Prospective assessment of AR splice variant and PSMA detection on circulating tumor cells of mCRPC patients: preliminary analysis of patients enrolled in PRIMERA trial (NCT04188275).

In our institution, a prospective observational trial testing micro-RNA (miRNA) and ARV7 mutational status in metastatic, castration resistant prostate cancer (mCRPC), is currently recruiting (PRIMERA trial, NCT04188275). A pre-planned interim analysis was performed when 50% of the planned accrual was reached. In this report, we explored the predictive value of Circulating Tumor Cell (CTC) detection in mCRPC patients undergoing 1st line therapy. Moreover, ARV7, ARFL, PSMA and PSA expression on CTC was reported to explore potential correlation with patient prognosis and response to therapy. PRIMERA is a prospective observational trial enrolling mCRPC patients undergoing standard treatment (ARTA + ADT) after I line ADT failure. Clinical and pathological features were collected. Outcomes selected for this preliminary analysis were time to castration resistance (TTCR), PSA at 8 weeks after ARTA therapy start, PSA drop at 8 weeks, Overall PSA drop, PSA nadir. Correlation between these outcomes and CTC detection was tested. Expression of ARV7, ARFL, PSA and PSMA was explored in CTC+ patients to assess their prevalence in this cohort and their impact on selected outcomes. Median TTCR was significantly shorter in CTC+ vs CTC- patients (32.3 vs 75 months, respectively, p = 0.03) and in ARFL+ vs ARFL- patients (30.2 vs 51.1 months, respectively, p = 0.02). ARV7, PSMA and PSA expression on CTC had no impact on median TTCR, nor on biochemical response to therapy. Patients in whom CTC and ARFL expression were detected had significant reduced TTCR. However, PSA response was not influenced by CTCs detection and specific biomarkers expression.

Cognitive and functional status in the extreme longevity.

Usually, the effects of cognitive decline are not noted before the age of 70 years, which involve the intellectual capacities, the attention, the processes of elaboration and the memory. The studies on the cognitive disturbances of the elderly are numerous, and document the progressive increase of cerebral deterioration with advancing age. However, only a few studies refer to the significance of the cognitive disturbances in the clinical conditions and autonomy of the long living subjects. For this reason, we studied the cerebral deterioration of an adequate number of centenarians in correlation with their clinical conditions and autonomy. Our centenarian sample derived from the Italian multi-center study on centenarians (IMUSCE), which was an epidemiological study which identified 1173 centenarians (202 males, 971 females) in the age range of 100-109 years. From this sample, we analyzed 346 subjects as far as the cognitive functions and the degree of autonomy by using the psychometric tests of the mini-mental state examination (MMSE) and the instrumental activities of daily living (IADL) for the functional evaluations. In addition, we evaluated the clinical conditions of the subjects dividing them in three groups: Group A (those in good clinical conditions), Group B (those in discrete clinical conditions), and Group C (those in deteriorated clinical conditions). These analyses revealed that 187 (54.1%) of the 346 examined centenarians have shown an MMSE score in the normal range (score ratio from 1.0 to 0.63). The cognitive disorders are present in the centenarians in a clearly higher frequency (13.1%), than found in the common elderly (5.1%). The severe cognitive disorders do not allow a total autonomy or even a slight dependency. Only six subjects (1.7%) of the total sample were totally independent. These subjects had no cognitive disorders, and were in good clinical conditions. The results show that having an MMSE score in the normal range, and being in good clinical conditions are necessary but not sufficient prerequisites for a total autonomy in the IADL scores.

Physical performance and creative activities of centenarians.

The human functional autonomy is reduced progressively with advancing age, although a considerable proportion of the centenarians maintain a good level of autosufficiency for the basic performance of the everyday life. Even if males have a lower probability to reach the centenarian age than females do, the male centenarians display generally better functional conditions than the females. Actually, there are no systemic studies on centenarian works or activities; nevertheless, the examples of several representative persons (artists, scientists, explorers, etc.) who remained active even after this age indicate that such a possibility is realistic, and that the maintenance of vital interest and passions, thus preserving competence and professional attitudes, is not in conflict with the reaching of extreme longevity.

Human gastric epithelium produces IL-4 and IL-4delta2 isoform only upon Helicobacter pylori infection.

