Diagnosis of post-surgical fine-needle aspiration biopsies of thyroid lesions with indeterminate cytology using HRMAS NMR-based metabolomics.

INTRODUCTION: Ultrasound examination coupled with fine-needle aspiration (FNA) cytology is the gold standard for the diagnosis of thyroid cancer. However, about 10-40% of these analyses cannot be conclusive on the malignancy of the lesions and lead to surgery. The cytological indeterminate FNA biopsies are mainly constituted of follicular-patterned lesions, which are benign in 80% of the cases. OBJECTIVES: The development of a FNAB classification approach based on the metabolic phenotype of the lesions, complementary to cytology and other molecular tests in order to limit the number of patients undergoing unnecessary thyroidectomy. METHODS: We explored the potential of a NMR-based metabolomics approach to improve the quality of the diagnosis from FNABs, using thyroid tissues collected post-surgically. RESULTS: The NMR-detected metabolites were used to produce a robust OPLSDA model to discriminate between benign and malignant tumours. Malignancy was correlated with amino acids such as tyrosine, serine, alanine, leucine and phenylalanine and anti-correlated with myo-inositol, scyllo-inositol and citrate. Diagnosis accuracy was of 84.8% when only indeterminate lesions were considered. CONCLUSION: These results on model FNAB indicate that there is a clear interest in exploring the possibility to export NMR metabolomics to pre-surgical diagnostics.

Mutant cohesin drives chromosomal instability in early colorectal adenomas.

Chromosome missegregation leads to chromosomal instability (CIN), thought to play a role in cancer development. As cohesin functions in guaranteeing correct chromosome segregation, increasing data suggest its involvement in tumorigenesis. In a screen of a large series of early colorectal adenomas, a precocious step during colorectal tumorigenesis, we identified 11 mutations in SMC1A core cohesin subunit. In addition, we sequenced the SMC1A gene in colorectal carcinomas and we found only one mutation. Finally, the transfection of the SMC1A mutations identified in early adenomas and wild-type SMC1A gene silencing in normal human fibroblasts led to CIN. Our findings that SMC1A mutations decrease from early adenomas to colorectal cancers and that mutations lead to CIN suggest that mutant cohesin could play a pivotal role during colorectal cancer development.

P2X7 protein expression and polymorphism in non-small cell lung cancer (NSCLC).

BACKGROUND: P2X7, a purinergic receptor, plays important roles in inflammatory diseases, but recently its expression has been found in several tumors, suggesting a potential role as a cancer cell biomarker. Moreover, the relative amount of P2X7 varies among human individuals due to numerous single nucleotide polymorphisms resulting in either a loss- or gain-of-function; the P2X7 gene is highly polymorphic, and polymorphisms in the promoter or coding region may modify its expression or function. A polymorphism in exon 13 of the P2X7 receptor gene at the +1513 position (Glu496Ala substitution, corresponding to SNP rs3751143) has been shown to eradicate the function of this receptor and has been correlated with histological variants and clinical parameters in thyroid cancer. Until now, no data regarding P2X7 expression and polymorphisms in lung cancer have been published; based on these premises, we decided to evaluate the impact of the P2X7 expression and polymorphisms in ninety-seven cases of non-small cell lung cancer (NSCLC). RESULTS: No significant difference in the genotype frequency of the A1513C polymorphism was found between the two histological variants of NSCLC, adenocarcinoma and squamous cell carcinoma, and no statistically significant associations were observed between P2X7 protein expression and the main clinico-pathological characteristics of the NSCLC patients. CONCLUSIONS: Based on our results, P2X7 expression and polymorphisms seem to have no potential impact in patients with non-small cell lung cancer; however, further studies will surely provide deeper insights regarding the role of this receptor at the clinical level in NSCLC.

Epidermal growth factor receptor and K-RAS mutations in 411 lung adenocarcinoma: a population-based prospective study.

