thyroid cancer is usually treated with primary surgery ( near - total or total thyroidectomy and lymph nodes dissection if necessary ) , ablation of the thyroid remnant with radioactive iodine ( rai ) ( based on the tumor stage ) and thyroid - stimulating hormone ( tsh ) suppressive therapy [ 1 - 3 ] . follow - up consists of neck ultrasonography ( us ) , basal and after tsh - stimulated thyroglobulin assay [ 1 , 2 , 4 , 5 ] . though thyroid cancer has generally a good prognosis , 10% to 15% of patients with thyroid cancer have recurrent disease , and about 5% will develop metastatic disease not responsive to rai , and eventually will die from this disease . the knowledge of cancer cell signaling could help us to develop novel anticancer therapies [ 7 - 11 ] . fine - needle aspiration cytology ( fnac ) is currently the most common and efficient method used for the preoperative diagnosis of thyroid nodules . in the majority of cases , the results obtained by fnac allow a good discrimination between the nodules that can be managed conservatively ( thy2 following the american thyroid association classification , about 70% of all fnacs ) and those requiring surgery ( thy4 and thy5 , about 10% ) . the most important limitation of fnac is its low accuracy in the evaluation of   follicular - patterned lesions ( thy3 ) , a situation where the cytologycannot differentiate benign from malignant lesions ( follicular adenomas vs follicular thyroid carcinomas / follicular variant of papillary thyroid carcinomas , respectively ) . this situation leads to a diagnosis of   undetermined nodule in an average 20% of all fnacs , a situation that indicates the necessity of a  diagnostic  surgery , with the only purpose to perform a definitive histology .   with these premises , it is clear that techniques other than cytology , e.g. ; molecular biology , flow cytometry , and immunocytochemistry , have been investigated to overcome the limitations of fnac . other researchers have investigated the possible presence of biomarkers of thyroid malignancy by using 2d - gel and mass spectrometry - based proteomics . a few studies have analyzed the possible role of different magnetic resonance ( mr ) techniques , such as spectroscopy ( mrs ) , in the differentiation between normal and malignant thyroid tissue . these techniques have been firstly used with the aim of identifying the morphologic features of malignancies , and then , with the improvement in the technology behind the mr , to identify the metabolic differences between thyroid neoplasms ( benign or malignant ) and normal thyroid tissue . in a second step , the utility of these approaches could be tested with the aim of differentiating between benign and malignant neoplasms . these experiments had generally been performed on operative specimens coming from surgically removed thyroids : the analyses produced the promising results that led to the subsequent phase of the application of the method in the preoperative work - up of thyroid nodules . a pre - clinical application of the mrs has been then studied , testing its application on specimens obtained from fnacs performed after the removal of the thyroid gland ( ex - vivo ) , an attempt to determine whether this technology could give a significant improvement in the preoperative diagnosis of the so - called  undetermined  thyroid lesions . in this paper , we will deal with the historical development and current state of the art of the mrs analysis of thyroid lesions , discussing the results obtained by the different techniques of nuclear magnetic resonance ( nmr ) and thyroid cancer diagnostics . molecular diagnosis of cancer requires analytical tools capable of distinguishing the key biomarkers , that are secondary metabolites , either directly linked to the disease or derived from its induced perturbations .   in recent years , several studies in this field have been started for two main reasons : the improved availability of analytical techniques such as mrs and mass spectrometry ( ms ) , and the consequent development of appropriate statistical tools capable of dealing with the huge mass of spectroscopic data associated to the metabolites presence and variation in concentration . in particular , ex - vivo mrs analysis plays a role that is complementary to that of both inner magnetic resonance ( imr ) and in vivo mrs since these latter tools , although providing excellent morphologic information , are unlikely to capture fine variations in the molecular composition of the lesions , i.e. in their lipid profile . the first report dealing with the application of proton mrs to cancer diagnosis is the article by mountford et al . , while the methodological aspects about the current protocols for the mrs analysis of biopsies can be found in the study by beckonert et al . . an overview of this progressively growing field of application can be found in other studies [ 15 - 18 ] . thyroid cancer has been the first field where molecular diagnosis was performed using ex - vivo mrs [ 19 - 23 ] . the main issue behind mrs analysis of ex - vivo specimens is the quality of the spectra obtained , that is directly related to the homogeneity of the sample and to its degree of molecular mobility . for this reason , biopsies are not expected to produce high - resolution mrs spectra , if specific techniques are not applied . in the case of thyroid tissue , even if the observed resolution was less than that usually obtained through mrs , the 2d - nmr ( cosy ) experiment allowed to detect several signals that were assigned to specific molecular features .   