Recent evidence suggests that interleukin-4 (IL-4) is related to mucosal tolerance by which an injurious immune response is prevented, suppressed or shifted to a non-injurious response. We investigated the expression of IL-4 and its splice variant isoform IL-4delta2 in gastric epithelial cells of healthy subjects and gastritis patients infected with Helicobacter pylori (H. pylori) with or without the cag pathogenicity island (cag-PAI). IL-4 and IL-4delta2 mRNAs were evaluated in microdissected gastric epithelium and in AGS cell lines co-cultured with H. pylori B128 or SS1 strains. IL-4 mRNA was consistently detected in microdissected gastric epithelial cells from healthy subjects. The IL-4 mRNA expression was low in H. pylori?infected patients, and markedly reduced in cag-PAI-positive ones. IL-4delta2 mRNA was expressed on gastric epithelium of H. pylori-infected patients, but not in healthy subjects. The IL-4delta2 expression was lower in cag-PAI-positive than in cag-PAI-negative H. pylori infected patients. AGS cells also produced IL-4 mRNA upon SS1 strain stimulation, whereas IL-4delta2 mRNA expression was detected in AGS co-cultured with either SS1 or B128 strains. An inverse correlation was documented between IL-4 and IL-4delta2 mRNA expression by microdissected gastric epithelial cells and the score of gastritis. IL-4, but not IL-4delta2, is expressed by gastric epithelium of healthy subjects, whereas IL-4delta2 and lesser IL-4 mRNA are detectable in the gastric epithelium of H. pylori-infected patients. Data suggest that gastric epithelial cells might regulate the balance between tolerance and immune response by the fine tuning of IL-4 and IL-4delta2 expression.

Longevity index (LI%) and centenarity index (CI%): new indicators to evaluate the characteristics of aging process in the Italian population.

Centenarians represent a group of population displaying the most rapid expansion. This progressive increase of the presence of centenarians is a multi-factorial phenomenon, which is due to the improvements of the environmental conditions and the life style and also to the progress of the medical science. In order to obtain a more reliable estimate of the longevity per gender and territorial entities, we propose two new indicators. (i) The ratio between the ultranonagenarians and the total population above 65 years old (called longevity index: LI%); (ii) the ratio between the centenarians and the total population above 90 years old (called centenarity index: CI%). An analysis of the data of the Italian National Office of Statistics (ISTAT, ) using these two indicators demonstrates that the subjects above the age of 90 are more frequent in the regions of Central and Northern Italy, which are more developed regarding the economic conditions and technological progress. Nevertheless, the Southern and Insular regions of Italy have a higher occurrence of centenarians among the ultranonagenarian population, and also a higher prevalence of male centenarians, as compared to the northern regions. This demonstrates that achievement of the threshold of 100 years old does not require only particular socio-economic conditions, but also an adequate climate and environment, as well as a favorable genetic composition.

Characterization of the functional and growth properties of long-term cell cultures established from a human somatostatinoma.

In somatostatinoma, a rare malignant somatostatin (SST)-secreting neoplasia, tumour regression is rarely observed, implying the need for novel antiproliferative strategies. Here, we characterized a long-term culture (SST-secreting cancer (SS-C cells)) established from a human somatostatinoma. High concentrations of SST and chromogranin A were released by SS-C cells and SST release was stimulated by depolarizing stimuli and inhibited by the SST analogue, octreotide. SS-C cells expressed mRNA for SST receptor (SSTR) subtypes 1, 2 and 4, being also able to bind native SST. Moreover, SS-C cells were positively stained with an antibody to SSTR2. SS-C cells also expressed interferon-gamma (IFN-gamma) receptor mRNA and measurable telomerase activity. Our findings indicate that in vitro exposure of SS-C cells to native SST-28, to octreotide, to IFN-gamma, or to 3'-azido-3'deoxythymidine (AZT), a telomerase inhibitor, results in inhibition of SS-C cell proliferation. Concomitant with growth inhibition, apoptosis was detected in SST-, octreotide-, IFN-gamma- or AZT-treated SS-C cell cultures. Taken together our results characterized native SST, SST analogues, IFN-gamma and a telomerase inhibitor as growth-inhibiting and proapoptotic stimuli in cultured human somatostatinoma cells. Based on these findings, the potential of SST analogues, IFN-gamma and AZT, alone or in combination, should be further explored in the medical treatment of somatostatinoma.

