Brian C. Netzel

Detailed Molecular Fingerprinting of Four New Anaplastic Thyroid Carcinoma Cell Lines and Their Use for Verification of RhoB as a Molecular Therapeutic Target

CONTEXT Anaplastic thyroid carcinoma (ATC) is a highly aggressive carcinoma in need of therapeutic options. One critical component of drug discovery is the availability of well-characterized cell lines for identification of molecular mechanisms related to tumor biology and drug responsiveness. Up to 42% of human thyroid cancer cell lines are redundant or not of correct tissue origin, and a comprehensive analysis is currently nonexistent. Mechanistically, RhoB has been identified as a novel molecular target for ATC therapy. OBJECTIVE The aim was to develop four ATC cell lines detailing genetic, molecular, and phenotypic characteristics and to test five classes of drugs on the cell lines to determine whether they inhibited cell proliferation in a RhoB-dependent fashion. DESIGN Four cell lines were derived from ATC tumors. Short tandem DNA repeat and mutational status of the originating tumors and cell lines were performed along with molecular and phenotypic characterizations. Compounds were tested for growth inhibition and ability to up-regulate RhoB. RESULTS Cell line authenticity was confirmed by DNA short tandem repeat analysis. Each proved unique regarding expression of thyroid markers, oncogene status, amplified and deleted genes, and proliferative growth rates. FTI-277, GGTI-286, lovastatin, romidepsin, and UCN-01 up-regulated RhoB and inhibited cell proliferation in a dose-responsive fashion with only romidepsin and FTI-277 being RhoB dependent. CONCLUSIONS Molecular descriptions of thyroid lines were matched to the originating tumors, setting a new standard for cell line characterization. Furthermore, suppressed RhoB is implicated as a molecular target for therapy against ATC because five classes of drugs up-regulate RhoB and inhibit growth dose-responsively.

Increasing Liquid Chromatography–Tandem Mass Spectrometry Throughput by Mass Tagging: A Sample-Multiplexed High-Throughput Assay for 25-Hydroxyvitamin D2 and D3

BACKGROUND The limits of chromatographic speed and mechanical frontend capabilities have been reached for many high-volume liquid chromatography-tandem mass spectrometry (LC-MS/MS) tests, curtailing the maximal achievable sample throughput. To overcome these boundaries, we developed and validated a derivatization-based sample-multiplex LC-MS/MS assay for detection of 25-hydroxyvitamins D2 and D3 [25(OH)D2 and 25(OH)D3], which increased sample throughput 5-fold. METHODS After separate derivatization with 1 of 5 different triazoline-diones (TADs), 5 calibrators, controls, or patient specimens were combined and injected together into an LC-MS/MS. On the basis of mass differences between TADs, the MS/MS quantified analyte and stable isotope internal standards for 25(OH)D2 and 25(OH)D3 for each respective multiplexed sample within the injection. Limits of detection and quantification, spiked recovery, linearity, imprecision, and patient results were determined and compared against our standard LC-MS/MS assay. RESULTS TAD multiplexing increased throughput on an LC-quadruplexed LC-MS/MS system from 60 samples/h to 300 samples/h. Limits of detection and quantification were 4.9 nmol/L [2 μg/L, 25(OH)D2], 2.2 nmol/L [0.9 μg/L, 25(OH)D3], and 10 nmol/L [4 μg/L, 25(OH)D2], 5 nmol/L [2 μg/L, 25(OH)D3], respectively. The assay was linear to 250 nmol/L (100 μg/L). Interassay CVs across the reportable range were 3.7%-15.2%. Spiked recoveries were 94%-119%. The method comparison with the standard LC-MS/MS method showed slopes of 0.96 and 0.97 (Deming regression) for 25(OH)D2 (n=1733) and 25(OH)D3 (n=7614) (R2=0.96 and 0.97), respectively. CONCLUSIONS Multiplexing samples by differential mass tagging in LC-MS/MS measurement of 25(OH)D2 and 25(OH)D3 allows for reliable quantification, with throughput increased over standard methods by the multiplexing factor.

Thyroglobulin (Tg) Testing Revisited: Tg Assays, TgAb Assays, and Correlation of Results With Clinical Outcomes.

