Can dedicated breast PET help to reduce overdiagnosis and overtreatment by differentiating between indolent and potentially aggressive ductal carcinoma in situ?

OBJECTIVES: To analyze the utility of metabolic imaging, and specifically of dedicated breast positron emission tomography (dbPET) to differentiate between indolent and potentially aggressive ductal carcinoma in situ (DCIS). METHODS: After institutional review board approval, we retrospectively reviewed the cases of pure DCIS who underwent dbPET before biopsy and surgery in Lucus Augusti Universitary Hospital (Lugo, Spain) and in Fudan Cancer Institute (Shanghai, China) between January 2016 and May 2018. Grade 1 and "non-comedo" grade 2 DCIS were considered low-risk disease, while intermediate-grade with necrosis or grade 3 cases were included in the high-risk group. DbPET sensitivity and specificity to differentiate between indolent and potentially aggressive DCIS were determined along with their respective 95% confidence intervals. RESULTS: We enrolled 139 surgery-confirmed pure DCIS cases. Fifty were high-risk neoplasms and 89 low-risk DCIS. Only seven low-risk lesions were positive at dbPET and five of potentially aggressive neoplasms did not show FDG uptake, all included into the field of view (FOV). Sensitivity and specificity of dbPET to differentiate between indolent and potentially aggressive DCIS were 90% (95% CI, 77-96%) and 92% (95% CI, 84-97%), respectively. CONCLUSION: Metabolic imaging could help to identify the subgroup of indolent lesions from those potentially aggressive ones that may be managed by active surveillance. KEY POINTS: • Low- and high-grade DCIS likely arise from two distinct evolutionary paths and when low-grade lesions progress to invasive cancer, the tumor is frequently low grade and well differentiated. • Ongoing clinical trials evaluate whether patients with low-risk DCIS could be safely managed by an active surveillance approach, with avoidance of unnecessary treatments and without impact on ipsilateral invasive breast cancer free survival time. • Dedicated breast PET may differentiate harmless from potentially hazardous DCIS, supporting active surveillance for the management of those women with low-grade DCIS, decreasing the rate of the upgrade to invasive carcinoma at surgical excision.

Texture analysis of high-resolution dedicated breast 18 F-FDG PET images correlates with immunohistochemical factors and subtype of breast cancer.

PURPOSE: This study aims to determine whether PET textural features measured with a new dedicated breast PET scanner reflect biological characteristics of breast tumors. METHODS: One hundred and thirty-nine breast tumors from 127 consecutive patients were included in this analysis. All of them underwent a 18F-FDG PET scan before treatment. Well-known PET quantitative parameters such as SUV m a x , SUV m e a n , metabolically active tumor volume (MATV) and total lesion glycolysis (TLG) were extracted. Together with these parameters, local, regional, and global heterogeneity descriptors, which included five textural features (TF), were computed. Immunohistochemical classification of breast cancer considered five subtypes: luminal A like (LA), luminal B like/HER2 - (LB -), luminal B like/HER2+ (LB+), HER2-positive-non-luminal (HER2pnl), and triple negative (TN). Associations between PET features and tumor characteristics were assessed using non-parametric hypothesis tests. RESULTS: Along with well-established associations, new correlations were found. HER2-positive tumors had significantly higher uptake (p < 0.001, AUCs > 0.70) and presented different global and regional heterogeneity (p = 0.002, p = 0.016, respectively, AUCs < 0.70). Nine out of ten analyzed features were significantly associated with immunohistochemical subtype. Uptake was lower for LA tumors (p < 0.001) with AUCs ranging from 0.71 to 0.88 for each subgroup comparison. Heterogeneity metrics were significantly associated when comparing LA and LB - (p < 0.01), being regional heterogeneity metrics more discriminative than any other parameter (AUC = 0.80 compared to AUC = 0.71 for SUV). LB+ and HER2pnl tumors also showed more regional heterogeneity than LA tumors (AUCs = 0.79 and 0.84, respectively). After comparison with whole-body PET studies, we observed an overall improvement in the classification ability of both non-heterogeneity metrics and textural features. CONCLUSIONS: PET parameters extracted from high-resolution dedicated breast PET images showed new and stronger correlations with immunohistochemical factors and immunohistochemical subtype of breast cancer compared to whole-body PET.

