured in an identical mouse brain. Moreover, [(123)I]5IA-SPECT was able to detect the up-regulation of α4ß2 nAChRs in the brains of Tg2576 transgenic mice. CONCLUSIONS: [(123)I]5IA-SPECT imaging would be a promising tool for evaluating α4ß2 nAChR availability in the mouse brain and may be useful in translational research focused on nAChR-related diseases.

Enhanced stability of Cu(2+)-ATCUN complexes under physiologically relevant conditions by insertion of structurally bulky and hydrophobic amino acid residues into the ATCUN motif.

Copper complexes formed by an amino terminal Cu(2+)- and Ni(2+)-binding (ATCUN) motif have attracted attention as metallodrug candidates that cleave DNA or RNA and inactivate enzymes. Although the stability of the Cu(2+)-ATCUN complex under physiologically relevant conditions is a key factor for medical applications, it has remained unclear. Here we prepared a series of ATCUN peptides by inserting various amino acid residues into positions 1 and 2, and investigated the stability of the Cu(2+)-ATCUN complexes in aqueous solution, blood plasma, and living animals. Systematic pH titration showed that the low basicity of the N-terminal amine of the peptide stabilized the Cu(2+)-ATCUN complex in aqueous solution. Interestingly, the stability of (64)Cu-labeled ATCUN complexes in blood plasma was significantly enhanced by the structural bulkiness and hydrophobicity of the amino acid residues at positions 1 and 2. To validate the in vivo stability, six ATCUN motifs (YYH, VVH, NNH, TTH, GGH, and DDH) were conjugated to a tumor-targeting peptide, octreotide (Oct). The stability of the (64)Cu-ATCUN-Oct complexes in blood plasma showed a similar trend to that of the (64)Cu-ATCUN complexes. The (64)Cu-YYH-Oct complex exhibited the highest stability in blood plasma. According to the positron emission tomography and competitive blocking studies of a tumor-bearing mouse model, (64)Cu-YYH-Oct specifically accumulated in tumors, suggesting that the complex was sufficiently stable to reach its target in vivo. The results show that the structural bulkiness and hydrophobicity of the residues at positions 1 and 2 are key parameters for designing metallodrugs on the basis of the Cu(2+)-ATCUN complex.

[Development of Radiolabeled Molecular Imaging Probes for in Vivo Analysis of Biological Function].

Molecular imaging is a newly emerging field aimed at advancing our understanding of biology and medicine through the noninvasive in vivo investigation of cellular molecular events involved in normal and pathologic processes. In this field, researchers and/or clinicians are combining modern tools of molecular and cell biology with state of the art technology in order to noninvasively image living subjects. Various imaging modalities such as optics (fluorescence and luminescence), nuclear magnetic resonance imaging, ultrasound, and radiation are being used to visually capture and study molecular and cellular events in living organisms. Among these modalities, nuclear medical molecular imaging uses radionuclides [i.e., positron emission tomography (PET) and single-photon emission computed tomography (SPECT)], and has characteristic properties that allow researchers and/or clinicians to obtain functional images of living subjects with high sensitivity. Translational molecular imaging, a research step between animal experiments and the clinical setting, has been successful when using nuclear medical molecular imaging. This approach leads to better methods for studying biological processes, as well as for diagnosing and managing diseases. In this review, two topics associated with our research on nuclear medical molecular imaging are summarized: (1) the development of a nuclear medical molecular imaging probe that targets cerebral nicotinic acetylcholine receptors (nAChRs), and the translational molecular imaging research conducted using this nAChR imaging probe; and (2) the development of oxygen-dependent degradable nuclear medical molecular imaging probes that target hypoxia-inducible factor-1-active tumor microenvironments.

Expanded polyglutamine embedded in the endoplasmic reticulum causes membrane distortion and coincides with Bax insertion.

The endoplasmic reticulum (ER) is important in various cellular functions, such as secretary and membrane protein biosynthesis, lipid synthesis, and calcium storage. ER stress, including membrane distortion, is associated with many diseases such as Huntington's disease. In particular, nuclear envelope distortion is related to neuronal cell death associated with polyglutamine. However, the mechanism by which polyglutamine causes ER membrane distortion remains unclear. We used electron microscopy, fluorescence protease protection assay, and alkaline treatment to analyze the localization of polyglutamine in cells. We characterized polyglutamine embedded in the ER membrane and noted an effect on morphology, including the dilation of ER luminal space and elongation of ER-mitochondria contact sites, in addition to the distortion of the nuclear envelope. The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. These results demonstrated that the ER membrane may be a target of polyglutamine, which triggers cell death through Bax.

