Neoadjuvant and adjuvant therapy in esophageal cancer.

Esophageal cancer is an aggressive malignancy that carries a high mortality rate. The treatment of locally advanced resectable esophageal cancer requires a multimodal approach involving chemotherapy, radiation therapy, and surgical resection. Optimal treatment combinations and sequences for squamous cell carcinoma (SCC) versus adenocarcinoma (AC) histological subtypes are still being determined. For very early stage esophageal cancers, endoscopic therapies or surgical resection without chemotherapy and radiation are preferred. Neoadjuvant chemoradiation followed by surgical resection has been the standard in locally advanced resectable esophageal cancer based on the landmark CROSS trial. Definitive chemoradiation is recommended for patients who are not surgical candidates or decline surgery. Perioperative chemotherapy without radiation can be considered for lower esophageal AC and gastroesophageal (GE)-junction AC based on landmark MAGIC and FLOT4 trials. Additional trials are underway to compare preoperative chemoradiation to perioperative chemotherapy in esophageal and GE-junction ACs. Thus far, targeted therapies against vascular endothelial growth factor (VEGF) and human epidermal growth factor receptor 2 (HER2) have not been successful in the neoadjuvant/adjuvant setting. The roll of immunotherapy in perioperative/adjuvant setting is promising. Based on the CheckMate 577 trial, adjuvant nivolumab should be considered for all patients following neoadjuvant chemoradiation and R0 resection with residual pathologic disease. Additional trials involving various immunotherapy agents are underway.

Clinical Outcomes for Patients With Metastatic Breast Cancer Treated With Immunotherapy Agents in Phase I Clinical Trials.

BACKGROUND: Immuno-oncology (IO) agents have demonstrated efficacy across many tumor types and have led to change in standard of care. In breast cancer, atezolizumab and pembrolizumab were recently FDA-approved in combination with chemotherapy specifically for patients with PD-L1-positive metastatic triple-negative breast cancer (TNBC). However, the single agent PD-1/PD-L1 inhibitors demonstrate only modest single agent efficacy in breast cancer. The purpose of this study was to investigate the efficacy of novel IO agents in patients with metastatic breast cancer (MBC), beyond TNBC, treated in phase I clinical trials at the University of Colorado. METHODS: We performed a retrospective analysis using a database of patients with MBC who received treatment with IO agents in phase I/Ib clinical trials at the University of Colorado Hospital from January 1, 2012 to July 1, 2018. Patient demographics, treatments and clinical outcomes were obtained. RESULTS: We identified 43 patients treated with an IO agent either as a single agent or in combination. The average age was 53 years; 55.8% had hormone receptor-positive/HER2-negative breast cancer, 39.5% TNBC and 4.7% HER2-positive. Patients received an average of 2 prior lines of chemotherapy (range 0-7) in the metastatic setting. Most patients (72.1%) received IO alone and 27.9% received IO plus chemotherapy. Median progression-free survival (PFS) was 2.3 months and median overall survival (OS) was 12.1 months. Patients remaining on study ≥ 6 months (20.9%) were more likely to be treated with chemotherapy plus IO compared to patients with a PFS < 6 months (77.8% v. 14.7%). No differences in number of metastatic sites, prior lines of chemotherapy, breast cancer subtype, absolute lymphocyte count, or LDH were identified between patients with a PFS ≥ 6 months vs. < 6 months. CONCLUSIONS: Our phase I experience demonstrates benefit from IO therapy that was not limited to patients with TNBC and confirms improved efficacy from IO agents in combination with chemotherapy. A subset of patients with MBC treated in phase I clinical trials with an IO agent derived prolonged clinical benefit. Predictors of response to immunotherapy in breast cancer remain uncharacterized and further research is needed to identify these factors.

Clinical outcomes of breast cancer patients treated in phase I clinical trials at University of Colorado Cancer Center.

Patients with metastatic breast cancer (MBC) refractory to standard of care therapies have a poor prognosis. The purpose of this study was to assess patient characteristics and clinical outcomes for patients with MBC treated on phase I clinical trials. We performed a retrospective review of all patients with MBC who were enrolled in phase I clinical trials at the University of Colorado Cancer Center from January 2012 to June 2018. A total of 208 patients were identified. Patients had a mean age of 57 years and received on average 2.1 (range 0-10) prior lines of chemotherapy. The majority of patients had hormone receptor-positive/HER2-negative breast cancer (58.6%) and 30.3% had triple-negative breast cancer. The median progression free survival (PFS) was 2.8 months (95% CI, 2.3-3.9) and median overall survival (OS) was 11.5 months (95% CI, 9.6-13.2). Independent factors associated with longer PFS in multivariable analysis were treatment in a breast cancer-selective trial or cohort (p = 0.016), age >50 years (p = 0.002), and ≤2 prior lines of chemotherapy in the metastatic setting (p = 0.025). Phase I clinical trials remain a valuable option for select patients with MBC and enrollment should be encouraged when available.

