Edition 68 - 2018, June / Bibliographic Reviews

Bibliographic Reviews – Ed. 68

Marco A. Rivarola y Alicia Belgorosky

Servicio de Endocrinología, Hospital de Pediatria Garrahan, Buenos Aires, Argentina

For this issue of Endocrinologia Pediatrica On line, we have selected the following publications: 


Clinical Endocrinology 87(6) 660-664, 2017. Corticotrophin-releasing hormone stimulation tests for the infants with relative adrenal insufficiency. Iwanaga K1, Yamamoto A1, Matsukura T1, Niwa F1, Kawai M1. Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.


BACKGROUND: Very low birthweight (VLBW) infants are considered to be vulnerable to relative adrenal insufficiency (RAI); however, diagnosis is difficult in some clinical settings. Considering this background, it is necessary to establish a diagnosis of RAI in preterm infants. OBJECTIVE: In this study, we attempted to clarify the difference in response to CRH stimulation tests for preterm infants with or without RAI. METHODS: Between June 2009 and December 2015, we performed CRH stimulation tests for preterm infants born at a gestational age of


Significant increases are reported in morbidities of bronchopulmonary dysplasia (BPD), necrotizing enterocolitis, symptomatic patent ductus arteriosus (PDA) and late-onset circulatory collapse (LCC). Some of these conditions were considered to be strongly associated with relative adrenal insufficiency (RAI). Among them, the association between RAI and BPD or LCC has been discussed intensively. RAI is considered as an etiology for respiratory or hemodynamic instability, not only for adults but also for preterm infants. As a concept, most neonatologists agree on RAI but it’s difficult to diagnose. In adults, Cooper described a flow chart to diagnose corticoid insufficiency; however, there are no reliable tools for diagnosis in preterm infants. In 2012, these authors reported the results of corticotrophin-releasing hormone (CRH) stimulation tests at 2 weeks of age in healthy preterm infants born at a gestational age (GA) of


J Clin Endocrinol Metab 103: 320–327, 2018. Adrenocorticotropin Acutely Regulates Pregnenolone Sulfate Production by the Human Adrenal In Vivo and In Vitro.

Juilee Rege,1* Aya T. Nanba,2* Richard J. Auchus,2,3 Jianwei Ren,Hwei-Ming Peng,2 William E. Rainey,1,2 and Adina F. Turcu2 1Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109; 2Division of Metabolism, Endocrinology, and Diabetes, University of Michigan, Ann Arbor, Michigan 48109; and 3Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109


BACKGROUND: Dehydroepiandrosterone sulfate (DHEAS) is the most abundant steroid in human circulation, and adrenocorticotropic hormone (ACTH) is considered the major regulator of its synthesis. Pregnenolone sulfate (PregS) and 5-androstenediol-3-sulfate (AdiolS) have recently emerged as biomarkers of adrenal disorders. OBJECTIVE: To define the relative human adrenal production of D5-steroid sulfates under basal and cosyntropin-stimulated conditions. METHODS: Liquid chromatography-tandem mass spectrometry was used to quantify three unconjugated and four sulfated D5-steroids in (1) paired adrenal vein (AV) and mixed venous serum samples (21 patients) and (2) cultured human adrenal cells both before and after cosyntropin stimulation, (3) microdissected zona fasciculata (ZF) and zona reticularis (ZR) from five human adrenal glands, and (4) a reconstituted in vitro human 17a-hydroxylase/17,20-lyase/(P450 17A1) system. RESULTS: Of the steroid sulfates, PregS had the greatest increase after cosyntropin stimulation in the AV (32-fold), whereas DHEAS responded modestly (1.8-fold). PregS attained concentrations comparable to those of DHEAS in the AV after cosyntropin stimulation (AV DHEAS/PregS, 24 and 1.3 before and after cosyntropin, respectively). In cultured adrenal cells, PregS demonstrated the sharpest response to cosyntropin, whereas DHEAS responded only modestly (21-fold vs 1.8-fold higher compared with unstimulated cells at 3 hours, respectively). Steroid analyses in isolated ZF and ZR showed similar amounts of PregS and 17a-hydroxypregnenolone in both zones, whereas DHEAS and AdiolS were higher in ZR (P, 0.05). CONCLUSION: These studies demonstrated that unlike DHEAS, PregS displayed a prominent acute response to cosyntropin. PregS could be used to interrogate the acute adrenal response to ACTH stimulation and as a biomarker in various adrenal disorders.


