Enrichment of high-grade tumors in breast cancer gene expression studies.

PURPOSE: Gene expression (GE) profiling for breast cancer classification and prognostication has become increasingly used in clinical diagnostics. GE profiling requires a reasonable tumor cell percentage and high-quality RNA. As a consequence, a certain amount of samples drop out. If tumor characteristics are different between samples included and excluded from GE profiling, this can lead to bias. Therefore, we assessed whether patient and tumor characteristics differ between tumors suitable or unsuitable for generating GE profiles in breast cancer. METHODS: In a consecutive cohort of 738 breast cancer patients who received neoadjuvant chemotherapy at the Netherlands Cancer Institute, GE profiling was performed. We compared tumor characteristics and treatment outcome between patients included and excluded from GE profiling. Results were validated in an independent cohort of 812 patients treated with primary surgery. RESULTS: GE analysis could be performed in 53% of the samples. Patients with tumor GE profiles more often had high-grade tumors [odds ratio 2.57 (95%CI 1.77-3.72), p < 0.001] and were more often lymph node positive [odds ratio 1.50 (95%CI 1.03-2.19), p = 0.035] compared to the group for which GE profiling was not possible. In the validation cohort, tumors suitable for gene expression analysis were more often high grade. CONCLUSIONS: In our gene expression studies, tumors suitable for GE profiling had more often an unfavorable prognostic profile. Due to selection of samples with a high tumor percentage, we automatically select for tumors with specific features, i.e., tumors with a higher grade and lymph node involvement. It is important to be aware of this phenomenon when performing gene expression analysis in a research or clinical context.

Intraoperative verification of resection margins of maxillary malignancies by cone-beam computed tomography.

Resection of maxillary cancer often results in incomplete excision because of the tumour's proximity to important structures such as the orbit. To deal with this problem we prospectively investigated the feasibility of intraoperative imaging during maxillectomy to verify the planned resection margins. In total, six patients diagnosed with maxillary cancer listed for maxillectomy were included, irrespective of the histological type of tumour. Before resection, an accurate intended resection volume was delineated on diagnostic images. At the end of the operation we took a cone-beam computed tomographic (CT) scan of the treated maxilla, after which the accuracy of the resection was quantitatively evaluated by comparing the preoperative resection plan and the images acquired intraoperatively, based on the anatomy. Further resection was then done if necessary and quantitatively evaluated with a second cone-beam CT scan. Postoperatively we compared the results of the scan with those of the histological examination. Of the six, two resections were reported pathologically as less than radical, each of which was detected by intraoperative CT and resulted in extensions of the original resections. The mean (SD) distance between the planned and the actual resection was 1.49 (2.78)mm. This suggests that intraoperative cone-beam CT imaging is a promising way to make an adequate intraoperative assessment of planned surgical margins of maxillary tumours. This allows for intraoperative resection margins to be improved, possibly leading to a better prognosis for the patient.

The hidden sentinel node in breast cancer: Reevaluating the role of SPECT/CT and tracer reinjection.

INTRODUCTION: Lymphoscintigraphy with planar imaging is considered a helpful tool to depict lymph node drainage in patients with invasive breast cancer. Single Photon Emission Computed Tomography with integrated CT (SPECT/CT) is usually performed to detect sentinel nodes (SN)s in breast cancer patients showing non-visualisation on lymphoscintigraphy. Incorporation of new SN indications (recurrent surgery, previous radiotherapy, or neo-adjuvant chemotherapy) has led to an increase of non-visualisation rates. The present study evaluates the contribution of SPECT/CT and tracer reinjection for SN-visualisation in breast cancer patients without drainage on lymphoscintigraphy. METHODS: Between 1st of July 2008 and 6th of November 2014 in total 1968 patients underwent a SN breast procedure, using intra-tumoural tracer administration. SPECT/CT was performed in 284 breast cancer patients with non-visualisation of SNs on lymphoscintigraphy. If SN non-visualisation persisted, a second radiotracer injection with repeated imaging was performed when logistics allowed this. Univariate analysis was applied to evaluate SPECT/CT visualisation rates in specific subgroups. RESULTS: The SPECT/CT visualisation rate was 23.2% (66/284). Univariate analysis revealed no significant subgroups influencing SPECT/CT visualisation. In patients receiving reinjection after persistent SPECT/CT non-visualisation the SN-visualisation rate reached 62.1% (36/58). Intraoperatively, the SN-identification rate using a gamma probe and blue dye was 87.9% (175/199) and 32.9% (28/85) for, respectively, primary and recurrent surgery after non-visualisation on lymphoscintigraphy. CONCLUSION: In this evaluation including new breast cancer SN indications, SPECT/CT scored lower than reinjection to visualise SNs in patients with non-visualisation on lymphoscintigraphy. Consequently, our institutional protocol has been readjusted.