Indirect chiral separation of 8 novel amphetamine derivatives as potential new psychoactive compounds by GC-MS and HPLC.

Amphetamine and its derivatives gained high popularity on the illegal drug market. In the last few years, a lot of new psychoactive compounds structurally related to amphetamine, such as 4-fluoroamphetamine and 4-fluoromethamphetamine swamped the drug market. They were designed to circumvent prohibition of amphetamine and N-methylamphetamine and are distributed via the Internet. Often, a halogen atom is introduced into the phenyl ring of amphetamine to turn the illegal amphetamine legal. Since amphetamines possess a chiral centre, two enantiomers are available, which might differ in activity. Since most of them are partially not commercially available to date, synthesis and characterisation of amphetamine derivatives might help authorities to identify these substances of abuse. The aim of this study was to investigate self-synthesized amphetamines concerning their identity and their enantiomeric status either by GC-MS or by HPLC. For GC-MS, derivatization with (R)-(+)-α-methoxy-α-trifluoromethylphenylacetic acid (MTPA) or (1R)-(-)-menthylchloroformate prior to analysis on a HP-5MS column was done. For chiral separation by HPLC a LiChrospher 100 RP-18e column and sulfated beta-cyclodextrin added to the mobile phase as chiral selector were used. Enantioseparation was accomplished successfully by both methods. Furthermore, simultaneous chiral separation of three positions isomers, namely 2-fluoroamphetamine, 3-fluoroamphetamine and 4-fluoroamphetamine, was shown successfully by HPLC.

Indirect chiral separation of new recreational drugs by gas chromatography-mass spectrometry using trifluoroacetyl-L-prolyl chloride as chiral derivatization reagent.

New recreational drugs such as amphetamine-, cathinone, and benzofury derivatives gained high popularity on the drug market in recent years. They can be purchased via the Internet from different providers and online portals. Most of these compounds are chiral, which makes the development of chiral separation methods necessary. Besides this, it is useful to find out if the compounds were sold as racemic mixtures. Also, it is important to check whether the new psychoactive compounds contain further ingredients or impurities. The aim of this research was the continuation of the application of a method for indirect chiral separation of 24 new psychoactive compounds recently purchased via the Internet. After derivatization with the chiral derivatization reagent trifluoroacetyl-L-prolyl chloride, chromatographic separation of diastereomers was achieved using a 30 m HP5-MS capillary column. As carrier gas, helium was used with a constant flow of 1.0 ml/min. Three different column temperature programs were tested. Under optimum conditions 13 out of 24 compounds were successfully resolved into their enantiomers obtaining Rs values up to 7.0. The use of a single quadrupole mass spectrometer as the detector allowed the identification of the compounds in multicomponent samples.

Analysis of a new drug of abuse: cathinone derivative 1-(3,4-dimethoxyphenyl)-2-(ethylamino)pentan-1-one.

Recently, novel psychoactive drugs for human abuse such as amphetamines, phenethylamines, benzofuries, and tryptamines, cathinones have gained high popularity. These designer drugs are mainly sold via online stores as "bath salts" and are labeled "not for human consumption." Due to the novelty of the compounds, only a little information about pharmacology, toxicology, and the long-term damage they may cause is available. Moreover, there are only few analytical methods for their identification and analysis. Among new cathinone derivatives, 1-(3,4-dimethoxyphenyl)-2-(ethylamino)pentan-1-one (DL-4662), became available via an internet shop. A sample of this compound was purchased and investigated. The first aim of our study was an identity check by NMR spectroscopy and gas chromatography with mass spectrometry. As many of the recreational drugs are chiral and are mainly sold as racemates, a further goal of our research was enantioseparation by gas chromatography with mass spectrometry and high-performance liquid chromatography with UV detection, to prove whether DL-4662 was traded enantiomerically pure or as racemic mixture. Both chiral separation methods showed the presence of a racemate.

Analysis and characterization of the novel psychoactive drug 4-chloromethcathinone (clephedrone).