Additional information gained by positron emission tomography with (68)Ga-DOTATOC for suspected unknown primary or recurrent neuroendocrine tumors.

OBJECTIVE: Positron emission tomography (PET)/computed tomography (CT) using (68)Ga-labeled 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid-D-Phe(1)-Tyr(3)-octreotide (DOTATOC) has been used to detect neuroendocrine tumors (NETs). The purpose of this study was to investigate the clinical efficacy of DOTATOC-PET/CT for detecting clinically suspected NETs when conventional imaging modalities were negative or inconclusive, in terms of additional value. METHODS: A total of 46 patients were analyzed retrospectively. Among them, 14 patients underwent a DOTATOC-PET/CT scan for detecting unknown primary tumors after histopathological confirmation of a NET at metastatic sites (group A): 7 patients for detecting metastasis or recurrence after surgery for NET because of their high hormone levels but with no recurrence detected by other imaging modalities (group B); the remaining 25 patients for detecting suspected NETs because their hormone levels were high with no history of histopathologically proven NET (group C). Additional information was assessed, according to each situation. RESULTS: In group A, unknown primary tumors were suspected by DOTATOC-PET/CT in 8 of 14 patients (gastrointestinal/pancreatic NET in 7 patients, prostatic cancer in 1 patient), but prostatic cancer was not confirmed by histopathology (i.e., false positive). In group B, DOTATOC-PET/CT depicted lesions in six of seven patients, including nodal metastasis (n = 5) and liver metastasis (n = 1). In group C, DOTATOC-PET/CT did not demonstrate any abnormal foci except in one case of pancreatic NET. Additional information was obtained in 50, 86, and 4 % of cases, in groups A, B, and C, respectively. CONCLUSIONS: DOTATOC-PET/CT was useful for detecting NETs, especially when recurrence or metastases were suspected because of high hormone levels after surgery for a NET. It is unlikely, however, that additional information can be acquired in patients with no history of NET simply based on high hormone levels.

Gallium-68-labeled anti-HER2 single-chain Fv fragment: development and in vivo monitoring of HER2 expression.

PURPOSE: We aimed to develop a gallium-68 (Ga-68)-labeled single-chain variable fragment (scFv) targeting the human epidermal growth factor receptor 2 (HER2) to rapidly and noninvasively evaluate the status of HER2 expression. PROCEDURES: Anti-HER2 scFv was labeled with Ga-68 by using deferoxamine (Df) as a bifunctional chelate. Biodistribution of [(68)Ga]Df-anti-HER2 scFv was examined with tumor-bearing mice and positron emission tomography (PET) imaging was performed. The changes in HER2 expression after anti-HER2 therapy were monitored by PET imaging. RESULTS: [(68)Ga]Df-anti-HER2 scFv was obtained with high radiochemical yield after only a 5-min reaction at room temperature. The probe showed high accumulation in HER2-positive xenografts and the intratumoral distribution of radioactivity coincided with HER2-positive regions. Furthermore, [(68)Ga]Df-anti-HER2 scFv helped visualize HER2-positive xenografts and monitor the changes in HER2 expression after anti-HER2 therapy. CONCLUSION: [(68)Ga]Df-anti-HER2 scFv could be a promising probe to evaluate HER2 status by in vivo PET imaging, unless trastuzumab is prescribed as part of the therapy.