in this study , the authors reported that two specific signals play a relevant role in the discrimination of lesions of different nature . these two signals are : a methylene ( -ch2- ) peak at 1.7 ppm , deriving from lipids and lysine , and one at 0.9 ppm , regrouping essentially all the methyl ( -ch3 ) groups from all molecules . based on the observation that the mrs-1d spectra of malignant tissue showed a lower content of lipids and higher concentrations of aminoacids when compared to their benign counterpart , the authors assumed the ratio between the two peaks ( 1.7 and 0.9 ppm ) as a tentative indicator for malignancy . after the analysis of the results obtained from the mrs spectra , and the revision of both cytologic and histologic results obtained from different thyroid lesions , a line was drawn at the value of 1.1 , allowing to discriminate the benign tissue ( when the ratio is > 1.1 ) from the malignant one ( when lower than the threshold ) . this method allowed a 100% discrimination between benign and malignant tissues , in specimens obtained from surgically removed thyroid glands . another study   aimed at verifying the possibility of obtaining the same results from material obtained from fna , using the same spectral markers to compare the results in cases of citologically undetermined lesions ( thy3 ) . this study allowed to elucidate the statistical relevance of analysing small amounts of cells coming from a lesion , a great possibility , in the future , to perform a thorough diagnosis with a relatively non - invasive method . in this study , among the 92 analysed cases , the results obtained from fnac were discordant from the final histology in only 6 . on the other hand , the analysis produced a low specificity in carcinoma detection ( 54% ) , which raised the question about the possible limits and opportunities of establishing the degree of aggressiveness of a lesion before the infiltration has started .   in another study , the analysis was performed with the 2d - nmr ( cosy ) with the aim of achieving a better resolution : the results showed that a second spectral marker , linked to cholesterol , was found at 2.05 ppm . the same group tried to extend this method to material obtained from fnac , aiming at avoiding surgery performed with the only purpose of obtaining a final diagnosis . however , the quality of the results obtained with the use of these specimens was not significant enough to draw any conclusion . the authors ascribed their unsatisfying results to the high blood content observed in the material obtained from the aspirations . the blood was virtually absent in samples obtained from surgically removed thyroids , that were devoid of circulating blood . the fnac samples analysed through the mrs produced spectra in which the discriminatory signals were biased by the ones given by blood cells . the authors concluded that , at that time , it was unclear whether their approach could be ever exported to cells coming from a fnac performed in the common preoperative workup . these results were obtained with a very simple spectral indicator and thus the same group , in order to expand their results , set up a sophisticated statistical analysis to evaluate if other and more discriminating markers could have been detected . the information in the mrs spectra was compressed using principal component analysis ( pca ) , and saving the first 10 principal components ( pcs ) , explaining 97% of the variance . then , three supervised statistics were created on this reduced dataset : linear and quadratic discriminant analysis , neural networks and genetic programming , which were used in conjunction to assign cases to histological results . this work was a pioneer study in the use of the advanced statistics that will eventually become the core of metabolomics , and thus provided more indication on potential ways of analysis rather than molecular biomarkers , as part of the spectral information ended up scrambled due to pca . to try to overcome the insufficient accuracy of pre - surgical fnac - based diagnosis using a non - invasive method , in vivo characterization of malignant thyroid lesions has been developed over the past decade using mrs . king et al .   