Heterogeneous drug penetrance of veliparib and carboplatin measured in triple negative breast tumors.

BACKGROUND: Poly(ADP-ribose) polymerase inhibitors (PARPi), coupled to a DNA damaging agent is a promising approach to treating triple negative breast cancer (TNBC). However, not all patients respond; we hypothesize that non-response in some patients may be due to insufficient drug penetration. As a first step to testing this hypothesis, we quantified and visualized veliparib and carboplatin penetration in mouse xenograft TNBCs and patient blood samples. METHODS: MDA-MB-231, HCC70 or MDA-MB-436 human TNBC cells were implanted in 41 beige SCID mice. Low dose (20 mg/kg) or high dose (60 mg/kg) veliparib was given three times daily for three days, with carboplatin (60 mg/kg) administered twice. In addition, blood samples were analyzed from 19 patients from a phase 1 study of carboplatin + PARPi talazoparib. Veliparib and carboplatin was quantified using liquid chromatography-mass spectrometry (LC-MS). Veliparib tissue penetration was visualized using matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) and platinum adducts (covalent nuclear DNA-binding) were quantified using inductively coupled plasma-mass spectrometry (ICP-MS). Pharmacokinetic modeling and Pearson's correlation were used to explore associations between concentrations in plasma, tumor cells and peripheral blood mononuclear cells (PBMCs). RESULTS: Veliparib penetration in xenograft tumors was highly heterogeneous between and within tumors. Only 35% (CI 95% 26-44%), 74% (40-97%) and 46% (9-37%) of veliparib observed in plasma penetrated into MDA-MB-231, HCC70 and MDA-MB-436 cell-based xenografts, respectively. Within tumors, penetration heterogeneity was larger with the 60 mg/kg compared to the 20 mg/kg dose (RSD 155% versus 255%, P = 0.001). These tumor concentrations were predicted similar to clinical dosing levels, but predicted tumor concentrations were below half maximal concentration values as threshold of response. Xenograft veliparib concentrations correlated positively with platinum adduct formation (R 2 = 0.657), but no PARPi-platinum interaction was observed in patients' PBMCs. Platinum adduct formation was significantly higher in five gBRCA carriers (ratio of platinum in DNA in PBMCs/plasma 0.64% (IQR 0.60-1.16%) compared to nine non-carriers (ratio 0.29% (IQR 0.21-0.66%, P < 0.0001). CONCLUSIONS: PARPi/platinum tumor penetration can be measured by MALDI-MSI and ICP-MS in PBMCs and fresh frozen, OCT embedded core needle biopsies. Large variability in platinum adduct formation and spatial heterogeneity in veliparib distribution may lead to insufficient drug exposure in select cell populations.

Estimation of tamoxifen metabolite concentrations in the blood of breast cancer patients through CYP2D6 genotype activity score.

Tamoxifen, a prodrug used for adjuvant breast cancer therapy, requires conversion to the active metabolite endoxifen through CYP 2D6. We aimed to construct an algorithm to predict endoxifen concentrations based on a patient's CYP 2D6 genotype, demographic factors, and co-medication use. Eighty-eight women enrolled in the UCSF TamGen II study and 81 women enrolled in a prospective study at Dana-Farber Cancer Institute were included in this analysis. All the women had been on tamoxifen for at least 3 months before blood collection. Demographic information included the patient's age, race/ethnicity, body mass index (where available), and self-reported and measured medications and herbals that affect 2D6 activity. DNA was extracted and genotyped for 2D6 (Amplichip, Roche Diagnostics). An activity score was calculated based on genotypes and adjusted for use of medications known to inhibit 2D6. Serum was tested for tamoxifen and metabolite concentrations and for the presence of drugs by liquid chromatography/mass spectrometry. Univariate and multivariate regression analysis were computed for age, body mass index, ethnicity, and adjusted activity score to predict tamoxifen metabolite concentrations in the training data-set of UCSF patients, and the resulting algorithm was validated in the Dana-Farber patients. For the training set, the correlation coefficient (r2) for log endoxifen and N-desmethyltamoxifen:endoxifen ratio to activity score, age, and race, were 0.520 and 0.659, respectively; 0.324 and 0.567 for the validation; and 0.396 and 0.615 for both the datasets combined. An algorithm that incorporates genotype and demographic variables can be used to predict endoxifen concentrations for women on tamoxifen therapy. If endoxifen levels are confirmed to be predictive of tamoxifen benefit, then this algorithm may be helpful to determine which women warrant endoxifen testing.

