Pietro Iacconi

Fine-needle aspiration of thyroid nodules: Proteomic analysis to identify cancer biomarkers

At present, the clinical and pathological analysis used in the diagnosis of papillary thyroid cancer (PTC) are insufficient to discern tumor behavior, and new diagnostic and prognostic markers need to be identified. In this study, we performed a comparative proteome analysis to examine the global changes of fine needle aspiration fluid (FNA) protein patterns of two variants of malignant PTC (classical variant PTC (cPTC) and tall cell variant PTC (TCV)) with respect to the controls. Changes in protein expression were identified using two-dimensional electrophoresis (2DE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS), as well as Western blot analysis. A statistical significant up-regulation of 17 protein spots in cPTC and/or TCV with respect to controls was demonstrated. These proteins included transthyretin precursor (TTR), ferritin light chain (FLC), proteasome activator complex subunit 1 and 2, alpha-1-antitrypsin precursor, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase chain B (LDH-B), apolipoprotein A1 precursor (Apo-A1), annexin A1, DJ-1 protein and cofilin-1. In addition, 12 protein spots were found exclusively in cPTC and three exclusively in TCV. These latter proteins (ferritin heavy chain (FHC), peroxiredoxin 1 (PRX1) and 6-phosphogluconate dehydrogenase (6-PDGH)) correspond to stress response proteins and, until now, had not been described in thyroid tumors. These findings illustrate the potential use of FNA proteomics to identify protein changes associated with thyroid cancer and to advance potential protein biomarkers in the diagnostic classification of the disease.

Early treatment of hereditary medullary thyroid carcinoma after attribution of multiple endocrine neoplasia type 2 gene carrier status by screening for ret gene mutations.

BACKGROUND Germline missense point mutations of the ret proto-oncogene have been shown as causative in multiple endocrine neoplasia type 2 (MEN 2A and 2B) and in familial medullary thyroid carcinoma (FMTC). Most of the mutations are found in exon 10, 11, or 16 of the gene and are easily recognized by restriction analysis. METHODS Using restriction analysis, we screened 58 subjects from nine kindreds. RESULTS Family members (n = 16) already known to be affected with the disease carried the germline mutation. Among the 42 subjects apparently unaffected, 37 were not gene carriers and 5 were gene carriers. Basal and pentagastrin-stimulated serum calcitonin levels were normal in two patients and abnormal in three. All patients were treated with total thyroidectomy and central node dissection. In all cases multiple foci of MTC were shown at histologic examination. CONCLUSIONS Our data indicate that genetic screening of MEN2 pedigrees allows the early identification of gene carriers. Because surgery of MTC in the preclinical phase has high probability of curing these patients, we suggest genetic screening soon after birth and total thyroidectomy in gene carriers as early as possible.

Disappearance rate of serum calcitonin after total thyroidectomy for medullary thyroid carcinoma.

We studied the half-life of serum calcitonin (CT) in patients subjected to total thyroidectomy for medullary thyroid carcinoma (MTC). One patient showed a rapid serum CT component with a half-life of 3 hours and a slow component with a half-life of 30 hours; in another case only the 30-hour component was found. By chromatography of tumor extracts, we found that all the immunoreactive CT had a molecular weight of 3,600. After surgery, normalization of serum CT was achieved within 15 days in 4 patients, at 3 months and at 6 months in 2 other patients, while 1 patient never normalized. Normalization of serum CT after surgery is not an index of definitive cure in MTC, as demonstrated by one patient who relapsed 3 months after normalization of serum CT. However, as a general rule, patients who reach undetectable serum CT levels soon after surgery, are those having the best prognosis.

Thyroid carcinoma in intrathoracic goiter

Introduction: Most cases of intrathoracic goiter can be managed by cervical incision alone. A thoracic approach may be needed when adhesions or an anomalous blood supply are present or carcinoma is suspected. Patients and methods: Only 44 patients out of 5263 operated on for goiter needed a thoracic incision. A sternotomy was performed in 29 cases and a thoracotomy in 15; a malignancy was present in 9 cases. Symptoms, surgical approach, histology, survival and pTN staging of these 9 patients were reviewed and discussed; no perioperative mortality was observed. Discussion: A thoracic approach is more frequently needed for treatment of intrathoracic thyroid carcinoma as it offers a greater chance of radical excision and better control of intraoperative bleeding. Histologically, thyroid carcinoma in intrathoracic goiter is often anaplastic or rare and has a poor long-term survival rate when compared to cervical forms.

Prevalence of cancer in follicular thyroid nodules: is there still a role for intraoperative frozen section analysis?