succeeded in discriminating thyroid carcinomas from normal thyroid tissue based on the comparison of their respective h mrs spectra : 8 patients with histologically confirmed thyroid cancerous tumors larger than 1 cm ( 3 anaplastic , 2 papillary , 1 follicular carcinomas and 2 metastatic nodes from papillary carcinomas ) , and 5 lesion - free volunteers participated to this study . proton mrs spectrum of each patient was recorded using a 1.5 t whole - body imaging system ( gyroscan acs - nt , philips , best , the netherlands ) , and significant differences in the spectral profiles were observed in 5 malignant samples . in addition to an increase of aminoacids and di- and tri - glycerides in carcinomas , the authors found that choline and creatine signals were only present in material coming from carcinomas , whereas no trace of such markers were observed in patients with normal thyroids . therefore , choline / creatine ratio was chosen as a marker of malignancy : this ratio ranged from 1.6 ( in cases of well - differentiated follicular carcinoma ) to 9.4 ( in thyroids with anaplastic thyroid cancer ) . however , in 2 patients with cancer ( 1 anaplastic cancer and 1 metastatic lesion , 25% of the whole study population ) , these signals could not be detected either . creatine signal was also not detected in one patient with anaplastic carcinoma .   according to the authors , this was due to mere technical issues , such as the low sensitivity of the technique , and they also pointed out the possible limitation of their method in the analysis of cystic lesions . gupta et al .   used the same approach to differentiate between malignant ( n=8 , papillary carcinomas ) and benign lesions ( n= 18 , with 14 colloid lesions , 2 lymphocytic thyroiditis , 1 follicular adenoma , and 1 cyst ) in patients with solitary thyroid nodules . as previously reported , the discrimination was based on the presence of choline signal on the h - mrs spectra of malignant samples , leading to a method that demonstrated a sensitivity of 100% and a specificity of 88% . yoshioka et al .   were one of the first research groups to use hr - mrs with the aim of characterizing thyroid cancer . using an 80mhz wp-80sy - wg - nmr spectrometer ( bruker biospin , karlsruhe , germany ) , they extracted the total lipid content of 15 papillary thyroid carcinomas , 13 follicular adenomas and their corresponding normal tissues ( n=27 ) , and their results showed a significantly different spectrum between normal thyroid tissue and papillary carcinoma than that obtained through h - mrs . the average concentrations of the main lipids were then compared between each group and kruskal - wallis one - way anova were used to highlight significant differences . the results demonstrated that thyroid cancer was characterized by higher concentrations of cholesterol and lower levels of dolichol , when compared to normal thyroid tissue . thyroid carcinoma and adenoma could be then differentiated on the basis of their dolichol content , which was at the level of normal tissues in adenoma samples . according to the authors , the low level of dolichol and the high level of cholesterol in cancer might be the consequence of the perturbation of the isoprenoids biosynthesis pathways ( in this pathway dolichol and cholesterol are major products ) in cancer metabolism ; on the opposite side , their concentrations are preserved in thyroid adenomas . moreover , as a parallel result , it was found that phosphatidylcholine and sphyngomyelin contents were the only significant markers for the discrimination between adenoma and normal tissue , even if they failed to differentiate between follicular adenoma and papillary cancer . more recently , three research groups investigated the potential of nmr - based metabolomics in the diagnosis of thyroid cancer . the advantage of the metabolomics approach is that it allows an untargeted analysis of the overall metabolites that constitutes a biological organism . the highest molecular discrimination of the nmr analysis performed on ex - vivo thyroid tissues had to wait for the development of hrmas and its application to medical studies , firstly demonstrated in 1996 on lymph node tissues . in the following lines we will briefly discuss the specificities of this method , before reviewing its contribution to the diagnosis of thyroid lesions . when using the hrmas , the sample is span around an axis oriented at 54.7 degrees ( the  magic angle  ) , with respect to the magnetic field of the mrs instrument . this method was introduced for the analysis of solids , where it provides a significant enhancement of the resolution due to the removal of the broadening effects that arise from multiple sources . indeed , a mrs spectrum achieves its optimal resolution only in those specific cases when the molecules of the sample are under very high symmetry , e.g. in liquids in molecular motion , but not in solids . thus , the mrs signal without the hrmas can easily loose a significant part of its resolution . the sample holder is a ceramic hollow cylinder , deriving from the equipment that was originally devised for the study of hard solids . the volume of the samples that can be analyzed in this tool is about 80 l , thus making it available for material obtained from a common biopsy . alternatives to this sample holder are represented by the cheaper but more fragile pyrex sample holder , that have been developed for specific manufactures , and by a plastic disposable