A mixed (long- and medium-chain) triglyceride lipid emulsion extracts local anesthetic from human serum in vitro more effectively than a long-chain emulsion.

BACKGROUND: Lipid emulsion infusion reverses cardiac toxicity of local anesthetics. The predominant effect is likely creation of a "lipid sink." This in vitro study determined the extent to which Intralipid® (Fresenius Kabi, Uppsala, Sweden) and Lipofundin® (B. Braun Melsungen AG, Melsungen, Germany) sequester anesthetics from serum, and whether it varies with pH. METHODS: Bupivacaine, ropivacaine, and mepivacaine were added to human drug-free serum (pH 7.4) at 10 µg/ml. The lipid emulsions were added, and the mixture shaken and incubated at 37°C. Lipid was removed by ultracentrifugation and drug remaining in the serum measured. Additional experiments were performed using 100 µg/ml bupivacaine and at pH 6.9. RESULTS: Lipofundin® extracted all three anesthetics to a greater extent than Intralipid® (34.7% vs..22.3% for bupivacaine, 25.8% vs..16.5% for ropivacaine, and 7.3% vs..4.7% for mepivacaine). By increasing either concentration of bupivacaine or lipid, there was an increase in drug extraction from serum. Adjusting the pH to 6.9 had no statistically significant effect on the percentage of bupivacaine sequestered. CONCLUSIONS: Bupivacaine, ropivacaine, and mepivacaine were sequestered to an extent consistent with their octanol:water partition constants (logP). In contrast with previous studies of extraction of lipids from buffer solutions, an emulsion containing 50% each of medium- and long-chain triglycerides extracted local anesthetics to a greater extent from human serum than one containing exclusively long-chain triglycerides, calling into question recent advanced cardiac life support guidelines for resuscitation from anesthetic toxicity that specify use of a long-chain triglyceride. The current data also do not support recent recommendations to delay administration until pH is normalized.

Alternative splicing of 3-hydroxy-3-methylglutaryl coenzyme A reductase is associated with plasma low-density lipoprotein cholesterol response to simvastatin.

BACKGROUND: HMGCR(3-Hydroxy-3-methylglutaryl coenzyme A reductase), the direct target of statin inhibition, undergoes alternative splicing of exon 13, which encodes part of the statin-binding domain of the enzyme. We hypothesized that HMGCR alternative splicing might be related to the interindividual variation in plasma low-density lipoprotein cholesterol response to statin treatment. METHODS AND RESULTS: We measured mRNA expression of both the full-length and the alternatively spliced HMGCR transcript lacking exon 13 (HMGCRv_1) in 170 simvastatin-incubated immortalized lymphocyte cell lines derived from participants in the Cholesterol and Pharmacogenetics (CAP) study who were treated with simvastatin 40 mg/d for 6 weeks. Greater upregulation of HMGCRv_1 in vitro was significantly correlated (P

Plaque array method and proteomics-based identification of biomarkers from Alzheimer's disease serum.

BACKGROUND: Progressive accumulation of amyloid plaques in the regions of brain, carotid and cerebral arteries is the leading cause of Alzheimer's disease (AD) and related dementia in affected patients. The early identification of individuals with AD remains a challenging task relying on symptomatic events and thus the development of a biomarker-based approach will significantly aid in the diagnosis of AD. METHODS: Here we describe a flow cytometer-based serum biomarker identification method using plaque particles, and applying mass spectrometry based proteomic analysis of the isolated plaque particles for the identification of serum proteins present in the plaque particles. RESULTS: We identified 195 serum proteins that participate in the process of plaque particle formation. Among the 195 proteins identified, 68.2% of them overlapped in abeta-42, cholesterol, tau-275 and α-synuclein plaque particles. Significantly, 22.5% of the proteins identified as bound to abeta-42 plaque particles generated in AD serum were unique when compared with cholesterol, α-synuclein and tau plaque particles. In age-matched control experiments, 15% of them showed in vitro insoluble abeta-42 particle formation and 59% of the identified plaque particle constituents from AD serum were also present in the insoluble plaque particles derived from control. CONCLUSIONS: We have developed an in vitro method for plaque particle detection and identified serum protein markers that are associated with AD-related plaque particle formation. With further clinical validation, this assay may provide a novel, non-invasive means for the early detection of AD.

