In vivo and ex vivo techniques using elastic scattering spectroscopy for diagnosis of malignancy in the thyroid gland

Thesis (M.A.)--Boston University, 2011.OBJECTIVE: Thyroid cancer is the most common endocrine malignancy and patients presenting with thyroid nodules often undergo surgery solely for diagnostic purposes. The goal of our study was to examine the accuracy of Elastic Scattering Spectroscopy (ESS) in distinguishing between benign and malignant thyroid nodules in fresh ex vivo specimens and to design an in vivo ESS probe and device, manufacture it and conduct a clinical trial. METHODS: Patients already undergoing thyroidectomy surgery were consented for the ex vivo study. ESS data was obtained from ex vivo specimens by recording 5 readings per nodule with five repetitive readings per each site. Final pathology reports were used to confirm the diagnosis. The spectra were analyzed using principal component analysis, linear discriminant analysis and leave one out technique. The in vivo ESS study was conceptually designed and IRB approval from Boston Medical Campus was obtained. RESULTS: The ex vivo study showed that ESS could predict the difference between benign and malignant tumors with a sensitivity of 74%, specificity of 90%, positive predictive value of 82% and negative predictive value of 85%. 193 spectra were analyzed from 64 patients, 120 spectra were from benign nodules and 73 from malignant nodules. Subanalysis examined only indeterminate nodules showed sensitivity of 65%, specificity of 79%, PPV 77% and NPV 67%. The in vivo ESS probe was designed and 12 identical instruments were manufactured. Initial experimental readings were taken and parameters were adjusted for the in vivo tissue environment. The clinical trial is underway. CONCLUSIONS: ESS is a practical tool that can accurately identify malignancy in ex vivo thyroid specimens with high specificity and sensitivity. Initial in vivo experimental trials have been conducted and show promise for similar results

Cell-free Embryonic Stem Cell Extract-mediated Derivation of Multi-potent Stem Cells from NIH3T3 Fibroblasts for Functional and Anatomical Ischemic Tissue Repair

The oocyte-independent generation of multipotent stem cells is one of the goals in regenerative medicine. We report that upon exposure to mouse ES cell (ESC) extracts, reversibly permeabilized NIH3T3 cells undergo de-differentiation followed by stimulus-induced re-differentiation into multiple lineage cell types. Genome-wide expression profiling revealed significant differences between NIH3T3 and ESC-extract treated NIH3T3 cells including re-activation of ESC specific transcripts. Epigenetically, ESC extracts induced CpG de-methylation of Oct4 promoter, hyper-acetylation of histones 3 and 4 and decreased lysine 9 (K-9) dimethylation of histone 3. In mouse models of surgically-induced hind limb ischemia (HLI) or acute myocardial infarction (AMI) transplantation of reprogrammed NIH3T3 cells significantly improved post-injury physiological functions and showed antomical evidence of engraftment and trans-differentiation into skeletal muscle, endothelial cell and cardiomyocytes. These data provide evidence for the generation of functional multi-potent stem like cells from terminally differentiated somatic cells without the introduction of trans-genes or ESC fusion

SUN-LB35 Using Chromogranin A to Unmask the Great Masquerader: A Case Reportof a Minimally Symptomatic Pheochromocytoma

Abstract Introduction/Background: Pheochromocytomas are rare neoplasms, occurring in less than 0.2% of the population. The classic triad of symptoms includes hypertension, headaches and sweating. An increasing number of patients with pheochromocytomas that are diagnosed at a pre-symptomatic stage.Clinical Case A 69 yo male being worked up for hematuria and possible bladder cancer was referred to endocrinology due to a 3 cm x 3 cm right adrenal “incidentaloma.” The mass was noted to be heterogeneously enhancing on CT adrenal protocol, suggesting pheochromocytoma, adrenal carcinoma or metastasis. He endorsed no clinical symptoms of hormone excess and blood pressure was controlled HCTZ. He was found to have elevated serum metanephrines (184 pg/mL, nl &amp;lt;57) and noremetanephrine (608 pg/dL, nl &amp;lt;148) and total metanephrines (792 pg/dL, nl &amp;lt;205). Other hormonal determinations were normal including cortisol (9AM, 8.97 ug/dL, nl 3-22), DHEAS 66 mcg/dL (25-240), estradiol 23 pg/mL (&amp;lt;39), 17 OHD 60 ng/dL, ACTH 37 (6-50), free testosterone 43.6, total testosterone 396, androstenedione 63 (20-220). Further testing revealed 24 hour urine metanephrine and catecholeamines; urinary metanehrine 2025, noremetanephrine 1729, total 2442 and epinephrine 48, norepinephrine 336 and dopamine 112 which were 2-3 times the upper limit of normal. Because the patient consumed significant amount of caffeine (10 cups of coffee and cola daily), these studies were repeated after caffeine washout. Repeat serum testing revealed metanephrine at the upper limit of normal (metanephrine 47, noremetanphrine 146, respectively). Chromogranin A levels were elevated before (626 units) and after PPI discontinuation, (539 units, nl 25-140). Given the biochemical results and size of mass, he was referred him surgical resection. He was treated pre-operatively with doxazosin and underwent an uncomplicated right adrenalectomy via posterior right retroperitoneoscopic. Pathology was consistent with pheochromocytoma without capsular invasion. Immunohistochemical stains were characteristic of pheochromocytoma, positive for synaptophysin, chromogranin, and S100 but negative for Cytokeratin AE1/AE3 and calretinin. Conclusion: We present the case of a patient with a clinically asymptomatic pheochromocytoma. The diagnosis was supported by elevations in chromogranin and catecholamine metabolites, although the latter results were mixed and clouded by potential confounding factors such as heavy caffeine intake. The case demonstrates that with early detection, screening should include patients with no symptoms and otherwise low risk of disease. Decisions on surgical resection should be based on clinical suspicion, symptoms, imaging, tumor size and biochemical findings given the aggressive nature of these tumors and their malignant potential.</jats:p