CONTEXT Measurement of thyroglobulin (Tg) by mass spectrometry (Tg-MS) is emerging as a tool for accurate Tg quantification in patients with anti-Tg autoantibodies (TgAbs). OBJECTIVE The objective of the study was to perform analytical and clinical evaluations of two Tg-MS assays in comparison with immunometric Tg assays (Tg-IAs) and Tg RIAs (Tg-RIAs) in a cohort of thyroid cancer patients. METHODS A total of 589 samples from 495 patients, 243 TgAb-/252 TgAb+, were tested by Beckman, Roche, Siemens-Immulite, and Thermo-Brahms Tg and TgAb assays, two Tg-RIAs, and two Tg-MS assays. RESULTS The frequency of TgAb+ was 58%, 41%, 27%, and 39% for Roche, Beckman, Siemens-Immulite, and Thermo-Brahms, respectively. In TgAb- samples, clinical sensitivities and specificities of 100% and 74%-100%, respectively, were observed across all assays. In TgAb+ samples, all Tg-IAs demonstrated assay-dependent Tg underestimation, ranging from 41% to 86%. In TgAb+ samples, the use of a common cutoff (0.5 ng/mL) for the Tg-MS, three Tg-IAs, and the USC-RIA improved the sensitivity for the Tg-MSs and Tg-RIAs when compared with the Tg-IAs. In up to 20% of TgAb+ cases, Tg-IAs failed to detect Tg that was detectable by Tg-MS. In Tg-RIAs false-high biases were observed in TgAb+ samples containing low Tg concentrations. CONCLUSIONS Tg-IAs remain the method of choice for Tg quantitation in TgAb- patients. In TgAb+ patients with undetectable Tg by immunometric assay, the Tg-MS will detect Tg in up to 20% additional cases. The Tg-RIA will detect Tg in approximately 35% cases, but a significant proportion of these will be clinical false-positive results. The undetectable Tg-MS seen in approximately 40% of TgAb+ cases in patients with disease need further evaluation.

Renal medullary 11β-hydroxysteroid dehydrogenase type 1 in Dahl salt-sensitive hypertension

The Dahl salt-sensitive rat is a widely used model of human salt-sensitive forms of hypertension. The kidney plays an important role in the pathogenesis of Dahl salt-sensitive hypertension, but the molecular mechanisms involved remain a subject of intensive investigation. Gene expression profiling studies suggested that 11 beta-hydroxysteroid dehydrogenase type 1 might be dysregulated in the renal medulla of Dahl salt-sensitive rats. Additional analysis confirmed that renal medullary expression of 11 beta-hydroxysteroid dehydrogenase type 1 was downregulated by a high-salt diet in SS-13BN rats, a consomic rat strain with reduced blood pressure salt sensitivity, but not in Dahl salt-sensitive rats. 11 beta-Hydroxysteroid dehydrogenase type 1 is known to convert inactive 11-dehydrocorticosterone to active corticosterone. The urinary corticosterone/11-dehydrocorticosterone ratio as well as urinary excretion of corticosterone was higher in Dahl salt-sensitive rats than in SS-13BN rats. Knockdown of renal medullary 11 beta-hydroxysteroid dehydrogenase type 1 with small-interfering RNA attenuated the early phase of salt-induced hypertension in Dahl salt-sensitive rats and reduced urinary excretion of corticosterone. Knockdown of 11 beta-hydroxysteroid dehydrogenase type 1 did not affect blood pressure in SS-13BN rats. Long-term attenuation of salt-induced hypertension was achieved with small hairpin RNA targeting renal medullary 11 beta-hydroxysteroid dehydrogenase type 1. In summary, we have demonstrated that suppression of 11 beta-hydroxysteroid dehydrogenase type 1 expression in the renal medulla attenuates salt-induced hypertension in Dahl salt-sensitive rats.

Pharmacokinetics of methylene blue dye for lymphatic mapping in breast cancer-implications for use in pregnancy.