The interface of a membrane-spanning leucine zipper mapped by asparagine-scanning mutagenesis.

An oligo-leucine sequence has previously been shown to function as an artificial transmembrane segment that efficiently self-assembles in membranes and in detergent solution. Here, a novel technique, asparagine-scanning mutagenesis, was applied to probe the interface of the self-assembled oligo-leucine domain. This novel approach identifies interfacial residues whose exchange to asparagine leads to enhanced self-interaction of transmembrane helices by interhelical hydrogen bond formation. As analyzed by the ToxR system in membranes, the interface formed by the oligo-leucine domain is based on a leucine-zipper-like heptad repeat pattern of amino acids. In general, the strongest impacts on self-assembly were seen with asparagines located around the center of the sequence, indicating that interaction is be more efficient here than at the termini of the transmembrane domains.

Biological variation of ß-sitosterol, campesterol, and lathosterol as cholesterol absorption and synthesis biomarkers.

BACKGROUND: The analysis of blood for ß-sitosterol and campesterol is the measures of cholesterol absorption while lathosterol is a measure of cholesterol synthesis. METHODS: The biological variability of ß-sitosterol, campesterol, and lathosterol was measured using liquid-chromatography tandem mass spectrometry from a cohort of 25 apparently healthy subjects, where blood was taken once every weeks for 6 weeks. The analytical, intra-individual, and group inter-individual variations (CVA, CV(I), and CV(G), respectively) were calculated. RESULTS: Using absolute values, the CVI for ß-sitosterol, campesterol, and lathosterol was 11.8%, 11.8%, and 22.5%, respectively, and the CV(G) was 28.5%, 28.8%, and 52.0%, respectively. This produced reference change values of about 24-36% for declining values and 32-47% for increasing values. The index of individuality was between 0.41 and 0.58, indicating that population based reference values are of little use for these biomarkers. The number of points needed for a homeostatic setpoint was 5 samples for ß-sitosterol and campesterol, and 19 samples for lathosterol. Similar findings were observed for values when normalized to total cholesterol. These results were higher than the biological variation for total, low density and high density cholesterol obtained from the literature. Results were essentially identical when sterol values were corrected to their respective total cholesterol concentration. CONCLUSIONS: The establishment of the biological variation for these biomarkers enables their use in the interpretation of results from clinical trials and lipid lowering treatment of patients at risk for cardiovascular disease in clinical practice.

[Extracapsular arthroscopic excision of popliteal cysts through anterior combined with posterior approach].

OBJECTIVE: To study the clinical effect of arthroscopic excision of popliteal cysts through a combined anterior and posterior approach. METHODS: From January 2010 to December 2012,20 patients with popliteal cysts were treated with arthroscopic excision. There were 14 males and 6 females, with an average age of 49.5 years old, ranging from 45 to 65 years old. The lump has been found for 4 to 18 months,with a mean time of 12 months. Their mean sagittal diameter was 4.5 cm (ranged from 3 to 7 cm). There were 12 popliteal cysts in the left and 8 popliteal cysts in the right. The main clinical manifestation included lump at popliteal fossa, swelling and pain at knee joint and some extent of dysfunction. All diagnoses were determined according to MRI, which clearly showed the communication of cyst and articular cavity. The cyst was removed under arthroscopy, through the posterior approach and then the intra-articular lesion was treated via the anterior approach. According to Rauschning and Lindgren classification, 2 patients were grade I, 6 patients were grade II, and 12 patients were grade III. The guidelines of Rauschning and Lindgren were used to evaluate the clinical effects. RESULTS: No complications such as the injury of blood vessel and nerve, or incision infection occurred in all patients. All the patients were followed up, and the duration ranged from 8 to 24 months, with a mean time of 16 months. According to the criteria of Rauschning and Lindgren, there were 14 cases of grade 0, and 6 cases of grade I after operation, which was improved obviously compared with that pre-operation. No cyst reoccurred and the knee joint pain was relieved. CONCLUSION: Treatment of popliteal cysts with arthroscopic excision through a combined anterior and posterior approach is effective to remove the cyst sac and treat intra-articular diseases simultaneously, resulting in the decrease recurrence rate of cyst.