A truncating mutation of HDAC2 in human cancers confers resistance to histone deacetylase inhibition.

Disruption of histone acetylation patterns is a common feature of cancer cells, but very little is known about its genetic basis. We have identified truncating mutations in one of the primary human histone deacetylases, HDAC2, in sporadic carcinomas with microsatellite instability and in tumors arising in individuals with hereditary nonpolyposis colorectal cancer syndrome. The presence of the HDAC2 frameshift mutation causes a loss of HDAC2 protein expression and enzymatic activity and renders these cells more resistant to the usual antiproliferative and proapoptotic effects of histone deacetylase inhibitors. As such drugs may serve as therapeutic agents for cancer, our findings support the use of HDAC2 mutational status in future pharmacogenetic treatment of these individuals.

Cell membrane CD44v6 levels in squamous cell carcinoma of the lung: association with high cellular proliferation and high concentrations of EGFR and CD44v5.

Membranous CD44v6 levels in tumors and surrounding samples obtained from 94 patients with squamous cell lung carcinomas were studied and compared to clinical stage, cellular proliferation, membranous CD44v5 levels, epidermal growth factor receptor EGFR and cytoplasmatic concentrations of CYFRA 21.1. CD44v6 positive values were observed in 33/38 non-tumor samples and in 76/94 tumor samples, but there were not statistically significant differences between both subgroups. In CD44v6 positive tumor samples, CD44v6 was not associated with clinical stage, histological grade, ploidy and lymph node involvement, but significant association was found with high cellular proliferation. Likewise, CD44v6 positive tumors had significantly higher levels of EGFR and CD44v5. In patients with squamous cell lung carcinomas and clinical stage I, positive CD44v6 cases were associated with the same parameters. Furthermore, positive CD44v5 squamous tumors were associated significantly with histological grade III and lower levels of CYFRA21.1. Our findings support the value of CD44v6 as a possible indicator of poor outcome in patients with squamous lung carcinomas.

CA15.3 serum concentrations in older women with infiltrating ductal carcinomas of the breast.

Breast cancer is currently becoming a disease of the elderly. We have studied the relation between CA 15.3 serum concentrations and clinical-pathological parameters in 69 women with IDC aged over 70 years (76.3±4.2; range: 71-88; median 76). A group of 205 women with the same tumor but aged <70 years (62.8±4.0; range: 55-70; median 63) was also considered for comparison. Tumor size, axillary lymph node involvement, distant metastasis and histological grade were taken account. Serum CA 15.3 was determined by luminescence assay. CA 15.3 serum concentrations ranged between 6 and 85 U/mL (median 22.9 U/mL), and were higher only in patients with greater (qualitative and quantitative; p: 0.041) tumor size. Our results show that in women with IDCs, and aged over 70 years, serum CA 15.3 serum concentrations are associated exclusively with a greater tumor size, being these findings different to those described in women with the same subtype of tumor considered as a whole or with lower age.

A novel in silico reverse-transcriptomics-based identification and blood-based validation of a panel of sub-type specific biomarkers in lung cancer.

Lung cancer accounts for the highest number of cancer-related deaths worldwide. Early diagnosis significantly increases the disease-free survival rate and a large amount of effort has been expended in screening trials and the development of early molecular diagnostics. However, a gold standard diagnostic strategy is not yet available. Here, based on miRNA expression profile in lung cancer and using a novel in silico reverse-transcriptomics approach, followed by analysis of the interactome; we have identified potential transcription factor (TF) markers that would facilitate diagnosis of subtype specific lung cancer. A subset of seven TF markers has been used in a microarray screen and was then validated by blood-based qPCR using stage-II and IV non-small cell lung carcinomas (NSCLC). Our results suggest that overexpression of HMGA1, E2F6, IRF1, and TFDP1 and downregulation or no expression of SUV39H1, RBL1, and HNRPD in blood is suitable for diagnosis of lung adenocarcinoma and squamous cell carcinoma sub-types of NSCLC. Here, E2F6 was, for the first time, found to be upregulated in NSCLC blood samples. The miRNA-TF-miRNA interaction based molecular mechanisms of these seven markers in NSCLC revealed that HMGA1 and TFDP1 play vital roles in lung cancer tumorigenesis. The strategy developed in this work is applicable to any other cancer or disease and can assist in the identification of potential biomarkers.