Laser-assisted microdissection for real-time PCR sample preparation.

Laser-assisted microdissection (LMD) has been developed to procure precisely the cells of interest in a tissue specimen, in a rapid and practical manner. Together with real-time PCR and RT-PCR techniques, it is now feasible to study genetic alterations, gene expression features and proteins in defined cell populations from complex normal and diseased tissues. The process that brings from sample collection to the final quantitative results is articulated in several steps, each of which requires optimal choices in order to end up with high-quality nucleic acid or protein that allows successful application of the final quantitative assays. This review will describe shortly the development of LMD technologies and the principles they are based on. Trying to highlight the advantages and disadvantages of LMD, the main problems related to specimens collection and processing, section preparation and extraction of bio-molecules from microdissected tissue samples have been analysed.

SDH mutations in patients affected by paraganglioma syndromes: a personal experience.

Mutations in genes encoding mitochondrial succinate dehydrogenase (SDH) are frequently involved in the development of neural crest-derived (NCD) tumors, such as pheochromocytomas (PHEOs) or paragangliomas (PGLs). In this study we report the results of sequencing analysis in leukocyte DNA of patients affected by PHEO/PGL who turned out to be SDH mutation carriers. A nonsense germline heterozygous mutation (Q109X) was found in the exon 4 of the SDHD gene in the index cases of six unrelated families affected by PHEO/PGL. Haplotype analysis showed the presence of a founder effect. Affected patients showed high clinical variability, ranging from monolateral to bilateral glomus tumors, variably associated or not with PGLs or PHEOs. A novel missense SDHD variant, T112I, was also found in one of our families. A new missense G106D mutation, involving a highly conserved amino acid, was found in two sisters affected by bilateral glomus tumors. A P81L mutation associated with abdominal and head and neck PGL was detected in three families. A G12S variant of the SDHD gene was found in one patient affected by a PHEO. The finding of this variant in 3 of 100 control subjects suggests that it is a polymorphism and not a mutation. A novel IVS2-1G>T variant was found at intron 2 of SDHD gene in one patient affected by a glomus tumor. All the tumors associated with SDHD mutations were benign. Conversely, the only mutation we found in SDHB gene (IVS3+1G>A) was associated with a malignant PHEO.

Phenotype variability of neural crest derived tumours in six Italian families segregating the same founder SDHD mutation Q109X.

BACKGROUND: Mutations in genes coding for the mitochondrial complex II succinate dehydrogenase (SDH) subunits cause familial neural crest derived (NCD) tumours. METHODS: Index cases from six apparently unrelated families affected by NCD tumours were analysed for mutations in the SDHB, SDHC, and SDHD genes. RESULTS: The same nonsense germline heterozygous mutation (Q109X) in exon 4 of the SDHD gene was found in each of the six families. Overall, 43 heterozygotes were identified. These were evaluated for the presence of NCD tumours through radiological examination of the neck, thorax, and abdomen, and measurement of urinary metanephrines and plasma chromogranin A. A novel missense SDHD variant, T112I, which did not segregate with the Q109X mutation and was not associated with phenotypic manifestations, was observed in one of the families. Microsatellite analysis showed a common haplotype in all individuals heterozygous for the Q109X mutation, indicating a founder effect. Overall, 18 heterozygotes were clinically affected by at least one NCD tumour. Every affected patient inherited the germline mutation from the father, confirming SDHD maternal genomic imprinting. Penetrance of the paternally inherited mutation progressively increased from 33% to 83% at 30 and 60 years, respectively. Affected patients showed high clinical variability, ranging from monolateral to bilateral glomus tumours variably associated or not with paragangliomas or phaeochromocytomas. Loss of heterozygosity was observed in tumour cells isolated by laser capture microdissection. CONCLUSIONS: This study shows that a single founder SDHD mutation is present in an area of central Italy and that this mutation is associated with widely variable interfamilial and intrafamilial expressivity.

Effect of diet and red wine consumption on serum total antioxidant capacity (TAC), dehydroepiandrosterone-sulphate (DHEAS) and insulin-like growth factor-1 (IGF-1) in Italian centenarians.