The process of sulfation enhances solubility and protein binding for steroids in the circulation, where they serve as inactive reservoirs for more hydrophobic, unconjugated precursors to active steroids. In addition, considerable evidence demonstrates that steroid sulfates directly activate cell surface receptors and thus influence neuromodulation and possibly reproduction. In the adrenal gland, sulfotransferase type 2A (SULT2A1) sulfates the 3b-hydroxyl group of the D5 -steroids pregnenolone (Preg), 17a-hydroxypregnenolone (17OHPreg), dehydroepiandrosterone (DHEA), and androsta-5-ene-3b,17b-diol to form Preg sulfate (PregS), 17OHPreg sulfate (17OHPregS), DHEA sulfate (DHEAS), and 5-androstenediol-3-sulfate (AdiolS), respectively. SULT2A1 deficiency has not been described to date, but mutations in the enzyme PAPS synthase 2, which provides the mandatory sulfate donor of all sulfotransferases, 30-phosphoadenosine-50-phosphosulfate, cause elevated DHEA and downstream androgen levels. Steroid sulfates can be hydrolyzed to their respective unconjugated D5-steroids by the enzyme steroid sulfatase. Nevertheless, DHEAS concentrations in human serum are 100-fold higher than those of DHEA. Circulating DHEAS has a much lower rate of clearance than DHEA and, as a result of its extended half-life, demonstrates little diurnal rhythm. Chronically, however, DHEAS is thought to be regulated by adrenocorticotropic hormone (ACTH) and is viewed as a reflection of the hypothalamic pituitary-adrenal axis integrity. DHEAS has been proposed as an auxiliary tool for the diagnosis of secondary adrenal insufficiency and autonomous adrenal hypercortisolism, although studies have disagreed over both applications. Remarkably, DHEAS is paradoxically low in patients with congenital adrenal hyperplasia due to classic 21-hydroxylase deficiency (21OHD), including those in poor control despite chronic ACTH stimulation and elevation of other 19-carbon steroids. In contrast, authors recently found that patients with classic 21OHD had higher PregS levels than sex- and age-matched controls. In a subsequent study of children and adults with classic 21OHD, they showed that PregS correlated with ACTH, whereas DHEAS did not. Given the emerging data supporting promising clinical applications of PregS, 17OHPregS, and AdiolS as biomarkers of adrenal disease and maturation, (Turcu AF et al, Eur J Endocrinol. 2016;174(5): 601–609; Turcu AF et al. J Clin Endocrinol Metab. 2017;102(8): 2701–2710; Rege J, et al J Clin Endocrinol Metab. 2016; 101(12):4585–4593) they compared the synthesis of these three adrenal-derived steroid sulfates with DHEAS in humans under basal and cosyntropin-stimulated conditions both in vivo and in vitro. For this, they simultaneously quantified the baseline and cosyntropin-stimulated production of PregS, 17OHPregS, DHEAS, and AdiolS by the human adrenal both in vivo and in vitro. The acute and sharp response of PregS to cosyntropin stimulation suggests that PregS can be used to acutely interrogate the hypothalamic pituitary-adrenal axis and serve as biomarker in various adrenal disorders.



J Clin Endocrinol Metab. 2017 Aug 1;102(8):2701-2710.

11-Oxygenated Androgens Are Biomarkers of Adrenal Volume and Testicular Adrenal Rest Tumors in 21-Hydroxylase Deficiency. Turcu AF1Mallappa A2, Elman MS2, Avila NA3,4, Marko J2, Rao H2, Tsodikov A5, Auchus RJ1,6, Merke DP2,7. 1 Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, Michigan 48109. 2 National Institutes of Health Clinical Center, Bethesda, Maryland 20892. 3 National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892. 4 Washington DC Veterans Affairs Medical Center, Radiology Service, Washington, DC 20422. 5 School of Public Health, University of Michigan, Ann Arbor, Michigan 48109. 6 Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109. 7 Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland 20892.


