Identification of novel BRCA1 large genomic rearrangements by a computational algorithm of amplicon-based Next-Generation Sequencing data.

BACKGROUND: Genetic testing for BRCA1/2 germline mutations in hereditary breast/ovarian cancer patients requires screening for single nucleotide variants, small insertions/deletions and large genomic rearrangements (LGRs). These studies have long been run by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). The recent introduction of next-generation sequencing (NGS) platforms dramatically improved the speed and the efficiency of DNA testing for nucleotide variants, while the possibility to correctly detect LGRs by this mean is still debated. The purpose of this study was to establish whether and to which extent the development of an analytical algorithm could help us translating NGS sequencing via an Ion Torrent PGM platform into a tool suitable to identify LGRs in hereditary breast-ovarian cancer patients. METHODS: We first used NGS data of a group of three patients (training set), previously screened in our laboratory by conventional methods, to develop an algorithm for the calculation of the dosage quotient (DQ) to be compared with the Ion Reporter (IR) analysis. Then, we tested the optimized pipeline with a consecutive cohort of 85 uncharacterized probands (validation set) also subjected to MLPA analysis. Characterization of the breakpoints of three novel BRCA1 LGRs was obtained via long-range PCR and direct sequencing of the DNA products. RESULTS: In our cohort, the newly defined DQ-based algorithm detected 3/3 BRCA1 LGRs, demonstrating 100% sensitivity and 100% negative predictive value (NPV) (95% CI [87.6-99.9]) compared to 2/3 cases detected by IR (66.7% sensitivity and 98.2% NPV (95% CI [85.6-99.9])). Interestingly, DQ and IR shared 12 positive results, but exons deletion calls matched only in five cases, two of which confirmed by MLPA. The breakpoints of the 3 novel BRCA1 deletions, involving exons 16-17, 21-22 and 20, have been characterized. CONCLUSIONS: Our study defined a DQ-based algorithm to identify BRCA1 LGRs using NGS data. Whether confirmed on larger data sets, this tool could guide the selection of samples to be subjected to MLPA analysis, leading to significant savings in time and money.

Next-generation sequencing of BRCA1 and BRCA2 genes for rapid detection of germline mutations in hereditary breast/ovarian cancer.

BACKGROUND: Conventional methods used to identify BRCA1 and BRCA2 germline mutations in hereditary cancers, such as Sanger sequencing/multiplex ligation-dependent probe amplification (MLPA), are time-consuming and expensive, due to the large size of the genes. The recent introduction of next-generation sequencing (NGS) benchtop platforms offered a powerful alternative for mutation detection, dramatically improving the speed and the efficiency of DNA testing. Here we tested the performance of the Ion Torrent PGM platform with the Ion AmpliSeq BRCA1 and BRCA2 Panel in our clinical routine of breast/ovarian hereditary cancer syndrome assessment. METHODS: We first tested the NGS approach in a cohort of 11 patients (training set) who had previously undergone genetic diagnosis in our laboratory by conventional methods. Then, we applied the optimized pipeline to the consecutive cohort of 136 uncharacterized probands (validation set). RESULTS: By minimal adjustments in the analytical pipeline of Torrent Suite Software we obtained a 100% concordance with Sanger results regarding the identification of single nucleotide alterations, insertions, and deletions with the exception of three large genomic rearrangements (LGRs) contained in the training set. The optimized pipeline applied to the validation set (VS), identified pathogenic and polymorphic variants, including a novel BRCA2 pathogenic variant at exon 3, 100% of which were confirmed by Sanger in their correct zygosity status. To identify LGRs, all negative samples of the VS were subjected to MLPA analysis. DISCUSSION: Our experience strongly supports that the Ion Torrent PGM technology in BRCA1 and BRCA2 germline variant identification, combined with MLPA analysis, is highly sensitive, easy to use, faster, and cheaper than traditional (Sanger sequencing/MLPA) approaches.

Optimizing the identification of risk-relevant mutations by multigene panel testing in selected hereditary breast/ovarian cancer families.

The introduction of multigene panel testing for hereditary breast/ovarian cancer screening has greatly improved efficiency, speed, and costs. However, its clinical utility is still debated, mostly due to the lack of conclusive evidences on the impact of newly discovered genetic variants on cancer risk and lack of evidence-based guidelines for the clinical management of their carriers. In this pilot study, we aimed to test whether a systematic and multiparametric characterization of newly discovered mutations could enhance the clinical utility of multigene panel sequencing. Out of a pool of 367 breast/ovarian cancer families Sanger-sequenced for BRCA1 and BRCA2 gene mutations, we selected a cohort of 20 BRCA1/2-negative families to be subjected to the BROCA-Cancer Risk Panel massive parallel sequencing. As a strategy for the systematic characterization of newly discovered genetic variants, we collected blood and cancer tissue samples and established lymphoblastoid cell lines from all available individuals in these families, to perform segregation analysis, loss-of-heterozygosity and further molecular studies. We identified loss-of-function mutations in 6 out 20 high-risk families, 5 of which occurred on BRCA1, CHEK2 and ATM and are esteemed to be risk-relevant. In contrast, a novel RAD50 truncating mutation is most likely unrelated to breast cancer. Our data suggest that integrating multigene panel testing with a pre-organized, multiparametric characterization of newly discovered genetic variants improves the identification of risk-relevant alleles impacting on the clinical management of their carriers.