Targeting the epidermal growth factor receptor has played a central role in advanced non-small cell lung cancer research, treatment, and patient outcomes over the last several years; however, a number of questions about this approach remain to be addressed. Through the Istituto Toscano Tumori and the Italian Association of Women Against Lung Cancer Project, we collected 411 lung adenocarcinomas from several clinical centers in Tuscany. Mutations were assessed by sequencing exons 18-21 of the epidermal growth factor receptor gene, and by restriction fragment length polymorphism analysis of codons 12 and 13 of the K-RAS gene. Epidermal growth factor receptor mutations (12.6%) were more frequently observed in females (p<0.0001), in non-smokers (p=0.005), and in the presence of bronchioloalveolar features (p=0.0004). K-RAS mutations (17.9%) were more frequent in males (p=0.0007) and were associated with smoking habits (p=0.005). Epidermal growth factor receptor and K-RAS mutations were mutually exclusive (p=0.001). We focused on 21 female patients with advanced/metastatic lung adenocarcinoma who received gefitinib 250 mg/day (expanded access) or erlotinib 150 mg/die as second/third-line therapy; partial response was associated with classic epidermal growth factor receptor mutations (p=0.006) and with a non-smoking history (p=0.02). None of the female patients with partial response and/or stable disease showed K-RAS alterations. Although the data obtained in our study have yet to be analyzed and confirmed with a larger number of patients treated with tyrosine kinase inhibitors, they should provide useful information for targeted therapy, in particular for non-smoking female lung cancer patients.

Overexpression of the cohesin-core subunit SMC1A contributes to colorectal cancer development.

BACKGROUND: Cancer cells are characterized by chromosomal instability (CIN) and it is thought that errors in pathways involved in faithful chromosome segregation play a pivotal role in the genesis of CIN. Cohesin forms a large protein ring that binds DNA strands by encircling them. In addition to this central role in chromosome segregation, cohesin is also needed for DNA repair, gene transcription regulation and chromatin architecture. Though mutations in both cohesin and cohesin-regulator genes have been identified in many human cancers, the contribution of cohesin to cancer development is still under debate. METHODS: Normal mucosa, early adenoma, and carcinoma samples deriving from 16 subjects affected by colorectal cancer (CRC) were analyzed by OncoScan for scoring both chromosome gains and losses (CNVs) and loss of heterozygosity (LOH). Then the expression of SMC1A was analyzed by immunochemistry in 66 subjects affected by CRC. The effects of SMC1A overexpression and mutated SMC1A were analyzed in vivo using immunocompromised mouse models. Finally, we measured global gene expression profiles in induced-tumors by RNA-seq. RESULTS: Here we showed that SMC1A cohesin core gene was present as extra-copies, mutated, and overexpressed in human colorectal carcinomas. We then demonstrated that cohesin overexpression led to the development of aggressive cancers in immunocompromised mice through gene expression dysregulation. CONCLUSION: Collectively, these results support a role of defective cohesin in the development of human colorectal cancer.

Differential microRNA expression profiles between young and old lung adenocarcinoma patients.

Lung cancer is the leading cause of cancer-related mortality, and approximately 80% of cases are non-small cell lung cancer (NSCLC). Recently, the incidence of NSCLC has been quickly increasing, while the age of patients at diagnosis is decreasing. To date, it is still controversial whether younger patients have better or worse outcomes compared with their older counterparts. MicroRNAs (miRNAs) have been defined to play a key role in cancer pathogenesis, and their aberrant expression has been suggested as a potential biomarker of prognosis in lung adenocarcinoma. To understand the molecular features of young and old adenocarcinoma patients, we investigated the expression level of a panel of miRNAs selected after a mini-literature review. The expression analysis was performed by the nCounter System® (NanoString Technologies) directly on RNA, including small RNAs. The analysis revealed that 7 miRNAs (miR-25-3p, miR-29c-3p, miR-33a-5p, miR-144-3p, miR-153-3p, miR-342-5p and miR-485-3p) were differentially expressed in the two groups (P<0.05). All of these miRNAs showed higher expression levels in young compared to old patients, and their predicted targets included EGFR, MET, VEGF-A, TP53 and PDGFRa. miR-144-3p had an opposite influence on overall survival since its upregulation was associated with a worse prognosis in young patients (P=0.01) and with a better outcome in the older group (P=0.03). We observed that lung cancer in young and old patients may be influenced by different regulatory mechanisms. Moreover, one of the down-regulated miRNAs showed a different prognostic impact in the two groups, confirming that young and old patients deserve a specific clinical approach.