The traditional mediterranean diet is associated with a hope for longer survival. It has also been shown that the red wine possesses a protective effect against the oxidative stress. We studied TAC, the DHEAS and the IGF-1 in a group of 26 healthy centenarians, 17 women and 9 men, of the age range of 100--105 years. Furthermore, we analyzed also serum urate and bilirubin levels between drinkers and abstainers. Most of centenarian subjects have been moderate wine consumers (<500 ml/day of red wine). These subjects were subdivided as follows: (i) Group A: those who had maintained the style of their dietary habits as compared to the previous years (n=3 males, 10 females); (ii) Group B: those who actually consumed a diet being deficient compared to that of the previous years, but remained moderate drinkers of red wine (n=3 males, 4 females); and (iii) Group C: those who actually consumed a diet being deficient compared to that of the previous years, and at the same time, were abstainers in wine consumption (n=3 males, 3 females). The results show that in men three of the studied parameters decreased from Group A to C to considerable extents, as follows (mean+/-S.D.). TAC: 302.4+/-32.3; 142.0+/-24.1 and 96.4+/-20.1 micromol/l; DHEAS: 3.35+/-0.81; 2.52+/-0.18 and 1.34+/-0.14 micromol/l; IGF-1: 85.7+/-6.7; 76.6+/-6.7 and 65.6+/-2.6 ng/ml, respectively. For the same parameters, the results in the women were: TAC: 258.4+/-12.2; 182.1+/-14.0 and 107.6+/-10.0 micromol/l; DHEAS: 3.85+/-0.16; 2.34+/-0.19 and 2.05+/-0.04 micromol/l; IGF-1: 89.7+/-6.7; 76.6+/-4.7 and 64.2+/-2.7 ng/ml, respectively. We did not find any significant difference in the other serum parameters between drinkers (n=14) and abstainers (n=3) (urate: 267.6+/-52.9, and 289.5+/-80.1; bilirubin: 9.81+/-4.29 and 7.18+/-2.89 micromol/l, respectively). Our data suggest that the deteriorated diet caused a reduction of TAC, DHEAS and IGF-1 in the centenarians. However, red vine consumption exerted a protective effect against this trend, even if this protection is not reaching statistical significance in some cases (in men), which is due most probably to the lower number of male subjects in the study.

Measurement of somatostatin receptor subtype 2 mRNA in breast cancer and corresponding normal tissue.

Somatostatin analogs are effective in inhibiting growth of human breast cancer cell lines. These antiproliferative effects are mediated by specific receptors located on cell membranes. The somatostatin receptor subtype 2 (sst2) is the principal mediator of somatostatin effects in normal and cancer cells, and its presence has already been demonstrated in breast cancer. The purpose of our study was to evaluate the clinical relevance of the expression of sst2 by quantifying its mRNA in a large group of infiltrating breast cancers and their corresponding normal tissues. The expression of sst2 mRNA was measured with quantitative real time RT-PCR in 169 breast cancers and in their corresponding unaffected tissues. We evaluated the association of sst2 expression with the commonest clinical-pathologic features of breast cancer. The correlation with a marker of cell proliferation (Ki-67) and with receptor concentration was also evaluated. In cancer tissues, we found that the absolute concentrations of sst2 mRNA were significantly higher in estrogen receptor (ER)-positive samples (P=0.002) as well as in lymph-node-negative cancers (P=0.04) (Student's t-test or one-way ANOVA). In addition, sst2 mRNA was significantly higher in breast cancers than in corresponding unaffected tissues (P=0.0002). However, when the clinical-pathologic parameters were considered, this gradient maintained its statistical significance only in tumors expressing positive prognostic markers, such as the presence of ER (P=0.0005) and progesterone receptors (PgR) (P=0005), and the lack of lymph-node involvement (P=0.0003). The same difference was also significant in postmenopausal women (P=0.001) and in T1 patients (P=0.001). In addition, sst2 mRNA expression was significantly higher (P=0.008) in low-proliferating breast cancers. Finally, we found that the quantitative expression of sst2 mRNA was directly related to the PgR concentration in breast cancer tissues (P<0.001). Our data seem to indicate that an upregulation of sst2 gene expression is a common feature of breast cancers which, on the basis of conventional predictive parameters, are expected to have a better prognosis. Featuring a possible role of somatostatin analogs in combined endocrine therapies for breast cancer, our results seem to confirm that the sst2 status of the tumor should be previously investigated.