CONTEXT: Patients with 21-hydroxylase deficiency (21OHD) have long-term complications, resulting from poor disease control and/or glucocorticoid overtreatment. Lack of optimal biomarkers has made it challenging to tailor therapy and predict long-term outcomes. OBJECTIVE: To identify biomarkers of disease control and long-term complications in 21OHD. SETTING AND PARTICIPANTS: Cross-sectional study of 114 patients (70 males), ages 2 to 67 years (median, 15 years), seen in a tertiary referral center. METHODS: We correlated a mass-spectrometry panel of 23 steroids, obtained before first morning medication, with bone age advancement (children), adrenal volume (adults), testicular adrenal rest tumors (TART), hirsutism, menstrual disorders, and pituitary hormones. RESULTS: Total adrenal volume correlated positively with 18 steroids, most prominently 21-deoxycortisol and four 11-oxygenated-C19 (11oxC19) steroids: 11β-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11ketoA4), 11β-hydroxytestosterone (11OHT), and 11-ketotestosterone (11ketoT) (r ≈ 0.7, P < 0.0001). Nine steroids were significantly higher (P ≤ 0.01) in males with TART compared with those without TART, including 11OHA4 (6.8-fold), 11OHT (4.9-fold), 11ketoT (3.6-fold), 11ketoA4 (3.3-fold), and pregnenolone sulfate (PregS; 4.8-fold). PregS (28.5-fold) and 17-hydroxypregnenolone sulfate (19-fold) levels were higher (P < 0.01) in postpubertal females with menstrual disorders. In males, testosterone levels correlated positively with all 11oxC19 steroids in Tanner stages 1 and 2 (r ≈ 0.7; P < 0.001) but negatively in Tanner stage 5 (r = -0.3 and P < 0.05 for 11ketoA4 and 11ketoT). In females, testosterone level correlated positively with all four 11oxC19 steroids across all Tanner stages (r ≈ 0.8; P < 0.0001). CONCLUSION: 11oxC19 steroids and PregS might serve as clinically useful biomarkers of disease control and long-term complications in 21OHD.


