Richard J. Robbins

Hippocampal interneuron loss and plasticity in human temporal lobe epilepsy

It has been hypothesized on the basis of animal models of epilepsy that abnormal neural activity in epilepsy may be related to reorganized neural circuits that facilitate epileptogenesis. Little evidence of this was available for human epilepsy. This paper provides the first evidence of such reorganization of a hippocampal seizure focus in human temporal lobe epilepsy (TLE). This reorganization involves the selective loss of somatostatin and neuropeptide Y immunoreactive interneurons, and axonal sprouting of other neuropeptide Y neurons and dynorphin-A immunoreactive granule cells. This set of changes is not exactly like those that are reported in animal models.

Serum Peptidome Patterns That Distinguish Metastatic Thyroid Carcinoma from Cancer-free Controls Are Unbiased by Gender and Age

Serum peptidomics is a special form of functional proteomics. The small number of blood proteins that are the source of most prominent peptides in human serum serve as a substrate pool for commonly occurring and/or cancer-derived proteases. Exoprotease activities in particular, when superimposed on the ex vivo coagulation and complement degradation pathways, contribute to generation of not only cancer-specific but also “cancer type”-specific serum peptides. Following development of a unique, semiautomated serum peptide profiling platform and after completing investigations to eliminate common experimental bias, we have now studied possible effects of gender and age on serum peptidomes of 200 healthy men and women, ages 20–80, and of 60 patients (30 men and 30 women) with metastatic thyroid carcinomas. Extensive MALDI-TOF MS and data analysis suggested negligible contributions of both age and gender to the serum peptidome patterns except that healthy men and women under 35 years, but not older individuals, could be distinguished with ∼70% accuracy. Considering the more advanced age of most patients, this finding is unlikely to interfere with peptidomics analysis of most cancers. By examining patient samples and age/gender-matched controls followed by variability analysis of either demographic or disease (versus control) groups, we could conclusively rule out demographic bias. An optimized, 12-peptide ion thyroid cancer signature was then developed, enabling classification of an independent validation set with 95% sensitivity and 95% specificity (binomial confidence intervals, 75.1–99.9%). Ten of these peptides had previously been assigned to signature patterns of other solid tumor cancers. One of the two newly discovered peptides was dehydro-Ala3-fibrinopeptide A. As we expand this study to include hundreds of thyroid cancer patients, the peptide signature will be adjusted, further validated, and then evaluated in a clinical setting used either independently or in combination with existing markers.

Resistance of [18F]-Fluorodeoxyglucose-Avid Metastatic Thyroid Cancer Lesions to Treatment with High-Dose Radioactive Iodine

Radioactive iodine (131I) is an important therapeutic option for the treatment of metastatic thyroid carcinoma. Survival in patients with metastases that concentrate radioiodine is better than those whose metastatic lesions do not take up radioiodine. Survival is markedly reduced in patients who have metastatic lesions that concentrate 18F-fluorodeoxyglucose (FDG) on positron emission tomography (PET). In this retrospective study, we evaluated the ability of 131I to destroy FDG-avid metastatic lesions in thyroid cancer patients. Twenty-five patients with positive FDG-PET scans received at least one dose of 131I treatment before a second FDG-PET was performed. The average interval between the two PET scans was 12.9 months. The average interval between the 131I treatment and the follow-up FDG-PET was 10.1 months. We measured posttherapy changes in lesional volume, in standard uptake values (SUV) of FDG, and in serum thyroglobulin (Tg) levels. The total volume of FDG-avid metastases rose significantly (p = 0.036) from a mean of 159 mL to 235 mL after 131I therapy, the maximum SUV rose from 9.3 to 11.9, the median Tg at the time of the second PET scan was 132% of that at baseline. Statistical analyses demonstrated no significant changes in maximum SUV, or serum Tg levels after 131I in the FDG-PET-positive group. In a control group of FDG-PET-negative patients, the serum Tg decreased to 38% of baseline after 131I therapy (p < 0.001). We conclude that high-dose 131I therapy appears to have little or no effect on the viability of metastatic FDG-avid thyroid cancer lesions.