BACKGROUND although blue dye is routinely used for lymphatic mapping, it is not used for lymphatic mapping in pregnancy-associated breast cancer, because of concern of fetal risk. METHODS to investigate the safety of blue dye for lymphatic mapping in pregnant women, the pharmacokinetics of methylene blue dye were examined in 10 nonpregnant women, and the results were extrapolated to estimate maximal fetal exposure to the dye. RESULTS plasma and urine measurements indicated that the dye quickly distributed from the breast injection site to the circulation, with 32% of the total dose excreted in urine within 48 hours. Combined with existing data on organ distribution of methylene blue, the estimated maximal dose to the fetus is 0.25 mg (5% of the administered dose), likely further reduced by other physiologic factors related to pregnancy. CONCLUSIONS the analysis suggests that methylene blue dye can be used for lymphatic mapping in pregnancy-associated breast cancer with minimal fetal risk.

First Steps toward Harmonization of LC-MS/MS Thyroglobulin Assays

To the Editor: Antithyroglobulin autoantibodies (TgAbs)1 frequently cause falsely low measurements of serum thyroglobulin (Tg) when immunometric assays (TgIAs) are used (1). This problem can be overcome by tryptic digestion of patient serum with subsequent measurement of Tg-proteotypic peptides by LC-MS/MS (TgMS) (2–4). However, it remains uncertain how different TgMS assays compare with each other, a crucial question in todays laboratory testing environment, wherein concern has been raised over patient samples being tested by different methods or in different laboratories over time (5). We therefore performed a pilot study to assess the intermethod concordance between 4 independently developed TgMS methods. The study was approved by the Mayo Clinic institutional review board. We tested 40 patient serum samples spanning Tg concentrations from <0.1 to 10 ng/mL (assigned by DxI TgIA, Beckman Coulter). We classified the samples as TgAb positive (n = 22) or TgAb negative (n = 18) on the basis of whether they contained TgAb concentrations that exceeded the functional sensitivity of the DxI TgAB assays (1.8 IU/mL). Samples were deidentified and shared between 4 laboratories for TgMS testing: ARUP Laboratories, Laboratory Corporation of America Holdings (LabCorp), Mayo Clinic, and the University of Washington (UWash). Each assay uses different sample preparation methodologies: ARUP and Mayo use different protein precipitation steps to enrich for thyroglobulin before proteolysis, and LabCorp and UWash digest serum samples directly after different denaturing steps. …

Comparison of multiple steroid concentrations in serum and dried blood spots throughout the day of patients with congenital adrenal hyperplasia

Background/Aim: Periodic measurement of plasma concentrations of cortisol precursors on a clinic visit may be of limited value in patients with congenital adrenal hyperplasia because it does not reflect a patient’s circadian patterns of adrenal steroid secretion. Steroid profiling in dried blood spots (DBS) may allow for more frequent and sensitive monitoring. Methods: We compared the agreement between 17α-hydroxyprogesterone (17-OHP) and androstenedione (D4A) levels determined from DBS samples and concurrently collected serum samples. Blood was drawn from 9 congenital adrenal hyperplasia patients every 4 h over a 24-hour period. Serum and DBS steroid levels were measured by liquid chromatography tandem mass spectrometry. Results: DBS determinations of 17-OHP overestimated corresponding serum levels (mean difference 1.67 ng/ml), and underestimated D4A serum levels (mean difference 0.84 ng/ml). However, the DBS assay yielded excellent agreement (97%) with serum 17-OHP, but did considerably poorer for D4A (31%). Conclusions: Our results indicate an excellent agreement between DBS and serum 17-OHP measurements to identify the peaks and troughs associated with an individual’s circadian pattern. Larger-scale studies are required to evaluate the utility of DBS for home monitoring and to determine if more frequent monitoring leads to improved clinical outcomes.