container that simplifies the sample preparation , also avoiding the necessity of cleaning the expensive ceramic objects . the small vials containing the material are water - tight and fit the ceramic cylinder (  inserts  ) , so that they can contain the biopsy and a few microliter of heavy water , which helps setting up the experiment for optimal results . the sample is typically span at rates of 2 to 4 khz , to avoid spectral artefacts . fast spinning has been a point of concern due to sample changes that may be induced by the associated centrifugal forces . recently , a procedure was demonstrated that allows much slower spinning rates ( about 400hz ) , so that in the future this aspect will be no more a concern . the first study on thyroid lesions using the hrmas was led by an american research group from the harvard medical school , who assessed the potential of this technique ( 600mhz spectrometer ) for the diagnosis of papillary thyroid carcinoma , based on the analysis of cytological ( fine - needle aspiration biopsies  fnab ) and histological samples obtained from 13 patients ( 4 papillary carcinoma , 4 follicular adenoma and 5 normal tissues ) . as already discussed above , hrmas - mrs has the significant advantage that it needs only a limited amount of tissue to complete a sound analysis , thus allowing the collection of the samples without losing a significant amount of tissue that might interfere with a thorough histologic diagnosis .   nevertheless , in this study , each sample was also microscopically analyzed at the moment of its collection , to verify the viability of the specimens in terms of cells composition : this was done to avoid any bias that can be given by fibrous tissue , inflammation , colloid , normal thyroid cells , and blood cells . following a principal component analysis of the h - hrmas - nmr spectra of the biopsies , 4 pcs were selected according to their correlation with their quantified tissue concentrations ( pc1 = % fibrovascular ; pc3 = % tumor + % colloid ; pc5= % benign epithelia ; pc6=% inflammation ; pc being the linear combination of the nmr signal areas ) . a conventional supervised multivariate statistical analysis was then performed on these 4 pcs , and the percent composition of the five represented pathological features was obtained . the resulting canonical scores 1 ( linear combination of the pcs and % of pathological features ) was able to discriminate the three tissue types from each other . the same coefficients ( obtained from pca and canonical analysis on the biopsies ) was then applied to the nmr spectra of specimens obtained from fnac , leading to a significant discrimination between normal tissue and follicular adenoma only along the second canonical score , whereas it was able to discriminate between normal tissue , follicular adenoma , and papillary cancer in materials obtained from biopsies . in two separate studies , another group [ 30 , 31 ] developed a metabolomics approach based on hrmas - nmr spectroscopy of thyroid biopsies obtained during surgery , in order to characterize thyroid lesions from healthy tissues , and discriminate benign from malignant tumors , in patients undergoing surgery for citologically undetermined lesions ( follicular adenoma , follicular carcinoma , or follicular variants of papillary carcinoma at final histology ) .   since no extraction is needed to analyze tissues using hrmas - nmr spectroscopy , a higher concentration of mobile hydrophobic and hydrophilic metabolites therefore , the study showed that benign and malignant thyroid lesions not only demonstrated a higher level of several aminoacids ( alanine , cysteine , glutamine , glutamate , isoleucine , leucine , lysine , phenylalanine , serine , tyrosine , valine ) , lactate and taurine , but also a significantly lower content of fatty acids , with respect to normal tissues .   furthermore , when this approach was used to analyze the samples that cytology failed to diagnose preoperatively , follicular adenomas and carcinomas as well as papillary carcinomas were analyzed using an orthogonal projections on latent structures discriminant analysis ( oplsda ) .   with a relatively high number of follicular lesions analyzed ( 30 follicular adenoma and 10 follicular thyroid carcinoma out of the 72 samples ) , the authors managed to build a robust statistical model , which enabled to discriminate cancerous tissues from benign lesions .   the results obtained showed that thyroid cancer ( both papillary and follicular ) differed from follicular adenomas by higher concentrations of taurine , lactate , phenylalanine and tyrosine and lower concentrations of myo- and scyllo - inositol , choline , phosphocholine and/or glycerophosphocholine and unknown compounds .   