Secondary recurrent multiple EGIST of the mesentary: A case report and review of the literature

AbstractINTRODUCTIONGastrointestinal stromal tumors (GISTs) are rare intra-abdominal tumors arising from mesenchymal stromal cells. EGISTs are mesenchymal tumors that originate outside the GI tract and tend to have similar characteristics to GISTs. To the best of our knowledge, few cases of long standing recurrent EGIST have been reported.PRESENTATION OF CASEWe present the case of a rare recurrent EGIST in the mesentery of a 39 year old female patient. The tumor was symptomatic at the time of complaint and measured 8.4cm×7.7cm×7.6cm. Histological analysis revealed a spindled pattern with fusiform cells arranged in long fascicles and little atypia. Immunochemistry showed positivity for CD117 and was negative for CD34, S-100, Desmin, and MSA. B-catenin was weakly positive. A Ki-67 staining shows approximately 5% positivity revealing a low proliferative rate. The patient was doing well postoperatively and was discharged on 400mg imanitib regimen.DISCUSSIONWhile GISTs are the most common tumors of the GI tract, recurrent EGISTs of the mesentery are extremely rare. Factors that indicate poor prognosis include tumor size greater than 5cm, mitotic rate greater than 1–5/10 HPF, presence of tumor necrosis or metastasis and most recently the c-kit mutation. Our patient had a very long time between recurrence of disease.CONCLUSIONThe current literature on EGISTs is limited. Our patient presents a very interesting case due to the time elapsed between disease recurrence and lack of metastasis or excessive growth

Abstract 579: De-differentiation of Somatic Fibroblasts by Mouse Embryonic Stem Cell-free Extracts: Multi-lineage Re-differentiation and Therapeutic Efficacy of Re-programmed Cells in Mouse Models of Hind limb and Acute Myocardial Ischemia

Background: De-differentiation of adult somatic cells into multipotent progenitor cells might provide an attractive, oocyte-independent alternate source for therapeutic cloning to generate pluripotent, autologous stem cells for regenerative medicine. We tested the hypothesis that exposure of reversibly permeabilized NIH3T3 fibroblasts to mouse embryonic stem cell (mES) extracts may provide regulatory molecules required for epigenetic changes leading to re-programming of terminally differentiated somatic cells and that such reprogrammed cells may display multi-lineage differentiation. Methods and Results: Streptolysin O-permeabilized NIH3T3 cells were exposed to mESC extracts in ATP-regenerating system. Plasma membrane was resealed by culturing in medium 2mM CaCl2. We report that upon exposure to mES extracts 3T3 cells undergo dedifferentiation as evident from morphological changes, induction of mES specific and the loss of lamin A/C, a specific marker of the soma. At epigenetic level, mES extract treatment induced de-methylation of Oct4 promoter and histone3 hyperacetylation at lysine 9 residue, as evident from restriction enzyme mapping, bisulphite genomic sequencing and chromatin immuno-precipitation experiments. Under defined culture conditions in vitro, reprogrammed 3T3 cells can be differentiated into neuronal, adipocyte, cardiomyocyte (CMC) and endothelial cells (EC). Moreover, transplantation of labeled reprogrammed 3T3 cells into mouse hind limb ischemia and acute myocardial infarction models resulted in the gain of physiological functions and showed evidence of acquiring the expression of muscle, EC and CMC specific proteins, in vivo. Moreover, when injected in SCID mice, reprogrammed cells formed teratomas. Conclusion: Taken together our biochemical, molecular and functional data provide a novel and oocyte- independent approach for the generation of functional autologous stem like cells from terminally differentiated somatic cells which may potentially be of therapeutic application in regenerative medicine.</jats:p

Cell-free Embryonic Stem Cell Extract-mediated Derivation of Multi-potent Stem Cells from NIH3T3 Fibroblasts for Functional and Anatomical Ischemic Tissue Repair

AbstractThe oocyte-independent generation of multipotent stem cells is one of the goals in regenerative medicine. We report that upon exposure to mouse ES cell (ESC) extracts, reversibly permeabilized NIH3T3 cells undergo de-differentiation followed by stimulus-induced re-differentiation into multiple lineage cell types. Genome-wide expression profiling revealed significant differences between NIH3T3 and ESC-extract treated NIH3T3 cells including re-activation of ESC specific transcripts. Epigenetically, ESC extracts induced CpG de-methylation of Oct4 promoter, hyper-acetylation of histones 3 and 4 and decreased lysine 9 (K-9) dimethylation of histone 3. In mouse models of surgically-induced hind limb ischemia (HLI) or acute myocardial infarction (AMI) transplantation of reprogrammed NIH3T3 cells significantly improved post-injury physiological functions and showed antomical evidence of engraftment and trans-differentiation into skeletal muscle, endothelial cell and cardiomyocytes. These data provide evidence for the generation of functional multi-potent stem like cells from terminally differentiated somatic cells without the introduction of trans-genes or ESC fusion. </jats:p