Prognostic role of TPL2 in early­stage non­small cell lung cancer.

Non­small cell lung cancer (NSCLC) accounts for ~70% of all lung cancer­associated mortalities worldwide. The serine/threonine protein kinase tumor progression locus 2 [TPL2/MAP3 kinase 8 (MAP3K8)] may impact oncogenic events; however the role of TPL2 in lung carcinogenesis remains unclear. The present study was focused on the potential prognostic role of TPL2 in 101 patients with early­stage NSCLC. Since TPL2 is a potential target of miR­21, the association between TPL2 and miR­21 expression was also examined. TPL2 and miR­21 mRNA expression was quantified using reverse transcription quantitative polymerase chain reaction (RT­qPCR). TPL2 protein levels were evaluated by immunohistochemistry (IHC). The present study identified that the mRNA expression of TPL2 was low in 52/101 (51%) cases and high in 49/101 (49%) cases. IHC analysis of TPL2 protein expression often demonstrated identical mRNA results. No statistically significant associations were observed between the mRNA expression of TPL2 and the predominant clinicopathological characteristics of the patients with NSCLC, as well as identifying no association between TPL2 and miR­21. TPL2 mRNA expression was significantly higher in patients with NSCLC with good prognosis (disease­free interval P=0.009; overall survival P=0.024), when compared with those of poor prognosis. Focusing on the difference in mRNA expression of TPL2 among the adenocarcinomas in affected patients, TPL2 was more highly expressed in lepidic adenocarcinomas compared with in the other subtypes (P=0.012). The present study is the first examination, to the best of our knowledge, of TPL2 in early­stage NSCLC in relation to miR­21, and in different adenocarcinoma subtypes. Future studies must clarify the mechanism by which TPL2 is involved in lung carcinogenesis due to its important translational implications.

Urokinase-type plasminogen activator receptor (uPAR) expression enhances invasion and metastasis in RAS mutated tumors.

The urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored cell membrane receptor that focuses urokinase (uPA) proteolytic activity on the cell surface. Its expression is increased in many human cancers, including non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and correlates with a poor prognosis and early invasion and metastasis. uPAR is able to control, through a cross-talk with tyrosine kinase receptors, the shift between tumor dormancy and proliferation, that usually precedes metastasis formation. Therefore, we investigated the role of uPAR expression in RAS mutated NSCLC and CRC cells. In this study we provided evidence, for the first time, that RAS mutational condition is functionally correlated to uPAR overexpression in NSCLC and CRC cancer cell lines and patient-derived tissue samples. Moreover, oncogenic features related to uPAR overexpression in RAS mutated NSCLC and CRC, such as adhesion, migration and metastatic process may be targeted, in vitro and in vivo, by new anti-uPAR small molecules, specific inhibitors of uPAR-vitronectin interaction. Therefore, anti-uPAR drugs could represent an effective pharmacological strategy for NSCLC and CRC patients carrying RAS mutations.

EGFR and KRAS mutational analysis in a large series of Italian non-small cell lung cancer patients: 2,387 cases from a single center.