Fusion of Freehand SPECT and Ultrasound to Perform Ultrasound-Guided Fine-Needle Aspiration Cytology of Sentinel Nodes in Head and Neck Cancer.

BACKGROUND AND PURPOSE: Criteria for ultrasound-guided fine-needle aspiration cytology (USgFNAC) for the detection of occult lymph node metastasis in patients with clinically negative head and neck cancer are based on the morphology of cervical lymph nodes. To improve the selection of lymph nodes for USgFNAC, we examined the feasibility of fused freehand single-photon emission tomography ultrasound-guided fine-needle cytology (freehand SPECT-USgFNAC) of sentinel nodes in patients with early stage oral and head and neck skin cancer. MATERIALS AND METHODS: Six patients with early-stage head and neck cancer (4 oral and 2 head and neck skin cancers) and a clinically negative neck who were scheduled for transoral or local excision and a sentinel node procedure underwent USgFNAC and freehand SPECT-USgFNAC preoperatively. RESULTS: All freehand SPECT sonographic examinations were technically successful in terms of identifying sentinel nodes. All aspirates of sentinel nodes obtained by freehand SPECT-USgFNAC contained substantial radioactivity, confirming puncture of the sentinel nodes. USgFNAC evaluated 13 lymph nodes; freehand SPECT-USgFNAC, 19 nodes; and sentinel node biopsy, 13 nodes. Three sentinel nodes were histopathologically positive and were selected for aspiration cytology by freehand SPECT-USgFNAC, but not by conventional ultrasound. The cytologic examination findings of the aspirations were negative or inconclusive. CONCLUSIONS: Freehand SPECT ultrasound can identify sentinel nodes and could potentially improve USgFNAC in patients with head and neck cancer by better selection of lymph nodes at highest risk of having metastases (sentinel nodes), but its sensitivity is limited by sampling error and insufficient aspirated material for cytology.

Simultaneous use of an (125)I-seed to guide tumour excision and (99m)Tc-nanocolloid for sentinel node biopsy in non-palpable breast-conserving surgery.

PURPOSE: In the present study we describe patients with non-palpable breast lesions, in which an Iodine-125 ((125)I)-marker (or "seed") for excision of the primary tumour and Technetium-99m nanocolloid ((99m)Tc-nanocolloid) for sentinel node biopsy (SNB) are used simultaneously. The purpose was to investigate any interference between (125)I-seeds and (99m)Tc-nanocolloid by an in vitro and in vivo analysis. METHODS: Contrast/interference-ratios between (125)I and (99m)Tc count-rates were determined in vitro using a realistic simulation model. Measurements were performed with 3 gamma-probes with different crystal materials. In 25 consecutive patients (99m)Tc-nanocolloid was intratumourally administered at the site of a previously implanted (125)I-seed. Respectively, the (125)I-setting and (99m)Tc-setting of the gamma-probe guided the wide local excision and SNB and maximum counts-per-second (cps) were measured. RESULTS: In vitro the different probes varied in (125)I- and (99m)Tc-sensitivity. The contrast-ratio between (125)I and (99m)Tc in the (125)I-channel was 4.6 for a 3-month-old (125)I-seed using the most appropriate gamma-probe. In vivo the gamma-probe in the (125)I-setting measured a median of 16,300 cps at the tumour site compared to 4820 cps using the (99m)Tc-setting. The (125)I-seed could be well distinguished from the (99m)Tc-nanocolloid in 92% of the patients and 96% required a single operation. The SNB was successful in all patients. CONCLUSIONS: Simultaneous use of (125)I-seeds and (99m)Tc-nanocolloid is possible under well-standardised conditions. Non-palpable breast lesions can be safely excised using the (125)I-seed in combination with a SN procedure. Use of (125)I-seeds is a next step within fine-tuning breast-conserving surgery that should lead to further investigation to confirm its value.

Prevention of cocaine-induced convulsions and lethality in mice: effectiveness of targeting different sites on the NMDA receptor complex.