Recombinant Human TSH–Assisted Radioactive Iodine Remnant Ablation Achieves Short-Term Clinical Recurrence Rates Similar to Those of Traditional Thyroid Hormone Withdrawal

Recent studies have confirmed that radioactive iodine therapy after recombinant human TSH (rhTSH) stimulation effectively ablates the normal thyroid remnant. However, no published study has determined the effectiveness of rhTSH preparations on the important endpoint of disease recurrence. Methods: Disease recurrence was retrospectively assessed a median of 2.5 y after radioiodine remnant ablation (RRA) in 394 consecutive thyroid cancer patients (93% papillary, 71% female, 47 ± 15 y old [mean ± SD], median 131I dose of 3,996 MBq [108 mCi]). Results: Similar rates of clinically evident disease recurrence (4% rhTSH vs. 7% thyroid hormone withdrawal [THW], P = not statistically significant) and residual thyroid bed uptake without other evidence of persistent disease (4% rhTSH vs. 7% THW, P = not statistically significant) were seen in the 320 patients undergoing rhTSH-assisted RRA and the 74 patients prepared for RRA by THW. When the definition of no clinical evidence of disease included a suppressed thyroglobulin level of less than 1 ng/mL and a stimulated thyroglobulin level of less than 2 ng/mL, rhTSH-assisted RRA was associated with significantly higher rates of no clinical evidence of disease (74% rhTSH vs. 55% THW, P = 0.02) and significantly lower rates of persistent disease (19% rhTSH vs. 32% THW, P = 0.02) than was RRA after THW. Patients selected for rhTSH-assisted RRA were older (48 ± 15 vs. 44 ± 15 y, P = 0.03) and received a slightly higher administered activity of 131I (median, 4,033 MBq [109 mCi] vs. 3,811 MBq [103 mCi], P = 0.01) but did not differ with respect to sex, histology, disease stage, or mean time to recurrence (19 ± 9 mo for rhTSH vs. 20 ± 16 mo for THW). Conclusion: rhTSH-assisted RRA is associated with rates of clinically evident disease recurrence and persistent uptake in the thyroid bed that are similar to those for traditional THW.

Trophic effects of insulin-like growth factor-I on fetal rat hypothalamic cells in culture

The hypothesis that insulin-like growth factor-I is a trophic factor for primary fetal rat hypothalamic cells was tested, since we previously reported a potent mitogenic effect of this peptide on virally-transformed hypothalamic cells. It was found that insulin-like growth factor-I produced significant and dose-dependent increases in the survival of fetal hypothalamic neurons in primary mixed glial/neuronal cultures. By 48 h in vitro, cultures treated with insulin-like growth factor-I (6 nM) had twice as many neurite-bearing cells as controls, while by day 15 a five-fold difference was present. The peptide was similarly active in promoting neuronal survival in neuron-enriched (98% neurons) hypothalamic cultures. Mixed hypothalamic cultures had specific binding sites for insulin-like growth factor-I. In addition, the neurons grown in the presence of insulin-like growth factor-I had a more differentiated morphology and had significantly higher levels of protein kinase C, an enzyme that increases during neurite formation and synaptogenesis. Finally, glial-enriched cultures (greater than 99% glial cells) obtained from the fetal hypothalamus showed increased [3H]thymidine incorporation in response to insulin-like growth factor-I. These results further support the contention that insulin-like growth factor-I is a neurotrophic factor and suggest that it may participate in the normal development of the hypothalamus by increasing neuronal survival/differentiation and stimulating glial growth.

Radioiodine Ablation of Thyroid Remnants After Preparation with Recombinant Human Thyrotropin

Radioiodine ablation (RA) of normal thyroid remnants after thyroidectomy for differentiated thyroid carcinoma improves the sensitivity of subsequent radioiodine scans and serum thyroglobulin measurements for detection of residual thyroid carcinoma. Local cancer recurrences are also lower after RA. One standard preparation for RA involves rendering the patient hypothyroid in order to stimulate endogenous thyrotropin (TSH) secretion and sodium iodide symporter (NIS) activity. An alternative approach is to prescribe thyroxine after thyroidectomy and to stimulate NIS with exogenous recombinant human thyrotropin (rhTSH). This latter approach was used in 10 patients at our medical center. Complete resolution of all visible 131I thyroid bed uptake was achieved in all when follow-up scans were performed 5 to 13 months later. This approach has the potential to successfully ablate thyroid remnants without the need to induce hypothyroidism.

Detection of thyroglobulin, thyroid peroxidase, and RET/PTC1 mRNA transcripts in the peripheral blood of patients with thyroid disease.

PURPOSE Detection of mRNA transcripts for thyroglobulin (TG), thyroid peroxidase (TPO) and RET/PTC1 in the peripheral blood of patients with thyroid disease. PATIENTS AND METHODS TG, TPO, and RET/PTC1 mRNA were analyzed in 52 peripheral-blood samples from 44 patients diagnosed with thyroid carcinoma (24 patients), adenoma (five patients), and nodular hyperplasia (15 patients) by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS TG and TPO were identified in 13 patients (54.2%) with thyroid carcinoma, which includes five of eight patients with no clinical evidence of disease at the time of blood collection. Four of 5 patients had cervical lymph node metastases and/or extrathyroid extension at the time of the initial surgery. RET/PTC1 mRNA was detected in the peripheral blood of only one patient with papillary thyroid carcinoma. This sample was also positive for TG and TPO. TG and TPO were detected in two patients (10%) with benign thyroid nodules. All positive samples from patients with benign thyroid lesions were collected before surgery, whereas all samples collected after surgery were negative. RET/PTC1 mRNA was not detected in any of the patients with benign thyroid nodules. RT-PCR positivity for TG and TPO mRNA was higher in patients with carcinoma than in patients with benign lesions (P = .002). CONCLUSION TG, TPO, and RET/PTC1 mRNA are detectable in the peripheral blood of patients with thyroid disease, which correlates with a diagnosis of carcinoma.