Adenosine triphosphate release by osmotic shock and hemoglobin A1C in diabetic subjects' erythrocytes.

We investigated the significance of adenosine triphosphate (ATP) release from diabetic subjects' red blood cells (RBCs) following osmotic shock (OS) and its possible relationship with hemoglobin A1C (HbA1C) and with the RBC membrane protein skeleton. RBCs from type I (insulin-dependent [IDDM]) and type II (non-insulin-dependent [NIDDM]) diabetic subjects and age- and sex-matched control subjects were submitted to OS using NaCl solutions (from 0.9% to 0.045% final concentration). ATP release values were determined by the bioluminescent method. For pattern study, they were expressed both as absolute values and as percentages (%) of ATP maximum release (at 0.045% NaCl solution). Twenty-seven IDDM and 25 NIDDM subjects and two control groups were investigated. ATP content in RBCs was 2.08 +/- 0.19 pmol/10(4) RBC in IDDM and 1.23 +/- 0.20 pmol/10(4) RBC in NIDDM subjects. The ATP content of IDDM subjects' RBCs was significantly higher than that of the corresponding control group. ATP release at 0.49% NaCI OS, both as absolute value and as percentage value, was significantly lower in both diabetic groups, and ATP% was inversely correlated with HbA1" (IDDM: r = -.489, P < .01; NIDDM: r = -.654, P < .01), suggesting a possible relationship between Hb glycation, RBC membrane protein skeleton glycation, and its influence on ATP release by OS. In conclusion, the proposed method seems useful for measuring RBC ATP content and, at the same time, for monitoring the leak effect of the RBC membrane before it bursts.

Type-2 somatostatin receptor mRNA levels in breast and colon cancer determined by a quantitative RT-PCR assay based on dual label fluorogenic probe and the TaqMan technology.

We reported previously that the expression of type 2 somatostatin receptor (sst2) was positively related to patient outcome in the childhood tumor neuroblastoma. To quantitate the expression of mRNA sst2 expression, we used a competitive RT-PCR assay. To improve the practicability of this measurement and its applicability to large groups of patients, we present here an original 'real-time' quantitative RT-PCR method, based on a dual-labeled fluorogenic probe and the TaqMan technology. By this method, we have measured sst2 mRNA expression in 24 breast cancer samples and 26 colon carcinomas as well as on the corresponding non-adjacent non-neoplastic tissue of the same patients. The proposed method has a dynamic range of 4 x 10(4) to 4 x 10(8) molecules of sst2 mRNA. The intra-assay precision of the test, evaluated as signal detection variability, was 2.4%. Accuracy, evaluated by the addition of standard RNA to unknown samples, provided a mean recovery of 98+/-2%. A significant correlation has been observed in a study performed in 24 neuroblastoma samples measured both with the proposed method and with a competitive RT-PCR assay (r=0.913, p<0.001). In our preliminary clinical study, no significant differences were observed in sst2 mRNA levels between normal and tumor specimens in both colorectal (normal tissue 5.1 x 10(7)+/-2.0 x 10(7) molecules/microg total RNA, cancer tissue 9.7 x 10(7)+/-4.2 x 10(7)) and breast tumors (normal tissue 5.5 x 10(8)+/-2.0 x 10(8), cancer tissue 4.4 x 10(8)+/-3,7 x 10(8)).However, in colorectal cancer, sst2 mRNA values of subjects with high circulating carcinoembryonic antigen (CEA) levels (>5 ng/ml) were statistically lower (2.3 x 10(7)+/-6.2 x 10(6) molecules/, microg total RNA; p<0.05) than those of subjects with low CEA concentration (1.4 x 10(8)+/-6.7 x 10(7)). Also, the sst2 mRNA ratio between normal and tumor tissue (N/T ratio) resulted significantly inversely related to CEA levels. In breast cancer, a significant difference was found between the mean N/T ratio of negative (below 10 fmol/mg protein) and positive estrogen receptor tumors (p<0.05). Analogous results were found selecting breast tumors on the basis of the progesterone receptor status (p<0.05). The proposed method is accurate, precise, sensitive and less labor-intensive than the competitive RT-PCR assay. For a correct evaluation of sst2 mRNA expression, it seems very important to measure the sst2 expression both in tumor and in the non-tumoral non-adjacent tumor specimens.