N-methyl-D-aspartate (NMDA) receptors appear to be involved in the behavioral toxic effects of cocaine. Therefore, different classes of NMDA receptor antagonists were compared for their ability to attenuate cocaine-induced convulsions and lethality in male, Swiss Webster mice. The mice were pre-treated (i.p.) with vehicle or an antagonist from one of the following classes: NMDA/glycine site antagonist (7-chlorokynurenic acid, ACEA-1021, ACEA-1031, ACEA-1328, DCQX, R(+)-HA-966), competitive antagonist (CPP, D-AP7), channel blocker (MK-801, memantine), or allosteric modulator (ifenprodil, CP-101,606, Co 101022, haloperidol). After a 15 min pre-treatment period, the mice were administered a convulsive (60 mg/kg, i.p.) or lethal (125 mg/kg, i.p.) dose of cocaine, equivalent to the calculated ED/LD97 values. Pre-treatment with competitive or NMDA/glycine site antagonists dose-dependently attenuated cocaine-induced convulsions and lethality (P<0.05). Pre-treatment with channel blockers or allosteric modulators of the NMDA receptor protected against cocaine-induced convulsions (P<0.05), but were ineffective or less effective than the competitive and glycine site antagonists in preventing death. The glutamate release inhibitor riluzole failed to prevent both the convulsions and lethality induced by cocaine. Significantly, post-treatment with NMDA/glycine site antagonists (ACEA-1021, ACEA-1031, ACEA-1328) after a cocaine overdose prevented death in a significant number of animals. The data suggest that NMDA receptors are involved in the pathophysiology of a cocaine overdose.

Rimcazole analogs attenuate the convulsive effects of cocaine: correlation with binding to sigma receptors rather than dopamine transporters.

Cocaine interacts with dopamine transporters and sigma receptors at concentrations that are achievable in vivo, suggesting that they may both be viable targets for the development of anti-cocaine agents. Rimcazole binds to both of these targets and also attenuates cocaine-induced locomotor activity and sensitization. To further characterize the mechanism(s) underlying the attenuation of cocaine-induced convulsions and lethality, rimcazole and three analogs (SH3/24, SH2/21, SH1/57), with a range of affinities for dopamine transporters and sigma receptors, were evaluated. The highly selective and potent sigma receptor ligand LR176 was used as a reference. Competition binding studies confirmed that the rank order of the compounds at dopamine transporters vs. sigma receptors differed, thus enabling a correlation between the relative anti-cocaine activities of the compounds in behavioral studies and their affinities for dopamine transporters vs. sigma receptors. In behavioral studies, male Swiss Webster mice were pre-treated with one of the compounds (0-60 mg/kg, i.p.), then challenged 15 min later with either a convulsive (60 mg/kg, i.p.) or lethal (125 mg/kg, i.p.) dose of cocaine. When the compounds were ranked according to their protective effect, there was a significant correlation between their anticonvulsant actions and their affinities for sigma receptors, but not dopamine transporters. Although the rimcazole analogs were ineffective against the lethal effects of cocaine, the selective sigma receptor ligand LR176 provided significant protection. These data thus suggest that sigma receptors may mediate some of the toxic effects associated with cocaine and that sigma receptor antagonists may be developed as pharmacotherapeutic agents for this application.

Synthesis and ligand binding studies of 4'-iodobenzoyl esters of tropanes and piperidines at the dopamine transporter.

Four analogs and two homologs of cocaine, designed as potent cocaine antagonists, were synthesized. The SN2 reaction between ecgonine methyl ester (13) or appropriately substituted piperidinol (19, 21) and appropriately substituted 4-iodobenzoyl chloride gave 4-iodobenzoyl esters of tropanes and piperidines (5-8). 2'-Hydroxycocaine (9) was obtained from 2'-acetoxycocaine (12) by selective transesterification with MeOH saturated with dry HCl gas. 2'-Acetoxycocaine (12) was synthesized from acetylsalicyloyl chloride (23) and ecgonine methyl ester (13). The binding affinities of these compounds were determined at the dopamine transporter for the displacement of [3H]WIN-35428. An iodo group substitution at the 4'-position of cocaine decreased dopamine transporter binding potency, while a hydroxy or acetoxy group at the 2'-position exhibited increased binding potency for the dopamine transporter compared to cocaine (10- and 3.58-fold, respectively). 2'-Hydroxylation also enhanced the bidning potency of 4'-iodococaine (5) by 10-fold. Replacement of the tropane ring with piperidine led to poor binding affinities.