Activating EGFR mutations are important genetic alterations that have strong therapeutic implications for non-small cell lung cancer (NSCLC) patients. However, the role of KRAS mutations in this process is still under evaluation. Here, we report on the feasibility of a large­scale EGFR and KRAS mutation analysis in the daily routine of a single center. NSCLCs from 2,387 patients were screened for EGFR and KRAS mutations from January 2010 to September 2015. Mutational analyses were performed in a single laboratory using single strand conformation polymorphism (SSCP)-Sanger sequencing and matrix­assisted laser desorption ionization­time of flight (MALDI­TOF) on Sequenom platform for EGFR and pyrosequencing for KRAS. Activating EGFR mutations were found in 14.1% of all tumors, whereas KRAS mutations were found in 30.5% of all tumors. Direct sequencing showed analyzable cytological, small biopsy and surgical specimen percentages of 90.3, 90.9 and 98.1%, respectively, whereas the MALDI­TOF platform showed analyzable cytological samples, small biopsies and surgical specimens percentages of 94.6, 95.7 and 96.9%, respectively. The mean analytical turnaround times (TAT) were 4 and 3 days for direct sequencing and the MALDI­TOF platform, respectively. Our results confirm that small biopsy or cytological samples can be used for reliable EGFR and KRAS mutation testing and indicate that adopting the MALDI­TOF platform reduces the rate of missed samples among the samples. Moreover, the 3-day analytical TAT of the MALDI-TOF multi-target technique is appropriate for clinical management and reduces the overall treatment decision time.

A multi-parametric protocol to study exercise intolerance in McArdle's disease.

McArdle's disease is the most common metabolic myopathy of muscle carbohydrate metabolism, due to deficiency of myophosphorylase and alteration of glycogen breakdown in muscle. The clinical manifestations usually begin in young adulthood, with exercise intolerance, exercise-induced muscle cramps, pain and recurrent episodes of myoglobinuria. Many patients experience the second wind phenomenon, characterized by an improved tolerance for aerobic exercise approximately after eight minutes of motor activity, secondary to the increased availability of blood glucose and free fatty acids associated to an enhanced glucose uptake by muscle cells. In this study, we aimed to test a multi-parametric protocol in order to detect the impairment of muscular metabolism and motor performance in patients with McArdle's disease. We enrolled 5 patients and 5 age-matched healthy subjects, that were evaluated by: (01) monitoring of physical activity with an electronic armband; (02) testing of cardiopulmonary, metabolic and respiratory responses to exercise with a cardiopulmonary exercise test and analyzing muscle fatigue during exercise test by surface electromyography (04) evaluating blood lactate and oxidative stress biomarkers at rest and during exercise. The patients were tested at baseline and after three days of carbohydrate-rich diet integrated with tricarboxylic acid cycle intermediate and creatine. The multiparametric protocol proved to be useful to detect the oxidative capacity impairment and the second wind phenomenon of patients. We did not observe any significant differences of muscle metabolic response during the exercise test after three days of carbohydrate-rich diet.

Anaplastic lymphoma kinase gene rearrangements in cytological samples of non-small cell lung cancer: comparison with histological assessment.

BACKGROUND: Anaplastic lymphoma kinase (ALK) gene rearrangements are detected in approximately 5% of cases of non-small cell lung cancer (NSCLC). Patients who are positive for ALK rearrangements may be successfully treated with the ALK inhibitor crizotinib. Because advanced-stage lung cancers are not suitable for surgical resection, approximately 70% of patients are diagnosed via preoperative specimens. In the current study, the authors evaluated the suitability of stained cytologic direct smears and cell blocks for fluorescence in situ hybridization (FISH) to determine ALK status compared with small biopsies. METHODS: A total of 493 consecutive patients with NSCLC were analyzed by FISH for ALK gene rearrangements. The analyzed sample comprised 180 cytological samples, 94 direct smears, 86 cell blocks, and 313 preoperative small biopsies. Moreover, in the same series, 426 patients and 369 patients, respectively, were evaluated for epidermal growth factor receptor and KRAS mutations, respectively. RESULTS: Of the total of 493 patients, 18 patients who were positive for a gene rearrangement (4.4%) demonstrated ALK FISH rearrangements, whereas 387 patients (95.6%) were negative. All other cases were classified as inadequate (7.7%) and insufficient (10.1%). A strong statistical association was found between the cytology and the small biopsy with respect to ALK rearrangements (P=.0048). Fifty-three patients (12.4%) demonstrated an epidermal growth factor receptor mutation, whereas 90 patients (24.4%) were found to have KRAS mutations. None of these patients presented with ALK gene rearrangements. CONCLUSIONS: ALK gene rearrangements may be easily detected in cytological samples and particularly in direct smears, thereby yielding important improvements in the diagnostic approach to patients with advanced NSCLC. Cytological samples may be useful for ALK analysis when insufficient material is available in cell blocks or small biopsies.