The management of 21OHD has been limited by inadequate biomarkers of disease control. The advent of liquid chromatography-tandem mass spectrometry (LC-MS/MS) panels has expanded the repertoire of steroid biomarkers for 21OHD. Steroids synthesized with the participation of 11b-hydroxylase (CYP11B1), such as 11b-hydroxyandrostenedione (11OHA4) and 11-ketotestosterone (11ketoT), are abundant in patients with 21OHD, and these 11-oxygenated-C19 (11oxC19) steroids have been proposed to be clinically relevant androgens. Herein, authors examined the relationship between the serum steroid metabolome of children and adults with classic 21OHD and clinical findings reflecting longstanding poor disease control, such as increased adrenal volume, advanced bone age, presence of testicular adrenal rest tumors (TARTs) in males, and presence of hirsutism and menstrual disorders in females. This is apparently the first study to implement LC-MS/MS for comprehensively analyze the relationship between a wide spectrum of traditional and emerging serum steroid biomarkers and clinical outcomes, reflecting poor long-term disease control of classic 21OHD. Adverse effects solely deriving from iatrogenic Cushing syndrome were not within the scope of the study. They identified associations of several steroid biomarkers with adrenal volume, presence of TART, menstrual disturbances, and hirsutism, but not predictors of advanced bone age or TART size. In total, 18 of the 23 measured steroids correlated directly with adrenal volume. The tightest correlation of adrenal volume was with 21dF and the 11oxC19 steroids 11OHA4, 11ketoA4, 11OHT, and 11ketoT, followed closely by A4, 17OHP, and 16OHP. Along with 17OHP, 21dF and 16OHP have been found to be elevated in patients with 21OHD. The 21dF and all 11oxC19 steroids result from the further metabolism of precursors via CYP11B1. CYP11B1 catalyzes the final step in cortisol synthesis, and is predominantly expressed in the adrenal gland, with similar expression in zona fasciculata and zona reticularis. As they have recently shown, the 11oxC19 steroids 11OHA4, 11ketoA4, 11OHT, and 11ketoT are three- to fourfold higher in treated patients with classic 21OHD compared with sex- and age-matched control subjects. These results further demonstrate that the production of all four 11oxC19 steroids is proportional with adrenal volume. In addition, adrenal volume correlated inversely with LH, suggesting that the abundant production of adrenal androgens leads to hypothalamic-pituitary-gonadal axis suppression. In a cross-sectional study of 26 men with classic 21OHD, Reich and colleagues (Reisch N et al. Total adrenal volume but not testicular adrenal rest tumor volume is associated with hormonal control in patients with 21-hydroxylase deficiency. J Clin Endocrinol Metab 2010 95:2065–2072) found a similar positive correlation of the total adrenal volume with the established biomarkers, 17OHP and A4, as measured by immunoassays, and a negative correlation with LH. They identified nine steroid biomarkers to be significantly higher in males with TART as compared with their counterparts of similar ages without TART. Of these, 11OHA4 was the most significant. Interestingly, two conjugated steroids, PregS and 17OHPregS, were significantly higher in males with than in those of the same age without TART, whereas DHEAS and androst-5-ene-3b,17b-diol-3-sulfate were similar. We have previously shown that PregS is higher in patients with classic 21OHD than in age- and sex-matched control subjects, whereas DHEAS and androst-5-ene-3b, 17b-diol-3-sulfate were both dramatically lower in 21OHD. Taken together, these findings suggest that the upstream conjugated steroids PregS and 17OHPregS could be useful biomarkers of poor disease control, whereas the ordinarily major adrenal C19 steroid DHEAS cannot serve this purpose. Although adrenal volume and TART size were not correlated, the median adrenal volume was higher in the TART group compared with males without TART, suggesting poorer disease control in the preceding months to years. Collectively, these lines of evidence suggest that, although the presence and ultimate dimensions of TART depend on a suite of prenatal events, the chronicity and amplitude of ACTH elevation contributes to TART enlargement. These findings once again demonstrate that the gonadal capacity to synthesize 11ketoT is negligible. Because the androgenic activity of 11ketoT parallels that of T, they have previously proposed that 11ketoT might act as the major circulating androgen in many patients with 21OHD. Several findings from the present studies, including the positive correlation with adrenal volume, the higher concentration in males with TART, and the positive correlation with ACTH and lack of correlation with LH, further promote 11ketoT as a promising clinical biomarker in patients with 21OHD. This seems to be the first study to implement LC-MS/MS for an extensive analysis of the serum steroid metabolome across a wide range of ages and in relation with long-term complications in patients with 21OHD. They found a link between the morning serum steroid metabolome and long-term complications in patients with classic 21OHD. They identified a set of steroid biomarkers that are associated with increased adrenal volume and the presence of TART, but not TART size, in males and menstrual disorders in females. In particular, four 11oxC19 steroids and PregS emerged as biomarkers with promising clinical value for monitoring treatment of 21OHD. LC-MS/MS offers the benefit of sensitive and specific quantitation of multiple analytes simultaneously. This approach is likely to expand into clinical laboratories, thereby allowing the development of valuable biomarker tools for optimizing patient care. This study constitutes only an early step in the discovery of greatly needed biomarkers in 21OHD management. Longitudinal prospective studies that adopt a similar steroid metabolome approach are necessary for further selection of relevant biomarkers to guide treatment of patients with 21OHD.


FASEB J. 2018 Jan 24:fj201700769RR. doi: 10.1096/fj.201700769RR.

Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells. Neirijnck Y, Calvel P, Kilcoyne KR, Kühne F, Stévant I, Griffeth RJ, Pitetti JL, Andric SA, Hu MC, Pralong F, Smith LB, Nef S. *Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland; †Medical Research Council (MRC) Centre for Reproductive Health, University of Edinburgh, Edinburgh, Scotland, United Kingdom; ‡Laboratory for Reproductive Endocrinology and Signaling, Faculty of Sciences, University of Novi Sad, Novi Sad, Serbia; §Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan; {Department of Internal Medicine, University Hospital, Lausanne, Switzerland; and kSchool of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia


Authors show here that specific deletion of both Insr and Igf1r in steroidogenic cells in mice leads to severe alterations of adrenocortical and testicular development. Double-mutant mice display drastic size reduction of both adrenocortex and testes, with impaired corticosterone, testosterone, and sperm production. Detailed developmental analysis of the testes revealed that fetal Leydig cell (LC) function is normal, but there is a failure of adult LC maturation and steroidogenic function associated with accumulation of progenitor LCs (PLCs). Cell-lineage tracing revealed PLC enrichment is secondary to Insr and Igf1r deletion in differentiated adult LCs, suggesting a feedback mechanism between cells at different steps of differentiation. Taken together, these data reveal the cell-autonomous and non-autonomous roles of the IGF system for proper development and maintenance of steroidogenic lineages.


IGFs have critical roles in testicular function, but previous studies have been performed using either primary LCs in vitro, in vitro LC lines or constitutive Igf1 mutant animals. However, from those studies it cannot be differentiated whether the effects on LCs are direct or indirect (via other testicular cell lineages). Moreover, it is unclear whether redundancies with related factors, such as IGF2 and INS, underestimate the importance of the IGF family in regulating steroidogenesis and LC development and function. To investigate the functional relevance of INS and IGF1 receptors in the development of adrenocortical and testicular steroidogenic cells, they specifically deleted these receptors using a previously described Cre recombinase mouse line, in which expression is driven by the human Cyp11a1 (P450scc) gene promoter (SCC:Cre). For simplicity, SCC:Cre-mediated deletion of Insr (SCC:Cre; Insrflox/flox), Igf1r (SCC:Cre; Igf1rflox/flox), or both Insr and Igf1r (SCC:Cre; Insrflox/flox;Igf1rflox/flox) was abbreviated as SCC:Cre; Insr, SCC:Cre; Igf1r, and SCC:Cre; Insr;Igf1r, respectively. By breeding SCC:Cre with a Rosa26:tdTOMATO reporter line, they first demonstrated that CRE activity was initiated as early as E10.5 in the mouse adreno-gonadal primordium. In the developing testis and adrenal gland at E13.5, CRE-mediated deletion was observed in both 3bHSD+ adrenal cortex, and testicular LCs. Of note, 3bHSD+ cells were all positive for CRE activity, regardless of their morphology (round or spindle shaped), suggesting SSC:CRE is active in committed PLCs as well as differentiated LCs. Importantly, a strong signal is also visible in Sertoli cells (SCs), suggesting that SCC:Cre is/has been active in both cell types. The efficiency of the SCC:Cre transgene to eliminate both Insr and Igf1r transcripts specifically and completely in LCs was assessed by immunohistochemistry. Adrenal cortex development and function is substantially affected when both Insr and Igf1r are ablated.



Proc Natl Acad Sci U S A. 2014 May 6;111(18):E1924-32. Fetal programming of adult Leydig cell function by androgenic effects on stem/progenitor cells. Kilcoyne KR1, Smith LB, Atanassova N, Macpherson S, McKinnell C, van den Driesche S, Jobling MS, Chambers TJ, De Gendt K, Verhoeven G, O’Hara L, Platts S, Renato de Franca L, Lara NL, Anderson RA, Sharpe RM. 1 Medical Research Council Centre for Reproductive Health, The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, United Kingdom.