2beta-Substituted analogues of 4'-iodococaine: synthesis and dopamine transporter binding potencies.

A series of 2beta-substituted analogues of 4'-iodococaine (3) was synthesized and evaluated in an in vitro dopamine transporter (DAT) binding assay. Selective hydrolysis at the 2beta-position of 3 gave the carboxylic acid 15 that served as the intermediate for the synthesis of compounds 4, 5, and 6-11. The 2beta-alkyl derivatives were obtained from ecgonine methyl ester (17) through a series of reactions leading to the aldehyde 20. Wittig reaction of 20 with methyltriphenylphosphorane followed by hydrogenation and benzoylation gave the products 12 and 13. The binding affinity of 4'-iodococaine (3) was 10-fold less than that of cocaine. The hydroxymethane, acetate, amide, benzyl ester, oxidazole, and ethane derivatives of 3 exhibited decreased binding while the vinyl, phenyl, and ethyl esters showed a moderate increase in binding affinity. Only the isopropyl derivative 8 exhibited a 2-fold increase in binding affinity compared with 4'-iodococaine (3). Hydroxylation of 8 at the 2'-position gave 14 which enhanced not only the binding potency at the DAT by another 2-fold but also the selectivity at the DAT over the norepinephrine and serotonin transporters. Compound 14 failed to stimulate locomotor activity in C57BL/6J mice over a wide dose range and blocked cocaine-induced locomotor stimulant action.

Correlation between neuroleptic binding to sigma(1) and sigma(2) receptors and acute dystonic reactions.

Acute dystonic reactions are motor side effects that occur soon after the initiation of neuroleptic treatment. Although earlier studies indicate that these abnormal movements can be induced in animals and humans via activation of sigma receptors, the relative contribution of the different sigma receptor subtypes is unknown. Since sigma(1) and sigma(2) receptor are differentially represented in motor regions of the brain, the affinities of 17 neuroleptics for these sigma receptor subtypes were determined using competition binding studies. The results revealed that most neuroleptics do not exhibit selectivity for either of the sigma receptor subtypes, as reflected by a significant correlation between the affinities of the neuroleptics for sigma(1) vs. sigma(2) receptors. Moreover, when the sigma binding affinities of the neuroleptics were correlated with the tendency of the drugs to produce acute dystonic reactions in humans, there was a significant correlation for both subtypes. Together with earlier studies in animals, the data suggest that neuroleptic-induced motor side effects can be mediated through both sigma(1) and sigma(2) receptors.

Effects of AMPA/kainate glutamate receptor antagonists on cocaine-induced convulsions and lethality in mice.

Prior studies demonstrate that NMDA receptor antagonists attenuate cocaine-induced convulsions and lethality. Since glutamate is the primary neurotransmitter for NMDA receptors, pharmacological interventions to lower glutamatergic activity through non-NMDA ionotropic receptor-mediated mechanisms were evaluated for their ability to prevent the convulsive and lethal effects of cocaine. Pre-treatment of male, Swiss Webster mice with the alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA)/kainate receptor antagonists 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX; 10-80 mg/kg, i.p.) or 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2, 3-benzodiazepine hydrochloride (GYKI 52466; 10-20 mg/kg, i.p.) failed to significantly attenuate cocaine-induced convulsions or lethality. Although ineffective when administered alone, NBQX enhanced the protective effects of 5-nitro-6,7-dichloro-1, 4-dihydro-2,3-quinoxalinedione (ACEA-1021), an NMDA/glycine site antagonist, when administered in combination. The mixed NMDA/non-NMDA receptor competitive antagonist 5-chloro-7-trifluoromethyl-1,2,3,4-tetrahydroquinoxaline-2,3-dione (ACEA-1011) also protected against the convulsive effects of cocaine. The data suggest that AMPA/kainate receptors indirectly influence the pathophysiological changes that occur after a cocaine overdose through modulation of NMDA receptors.

N-alkyl substituted analogs of the sigma receptor ligand BD1008 and traditional sigma receptor ligands affect cocaine-induced convulsions and lethality in mice.

