Genetic testing for germline RET pathogenic variants, which cause the Multiple Endocrine Neoplasia Type 2 (MEN2) syndrome, has become crucial in managing patients with medullary thyroid carcinoma ...(MTC). Classically, RET heterozygous missense pathogenic variants are transmitted in a Mendelian autosomal dominant pattern, of which germline/gonadal mosaicism has never been reported. We report the novel occurrence of a MEN2A patient's family in which the siblings inherited three different RET 634 genotypes: wild type (p.Cys634), p.Cys634Gly or p.Cys634Arg heterozygous pathogenic variants. We hypothesized that germline/gonadal mosaicism, derived from an inherited + early somatic mutation in the mother or a double de novo mutation during maternal embryogenesis, led to this rare event in the RET gene. Exome analysis of the proband's deceased mother's paraffin‐embedded thyroid tissue confirmed the three nucleotides in the same 634 codon position. For the first time, we describe germline/gonadal mosaicism in RET, generating a second pathogenic amino acid change in the same codon causing MEN2A. Our finding shows that RET parental mosaicism, confirmed by somatic exome sequencing, might explain discrepant genotype cases in siblings with inherited cancers.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Different pathogenic germline mutations in the RET oncogene are identified in MEN 2, a hereditary syndrome characterized by medullary thyroid carcinoma (MTC) and other endocrine tumors. Although ...genetic predisposition is recognized, not all RET mutation carriers will develop the disease during their lifetime or, likewise, RET mutation carriers belonging to the same family may present clinical heterogeneity. It has been suggested that a single germline mutation might not be sufficient for development of MEN 2-associated tumors and a somatic bi-allelic alteration might be required. Here we investigated the presence of somatic second hit mutation in the RET gene in MTC.
We integrated Multiplex Ligation-dependent Probe Amplification (MLPA) and whole exome sequencing (WES) to search for copy number alteration (CNA) in the RET gene in MTC samples and medullary thyroid cell lines (TT and MZ-CR-1). We next found reads spanning exon-exon boundaries on RET, an indicative of retrocopy. We subsequently searched for RET retrocopies in the human reference genome (GRCh37) and in the 1000 Genomes Project data, by looking for reads reporting joined exons in the RET locus or distinct genomic regions. To determine RET retrocopy specificity and recurrence, DNA isolated from sporadic and MEN 2-associated MTC (n = 37), peripheral blood (n = 3) and papillary thyroid carcinomas with RET fusion (n = 10) samples were tested using PCR-sequencing methodology.
Through MLPA we have found evidence of CNA in the RET gene in MTC samples and MTC cell lines. WES analysis reinforced the presence of the CNA and hinted for a retroposed copy of RET not found in the human reference genome and 1.000 Genomes Project. Extended analysis confirmed the presence of a somatic MTC-related retrocopy of RET in both sporadic and hereditary tumors. We further unveiled a recurrent (28%) novel point mutation (p.G548 V) found exclusively in the retrocopy of RET. The mutation was also found in cDNA of mutated samples, suggesting it might be functional.
We here report a somatic specific RET retroposed copy in MTC samples and cell lines. Our results support the idea that generation of retrocopies in somatic cells is likely to contribute to MTC genesis and progression.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Summary
Objective
Staging systems applied to medullary thyroid cancer (MTC) rely on initial clinical and pathological features and do not consider the response to treatment. To determine whether MTC ...staging can be improved by incorporating the first postoperative calcitonin measurement.
Patients and measurements
Eighty‐five patients being monitored for MTC (median follow‐up 5 years) were retrospectively classified according to both the American Joint Committee on Cancer (AJCC) and the proposed combined risk stratification system (low, intermediate and high risk), which incorporates the first postoperative calcitonin measurement, using the outcomes no evidence of disease (NED), biochemical evidence of disease, structurally identifiable disease and death.
Results
Ninety per cent of AJCC I patients were classified as NED at final follow‐up. When we added a postoperative calcitonin measurement, 95% low‐risk patients were classified as NED at final follow‐up. AJCC stages I and IV were associated, respectively, with no occurrence and a high rate (63%) of structurally identifiable disease. Stages II and III yielded similar predictions of structurally identifiable disease, 13% and 14%, respectively. When we included the postoperative calcitonin level, the patients with structural evidence of disease included none from the low‐risk group, 10% from the intermediate group and 63% from the high‐risk group. The proportion of variance explained analysis (PVE) was better for the combined risk stratification system (54%) than for the AJCC system alone (32%).