Liquid chromatography–tandem mass spectrometry analysis of urinary fluticasone propionate-17beta-carboxylic acid for monitoring compliance with inhaled-fluticasone propionate therapy

BACKGROUND Inhaled corticosteroids including fluticasone propionate (FP) are the most effective treatment for persistent-asthma. Noncompliance ranging from 20% to 80% of treated patients is associated with substantial health care costs, morbidity and fatalities. A noninvasive test to assess FP treatment compliance is needed. The major metabolite of FP is FP-17beta-carboxylic acid (FP17betaCA) and is excreted in urine. This study demonstrates the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure FP17betaCA in urine and evaluation of FP17betaCA urinary elimination. EXPERIMENTAL Fluorometholone was used as the internal standard. After acetonitrile precipitation, samples were extracted with dichloromethane, washed and dried. Reconstituted extract (60 microL) was subjected to reversed-phase chromatography and positive-ion mode LC-MS/MS analysis. Assay precision, linearity, recovery and sample stability were determined. Elimination evaluation included measurement of FP17betaCA in urine collected daily from human subjects before (day 1), during treatment (days 2-5; dose FP-110 microg 2 puffs/day), and following cessation of FP therapy (days 6-14; n=4). RESULTS Linear range of the FP17betaCA assay was 10.3-9510pg/mL. Limit of quantitation (LOQ) was 10.3 pg/mL and recovery ranged from 85.8% to 111.9%. Inter-assay CVs were 7.4-12.0% for FP17betaCA concentrations of 11.1-5117 pg/mL. Urine FP17betaCA was absent in subjects prior to FP therapy, detectable (180-1991 ng FP17betaCA/g creatinine) throughout the dosing period and reached below the LOQ at 6 days after therapy cessation. CONCLUSIONS Measurement of FP17betaCA by LC-MS/MS has acceptable analytical performance for clinical use. These data support the clinical utility of measuring FP17betaCA in urine to monitor patient compliance with FP therapy.

Usefulness of a Thyroglobulin Liquid Chromatography–Tandem Mass Spectrometry Assay for Evaluation of Suspected Heterophile Interference

To the Editor: The accuracy of human serum thyroglobulin (Tg)1 measurement by immunoassays can be affected by anti-Tg autoantibodies (TgAB) (1). In immunometric Tg assays, which dominate the market, TgAB interference can result in false-low measurements. Tg quantification by liquid chromatography–tandem mass spectrometry (LC-MS/MS) was first described by Hoofnagle et al. (2) and subsequently shown by Clarke et al. to overcome TgAB interference by the use of proteotypic peptide quantitation after tryptic digestion, with peptide-specific immunocapture (3). In the June 2013 issue of Clinical Chemistry , Kushnir et al. presented a study that further solidified the usefulness of using LC-MS/MS in overcoming the effects of TgAB on accurate Tg quantification by demonstrating that 23% of TgAB-positive serum samples, which had undetectable Tg concentrations by immunoassay (<0.1 μg/L), had Tg values above the limit of quantification (LOQ) of their LC-MS/MS Tg assay (≥0.5 μg/L) (4). We read this study with great interest because we recently implemented a similar methodology in our laboratory. In TgAB-positive/Tg-negative samples by immunoassay, our results …

Companion-diagnostic testing limited to KRAS codons 12 and 13 misses 17% of potentially relevant RAS mutations in colorectal cancer

BACKGROUND KRAS codons 12 and 13 mutations are commonly used to identify colorectal carcinoma (CRC) patients who are unlikely to benefit from anti-EGFR therapy. However, humans have four different homologous RAS proteins and no routine screening is performed for the other mutation sites. Non-screened mutations may still be present in a significant subset of patients without KRAS codon 12 and 13 mutations. METHODS We developed a LightCycler screening assay that encompasses codons 12, 13 and 61 of all RAS genes. Screen-positive specimens were characterized by Sanger sequencing. 130 CRC specimens were screened for all RAS genes. The results for KRAS codons 12 and 13 were compared with an FDA approved method (Qiagen). RESULTS Twenty-nine of 130 specimens (22.3%) were positive for KRAS codons 12 and 13, with 100% congruence with the Qiagen method. Six additional specimens were identified to have mutations. One mutation in HRAS codon 61, two in KRAS codon 61, and three in NRAS codon 61. CONCLUSION Limiting RAS testing to only KRAS codons 12 and 13 in companion diagnostic testing of CRC results in nearly 1/5 of patients with RAS mutations not being excluded from costly EGFR antagonist treatment, despite likely futility. Inclusion of all RAS genes in companion diagnostic screening is warranted.