The optimal surgical management of follicular thyroid nodules and the effectiveness of frozen section (FS) analysis in planning the operation are still controversial. In this study, we reviewed the prevalence of cancer in 309 consecutive patients (230 females, 79 males, aged 42 +/- 13 years) with follicular nodules at fine-needle aspiration cytology (FNAC) and the efficacy of FS evaluation in selecting cancers. On the whole, the prevalence of cancer was 20.1%; the occurrence of follicular variants of papillary cancer (14.9%) was threefold higher than follicular cancers (5.2%). The presence of atypical features at FNAC selected nodules with a significantly higher prevalence of cancer (46.7%, p = 0.01). FS analysis was performed in 142 patients and recognized only 8 of 27 (30%) cancers, one fourth of them with atypia at FNAC. In conclusion, this study confirms a 20% overall prevalence of malignancy in patients with follicular thyroid nodules. The association of cell atypia with a follicular pattern may define a subgroup of nodules more likely to be malignant. FS was seldom effective in recognizing cancer confirming the doubt on its cost effectiveness in planning the surgical approach. Protocols aimed at a better cytologic identification of follicular variants of papillary cancer should be considered.

Sixty Adrenal Masses of Large Dimensions: Hormonal and Morphologic Evaluation

OBJECTIVES To evaluate the nature and function of adrenal masses of large dimensions (macrotumors). METHODS Sixty consecutive patients (31 women, 29 men, age range 15 to 84 years) with adrenal masses 4.0 cm in diameter or larger (range 4.0 to 15.0 cm) underwent morphologic study by computed tomography (CT); the majority also underwent 131-I-6beta-norcholesterol (131I-NC) or 131I-MIBG scintigraphy. Basal evaluation of glucocorticoids, mineralcorticoids, and catecholamines was performed in all patients, and in 38 cases determination of androgens was also made. In addition, on the basis of various clinical suspicions, a dynamic hormonal study was performed. RESULTS Macrotumors were benign in 78.3% of cases and included pheochromocytomas (n = 17), nonfunctioning cortical adenomas (n = 12), and cortisol-secreting tumors (n = 7, Cushings syndrome). Malignant forms were 21.7% of the total, including pheochromocytomas (n = 3), cortical carcinomas (n = 6), and metastases (n = 4). On CT, malignant masses were larger (8.4+/-0.9 cm) than benign ones (5.7+/-0.3 cm) (P < 0.0001) and the mass size was strictly related to malignancy (P < 0.03). CT did not offer other diagnostic criteria for malignancy, except irregular margins and regional lymph node enlargement, which were more frequently (P < 0.0001) found in malignant forms. 131I-MIBG scintigraphy showed tracer uptake in all pheochromocytomas, both benign and malignant. By contrast, on 131I-NC scintigraphy, cortical malignancies never accumulated the radiotracer, whereas uptake was observed in all cases of solid cortical benign adenomas. Patients with cortical carcinomas showed plasma sex steroids above the normal range, pheochromocytomas were asymptomatic in 15% of cases, and almost half of the patients with Cushings syndrome did not show clinical features of the disease (pre-Cushings syndrome). CONCLUSIONS Adrenal macrotumors frequently show endocrine activity and the medulla seems to be involved more than the cortex. Pheochromocytomas and cortisol-secreting adenomas are sometimes asymptomatic. Malignancy is often found in macromasses and, at least for the cortical forms, size of the tumor on CT, 131I-NC uptake on scintigraphy, and determination of levels of sex steroids seem to be useful criteria for predicting the nature of the mass.

Apoptosis control and proliferation marker in human normal and neoplastic adrenocortical tissues

We evaluated by immunohistochemistry the expression of the Bcl-2 and p53 proteins, as markers of apoptosis control, and of MIB-1, as a marker of cell proliferation, in a series of normal and neoplastic adrenocortical tissues. The specimens were 13 normal adrenals, 13 aldosterone-producing adenomas, 13 non-functioning adenomas and 16 carcinomas. Results were calculated as percentage of immunostained cells by using specific antibodies. No p53 protein was detected in any of the adrenocortical adenomas (functioning and non functioning) or normal adrenals, while p53 was overexpressed in 15 out of 16 carcinomas. In particular, 10 adrenal cancer specimens (62.5%) showed strong staining in a high percentage (range 10–50%) of the malignant cells. The percentage of Bcl-2 positive cells was higher (P<0.05 or less) in non-functioning adenomas (8.1±1.9%) and in carcinomas (14.9±5.6%) than in normals (2.9±0.9%) and aldosterone-producing adenomas (5.3±1.3%) since four specimens of the non-functioning adenomas-group (30.7%) and six of the carcinomas-group (37.5%) showed over 10% positivity (cut-off for normal values, set at 90th percentile of our controls). MIB-1 positivity was 0.50±0.36% in normals, 0.54±0.08% in non-functioning adenomas and 0.54±0.08% in aldosterone-producing adenomas. MIB-1 was expressed in all carcinomas with values (13.7±3.1%) significantly (P<0.0006) higher than in the other groups. In conclusion, the present data indicate that the apoptosis control and proliferation activity evaluated by the p53 and MIB-1 proteins are impaired in adrenal carcinomas but preserved in adenomas, independently of their functional status. Therefore, these immunohistochemical markers, overexpressed in carcinomas only, may be useful in the diagnosis of malignancy in adrenocortical tumours. Whether Bcl-2 positivity found in some carcinomas and non-functioning adenomas may constitute, in the latter, a negative prognostic marker is still unknown.