Quantitative RT-PCR assay for VEGF mRNA in human tumors of the kidney.

Angiogenesis is the formation of new capillaries from pre-existing vessels, and recent evidence has demonstrated that tumor growth is controlled mainly by angiogenesis. Vascular endothelial growth factor (VEGF) is an endothelium-specific growth factor which is strongly angiogenic in vitro and in vivo. We have developed a quantitative RT-PCR assay for the measurement of VEGF mRNA expression using a real-time procedure based on the use of fluorogenic probes and the ABI PRISM 7700 Sequence Detector System. The assay performance of this method in terms of practicability and reliability is reported with results that seem promising for its widespread use in the clinical laboratory. The method has been applied to the measurement of mRNA of VEGF in human renal cell carcinomas (RCC). Preliminary results show a significantly higher VEGF mRNA expression (ratio values between 181 and 2222) in tumor specimens compared to non-adjacent, non-tumoral tissue of the same subjects.

[Somatostatin receptors in non-endocrine tumours]. / Recettori per la somatostatina in tumori non endocrini.

The study of the antiproliferative action of somatostatin (ss) is important not only to understand the regulation of neuroendocrine tumours that express receptors (sst), but also non-endocrine tumours which express these receptors. We previously demonstrated the presence of sst2 in a wide panel of cell lines from human neuroblastoma. Although hypotheses have been put forward that treatment with ss or its analogs may be beneficial in oncological patients, this does not appear to be the case in neuroblastoma; patients with high sst2 levels (who are therefore sensitive to ss treatment) have per se a relatively positive outcome. Therefore, adjuvant treatment with ss is not necessary. Viceversa, patients with a poor prognosis are essentially characterized by a low expression of sst2 (and therefore are insensitive to a therapy with ss). In these patients adjuvant treatment with ss might be indicated, but would have little chance of success. Although the majority of neuroendocrine tumours expresses sst2, pancreas and prostate cancer express sst1 but not sst2, and are therefore insensitive to octreotide treatment which binds preferentially to sst2. Tumours like colorectal carcinoma and breast cancer also express sst2 in their more favourable forms. However, the concentration of sst2 in colorectal cancer is similar, if not lower than that in the surrounding normal tissue. Therefore, the probability of successful adjuvant therapy with ss is relatively low. In breast cancer, it is possible that sensitivity to estrogens may have a positive influence on the expression of sst2. This might justify clinical trials with ss in breast cancer.

Multidrug resistance in ovarian cancer: comparing an immunocytochemical study and ATP-tumor chemosensitivity assay.

The aim of our study was to evaluate the possible prognostic and predictive significance of the expression of P-glycoprotein, a transmembrane transport protein related to multidrug resistance, in previously untreated patients with FIGO stage III ovarian cancer; to compare the results of immunocytochemical analysis of tissue sections of tumors to the in vitro chemosensitivity to cytotoxic drug of fresh samples of the same tumors; and to evaluate survival in women who underwent the same surgical treatment and the same adjuvant chemotherapy.

Effects of gemcitabine in normal and transformed human lung cell cultures: cytotoxicity and increase in radiation sensitivity.

AIMS AND BACKGROUND: Several anticancer drugs increase cell sensitivity to irradiation. Gemcitabine (2', 2' difluorodeoxycytidine) decreases the cellular dNTP pools and thus significantly increases the sensitivity to the DNA damaging effects of low-dose radiation. In this study we have investigated whether gemcitabine may play a role as radiosensitizer also in lung adenocarcinoma treatment. METHODS & STUDY DESIGN: We studied this nucleoside analogue in normal and transformed human cell lines (fetal lung and lung adenocarcinoma). After drug treatment, cell lines were irradiated with different doses. Cell damage following drug treatment and/or irradiation was assessed by measuring intracellular ATP level and by the colony forming assay. RESULTS: The two cell lines significantly differed in their sensitivity to the toxic effects of the drug; the normal cell line was much more resistant than its transformed counterpart. This difference was observed in both assays, although it was more evident in the colony forming assay. A low radiation dose (50-100 cGy) did not cause any significant damage to transformed cells; normal cells were more resistant and doses up to 500 cGy caused little damage. However, when transformed cells were pretreated for three hours with gemcitabine, even a nontoxic concentration of the drug (1-10 nM) caused a marked sensitization of the cells to irradiation (50-100 cGy). The radiosensitizing effect of gemcitabine could be observed also in normal cells, although these cells were more resistant to the damaging effects of both anticancer treatments. CONCLUSIONS: This study demonstrates that gemcitabine, a chemotherapeutic agent already used in the clinic, could be proposed as a radiosensitizer for radiation therapy of lung adenocarcinoma, having a clearly potentiating effect on lowdose radiation.