Cocaine binds to sigma receptors with comparable affinity to its well-established interaction with dopamine transporters. Previous studies have shown BD1008 (N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine) to have high affinity and selectivity for sigma receptors, and to additionally attenuate the locomotor stimulatory effects of cocaine. Therefore, in the present study, three N-alkyl substituted analogs of BD1008 were characterized in receptor binding and behavioral studies: BD1060 (N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl)ethylamine), BD1067 (N-[2-(3,4-dichlorophenyl)ethyl]-N-ethyl-2-(1-pyrrolidinyl)ethylamine), and BD1052 (N-[2-(3,4-dichlorophenyl)ethyl]-N-allyl-2-(1-pyrrolidinyl)ethylamine). Similarly to BD1008, all three analogs exhibited high affinity and selectivity for sigma receptors. In behavioral studies, BD1008, BD1060 or BD1067 attenuated cocaine-induced convulsions and lethality in Swiss Webster mice. The protective effects appear to be mediated through sigma receptor antagonism because traditional sigma receptor antagonists with high to moderate affinity for these receptors also attenuated the behavioral toxicity of cocaine. In contrast, traditional and novel sigma receptor agonists such as di-o-tolylguanidine and BD1052 worsened the behavioral toxicity of cocaine. To further characterize the actions of the N-alkyl substituted compounds, they were microinjected into the rat red nucleus, a functional assay of sigma receptor activity, where they produced agonist vs. antagonist actions that were consistent with their effects on cocaine-induced behaviors. Together, the data demonstrate that BD1008, BD1060 or BD1067 can attenuate the behavioral toxicity of cocaine, most likely through functional antagonism of sigma receptors.

Conformationally restricted analogs of BD1008 and an antisense oligodeoxynucleotide targeting sigma1 receptors produce anti-cocaine effects in mice.

Cocaine's ability to interact with sigma receptors suggests that these proteins mediate some of its behavioral effects. Therefore, three novel sigma receptor ligands with antagonist activity were evaluated in Swiss Webster mice: BD1018 (3S-1-[2-(3,4-dichlorophenyl)ethyl]-1,4-diazabicyclo[4.3.0]nonane), BD1063 (1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine), and LR132 (1R,2S-(+)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl)cyclohexylamine). Competition binding assays demonstrated that all three compounds have high affinities for sigma1 receptors. The three compounds vary in their affinities for sigma2 receptors and exhibit negligible affinities for dopamine, opioid, GABA(A) and NMDA receptors. In behavioral studies, pre-treatment of mice with BD1018, BD1063, or LR132 significantly attenuated cocaine-induced convulsions and lethality. Moreover, post-treatment with LR132 prevented cocaine-induced lethality in a significant proportion of animals. In contrast to the protection provided by the putative antagonists, the well-characterized sigma receptor agonist di-o-tolylguanidine (DTG) and the novel sigma receptor agonist BD1031 (3R-1-[2-(3,4-dichlorophenyl)ethyl]-1,4-diazabicyclo[4.3.0]nonane) each worsened the behavioral toxicity of cocaine. At doses where alone, they produced no significant effects on locomotion, BD1018, BD1063 and LR132 significantly attenuated the locomotor stimulatory effects of cocaine. To further validate the hypothesis that the anti-cocaine effects of the novel ligands involved antagonism of sigma receptors, an antisense oligodeoxynucleotide against sigma1 receptors was also shown to significantly attenuate the convulsive and locomotor stimulatory effects of cocaine. Together, the data suggests that functional antagonism of sigma receptors is capable of attenuating a number of cocaine-induced behaviors.

Design and synthesis of isoxazole containing bioisosteres of epibatidine as potent nicotinic acetylcholine receptor agonists.

An efficient synthesis of isoxazole containing isosteres of epibatidine is described. The synthesis proceeded from N-tert-butoxycarbonyl (Boc)-exo-2-(methoxycarbonyl)-7-azabicyclo[2.2.1]heptane (9). Compound 9 was reacted with the dilithium salt of an appropriately substituted oxime in tetrahydrofuran (THF). Cyclodehydration of the resultant beta-keto oxime and deprotection of the N-Boc group in 5 N aqueous HCl afforded the isoxazole containing isosteres of epibatidine (6-8). The binding affinities of these compounds were determined at the nicotinic acetylcholine receptor for the displacement of [3H]cystisine. The unsubstituted isoxazole containing isostere (6) showed the lower binding potency compared to the 3'-methylisoxazole isostere (7). Substitution with a phenyl group at the 3'-position of the isoxazole significantly reduced the binding potency. The in vivo toxicological studies of these analogs were also performed. The LD50 of the analogs ranged in the order: Me > H > Ph.