Image-guided breast biopsy and localisation: recommendations for information to women and referring physicians by the European Society of Breast Imaging.

We summarise here the information to be provided to women and referring physicians about percutaneous breast biopsy and lesion localisation under imaging guidance. After explaining why a preoperative diagnosis with a percutaneous biopsy is preferred to surgical biopsy, we illustrate the criteria used by radiologists for choosing the most appropriate combination of device type for sampling and imaging technique for guidance. Then, we describe the commonly used devices, from fine-needle sampling to tissue biopsy with larger needles, namely core needle biopsy and vacuum-assisted biopsy, and how mammography, digital breast tomosynthesis, ultrasound, or magnetic resonance imaging work for targeting the lesion for sampling or localisation. The differences among the techniques available for localisation (carbon marking, metallic wire, radiotracer injection, radioactive seed, and magnetic seed localisation) are illustrated. Type and rate of possible complications are described and the issue of concomitant antiplatelet or anticoagulant therapy is also addressed. The importance of pathological-radiological correlation is highlighted: when evaluating the results of any needle sampling, the radiologist must check the concordance between the cytology/pathology report of the sample and the radiological appearance of the biopsied lesion. We recommend that special attention is paid to a proper and tactful approach when communicating to the woman the need for tissue sampling as well as the possibility of cancer diagnosis, repeat tissue sampling, and or even surgery when tissue sampling shows a lesion with uncertain malignant potential (also referred to as "high-risk" or B3 lesions). Finally, seven frequently asked questions are answered.

Vaginal residence and pharmacokinetic preclinical study of topical vaginal mucoadhesive W/S emulsions containing ciprofloxacin.

The aim of this work is to test the in vivo behavior of a mucoadhesive vaginal emulsion resistant to the clearance of vaginal fluids using ciprofloxacin (CPX) as an anti-infective model of drug. CPX is a broad-spectrum antibiotic used in the treatment of sexual tissues infections, as intravenous injection in a dose of 20 mg every 12 h. In this study, CPX was incorporated in water in silicone (W/S) mucoadhesive emulsions and the in vivo residence time and the CPX in vivo absorption and distribution to the sexual organs was studied using the rat as animal model. W/S emulsion shows excellent in vitro bioadhesion having high resistance to the vaginal fluids clearance. The drug release profiles show a constant release of CPX during at least 6 h according to a zero-order kinetics. In vivo computerized PET/CT Image Analysis after intravaginal administration to rats indicates that W/S emulsions remain in the vaginal area for a long time and shows a good absorption of the radiotracers used as markers through the vaginal mucosa. Ciprofloxacin pharmacokinetic studies developed after the single intravaginal administration of W/S emulsion shows a good absorption and distribution of CPX on the uterus and ovarian tissue. A significant concentration of CPX in the sexual tissues was observed after 24 h of administration of W/S emulsion. Therefore, W/S emulsions have a good in vivo residence and drug release in the vaginal mucosae showing a great potential for the treatment of sexual tissues infections, as vaginal bioadhesive delivery systems of antinfectious drugs.

Longitudinal PET/CT evaluation of TNBS-induced inflammatory bowel disease rat model.