Fetal growth plays a role in programming of adult cardiometabolic disorders, which in men, are associated with lowered testosterone levels. Fetal growth and fetal androgen exposure can also predetermine testosterone levels in men, although how is unknown, because the adult Leydig cells (ALCs) that produce testosterone do not differentiate until puberty. To explain this conundrum, we hypothesized that stem cells for ALCs must be present in the fetal testis and might be susceptible to programming by fetal androgen exposure during masculinization. To address this hypothesis, we used ALC ablation/regeneration to identify that, in rats, ALCs derive from stem/progenitor cells that express chicken ovalbumin upstream promoter transcription factor II. These stem cells are abundant in the fetal testis of humans and rodents, and lineage tracing in mice shows that they develop into ALCs. The stem cells also express androgen receptors (ARs). Reduction in fetal androgen action through AR KO in mice or dibutyl phthalate (DBP) -induced reduction in intratesticular testosterone in rats reduced ALC stem cell number by ∼40% at birth to adulthood and induced compensated ALC failure (low/normal testosterone and elevated luteinizing hormone). In DBP-exposed males, this failure was probably explained by reduced testicular steroidogenic acute regulatory protein expression, which is associated with increased histone methylation (H3K27me3) in the proximal promoter. Accordingly, ALCs and ALC stem cells immunoexpressed increased H3K27me3, a change that was also evident in ALC stem cells in fetal testes. These studies highlight how a key component of male reproductive development can fundamentally reprogram adult hormone production (through an epigenetic change), which might affect lifetime disease risk.


Men are defined by androgens (testosterone), which drive fetal masculinization (male development) and puberty and maintain masculinity in adulthood, including sex drive, erectile function, and fertility. Moreover, Western cardiometabolic diseases are all associated with lowered testosterone levels in men. Therefore, influences on testosterone levels in adulthood have pervasive importance for masculinity and health. This study shows, for the first time, that testosterone levels during fetal masculinization can (re)program adult testosterone levels through effects on stem cells, which develop into adult Leydig cells (the source of testosterone) after puberty. These stem cells are present in fetal testes of humans and animals, and using the latter, we show how these cells are reprogrammed to affect adult testosterone levels.


Evidence that altered fetal growth/development can fundamentally alter the risk of health disorders in adulthood and perhaps, future generations continues to grow. Such fetal programming applies to common disorders encapsulated in the metabolic syndrome, which are interlinked in adult men with low testosterone levels. Large studies from the United States and Europe also show that testosterone levels in men of all ages are declining with later year of birth. Aging is itself associated with declining testosterone levels and a high incidence of primary/compensated hypogonadism. Because low testosterone levels are also associated with generalized pro-inflammatory changes, frailty, and risk of dying in aging men, what determines an adult man’s testosterone level is of fundamental importance.

There is also evidence from human and animal experimental studies that fetal programming can influence adult testosterone levels, particularly that reduced fetal androgen exposure leads to lower adult male testosterone levels. This data fits with growing evidence that subtle deficiency in fetal androgens is a major determinant of adult male reproductive disorders, such as low sperm production, and might explain why low sperm counts are often associated with compensated Leydig cell failure in men. However, the mechanisms through which fetal events could influence adult testosterone levels are unknown.

The finding that fetal deficits in androgen action can result in compromised adult Leydig cell function in rodents fits with emerging evidence from humans. There is a similar connection between reduced fetal androgens and reduced adult sperm counts/sperm production in men and rats. What further ties these observations together is that men with low sperm counts commonly exhibit compensated adult Leydig cell failure, although why is unknown. It has been suggested that it may be indicative of a common underlying (fetal) cause, and the present animal experimental studies provide direct supporting evidence for this suggestion. This suggested connection has widespread implications, because one in six young men in northern European countries has a low sperm count (



J Clin Pathol. 2017 Jun;70(6):500-507. doi: 10.1136/jclinpath-2016-204089. Multicentre validation of a microRNA-based assay for diagnosing indeterminate thyroid nodules utilizing fine needle aspirate smears.