Conclusion
Including the first postoperative calcitonin measurement with the anatomical staging system can better predict the clinical outcome of patients with MTC and refine the follow‐up of these patients.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Summary
Objective
Reviewing the clinical outcomes of a large kindred with a RET p.Gly533Cys mutation, 10 years after the first description of this kindred, has provided an important set of clinical ...data for healthcare decision‐making.
Design and Patients
We identified 728 RET533 Brazilian relatives, spread out over 7 generations. We performed clinical examination, biochemical and imaging analyses in the proband and in 103 carriers.
Measurement and Results
The proband has been followed without evidence of structural disease in the last 10 years but with elevated calcitonin. The clinical and surgical features of 60 thyroidectomized RET533 relatives were also described. Forty‐six patients had MTC (21–72 years), and 11 patients had C‐cell hyperplasia (CCH) (5–42 years). Twelve MTC patients with lymph node metastases had a tumour size of 0·7–2·8 cm. Calcitonin level and CEA were correlated with disease stage, and none of the patients presented with an altered PTH or metanephrine. A 63‐year‐old woman developed pheochromocytoma and breast cancer. Two other RET533 relatives developed lung squamous cell carcinoma and melanoma.
Conclusions
A vast clinical variability in RET533 presentation was observed, ranging from only an elevated calcitonin level (3%) to local metastatic disease (25%). Many individuals were cured (42%) and the majority had controlled chronic disease (56%), reinforcing the need for individualized ongoing risk stratification assessment. The importance of this update relies on the fact that it allows us to delineate the natural history of RET 533 MEN2A 10 years after its first description.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Medullary thyroid carcinoma (MTC), a neuroendocrine tumor originating from thyroid parafollicular cells, has been demonstrated to be associated with mutations in RET, HRAS, KRAS and NRAS. However, ...the role of other genes involved in the oncogenesis of neural crest tumors remains to be fully investigated in MTC. The current study aimed to investigate the presence of somatic mutations in BRAF, CDKN2A and PI3KCA in MTC, and to investigate the correlation with disease progression. DNA was isolated from paraffin-embedded tumors and blood samples from patients with MTC, and the hotspot somatic mutations were sequenced. A total of 2 novel HRAS mutations, p.Asp33Asn and p.His94Tyr, and polymorphisms within the 3′ untranslated region (UTR) of CDKN2A (rs11515 and rs3088440) were identified, however, no mutations were observed in other genes. It was suggested that somatic point mutations in BRAF, CDKN2A and PI3KCA do not participate in the oncogenesis of MTC. Further studies are required in order to clarify the contribution of the polymorphisms identified in the 3′UTR of CDKN2A in MTC.
Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant genetic disease caused by RET gene germline mutations that is characterized by medullary thyroid carcinoma (MTC) associated with ...other endocrine tumors. Several reports have demonstrated that the RET mutation profile may vary according to the geographical area. In this study, we collected clinical and molecular data from 554 patients with surgically confirmed MTC from 176 families with MEN2 in 18 different Brazilian centers to compare the type and prevalence of RET mutations with those from other countries. The most frequent mutations, classified by the number of families affected, occur in codon 634, exon 11 (76 families), followed by codon 918, exon 16 (34 families: 26 with M918T and 8 with M918V) and codon 804, exon 14 (22 families: 15 with V804M and 7 with V804L). When compared with other major published series from Europe, there are several similarities and some differences. While the mutations in codons C618, C620, C630, E768 and S891 present a similar prevalence, some mutations have a lower prevalence in Brazil, and others are found mainly in Brazil (G533C and M918V). These results reflect the singular proportion of European, Amerindian and African ancestries in the Brazilian mosaic genome.
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.
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.
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.
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.
Abstract
Background: Tumor-associated macrophages (TAMs) are alternatively activated or M2 macrophages that infiltrate cancer tissues. It has been suggested that TAMs exert several pro-tumor ...functions by releasing several growth factors and proangiogenic cytokines that favor tumor progression. In fact, TAM density has been associated with poor prognosis. Although it is well known the pro-tumor roles of TAM in several tumor types, its role in medullary thyroid cancer (MTC) is still unknown. We here evaluated the M2 macrophage marker CD163 in MTC and correlated with RET mutational status and clinical and pathological features. Because it has been suggested TAMs could interact with CD44-positive tumor cells to during tumorigenesis process, we also investigated the expression of CD44 in MTC.
Patients and Methods: CD163 and CD44 expression we investigated in 78 MTC by immunohistochemistry (IHC). Because hereditary form is caused by germline mutations in the RET gene, all patients were screened for RET mutations (exons 8, 10, 11, 13-16) in the DNA isolated from blood samples. Those MTC that were negative for RET germline mutations, were also screened for somatic mutations in the RET gene (exons 10, 11 e 16).