Inflammatory bowel disease (IBD) is a group of chronic disorders of the gastrointestinal tract, which two main types are Crohn's disease and ulcerative colitis. It has multifactorial etiologies, being essential the use of animal models and disease activity measures to develop new therapies. With this aim, the use of animal models in combination with non-invasive molecular imaging can play an important role in the development of new treatments. In this study, IBD was induced in rats using 2,4,6-trinitrobenzenesulfonic acid (TNBS) and longitudinal [18F]FDG PET/CT scans were conducted to assess disease progression post-TNBS administration. Afterwards, [18F]FDG PET/CT scans were carried out after treatment with methylprednisolone to validate the model. In non-treated rats, SUVmax (Standardized Uptake Value) rapidly increased after IBD induction, being particularly significant (p < 0.01) on days 7-13 after induction. There were no significant differences between non-treated and treated IBD rats from days 0-3. Nevertheless, treated IBD rats showed a significant decrease in SUVmax between days 7-13 (p < 0.01). Histological examination showed descending and transverse colon as the most affected regions. There was a moderate (R2 = 0.61) and strong (R2 = 0.82) correlation of SUVmax with Nancy grade (parameter for histological assessment of disease activity) and weight changes, respectively. In this study, we have performed the first longitudinal [18F]FDG PET/CT assessment of TNBS-induced IBD in rats, demonstrating the potential role of preclinical molecular imaging for the evaluation of new therapies in combination with IBD rat models.

Preclinical characterization and clinical evaluation of tacrolimus eye drops.

Severe allergic ocular diseases as atopic keratoconjunctivitis can induce corneal damage due to inflammatory substances released from giant papillae. Tacrolimus eye drops are one of the current therapeutic alternatives for its treatment. This work is aimed at developing and characterizing a 0.03% tacrolimus ophthalmic formulation, which was introduced in three types of vehicles (BBS, PVA and Hyaluronic Acid). For this, we have performed in vitro (stability studies) and in vivo assays (corneal permanence time measured directly by Positron Emission Tomography) of three potential formulations. Next, the best formulation was selected, and its toxicological profile and clinical effectiveness have been evaluated. The biopermanence studies (direct measurements and PET/CT) showed that the formulations with PVA and Hyaluronic Acid present more retention time on the ocular surface of rats than PBS. From the stability study, we have determined that tacrolimus with PVA in cold storage is the best option. Tacrolimus with PVA has shown lower cytotoxicity than cyclosporine at early times. On the other hand, the pilot study performed has shown significant improvements in patients, with no noticeable adverse reactions. Based on stability, biopermanence, safety and clinical effectiveness studies, we concluded that tacrolimus-PVA eye drops are a suitable candidate for its clinical application in inflammatory ophthalmology diseases.

Ophthalmic Econazole Hydrogels for the Treatment of Fungal Keratitis.

Econazole is a feasible alternative treatment in the management of fungal keratitis. Nevertheless, its low water solubility is considered the main limitation to the incorporation into ophthalmic formulations. In this work, econazole nitrate is solubilized by using cyclodextrins to achieve an optimum therapeutic concentration. Phase solubility diagrams suggest α-cyclodextrin as the most effective cyclodextrin and later the inclusion complex formed with this one was characterized in solution by 1D, 2D-NMR, and molecular modeling. Econazole-α-cyclodextrin inclusion complex was included in 2 types of ocular hydrogels: a natural polysaccharides ion-sensitive hydrogel and a hyaluronic acid hydrogel. Both of them show no ocular irritation in the hen's egg test on chorioallantoic membrane assay and a controlled econazole release over time. Permeability studies suggest that hydrogels do not modify the econazole nitrate permeability through bovine cornea in comparison with an econazole-α-cyclodextrin inclusion complex solution. Finally, ocular biopermanence studies performed using positron emission tomography show these hydrogels present a high retention time on the eye. Results suggest the developed formulations have a high potential as vehicles for the econazole topical ocular administration as fungal keratitis treatment.

Medical chemistry to spy cancer stem cells from outside the body.

Accumulating evidence indicates that cancer is maintained by cancer stem cells (CSC). The goal of molecular imaging is to detect pathologic biomarkers, which can lead to early recognition of cancer, better therapeutic management, and improved monitoring for recurrence. The main focus of this review is to describe the different classes of tracers, contrast agents and dyers, and their putative application to improve cancer stem cells detection and follow-up. Although the in vivo cancer diagnosis has not significantly changed for the past three decades, however, in the future it might be possible to trace all cancer cells, including the cancer stem cells.