A Sequence-specific Exopeptidase Activity Test (SSEAT) for “Functional” Biomarker Discovery

One form of functional proteomics entails profiling of genuine activities, as opposed to surrogates of activity or active “states,” in a complex biological matrix: for example, tracking enzyme-catalyzed changes, in real time, ranging from simple modifications to complex anabolic or catabolic reactions. Here we present a test to compare defined exoprotease activities within individual proteomes of two or more groups of biological samples. It tracks degradation of artificial substrates, under strictly controlled conditions, using semiautomated MALDI-TOF mass spectrometric analysis of the resulting patterns. Each fragment is quantitated by comparison with double labeled, non-degradable internal standards (all-d-amino acid peptides) spiked into the samples at the same time as the substrates to reflect adsorptive and processing-related losses. The full array of metabolites is then quantitated (coefficients of variation of 6.3–14.3% over five replicates) and subjected to multivariate statistical analysis. Using this approach, we tested serum samples of 48 metastatic thyroid cancer patients and 48 healthy controls, with selected peptide substrates taken from earlier standard peptidomics screens (i.e. the “discovery” phase), and obtained class predictions with 94% sensitivity and 90% specificity without prior feature selection (24 features). The test all but eliminates reproducibility problems related to sample collection, storage, and handling as well as to possible variability in endogenous peptide precursor levels because of hemostatic alterations in cancer patients.

Trophic effects of basic fibroblast growth factor on fetal rat hypothalamic cells: interactions with insulin-like growth factor I.

The existence of different growth factors within a single brain region suggests that developing brain cells are exposed to a variety of trophic factors throughout neurogenesis. Cooperative interactions between growth factors are known to orchestrate growth and differentiation of various cell types. We explored the possibility that two growth factors may interact in promoting in vitro growth in fetal hypothalamic cells. We found that basic fibroblast growth factor (b-FGF) exerts trophic effects on primary mixed hypothalamic cell cultures, on enriched hypothalamic neuronal cultures, and on hypothalamic glial cultures. In addition, b-FGF increased the growth rate of two virally transformed hypothalamic cell lines. Since insulin-like growth factor I (IGF-I) also promotes growth of rat hypothalamic cells in vitro, we examined the combined effects of b-FGF and IGF-I on hypothalamic cells. Significantly higher numbers of neurite-bearing cells were present in primary mixed hypothalamic cultures when b-FGF and IGF-I were added together than were added separately. The effect was additive. These results establish b-FGF as a putative hypothalamic neurotrophic factor and demonstrate potential coordinate interactions between IGF-I and b-FGF in stimulating the growth or survival of developing hypothalamic cells.

Compassionate use of recombinant human thyrotropin to facilitate radioiodine therapy: case report and review of literature.

OBJECTIVE To describe the use of recombinant human thyrotropin (thyroid-stimulating hormone) (rhTSH) to assist in radioiodine therapy in a patient with thyroid carcinoma who was unable to produce sufficient endogenous thyrotropin when hypothyroid and to review the related literature. METHODS The study patient underwent formal dosimetric analysis and received radioiodine in conjunction with rhTSH. Follow-up scanning studies were performed. RESULTS We found good localization of radioiodine on the posttherapy scans after administration of (131)I while the patient continued to receive thyroxine replacement after two intramuscularly administered injections of rhTSH. Some of his metastatic lesions disappeared and his serum thyroglobulin level decreased after the first rhTSH-assisted dose of (131)I was administered. His blood radioiodine clearance rate was unexpectedly more rapid in the hypothyroid state than when he was euthyroid (taking thyroxine) after administration of rhTSH. His whole-body clearance rate was more delayed when he was hypothyroid. Swelling of some of the metastatic thyroid cancer lesions developed when the patient was hypothyroid and after rhTSH was administered, the latter being much more rapid in onset. CONCLUSION Therapeutic doses of radioiodine can be delivered with the assistance of rhTSH administration while patients continue to take suppressive doses of thyroxine. Metastatic thyroid carcinoma lesions can swell rapidly after administration of rhTSH. The commercially available form of rhTSH is approved only for diagnostic use. Its safety and efficacy in assisting radioiodine therapy have not been fully determined.