Gene expression profiling from formalin-fixed, paraffin-embedded tissue for tumor diagnosis.

BACKGROUND: Molecular profiling assays have emerged as a promising tool in tumor diagnosis. Recent advances have allowed the use of formalin-fixed, paraffin-embedded (FFPE) tissues in such assays involving the use of microarrays. The Pathwork Tissue of Origin (TOO) Test was developed for use with FFPE tissue to aid tumor diagnosis. We sought to determine the performance of the TOO test on routine specimens from an independent laboratory. METHODS: Forty-five blinded, archived clinical specimens from the UCSF Department of Pathology were tested. Total RNA was processed to prepare labeled cDNA for hybridization to Pathchip microarrays. Hybridization data was analyzed with a 2000-gene classification model to quantify similarity between RNA expression of the study specimens and the 15 tissues on the test panel. RESULTS: 44/45 (98%) specimens were successfully processed. 37 cases met study inclusion criteria. Of these, 35 (95%) gave results which were in agreement with the reference diagnosis. In no case was the reference diagnosis ruled out. CONCLUSIONS: The Tissue of Origin Test gave a high agreement with the reference diagnosis when archived clinical specimens from UCSF were assessed. Molecular profiling assays are highly accurate, and can be a useful tool in cancer diagnosis.

Partition constant and volume of distribution as predictors of clinical efficacy of lipid rescue for toxicological emergencies.

CONTEXT: Lipid infusion is useful in reversing cardiac toxicity of local anesthetics, and recent reports indicate it may be useful in resuscitation from toxicity induced by a variety of other drugs. While the mechanism behind the utility of lipid rescue remains to be fully elucidated, the predominant effect appears to be creation of a "lipid sink". OBJECTIVE: Determine whether the extraction of drugs by lipid, and hence the clinical efficacy of lipid rescue in toxicological emergencies can be predicted by specific drug properties. MATERIALS AND METHODS: Each drug investigated was added individually to human drug-free serum. Intralipid® was added to this drug-containing serum, shaken and then incubated at 37°C. The lipid was removed by ultracentrifugation and the concentration of drug remaining in the serum was measured by high-pressure liquid chromatography. RESULTS: In this in vitro model, the ability of lipid emulsion to bind a drug was largely dependent upon the drug's lipid partition constant. Additionally, using a multiple linear regression model, the prediction of binding could be improved by combining the lipid partition constant with the volume of distribution together accounting for approximately 88% of the variation in the decrease in serum drug concentration with the administration of lipid emulsion. CONCLUSIONS: The lipid partition constant and volume of distribution can likely be used to predict the efficacy of lipid infusion in reversing the cardiac toxicity induced by anesthetics or other medications.

Serum verapamil concentrations before and after Intralipid® therapy during treatment of an overdose.

CONTEXT. Intralipid® infusion is useful in reversing cardiac and central nervous system toxicity of local anesthetic drugs, and recent reports suggest utility in other drug overdoses. CASE DETAILS. A 47-year-old man presented to the emergency department with hypotension and complete heart block 3 h after a sustained-release verapamil overdose. He was given supportive care including calcium and hyperinsulinemia/euglycemia therapy. Nineteen and 29 h post-ingestion, Intralipid® was administered as a bolus, followed by an infusion. OBJECTIVE. The objective of this study was to determine the serum verapamil concentrations before and after Intralipid® administration and to ascertain its clinical effects. DISCUSSION. It was found that administration of Intralipid® was followed by a decrease in verapamil concentration once the lipid had been removed from the sample, demonstrating that Intralipid® was effective in sequestering verapamil, effectively removing it from the serum, and supporting its use in the treatment of verapamil overdose. Intralipid® administration was associated with an increase in the patient's blood pressure, but because other vasoactive drugs were given at the same time, it was difficult to determine its relative contribution to clinical improvement.

