The technological advancements in genetics produced a profound impact on the research and diagnostics of non-communicable diseases. The availability of next-generation sequencing (NGS) allowed the identification of novel candidate genes but also an in-depth modification of the understanding of the architecture of several endocrine diseases. Several different NGS approaches are available allowing the sequencing of several regions of interest or the whole exome or genome (WGS, WES or targeted NGS), with highly variable costs, potentials and limitations that should be clearly known before designing the experiment. Here, we illustrate the NGS scenario, describe the advantages and limitations of the different protocols and review some of the NGS results obtained in different endocrine conditions. We finally give insights on the terminology and requirements for the implementation of NGS in research and diagnostic labs.
Luca Persani, Tiziana de Filippis, Carla Colombo and Davide Gentilini
Guia Vannucchi, Michela Perrino, Stefania Rossi, Carla Colombo, Leonardo Vicentini, Davide Dazzi, Paolo Beck-Peccoz and Laura Fugazzola
Pregnancy represents a favorable condition for the development of thyroid nodules, likely due to the secretion of hormones with stimulatory activity. In particular, differentiated thyroid cancer (DTC) represents the second most frequent tumor among those diagnosed during pregnancy. However, few and discordant data are available about the impact of pregnancy on tumor outcome.
A total of 123 women with DTC were divided into three groups according to the timing of tumor diagnosis (group 1, at least 1 year after the delivery; group 2, during pregnancy or in the first year after delivery; and group 3, before pregnancy or nulliparity) and evaluated according to the international guidelines. Furthermore, immunohistochemical studies of estrogen receptor α (ERα) were performed in 38 papillary thyroid cancer tissues from the three groups.
Thyroid cancer diagnosed during pregnancy was associated with a poorer prognosis compared to tumors developed in nongravidic periods (P<0.0001). Accordingly, at the stepwise logistic regression analysis, the diagnosis of DTC during pregnancy or in the first year post partum was the most significant indicator of persistent disease (P=0.001). Interestingly, ERα expression significantly differed among tumors of the three groups, being detected in 31% of group 1, in 87.5% of group 2, and in 0% of group 3 (P=0.01).
Present data indicate that pregnancy has a negative impact on the outcome of thyroid cancer. The presence of ERα in the majority of tumors diagnosed during pregnancy indicates that the poorer outcome of these cases could be related to the estrogen-mediated growth stimulus.
Valentina Cirello, Roberta Rizzo, Milena Crippa, Irene Campi, Daria Bortolotti, Silvia Bolzani, Carla Colombo, Guia Vannucchi, Maria Antonia Maffini, Federica de Liso, Stefano Ferrero, Palma Finelli and Laura Fugazzola
The physiological persistence of fetal cells in the circulation and tissue of a previously pregnant woman is called fetal cell microchimerism (FCM). It has been hypothesized to play a role in systemic autoimmune disease; however, only limited data are available regarding its role in autoimmune thyroid disease (AITD).
Circulating FCM was analyzed in a large series of previously pregnant women with Graves' disease (GD), Hashimoto's thyroiditis (HT), or no disease (healthy controls (HCs)). To exclude the possible bias related to placental factors, the polymorphic pattern of human leukocyte antigen-G (HLA-G) gene, which is known to be involved in the tolerance of fetal cells by the maternal immune system, was investigated.
FCM was evaluated by PCR in the peripheral blood, and the Y chromosome was identified by fluorescence in situ hybridization in some GD tissues. HLA-G polymorphism typing was assessed by real-time PCR.
FCM was significantly more frequent in HC (63.6%) than in GD (33.3%) or HT (27.8%) women (P=0.0004 and P=0.001 respectively). A quantitative analysis confirmed that circulating male DNA was more abundant in HC than it was in GD or HT. Microchimeric cells were documented in vessels and in thyroid follicles. In neither GD/HT patients nor HC women was the HLA-G typing different between FCM-positive and FCM-negative cases.
The higher prevalence of FCM in HC as compared to GD and HT patients suggests that it plays a possible protective role in autoimmune thyroid disorders. Placental factors have been excluded as determinants of the differences found. The vascular and tissue localization of microchimeric cells further highlights the ability of those cells to migrate to damaged tissues.
Guia Vannucchi, Simone De Leo, Michela Perrino, Stefania Rossi, Delfina Tosi, Valentina Cirello, Carla Colombo, Gaetano Bulfamante, Leonardo Vicentini and Laura Fugazzola
Thyroid cancer is highly prevalent in women during the fertile age, which suggests a possible impact of hormonal and reproductive factors.
We studied the expression of estrogen receptor α (ERα or ESR1) and progesterone receptor (PR or PGR) in 182 female and male patients with papillary thyroid cancer and correlated it to clinical and molecular features.
ERα and PR expression was found in 66.5 and 75.8% of patients respectively and was significantly correlated with larger tumor size and with a non-incidental diagnosis. Moreover, a trend toward a higher prevalence of local metastases was observed in ER- and PR-expressing tumors, which possibly indicates a more aggressive behavior. Interestingly, the occurrence of the ‘receptor conversion’ phenomenon, which has already been reported to have a negative prognostic effect in breast cancer, was demonstrated for the first time in thyroid tumors. Indeed, almost all of the ERα-positive primary tumors analyzed had ERα-negative metastatic lymph nodes. At the genetic analyses, BRAF V600E mutation was detected in 23.2% of the tumors and had a higher prevalence in larger tumors and in those with a stronger ERα or PR staining.
The whole of the findings reported in the present study argue for an association between ERα and PR sex hormone receptor expression and a more aggressive presentation. Although no impact on outcome was found, the evaluation of ERα and PR receptor expression could add insights into the biological behavior of tumors and could modify the follow-up, particularly in fertile women affected with persistent disease.