finally analyzed extracts of homogenized tissue samples using liquid - state nmr - based metabolomics in order to understand the molecular mechanisms involved in the development of thyroid tumors and in particular , thyroid cancer . healthy tissues , benign lesions ( including non - neoplastic nodules ) , follicular adenomas , and thyroid carcinomas were analyzed , and their respective profiles compared using pair - wise oplsda . comparison between healthy tissues and each of the three other tissue types all led to statistically significant discrimination : a higher concentration of several aminoacids ( methionine , alanine , glutamate , glycine , tyrosine , phenylalanine ) and lactate in tumor samples was also demonstrated . in addition , increased level of hypoxanthine and decreased level of acetone in thyroid lesions were also described , that were not detected in torregrossa s samples . also , new markers such as 3-hydroxybutyrate and -glucose ( deregulated only in benign lesions ) , and phosphocholine and formate ( only present in non - neoplastic nodules ) , were reported . finally , four metabolites ( creatine , scyllo - inositol , myo - inositol and uracil ) were found to be selective biomarker candidates for thyroid cancer . interestingly , the authors commented about the metabolic specificity of follicular adenomas , which possess some of the metabolic features of normal thyroid tissue , while simultaneously displaying part of the metabolic profile associated with thyroid cancer . this , in addition with other evidences ( such as epidemiological and demographic data , cases of malignancies found within benign nodules , identical cytological appearance ) [ 34 , 35 ] , may indicate an intermediate nature of these benign tumors that could support the theory that follicular thyroid carcinoma might arise from preexisting follicular adenoma , or that , in some cases , a follicular adenoma can be a preneoplastic lesion . these metabolomics studies came at the final conclusion that specific markers of different tissues ( benign and malignant ) were correlated with the cell proliferation observed during the development of the tumor ( e.g. the increase in amino acid level and the decrease in lipids ) . moreover , both the warburg effect ( due to the lactate ) and the imbalanced osmolytic function ( due to the differences in myo - inositol and scyllo - inositol ) , in addition with other markers of tumor aggressiveness ( taurine ) , were characteristic features highlighted in thyroid cancer . used metabolite set enrichment analysis on the discriminant metabolites in order to highlight the more commonly involved metabolic pathways in the development of thyroid cancer . the outcomes obtained by this analysis pointed out that protein biosynthesis were the most affected pathways , as a consequence of the increased proliferation rate of cancer cells , which requires fast supplementation of protein machinery . it appears that the high - resolution of hrmas - mrs improved the overall accuracy of the analysis of thyroid lesions to a point that a significant improvement in the diagnosis of cytologically undetermined lesions can be expected . this analysis , in the meantime , allows a more precise comprehension of the alterations in the metabolic pathways induced by the development of the different tumors . although these results are promising , at the moment , a clinical application of the method to the common workup of thyroid nodules can not be used , due to both the limitation in the availability of this technology and the wide range of techniques , that are not uniformly used . the next future will certainly see a wider application of these methods to the clinical practice in patients affected with thyroid nodules and various other neoplastic diseases . =   radioactive iodine  =   thyroid - stimulating hormone  =   fine - needle aspiration cytology  =   magnetic resonance  =   magnetic resonance spectroscopy  =   nuclear magnetic resonance  =   mass spectrometry  =   inner magnetic resonance  =   principal components  =   principal component analysis  =   high - resolution magic - angle - spinning  =   fine - needle aspiration biopsies  =   orthogonal projections on latent structures discriminant analysis
we review the progress and state - of - the - art applications of studies in magnetic resonance spectroscopy ( mrs ) and imaging as an aid for diagnosis of thyroid lesions of different nature , especially focusing our attention to those lesions that are cytologically undetermined . 
 it appears that the high - resolution of high - resolution magic - angle - spinning ( hrmas ) mrs improves the overall accuracy of the analysis of thyroid lesions to a point that a significant improvement in the diagnosis of cytologically undetermined lesions can be expected . 
 this analysis , in the meantime , allows a more precise comprehension of the alterations in the metabolic pathways induced by the development of the different tumors . 
 although these results are promising , at the moment , a clinical application of the method to the common workup of thyroid nodules can not be used , due to both the limitation in the availability of this technology and the wide range of techniques , that are not uniformly used . 
 the coming future will certainly see a wider application of these methods to the clinical practice in patients affected with thyroid nodules and various other neoplastic diseases .