64Cu-DOTA-anti-CTLA-4 mAb enabled PET visualization of CTLA-4 on the T-cell infiltrating tumor tissues.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) targeted therapy by anti-CTLA-4 monoclonal antibody (mAb) is highly effective in cancer patients. However, it is extremely expensive and potentially produces autoimmune-related adverse effects. Therefore, the development of a method to evaluate CTLA-4 expression prior to CTLA-4-targeted therapy is expected to open doors to evidence-based and cost-efficient medical care and to avoid adverse effects brought about by ineffective therapy. In this study, we aimed to develop a molecular imaging probe for CTLA-4 visualization in tumor. First, we examined CTLA-4 expression in normal colon tissues, cultured CT26 cells, and CT26 tumor tissues from tumor-bearing BALB/c mice and BALB/c nude mice by reverse transcription polymerase chain reaction (RT-PCR) analysis and confirmed whether CTLA-4 is strongly expressed in CT26 tumor tissues. Second, we newly synthesized 64Cu-1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid-anti-mouse CTLA-4 mAb (64Cu-DOTA-anti-CTLA-4 mAb) and evaluated its usefulness in positron emission tomography (PET) and ex-vivo biodistribution analysis in CT26-bearing BALB/c mice. High CTLA-4 expression was confirmed in the CT26 tumor tissues of tumor-bearing BALB/c mice. However, CTLA-4 expression was extremely low in the cultured CT26 cells and the CT26 tumor tissues of tumor-bearing BALB/c nude mice. The results suggested that T cells were responsible for the high CTLA-4 expression. Furthermore, 64Cu-DOTA-anti-CTLA-4 mAb displayed significantly high accumulation in the CT26 tumor, thereby realizing non-invasive CTLA-4 visualization in the tumor. Together, the results indicate that 64Cu-DOTA-anti-CTLA-4 mAb would be useful for the evaluation of CTLA-4 expression in tumor.

Radiolabeled probes targeting hypoxia-inducible factor-1-active tumor microenvironments.

Because tumor cells grow rapidly and randomly, hypoxic regions arise from the lack of oxygen supply in solid tumors. Hypoxic regions in tumors are known to be resistant to chemotherapy and radiotherapy. Hypoxia-inducible factor-1 (HIF-1) expressed in hypoxic regions regulates the expression of genes related to tumor growth, angiogenesis, metastasis, and therapy resistance. Thus, imaging of HIF-1-active regions in tumors is of great interest. HIF-1 activity is regulated by the expression and degradation of its α subunit (HIF-1α), which is degraded in the proteasome under normoxic conditions, but escapes degradation under hypoxic conditions, allowing it to activate transcription of HIF-1-target genes. Therefore, to image HIF-1-active regions, HIF-1-dependent reporter systems and injectable probes that are degraded in a manner similar to HIF-1α have been recently developed and used in preclinical studies. However, no probe currently used in clinical practice directly assesses HIF-1 activity. Whether the accumulation of (18)F-FDG or (18)F-FMISO can be utilized as an index of HIF-1 activity has been investigated in clinical studies. In this review, the current status of HIF-1 imaging in preclinical and clinical studies is discussed.

Synthesis and evaluation of a radioiodinated peptide probe targeting αvß6 integrin for the detection of pancreatic ductal adenocarcinoma.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) remains a major cause of cancer-related death. Since significant upregulation of αvß6 integrin has been reported in PDAC, this integrin is a promising target for PDAC detection. In this study, we aimed to develop a radioiodinated probe for the imaging of αvß6 integrin-positive PDAC with single-photon emission computed tomography (SPECT). METHODS: Four peptide probes were synthesized and screened by competitive and saturation binding assays using 2 PDAC cell lines (AsPC-1, αvß6 integrin-positive; MIA PaCa-2, αvß6 integrin-negative). The probe showing the best affinity was used to study the biodistribution assay, an in vivo blocking study, and SPECT imaging using tumor bearing mice. Autoradiography and immunohistochemical analysis were also performed. RESULTS: Among the 4 probes examined in this study, (125)I-IFMDV2 showed the highest affinity for αvß6 integrin expressed in AsPC-1 cells and no affinity for MIA PaCa-2 cells. The accumulation of (125)I-IFMDV2 in the AsPC-1 xenograft was 3-5 times greater than that in the MIA PaCa-2 xenograft, consistent with the expression of αvß6 integrin in each xenograft, and confirmed by immunohistochemistry. Pretreatment with excess amounts of A20FMDV2 significantly blocked the accumulation of (125)I-IFMDV2 in the AsPC-1 xenograft, but not in the MIA PaCa-2 xenograft. Furthermore, (123)I-IFMDV2 enabled clear visualization of the AsPC-1 xenograft. CONCLUSION: (123)I-IFMDV2 is a potential SPECT probe for the imaging of αvß6 integrin in PDAC.