KRAS and BRAF genotyping of synchronous colorectal carcinomas.

v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) genotyping is required prior to anti-epidermal growth factor receptor monoclonal antibody therapy administered in cases of metastatic colorectal carcinoma (CRC). Thus, KRAS mutation screening is required for patient management. The present study reported the experience of KRAS/v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutational screening on synchronous CRC pairs from 26 patients, which were defined as index lesions (ILs) and concurrent lesions (CLs) on the basis of tumor grade and dimension and their respective lymph node and distant metastases. Overall, KRAS mutations were present in 38.4% of patients, whereas BRAF mutations were present at a frequency of 11.5%. The genotyping of paired synchronous carcinomas indicated that 11 patients (42.3%) exhibited discordant KRAS mutational statuses in terms of the presence of a mutation in only one lesion of the pair or of two different mutations harbored by each lesion. BRAF mutations were present in the synchronous tumors of two cases, whereas in two other cases, only the IL or CL harbored mutant BRAF. Overall, the mutational statuses of distant and lymph node metastases confirm the genetic heterogeneity of synchronous primary tumors. These results highlighted the fact that adequate sampling and comprehensive testing, when feasible, is likely to optimize the decision-making process for treatment approaches, even in the relatively rare event of multiple synchronous lesions.

Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens: correlation with physiological and pathological characteristics, and with biomarkers of one-carbon metabolism.

We evaluated the promoter methylation levels of the APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in 107 colorectal cancer (CRC) samples and 80 healthy adjacent tissues. We searched for correlation with both physical and pathological features, polymorphisms of folate metabolism pathway genes (MTHFR, MTRR, MTR, RFC1, TYMS, and DNMT3B), and data on circulating folate, vitamin B12 and homocysteine, which were available in a subgroup of the CRC patients. An increased number of methylated samples were found in CRC respect to adjacent healthy tissues, with the exception of APC, which was also frequently methylated in healthy colonic mucosa. Statistically significant associations were found between RASSF1A promoter methylation and tumor stage, and between hMLH1 promoter methylation and tumor location. Increasing age positively correlated with both hMLH1 and MGMT methylation levels in CRC tissues, and with APC methylation levels in the adjacent healthy mucosa. Concerning gender, females showed higher hMLH1 promoter methylation levels with respect to males. In CRC samples, the MTR 2756AG genotype correlated with higher methylation levels of RASSF1A, and the TYMS 1494 6bp ins/del polymorphism correlated with the methylation levels of both APC and hMLH1. In adjacent healthy tissues, MTR 2756AG and TYMS 1494 6bp del/del genotypes correlated with APC and MGMT promoter methylation, respectively. Low folate levels were associated with hMLH1 hypermethylation. Present results support the hypothesis that DNA methylation in CRC depends from both physiological and environmental factors, with one-carbon metabolism largely involved in this process.

An "inflammatory" mitochondrial myopathy. A case report.

We describe a case of an adult male patient with progressive external ophthalmoplegia and upper limb weakness, who presented with an episode of sudden respiratory failure. Muscle biopsy showed ragged-red and COX-negative fibers associated with discrete inflammatory infiltrates and necrotizing features. Apart from artificial ventilator support, he was treated with intravenous immunoglobulins and carnitine, with excellent clinical outcome. Mitochondrial DNA analysis revealed the 3251A>G mutation, previously reported in association with rapidly progressive mitochondrial myopathy and respiratory failure. Our case expands the spectrum of this mutation and suggests a therapeutic attempt with immunoglobulins in mitochondrial patients with acute respiratory failure, at least when this mutation and/or muscle inflammation is present. Moreover, this case supports the idea of a pathologic inflammatory response induced by mitochondrial disease; such an abnormal response may be a contributory factor in disease progression or acute exacerbation typical of some mitochondrial diseases, but further studies are needed.