Lithwick-Yanai G1, Dromi N1, Shtabsky A2,3, Morgenstern S3,4, Strenov Y3,4, Feinmesser M3,4, Kravtsov V3,5, Leon ME6, Hajdúch M7, Ali SZ8, VandenBussche CJ8, Zhang X9,10, Leider-Trejo L2,3, Zubkov A2, Vorobyov S11, Kushnir M1, Goren Y1,12, Tabak S1, Kadosh E1, Benjamin H13, Schnitzer-Perlman T1, Marmor H1, Motin M1, Lebanony D1, Kredo-Russo S1, Mitchell H13, Noller M13, Smith A13, Dattner O13, Ashkenazi K13, Sanden M13, Berlin KA13, Bar D1, Meiri E1. 1 Rosetta Genomics Ltd, Rehovot, Israel. 2 Pathology Institute, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel. 3 The Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. 4 Pathology Institute, Rabin Medical Center, Petach Tikva, Israel. 5 Pathology Institute, Meir Medical Center, Kfar Saba, Israel. 6 H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA. 7 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic. 8 The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 9 Temple University Hospital, Philadelphia, Pennsylvania, USA. 10 Cooper University Hospital, Cooper Medical School of Rowan University at Camden, New Jersey, USA. 11 National Centre of Clinical and Morphological Diagnostics, St Petersburg, Russia. 12 Geha Mental Health Center, Petach Tikva, Israel. 13 Rosetta Genomics Inc, Philadelphia, Pennsylvania, USA.


AIMS: The distinction between benign and malignant thyroid nodules has important therapeutic implications. The objective was to develop an assay that could classify indeterminate thyroid nodules as benign or suspicious, using routinely prepared fine needle aspirate (FNA) cytology smears. METHODS: A training set of 375 FNA smears was used to develop the microRNA-based assay, which was validated using a blinded, multicentre, retrospective cohort of 201 smears. Final diagnosis of the validation samples was determined based on corresponding surgical specimens, reviewed by the contributing institute pathologist and two independent pathologists. Validation samples were from adult patients (≥18 years) with nodule size >0.5 cm, and a final diagnosis confirmed by at least one of the two blinded, independent pathologists. The developed assay, RosettaGX Reveal, differentiates benign from malignant thyroid nodules, using quantitative RT-PCR. RESULTS: Test performance on the 189 samples that passed quality control: negative predictive value: 91% (95% CI 84% to 96%); sensitivity: 85% (CI 74% to 93%); specificity: 72% (CI 63% to 79%). Performance for cases in which all three reviewing pathologists were in agreement regarding the final diagnosis (n=150): negative predictive value: 99% (CI 94% to 100%); sensitivity: 98% (CI 87% to 100%); specificity: 78% (CI 69% to 85%). CONCLUSIONS: A novel assay utilising microRNA expression in cytology smears was developed. The assay distinguishes benign from malignant thyroid nodules using a single FNA stained smear, and does not require fresh tissue or special collection and shipment conditions. This assay offers a valuable tool for the preoperative classification of thyroid samples with indeterminate cytology.


These authors also use a genomic biomarker (a microRNA) to identify as thyroid papillary carcinoma a thyroid nodule biopsy collected by fine needle aspiration; similar to the publication commented in the Subject Review Section for another marker (microRNA-146b). Future statistical studies will define the best bio-markers for this important diagnosis


Thyroid. 2016 Jun; 26(6):785-93.

A genomic alternative to Identify medullary thyroid cancer preoperatively in thyroid nodules with indeterminate cytology. Kloos RT1Monroe RJ2Traweek ST3Lanman RB1Kennedy GC41 Department of Medical Affairs, Veracyte, Inc., South San Francisco, California. 2 CLIA, Veracyte, Inc., South San Francisco, California. 3 Thyroid Cytopathology Partners, Austin, Texas. 4 Department of Research and Development , Veracyte, Inc., South San Francisco, California.


BACKGROUND: The use of calcitonin screening for the rare medullary thyroid cancer (MTC) is controversial due to questions of efficacy, accuracy, and cost-effectiveness. This study reports the results of a large prospective validation using a machine-trained algorithm (MTC Classifier) to preoperatively identify MTC in fine-needle aspiration biopsies, in lieu of calcitonin measurements.