Results: Germline mutation in the RET gene were identified in 29 patients (p.G533C, p.C609Y, p.C611Y, p.C620Y, p.C634R, p.C634Y/Y791F, p.S891A and p.M918V). Approximately 49 cases are sporadic cases in which somatic RET mutations were identified in about 6 cases (p.M918T, p.C634R and p.C634Y). CD163 expression was mainly found in sporadic MTC when compared to hereditary MTC (p=0.021), suggesting that TAM may play a role in the progression of sporadic MTC. On the other hand CD44 expression was mainly found in hereditary MTC than in sporadic cases (p=0.017), particularly in patients with RET germlime mutation at codon C634.
Conclusion: TAM seems to be more important for sporadic MTC tumorigenesis when compared to hereditary MTC. On the other hand, CD44 expression correlated with hereditary MTC, particularly in patients with mutation at codon C634.
Citation Format: MARTA MIYAZAWA, MARIA JOSE C. P. SANTOS, LARISSA V. BIM, ERIKA S. L. ORTI-RADUAN, FLÁVIA O. F. VALENTE, ADAUTO F. NUNES, ROSANA DELCELO, RUI M. B. MACIEL, JANETE M. CERUTTI. TAM and CD44 in medullary thyroid carcinoma. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1072. doi:10.1158/1538-7445.AM2014-1072
Summary
Objective
Reviewing the clinical outcomes of a large kindred with a
RET
p.
G
ly533
C
ys mutation, 10 years after the first description of this kindred, has provided an important set of ...clinical data for healthcare decision‐making.
Design and Patients
We identified 728
RET
533 Brazilian relatives, spread out over 7 generations. We performed clinical examination, biochemical and imaging analyses in the proband and in 103 carriers.
Measurement and Results
The proband has been followed without evidence of structural disease in the last 10 years but with elevated calcitonin. The clinical and surgical features of 60 thyroidectomized
RET
533 relatives were also described. Forty‐six patients had
MTC
(21–72 years), and 11 patients had
C
‐cell hyperplasia (
CCH
) (5–42 years). Twelve
MTC
patients with lymph node metastases had a tumour size of 0·7–2·8 cm. Calcitonin level and
CEA
were correlated with disease stage, and none of the patients presented with an altered
PTH
or metanephrine. A 63‐year‐old woman developed pheochromocytoma and breast cancer. Two other
RET
533 relatives developed lung squamous cell carcinoma and melanoma.
Conclusions
A vast clinical variability in
RET
533 presentation was observed, ranging from only an elevated calcitonin level (3%) to local metastatic disease (25%). Many individuals were cured (42%) and the majority had controlled chronic disease (56%), reinforcing the need for individualized ongoing risk stratification assessment. The importance of this update relies on the fact that it allows us to delineate the natural history of
RET
533
MEN
2
A
10 years after its first description.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Calcitonin and carcinoembryonic antigen (CEA) doubling times are established prognostic markers in medullary thyroid cancer (MTC). On the other hand,
F-fluorodeoxyglucose (FDG) positron emission ...tomography/computed tomography (PET/CT) shows an increased rate of detection with high blood tumor marker levels in several cancers. This study aimed to analyze the ability of
F-FDG PET/CT to determine prognosis in the follow-up of patients with MTC.
Medical records of 17 patients with MTC who underwent
F-FDG PET/CT were analyzed retrospectively. All patients were classified into two groups: stable disease or progressive disease.
Eight patients presented with progressive disease, and all of them showed
F-FDG uptake (100%), compared to only 3 of 9 patients who presented in stable condition (33%).
F-FDG PET/CT results were able to distinguish progressive from stable disease (P = .009). Calcitonin levels >4,020 pg/mL (P = .0004), CEA levels >26.8 ng/mL (P = .04), and a calcitonin doubling time <24.1 months (P = .015) were associated with progressive disease in our cohort. The proportion of variance explained that predicted progressive disease was 32% for
F-FDG uptake, 27.1% for a calcitonin doubling time of 24.1 months, and 41.2% for doubling time plus
F-FDG PET/CT.
F-FDG uptake was able to distinguish progressive from stable disease. However, this tool should not replace the validated calcitonin doubling time, but rather the combination of information could improve the clinical re-assessment and better identify high-risk patients who require more careful surveillance.
CEA = carcinoembryonic antigen CT = computed tomography
F-FDG =
F-fluorodeoxyglucose MTC = medullary thyroid cancer PET = positron emission tomography PVE = proportion of variance explained sCT = serum calcitonin SUV = standard uptake value US = ultrasound.
PDF