Expression of p-AKT and p-mTOR in a large series of bronchopulmonary neuroendocrine tumors.

Bronchopulmonary neuroendocrine tumors (BP-NETs) are separated into four subgroups: typical carcinoid tumor (TC), atypical carcinoid tumor (AC), large-cell neuroendocrine carcinoma (LCNEC) and small-cell lung carcinoma (SCLC). The signaling pathway involving AKT/mammalian target of rapamycin (mTOR) is crucial to the regulation of cell growth, proliferation and survival, and is frequently activated in human cancers. Consequently, mTOR is considered an attractive target for anticancer agents. The present study aimed to evaluate the expression of phosphorylated AKT and mTOR in a series of BP-NETs, and to analyze the correlations with clinicopathological parameters. p-AKT and p-mTOR levels were determined by immunohistochemistry in a series of 210 BP-NETs, including 85 SCLCs, 17 LCNECs, 26 ACs, 75 TCs and 7 tumorlets. Higher p-AKT and p-mTOR expression levels were identified in the majority of tumorlets and carcinoids in comparison to the LCNECs (P=0.0001) and SCLCs (P=0.0002). Furthermore, a significant association was observed between p-mTOR expression and tumor size (T) in SCLCs (P=0.04) and LCNECs (P=0.03): T3-T4 tumors exhibited significantly lower p-mTOR expression compared to T1-T2 tumors. In conclusion, most of the BP-NETs examined in this study expressed p-AKT and p-mTOR, suggesting that the AKT/mTOR pathway plays an important role in these tumors. Additionally, our results confirm that low- to intermediate-grade tumors are more closely associated to each other than to high-grade tumors, despite sharing common classification and a common origin from neuroendocrine cells. These findings improve our knowledge of the biological characterization of these tumors and indicate new therapeutic opportunities for the treatment of BP-NETs.

Different estrogen receptor beta expression in distinct histologic subtypes of lung adenocarcinoma.

Adenocarcinoma is becoming the most common histologic type of lung cancer in both sex. Although most cases are seen in smokers, it develops more frequently than other histologic types in individuals who have never smoked. This evidence suggests that other putative etiologic factors, such as sex hormones, need to be investigated. Several subtypes of lung adenocarcinoma have been recently described with distinct clinicopathologic features and prognostic implications. The purpose of this study is to investigate the role of estrogen receptor beta in lung adenocarcinoma, with particular attention paid to its different histologic subtypes. Nuclear estrogen receptor beta expression was evaluated by immunohistochemistry in 112 lung adenocarcinomas, including both "single subtype" and "mixed subtype" samples. Using a 2-level (high/low) score system, estrogen receptor beta expression was high in most (75%) adenocarcinomas and turned out to be strongly related to the histologic subtypes. In fact, estrogen receptor beta expression was low or negative in 68.2% of solid subtypes, whereas it was high in 76.5% of nonmucinous bronchioloalveolar, in 69.4% of acinar, and in 61.2% of papillary patterns (P = .00004). Furthermore, a strong association between estrogen receptor beta expression and tumor histologic grade was observed: estrogen receptor beta was highly expressed predominantly in well- and moderately differentiated tumors (P = .0014). In conclusion, estrogen receptor beta expression has distinct patterns in lung adenocarcinoma, suggesting a specific role for estrogen receptor beta in the pathogenesis of different histologic subtypes of this type of cancer. Moreover, loss of estrogen receptor beta expression in poorly differentiated (G3) tumors could represent a crucial step in the dedifferentiation process of lung adenocarcinoma.