Nonrecurrent laryngeal nerve predicted before thyroidectomy by preoperative imaging.

A 48-year-old woman, complaining of dyspnea on exercise and dysphagia, was referred to us for a multinodular goiter. A CT scan excluded a substernal thyroid mass but demonstrated the abnormal course of the brachiocephalic artery in a very posterior position behind the esophagus. The angio-MRI (A) confirmed the presence of an abnormal right brachiocephalic artery, arising from the aortic arch beyond the left subclavian artery and crossing to the right behind the trachea and esophagus, just in front of the vertebrae (arteria lusoria). On the basis of preoperative imaging, the right nonrecurrent laryngeal nerve was predicted. The usual collar incision was made, the right lobe mobilized, and the upper pole vessels ligated. The tracheoesophageal groove was explored, and the nonrecurrent laryngeal nerve (B, arrowhead) was found crossing transversally from the carotid sheath and entering the larynx under the inferior border of the inferior constrictor muscle. A total thyroidectomy was carried out, the postoperative course was uneventful, and the patient was discharged 2 days after operation. The nonrecurrent inferior laryngeal nerve is a rare occurrence in thyroid surgery (0.6%); the risk of injury in the presence of this anatomic variant is high, even for experienced endocrine surgeons. The right nonrecurrent laryngeal nerve is strictly associated with an aberrant brachiocephalic artery that sometimes is symptomatic (dysphagia). Preoperative imaging, such as barium swallow, CT, or angio-MRI, easily predicts this rare anomaly. Careful, complete dissection of the nerve is always advocated during cervicotomy.

A proteomic profile of washing fluid from the colorectal tract to search for potential biomarkers of colon cancer

Washing fluid (WF) from the colon rectal tract after surgical resection might represent a first step in obtaining a mixture of proteins derived from the secretion of tumoral epithelial cells potentially involved in the pathological progression of tissue. In this study, we performed a proteomic analysis of colorectal WF to search for potential biomarkers of colon cancer. The outcome of this approach might open the possibility of using WF to screen for the precancerous and early stages of colorectal cancer (CRC). Samples of WFs were obtained during surgery from 35 patients submitted to colon resection for suspicious adenocarcinoma or carcinoma, while the respective controls were obtained by washing the healthy sections. WFs were immediately centrifuged, concentrated and trichloroacetic acid (TCA) was added to obtain protein pellets. After two-dimensional gel electrophoresis (2DE), the protein patterns of malignant samples were compared with respective normal samples. Forty-one protein spots were found to be differentially expressed exhibiting ≥2 fold-change of mean value spot intensities. After mass spectrometry, these protein spots collapsed into 38 different proteins. Interestingly, 19 of the differentially expressed proteins identified in the study corresponded to those suggested as being potential biomarkers of CRC. In accordance with the literature, these proteins showed the same direction of change (up or down for all proteins). Our results suggest that WF has the potential of being a method for the exploration of clinical samples for biomarker and drug target discovery.

Rapid Reversal of Severe Lactic Acidosis After Thiamine Administration in Critically Ill Adults A Report of 3 Cases

BACKGROUND Thiamine plays a critical role in energy metabolism. Critically ill patients may have thiamine deficiency and increased mortality due to potentially irreversible consequences. The aim of this study was to show the impact of thiamine deficiency in a series of patients and the rapid response to thiamine replacement, showing the changes in clinical and metabolic conditions over time. METHODS We described 3 cases of hospitalized patients who had received parenteral nutrition (PN) without vitamin supplementation. All the patients were admitted to the ICU between 2010 and 2011 with a severe form of lactic acidosis, an unstable circulatory state, and a different neurological disorder (a lethargic state, a severe form of impaired near-coma consciousness, and Wernicke encephalopathy). RESULTS Intravenous (IV) administration of thiamine was associated with a rapid and marked restoration of acid-base balance, hemodynamic stability and the disappearance of neurological disturbances, and normalization of the clinical and biochemical conditions of all the patients within the following hours. CONCLUSIONS The 3 cases demonstrated the rapidity of the reversal of severe thiamine deficiency, achieved by appropriate replacement in different hospitalized patients. The regression of clinical and biochemical disorders requires a prompt diagnosis and treatment based on the IV administration of thiamine and magnesium sulfate. In hospitalized patients at risk, thiamine deficiency is prevented by the integration of thiamine supplementation into PN and other forms of nutrition support.