Identification of clinically significant tumor antigens by selecting phage antibody library on tumor cells in situ using laser capture microdissection.

Much work has been done to develop tumor-targeting antibodies by selecting a phage antibody library on cancer cell lines. However, when tumor cells are removed from their natural environment, they may undergo genetic and epigenetic changes yielding different surface antigens than those seen in actual cases of cancer. We developed a strategy that allows selection of phage antibodies against tumor cells in situ on both fresh frozen and paraffin-embedded tissues using laser capture microdissection. By restricting antibody selection to binders of internalizing epitopes, we generated a panel of phage antibodies that target clinically represented prostate cancer antigens. We identified ALCAM/MEMD/CD166, a newly discovered prostate cancer marker, as the target for one of the selected antibodies, demonstrating the effectiveness of our approach. We further conjugated two single chain Fv fragments to liposomes and demonstrated that these nanotargeting devices were efficiently delivered to the interior of prostate cancer cells. The ability to deliver payload intracellularly and to recognize tumor cells in situ makes these antibodies attractive candidates for the development of targeted cancer therapeutics.

Qdot nanobarcodes for multiplexed gene expression analysis.

We report a quantum dot (Qdot) nanobarcode-based microbead random array platform for accurate and reproducible gene expression profiling in a high-throughput and multiplexed format. Four different sizes of Qdots, with emissions at 525, 545, 565, and 585 nm are mixed with a polymer and coated onto the 8-mum-diameter magnetic microbeads to generate a nanobarcoded bead termed as QBeads. Twelve intensity levels for each of the four colors were used. Gene-specific oligonucleotide probes are conjugated to the surface of each spectrally nanobarcoded bead to create a multiplexed panel, and biotinylated cRNAs are generated from sample total RNA and hybridized to the gene probes on the microbeads. A fifth streptavidin Qdot (655 nm or infrared Qdot) binds to biotin on the cRNA, acting as a quantification reporter. Target identity was decoded based on spectral profile and intensity ratios of the four coding Qdots (525, 545, 565, and 585 nm). The intensity of the 655 nm Qdot reflects the level of biotinylated cRNA captured on the beads and provides the quantification for the corresponding target gene. The system shows a sensitivity of < or =10(4) target molecules detectable with T7 amplification, a level that is better than the 10(5) number achievable with a high-density microarray system, and approaching the 10(3)-10(4) level usually observed for quantitative PCR (qPCR). The QBead nanobarcode system has a dynamic range of 3.5 logs, better than the 2-3 logs observed on various microarray platforms. The hybridization reaction is performed in liquid phase and completed in 1-2 hours, at least 1 order of magnitude faster than microarray-based hybridizations. Detectable fold change is lower than 1.4-fold, showing high precision even at close to single copy per cell level. Reproducibility for this proof-of-concept study approaches that of Affymetrix GeneChip microarray, with an R(2) value between two repeats at 0.984, and interwell CV around 5%. In addition, it provides increased flexibility, convenience, and cost-effectiveness in comparison to conventional gene expression profiling methods.

The interface between self-assembling erythropoietin receptor transmembrane segments corresponds to a membrane-spanning leucine zipper.

Structural and functional studies recently indicated that the erythropoietin receptor exists as a preassembled homodimer whose activation by ligand binding requires self-interaction of its transmembrane segment. Here, we probed the interface formed by the transmembrane segments by asparagine-scanning mutagenesis in a natural membrane. We show that this interface is based on a leucine zipper-like heptad repeat pattern of amino acids. The strongest impact of asparagine was observed at position 241, suggesting the highest packing density around this position, which is in agreement with results obtained upon mutation to alanine. Interestingly, the same face of the transmembrane helix had previously been shown to enter a heterophilic interaction with the transmembrane segment of gp55-P, a viral membrane protein that leads to ligand-independent receptor activation in infected cells. Further, functional characterization of an erythropoietin receptor mutant with asparagine at position 241 in a hematopoietic cell line showed that this protein could still be activated by erythropoietin yet was not constitutively active. This suggests that forced self-interaction of the transmembrane segments does not suffice to induce signaling of the erythropoietin receptor.