METHODS: Cytology analysis on a prospective consecutive series of 50,430 thyroid nodule biopsies yielded a total of 7815 indeterminate (Bethesda categories III/IV) cases, which were tested with the MTC classifier. A prospective, consecutively submitted series of 2673 Bethesda III-VI cases with cytology determined locally was also evaluated. RNA was isolated and tested for the MTC Classifier using microarrays. RESULTS: Forty-three cases were positive by the MTC Classifier among 10,488 tested nodules (0.41%), consistent with the low prevalence of MTC. Of these, all but one was histologically or biochemically confirmed as MTC, yielding a positive predictive value (PPV) of 98%. Of the positive cases, only 19 (44%) had been specifically suspected of MTC by cytology, highlighting the limitations of light microscopy to detect this disease. Three surgically confirmed MTC cases, that were detected by the MTC Classifier, had low basal serum calcitonin values, indicating these would have been missed by traditional calcitonin screening methods. A pooled analysis of three independent validation sets demonstrates high test sensitivity (97.9%), specificity (99.8%), PPV (97.9%), and negative predictive value (99.8%). CONCLUSIONS: A clinical paradigm is proposed, whereby cytologically indeterminate thyroid nodules being tested for common malignancies using gene expression can be simultaneously tested for MTC using the same genomic assay at no added cost.


Medullary thyroid cancer (MTC) accounts for only 2.2% of thyroid cancers, but is responsible for up to 13.5% of its mortality. Almost 50% of the more common sporadic MTC (sMTC) cases present at stage III or IV disease, with no evidence of increased survival in recent decades.

Challenges in interpreting cytology result in one-half of MTC cases being missed. A specific diagnosis of MTC is critical to planning the optimal surgery, which is more extensive than for thyroid nodules in which MTC is not specified.

Unfortunately, many sMTC patients undergo suboptimal preoperative evaluations and initial surgical interventions, resulting in additional ‘‘completion’’ surgeries and/or compromised patient outcomes. In addition, when MTC is not suspected, more than one-quarter of patients with cytologically indeterminate fine needle aspiration biopsies (FNABs) do not undergo surgical resection in the short term, an approach that would delay the diagnosis and treatment of MTCs missed by cytology. Conversely, a false diagnosis of MTC can lead to an overly aggressive surgery. As medullary thyroid tumors often overproduce calcitonin, screening for its production, either by immunohistochemistry, or by measurement in the serum, has been a focus of diagnostic efforts.

While serum calcitonin screening is accepted in some countries it is not uniformly accepted in the United States and elsewhere, resulting partly from its high false positive rate (60–90%) and low positive predictive value (PPV) of 10–40%. Thus, serum calcitonin screening thresholds with high sensitivity may drive unnecessary MTC-related testing and surgery in patients who do not have MTC.

It was reasoned that if there were a paradigm for testing FNAs from indeterminate nodules, an additional classifier could be incorporated to test for MTC without incurring the expense and effort of a separate test. Recently, machine learning was used to develop an algorithm that uses mRNA gene expression data, from thyroid FNAB samples, to reclassify cytologically indeterminate nodules as benign or suspicious, with high sensitivity and negative predictive value (NPV). This classification algorithm (Afirma_Gene Expression Classifier [GEC]) uses 142 genes to separate benign and suspicious nodules, but the presence of nearly 3000 genes on the microarray allows future tests to be added by developing algorithms on different gene sets for different diagnostic purposes.

This approach was used to develop a classifier for MTC, and as all genes are measured with one assay on one microarray, the MTC result is generated concurrently with the GEC result, at no added cost. The MTC portion of the classifier was trained by analyzing expression data on 22 known MTC cases from 220 surgical tissues, confirmed by histopathology. Using machine learning and feature selection on 283,927 gene transcripts measured in training, a group of five genes was selected (CALCA, CEACAM5, SCG3, SCN9A, and SYT4), which together have high accuracy for detecting MTC.

After the MTC Classifier was trained and locked, two independent FNAB sample sets with a combined total of 489 samples were tested to establish clinical validation. Those studies showed high specificity and high NPV of the MTC Classifier. Here, the MTC Classifier is tested on 10,488 thyroid nodule FNAB samples referred for GEC testing from a large prospective clinical series of 53,103 consecutively received thyroid nodule FNABs, the largest cohort tested to date A pooled analysis combining all three studies demonstrates exceptionally high diagnostic accuracy.

Posted in Edition 68 - 2018, June, Bibliographic Reviews
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