Thyroid : official journal of the American Thyroid Association.2013 Mar;23(3):308-316.

Measurement of calcitonin and calcitonin gene-related peptide mRNA refines the management of patients with medullary thyroid cancer and may replace calcitonin-stimulation tests.

Camacho CP1, Lindsey SC1, Melo MC1, Yang JH1, Germano-Neto F1, Valente FOF1, Lima TR2, Biscolla RP1, Vieira JG1, Cerutti JM2, Dias-da-Silva MR1, Maciel RM1.

1- Laboratory of Molecular and Translational Endocrinology, Department of Medicine, and 2- Department of Genetics, Escola Paulista de Medicina, Federal University of São Paulo, São Paulo, Brazil.

 

Abstract

Serum calcitonin (sCT) is the main tumor marker for medullary thyroid cancer (MTC), but it has certain limitations. Various sCT assays may have important intra-assay or interassay variation and may yield different and sometimes conflicting results. A pentagastrin- or calcium-stimulation calcitonin (CT) test may be desirable in some situations. Alternatively, or in the absence of the stimulation test, mRNA detection offers the advantages of being more comfortable and less invasive; it only requires blood collection and has no side effects. The objective of this study was to investigate the applicability of measuring calcitonin-related polypeptide alpha (CALCA) gene transcripts (CT-CALCA and calcitonin gene-related peptide [CGRP]-CALCA) in patients with MTC and in relatives diagnosed with a RET mutation and to test mRNA as an alternative diagnostic tool for the calcitonin-stimulation test. METHODS: Twenty-three healthy controls and 26 individuals evaluated for MTC were selected, including patients with sporadic or hereditary MTC and RET mutation-carrying relatives. For molecular analysis, RNA was extracted from peripheral blood, followed by cDNA synthesis using 3.5 μg of total RNA. Quantitative real-time polymerase chain reaction (RT-qPCR) was performed with SYBR Green and 200 nM of each primer for the two specific mRNA targets (CT-CALCA or CGRP-CALCA) and normalized with the ribosomal protein S8 as the reference gene. RESULTS: We detected CALCA transcripts in the blood samples and observed a positive correlation between them (r=0.946, p<0.0001). Both mRNAs also correlated with sCT (CT-CALCA, r=0.713, p<0.0001; CGRP-CALCA, r=0.714, p<0.0001). The relative expression of CT-CALCA and CGRP-CALCA presented higher clinical sensitivity (86.67 and 100, respectively), specificity (97.06 and 97.06), positive predictive value (92.86 and 93.75), and negative predictive value (94.29 and 100), than did sCT (73.33, 82.35, 64.71, and 87.50, respectively). In addition, the CALCA transcript measurement mirrored the response to the pentagastrin test. CONCLUSION: We demonstrate that the measurement of CALCA gene transcripts in the bloodstream is feasible and may refine the management of patients with MTC and RET mutation-carrying relatives. We propose considering the application of this diagnostic tool as an alternative to the calcitonin-stimulation test.

PMID: 23259706

 

Supplement:

The present study was elaborated during the follow-up of MTC patients (with sporadic or hereditary disease) and RET mutation-carrying relatives in the Multiple Endocrine Neoplasia outpatient clinic at the Federal University of Sao Paulo and it evaluates the use of calcitonin mRNA and CGRP mRNA as tumor markers in MTC, in comparison to basal and stimulated serum calcitonin. Calcitonin and CGRP mRNA are synthetized by an alternative splicing in the CALCA gene, as seen in Figure 1.

Although sCT is a good diagnostic marker, Giovanella and Suriano pointed out that during thyroid nodule evaluation, a false-positive sCT result is more frequent than a real MTC (1). On the other hand, the detection of free mRNA or miRNA in the plasma or urine is now largely accepted, as is the concept that mRNA detected in the blood may not necessarily reflect the cell itself or its physiology (2-4). Mittelbrunn and Sánchez-Madrid have emphasized the diagnostic potential of RNA-based communication, which is a non-invasive way to detect changes in the pathological process (5).

The major contributions of our study are the detection of CT and CGRP mRNA in the blood, the correlation of these mRNAs with serum calcitonin and the ability to distinguish thyroid-healthy controls, mutation-carrying relatives and patients with or without evidence of disease (6). Finally, the results of the statistical analysis presented in this work and the clinical accuracy of the RNA as a diagnostic tool, similar to the gold standard stimulation test, reinforce the value of persisting in the RNA strategy to create an easy, affordable and precise diagnostic tool.

Figure 1

FIGURE 1 – Schematic representation of the alternative splicing observed in CALCA gene, leading to the synthesis of calcitonin and CGRP.

 

References

  1.             Giovanella L, Suriano S 2011 Spurious hypercalcitoninemia and heterophilic antibodies in patients with thyroid nodules. Head Neck 33:95-97.
  2.             Liu R, Ma X, Xu L, Wang D, Jiang X, Zhu W, Cui B, Ning G, Lin D, Wang S 2012 Differential microRNA expression in peripheral blood mononuclear cells from Graves’ disease patients. J Clin Endocrinol Metab 97:E968-972.
  3.             Quackenbush JF, Cassidy PB, Pfeffer LM, Boucher KM, Hawkes JE, Pfeffer SR, Kopelovich L, Leachman SA 2014 Isolation of circulating microRNAs from microvesicles found in human plasma. Methods Mol Biol 1102:641-653.
  4.             Alvarez ML, Khosroheidari M, Kanchi Ravi R, DiStefano JK 2012 Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers. Kidney Int 82:1024-1032.
  5.             Mittelbrunn M, Sánchez-Madrid F 2012 Intercellular communication: diverse structures for exchange of genetic information. Nat Rev Mol Cell Biol 13:328-335.
  6.             Camacho CP, Lindsey SC, Melo MC, Yang JH, Germano-Neto F, Valente FeO, Lima TR, Biscolla RP, Vieira JG, Cerutti JM, Dias-da-Silva MR, Maciel RM 2013 Measurement of calcitonin and calcitonin gene-related peptide mRNA refines the management of patients with medullary thyroid cancer and may replace calcitonin-stimulation tests. Thyroid 23:308-316.

 

Acknowledgements:

The research of the authors is supported by the São Paulo State Research Foundation-FAPESP grants 2006/60402-1 and 2010/51547-1 (to R.M.B.M. and M.R.D.S), 2009/11257-7 and 2011/0787-2 (to J.M.C.), and 2011/20747-8 (to M.R.D.S.), by a grant from the Fleury Group (12518) and by a grant from the Brazilian Ministry of Health (25000.168513/2008-11). S.C.L.is a MD PhD scholar from FAPESP. M.C.C.M and F.G.N. are PhD students from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). R.M.B.M. and J.M.C. are investigators of the Brazilian Research Council. R.P.M.B., J.G.H.V., and R.M.B.M. are also investigators of the Fleury Group.

 

Contact:

Magnus R. Dias da Silva, MD, PhD

Laboratório de Endocrinologia Molecular e Translacional

Rua Pedro de Toledo, 669, 11o andar

Universidade Federal de São Paulo

04039-032, São Paulo, SP – Brazil

Email: mrdsilva@unifesp.br

 

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