DNA methylation and histone modifications in patients with cancer: potential prognostic and therapeutic targets.

Epigenetics, a combination of DNA modifications, chromatin organization, and variations in its associated proteins, configure a new entity that regulates gene expression throughout methylation, acetylation, and chromatin remodeling. In addition to silencing as a result of mutations, loss of heterozygosity, or classical genetic events epigenetic modification symbolizes essential early events during carcinogenesis and tumor development. The reversion of these epigenetic processes restoring normal expression of tumor-suppressor genes has consequently become a new therapeutic target in cancer treatment. Aberrant patterns of epigenetic modifications will be, in a near future, crucial parameters in cancer diagnosis and prognosis.

EMP3, a myelin-related gene located in the critical 19q13.3 region, is epigenetically silenced and exhibits features of a candidate tumor suppressor in glioma and neuroblastoma.

The presence of common genomic deletions in the 19q13 chromosomal region in neuroblastomas and gliomas strongly suggests the presence of a putative tumor suppressor gene for these neoplasms in this region that, despite much effort, has not yet been identified. In an attempt to address this issue, we compared the expression profile of 89 neuroblastoma tumors with that of benign ganglioneuromas by microarray analysis. Probe sets (637 of 62,839) were significantly down-regulated in neuroblastoma tumors, including, most importantly, a gene located at 19q13.3: the epithelial membrane protein 3 (EMP3), a myelin-related gene involved in cell proliferation and cell-cell interactions. We found that EMP3 undergoes hypermethylation-mediated transcriptional silencing in neuroblastoma and glioma cancer cell lines, whereas the use of the demethylating agent 5-aza-2-deoxycytidine restores EMP3 gene expression. Furthermore, the reintroduction of EMP3 into neuroblastoma cell lines displaying methylation-dependent silencing of EMP3 induces tumor suppressor-like features, such as reduced colony formation density and tumor growth in nude mouse xenograft models. Screening a large collection of human primary neuroblastomas (n = 116) and gliomas (n = 41), we observed that EMP3 CpG island hypermethylation was present in 24% and 39% of these tumor types, respectively. Furthermore, the detection of EMP3 hypermethylation in neuroblastoma could be clinically relevant because it was associated with poor survival after the first 2 years of onset of the disease (Kaplan-Meier; P = 0.03) and death of disease (Kendall tau, P = 0.03; r = 0.19). Thus, EMP3 is a good candidate for being the long-sought tumor suppressor gene located at 19q13 in gliomas and neuroblastomas.

Cysteamine polysaccharide hydrogels: Study of extended ocular delivery and biopermanence time by PET imaging.

Cystinosis is a rare autosomal recessive disorder in which cystine crystals accumulate within the lysosomes of various organs, including the cornea. Ocular treatment is based on the administration of cysteamine eye drops, requiring its instillation several times per day. We have introduced the cysteamine in two types of previously developed ocular hydrogels (ion sensitive hydrogel with the polymers gellan gum and kappa-carrageenan and another one composed of hyaluronic acid), aiming at increasing the ocular retention in order to extend the dosing interval. The biopermanence studies (direct measurements and PET/CT) show that these formulations present a high retention time on the ocular surface of rats. From the in vitro release study we determined that both hydrogels can control the release of cysteamine over time, showing a zero order kinetics during four hours. At the same time, these hydrogels could act as corneal absorption promoters, as they allow a higher permeation of cysteamine through bovine cornea compared to a solution. HET-CAM test and cytotoxicity assays show no irritation on the ocular surface. These results demonstrate that the developed formulations present a high potential as vehicles for the topical ocular administration of cysteamine.

In vivo eye surface residence determination by high-resolution scintigraphy of a novel ion-sensitive hydrogel based on gellan gum and kappa-carrageenan.