Novel psychoactive drugs, such as amphetamine-, cathinone-, benzofury- and tryptamine derivatives, gained high popularity on the global drug market in the last years. These drugs are sold via the Internet as for example "research chemicals", "room odorizers" or "lawn fertilizers" by different online suppliers. They are also known as "Legal Highs", among them, cathinone derivatives play an important role. Well known substituted cathinone derivatives are mephedrone, brephedrone and flephedrone. Since a couple of weeks, a chlorine substituted methcathinone derivative, namely clephedrone (4-chloromethcathinone), is commercially available via the Internet from www.deboralabs.com. The goal of this study was to confirm identity of this substance, which was done successfully by GC-MS and NMR. Since all cathinone derivatives are chiral, it was found out, whether the purchased sample was present as a racemic mixture. For this purpose, methods for enantioseparation by GC and CE were developed and applied successfully. In case of CE a chiral selector was added, whereas chiral separation with GC-MS was done indirectly, after derivatization of clephedrone with trifluoroacetyl-l-prolyl chloride.

PIWI homologs mediate histone H4 mRNA localization to planarian chromatoid bodies.

The well-known regenerative abilities of planarian flatworms are attributed to a population of adult stem cells called neoblasts that proliferate and differentiate to produce all cell types. A characteristic feature of neoblasts is the presence of large cytoplasmic ribonucleoprotein granules named chromatoid bodies, the function of which has remained largely elusive. This study shows that histone mRNAs are a common component of chromatoid bodies. Our experiments also demonstrate that accumulation of histone mRNAs, which is typically restricted to the S phase of eukaryotic cells, is extended during the cell cycle of neoblasts. The planarian PIWI homologs SMEDWI-1 and SMEDWI-3 are required for proper localization of germinal histone H4 (gH4) mRNA to chromatoid bodies. The association between histone mRNA and chromatoid body components extends beyond gH4 mRNA, since transcripts of other core histone genes were also found in these structures. Additionally, piRNAs corresponding to loci of every core histone type have been identified. Altogether, this work provides evidence that links PIWI proteins and chromatoid bodies to histone mRNA regulation in planarian stem cells. The molecular similarities between neoblasts and undifferentiated cells of other organisms raise the possibility that PIWI proteins might also regulate histone mRNAs in stem cells and germ cells of other metazoans.

RNA interference by feeding in vitro-synthesized double-stranded RNA to planarians: methodology and dynamics.

BACKGROUND: The ability to assess gene function is essential for understanding biological processes. Currently, RNA interference (RNAi) is the only technique available to assess gene function in planarians, in which it has been induced by means of injection of double-stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA. RESULTS: We describe a simple and robust RNAi protocol, involving in vitro synthesis of dsRNA that is fed to the planarians. Advantages of this protocol include the ability to produce dsRNA from any vector without subcloning, resolution of ambiguities in quantity and quality of input dsRNA, as well as time and ease of application. We have evaluated the logistics of inducing RNAi in planarians using this methodology in careful detail, from the ingestion and processing of dsRNA in the intestine, to timing and efficacy of knockdown in neoblasts, germline, and soma. We also present systematic comparisons of effects of amount, frequency, and mode of dsRNA delivery. CONCLUSIONS: This method gives robust and reproducible results and is amenable to high-throughput studies. Overall, this RNAi methodology provides a significant advance by combining the strengths of current protocols available for dsRNA delivery in planarians and has the potential to benefit RNAi methods in other systems.

Chiral separation of new cathinone- and amphetamine-related designer drugs by gas chromatography-mass spectrometry using trifluoroacetyl-l-prolyl chloride as chiral derivatization reagent.

Since cathinone derivatives gained high popularity on the recreational drugs market within the past 5 years the development of analytical methods for the achiral and chiral determination of this substance class is of great interest. Not at least because it is obvious that the pharmacological potency differs between both enantiomers. Cathinones are structurally closely related to amphetamines, which have similar stimulating effects and are somehow better investigated. The goal of this research was to perform indirect enantioseparation of novel psychoactive cathinone and amphetamine derivatives. Trifluoroacetyl-l-prolyl chloride was served as chiral derivatization agent (CDA). Chromatographic separation was performed using a commercially available HP5-MS capillary column with a length of 30 m. Helium was used as carrier gas with a constant flow of 1.0 ml/min. Under optimum conditions 14 amphetamine derivatives were successfully resolved into their enantiomers and detected with the single quadrupol detector. Racemic methcathinone derivatives analyzed with the same method showed different peak areas for each of the produced diastereomeric isomers, even if they are structurally closely related to the amphetamines. Derivatization experiments with the single isomers of methcathinone led to both diastereomers whereas the S(-) enantiomer seemed to racemize more likely. Based on comparative experiments with R-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA) as CDA, racemization due to the keto-enol-tautomerism of the cathinone derivatives seemed to be responsible for this phenomenon. Nevertheless, 18 cathinone derivatives were successfully enantioseparated and an approach of quantitative evaluation is demonstrated.