Clinical, biochemical and therapeutical aspects of amiodarone-induced hypothyroidism (AIH) in geriatric patients with cardiac arrhythmias.

The clinical features and the laboratory aspects of the amiodarone-induced hypothyroidism (AIH) in the elderly as well as the effects of amiodarone treatment in aged AIH people have not yet been well clarified. In the present paper, we evaluated 18 subjects of both sexes (7 females, 11 males), aged 65-83 years, affected by AIH, recruited in Central Tuscany, Italy. The patients were divided in two subsets on the basis of thyroid stimulating hormone (TSH) values: mild (TSH < 20 mU/l; Group A, n=11) and severe (TSH > 20 mU/l; Group B, n=7) hypothyroid patients. On the basis of clinical features, hypothyroidism was diagnosed only in two patients (out of Group B). Concerning the hormonal pattern, we found that free tetraiodothyronine (fT4) levels were significantly lower than the normal range only in Group B subjects; TSH and thyroglobulin were higher than normal in both groups; free triiodothyronine (fT3) were always in the normal range. Thyroid autoantibodies were found positive only in one patient out of Group A and in two patients out of Group B. In 5/18 patients T4 substitutive therapy was rapidly assigned, because of severe degree of hypothyroidism. In the remaining 13/18 patients, we evaluated the clinical behavior of AIH. After additional cardiac evaluation, amiodarone was withdrawn in 5/13 patients: during follow-up period (4-10 months) four patients became quickly euthyroid while one worsened. In 8/13 patients, amiodarone treatment had to be carried on; during follow-up (2-48 months), four patients remained mildly hypothyroid, while other four patients became severely hypothyroid. In conclusion, in amiodarone treated elderly people, diagnosis of hypothyroidism is reliable only on the basis of high values of TSH; clinical features and fT3 serum levels never enable diagnosis.

Circulating BRAFV600E in the diagnosis and follow-up of differentiated papillary thyroid carcinoma.

CONTEXT: Cell-free nucleic acids circulating in plasma are considered a promising noninvasive tool for cancer monitoring. BRAF(V600E) mutation in cell-free DNA (cfDNA) could represent an appropriate marker for papillary thyroid carcinoma (PTC). OBJECTIVE: Our aim is to investigate the role of BRAF(V600E)-mutated allele in cfDNA as a marker for the diagnosis and follow-up of PTC. STUDY DESIGN: BRAF(V600E) allele was detected and quantified by an allele-specific real-time quantitative PCR assay in plasma from 103 patients affected by nodular goiter. As control populations, we enrolled 49 healthy subjects and 16 patients with non-nodular thyroid diseases. RESULTS: The percentage of circulating BRAF(V600E) was significantly different between patients and controls and throughout different cytological categories of ultrasound-assisted fine-needle aspiration. Patients with a histopathological diagnosis of PTC showed a higher percentage of circulating BRAF(V600E) (P = .035) compared to those with benign histology. In 19 patients, a second blood draw, taken 3-6 months after surgery, showed a lower percentage of BRAF(V600E) in cfDNA than the presurgical sample (P < .001). The diagnostic performance of circulating BRAF(V600E) was assessed by receiver operating characteristic curve analysis resulting in an area under the curve of 0.797. A cutoff value was chosen corresponding to maximum specificity (65%) and sensitivity (80%). On this basis, we evaluated the predictive value of BRAF(V600E) in Thy 3 patients with a resulting positive predictive value of 33% and a negative predictive value of 80%. CONCLUSIONS: The results of the present study provide encouraging data supporting the possibility to take advantage of circulating BRAF(V600E) in the management of PTC.