In last years, sensitive hydrogels have become a breakthrough in ophthalmic pharmaceutical technology aimed at developing new strategies to increase the residence time of active substances. In a previous paper, we qualitatively demonstrated the capacity of a new ion sensitive hydrogel to increase the residence time. Nevertheless, the clearance of the gel from the ocular surface was not quantifiable with the used methodology. The aim of the present work was to use a well-established approach based on scintigraphy to quantitatively estimate the residence time of the previously proposed hydrogel. The rat corneal residence time of a topic ophthalmic formulation containing gellan gum and kappa carragenan (0.82% w/v) labeled with 99mTc-DTPA radiotracer was evaluated and compared with the residence of an aqueous solution. Ophthalmic safety studies such as eye irritation or passage through the cornea were also carried out. After 1.5h of contact, 77% of the hydrogel remained in the ocular surface, presenting kinetics of disappearance one-phase decay and a half time of 262min. We conclude that the novel ophthalmic hydrogel developed with kappa carrageenan and gellan gum remains for long periods of time on the corneal surface, presenting a drop that fits an exponential decay.

Preclinical PET Study of Intravitreal Injections.

Purpose: This work aimed at describing the time course of vitreous clearance through the use of positron emission tomography (PET) as a noninvasive tool for pharmacokinetic studies of intravitreal injection. Methods: The pharmacokinetic profile of intravitreal injections of molecules labeled with 18Fluorine (18F) was evaluated in adult Sprague Dawley rats by using a dedicated small-animal PET/computed tomography scanner. Different conditions were studied: three molecules radiolabeled with 18F (18F-FDG, 18F-NaF, and 18F-Choline), three volumes of intravitreal injections (7, 4, and 2 µL), and absence or presence of eye inflammation (uveitis). Results: Our results showed that there are significant pharmacokinetic differences among the radiolabeled molecules studied but not among the injected volumes. The presence or absence of uveitis was an important factor in vitreous clearance, since the elimination of the drug was clearly increased when this condition is present. Conclusions: Intravitreal pharmacokinetic studies based on the use of dedicated PET imaging can be of potential interest as noninvasive tools in ophthalmic drug development in small animals.

Positron Emission Tomography for the Development and Characterization of Corneal Permanence of Ophthalmic Pharmaceutical Formulations.

Purpose: This work is aimed at describing the utility of positron emission tomography/computed tomography (PET/CT) as a noninvasive tool for pharmacokinetic studies of biopermanence of topical ocular formulations. Methods: The corneal biopermanence of a topical ophthalmic formulation containing gellan gum and kappa carragenan (0.82% wt/vol) labeled with 18Fluorine (18F) radiotracers (18F-FDG and 18F-NaF) was evaluated by using a dedicated small-animal PET/CT, and compared with the biopermanence of an aqueous solution labeled with the same compounds. Regions of interest (ROIs) were manually drawn on the reconstructed PET images for quantifying the radioactivity concentration in the eye. The biopermanence of the formulations was determined by measuring the radioactivity concentration at different times after topical application. Additionally, cellular and ex vivo safety assays were performed to assess the safety of the performed procedures. Results: Differences were observed in the ocular pharmacokinetics of the two formulations. After 1.5 hours of contact, 90% of the hydrogel remained in the ocular surface, while only 69% of the control solution remained. Furthermore, it was observed that flickering had a very important role in the approach of the trial. The application of 18F-FDG in the eye was neither irritating nor cytotoxic for human corneal epithelial cells. Conclusions: The use of small-animal PET and 18F radiotracers in ocular pharmacokinetics of ophthalmic formulations is feasible and could be a safe method for future ocular pharmacokinetic studies in humans.

New therapeutic targets in cancer: the epigenetic connection.

Cancer is an epigenetic disease, a combination of DNA modifications, chromatin organization and variations in its associated proteins, configure a new entity that regulates gene function throughout methylation, acetylation and chromatin remodelling. Irregular de novo DNA methylation, mainly promoter hypermethylation, histone deacetylation or methylation are important means for the transcriptional repression of cancer-associated genes. Reverse these epigenetic processes restoring normal expression of malignancy- preventing-genes has consequently become a new therapeutic target in cancer treatment. Aberrant patterns of epigenetic modifications will be, in a near future, crucial parameters in cancer diagnosis, prognosis and therapy.