The role of peroxiredoxins in cancer.

Peroxiredoxins (PRDXs) are a ubiquitously expressed family of small (22-27 kDa) non-seleno peroxidases that catalyze the peroxide reduction of H2O2, organic hydroperoxides and peroxynitrite. They are highly involved in the control of various physiological functions, including cell growth, differentiation, apoptosis, embryonic development, lipid metabolism, the immune response, as well as cellular homeostasis. Although the protective role of PRDXs in cardiovascular and neurological diseases is well established, their role in cancer remains controversial. Increasing evidence suggests the involvement of PRDXs in carcinogenesis and in the development of drug resistance. Numerous types of cancer cells, in fact, are characterized by an increase in reactive oxygen species (ROS) production, and often exhibit an altered redox environment compared with normal cells. The present review focuses on the complex association between oxidant balance and cancer, and it provides a brief account of the involvement of PRDXs in tumorigenesis and in the development of chemoresistance.

Validation of the Ion Torrent PGM sequencing for the prospective routine molecular diagnostic of colorectal cancer.

OBJECTIVES: Treatment individualization based on specific molecular biomarkers is becoming increasingly important in oncology. In colorectal cancer (CRC), the molecular characterization of RAS and BRAF mutation status for prognostic and predictive purposes is commonly performed by different validated methods. However, as the number of clinically relevant mutations to be analyzed increases, the definition of new approaches for more sensitive, rapid and economic patient selection urges. To this aim, we evaluated the Ion Semiconductor sequencing using the Ion Torrent Personal Genome Machine (IT-PGM) in our routine molecular diagnostics for CRC in comparison with the gold standard direct Sanger sequencing. DESIGN AND METHODS: Formalin-fixed and paraffin-embedded tumor tissues obtained by surgery or biopsy of 66 CRCs were collected. DNA was extracted and sequenced by IT-PGM and Sanger method. RESULTS: The proposed IT-PGM sequencing strategy exceeded the 500 reads of coverage for all clinically relevant RAS/BRAF amplicons in most samples and thus guaranteed optimal determination. Indeed, the frequencies and the mutational spectrum of RAS and BRAF mutations were in agreement with literature data and revealed 100% concordance between the IT-PGM and routine Sanger sequencing approaches. Turnaround time and cost evaluation indicate that the IT-PGM sequencing permits the characterization of the clinically relevant mutational spots at lower cost and turnaround time compared to Sanger sequencing and allows inclusion of additional amplicons whose characterization may acquire significance in the very next future. CONCLUSION: The IT-PGM is a valid, flexible, sensitive and economical method alternative to the Sanger sequencing in routine diagnostics to select patients for anti-epidermal growth factor receptor therapy for metastatic CRC.

Novel and recurrent BRCA2 mutations in Italian breast/ovarian cancer families widen the ovarian cancer cluster region boundaries to exons 13 and 14.

Hereditary breast and ovarian cancer are mainly linked to mutations in BRCA1 and BRCA2 genes which confer a similar cumulative risk of developing breast cancer. Importantly, while BRCA2 mutation carriers generally have a lower cumulative risk for ovarian cancer, mutations clustered in the central portion of BRCA2 are associated with a higher proportion of ovarian compared with breast cancer cases. The boundaries of this ovarian cancer cluster region (OCCR) have been tentatively defined within a 3.3 kb region of BRCA2 exon 11, and herein, we reassessed these boundaries using our series of Italian breast/ovarian cancer families. We used direct sequencing to investigate BRCA mutations in 367 breast/ovarian cancer families. We also studied the association between the location of the mutations and the ovarian cancer phenotype in our cohort of BRCA2-mutated families. We observed the novel c.7309_7309delA frameshift mutation and the c.7007G>A deleterious mutation in BRCA2 exons 14 and 13, respectively, in five independent Italian families characterized by a high proportion of ovarian cancer cases. Of note, a significantly higher proportion of ovarian versus breast cancer cases was associated not only with mutations in the previously defined OCCR (OR = 5.91; p = 0.004), but also with the exon 13-14 region (OR = 7.37; p = 0.001) in our BRCA2-mutated families. Our data provide initial evidence for a novel putative OCCR in BRCA2 exons 13-14.

Peroxiredoxin 2 nuclear levels are regulated by circadian clock synchronization in human keratinocytes.

Circadian rhythms are highly conserved time tracking systems regulating important biological processes at both systemic and cellular levels. The present study was aimed to identify proteins and biological functions circadian regulated in human keratinocytes. HaCaT keratinocytes were entrained by temperature cycles, and a proteomic study was performed on cell fractions isolated under free running conditions at constant temperature. Bioinformatics analysis revealed that molecular clock entrainment was associated with changes in molecular components regulating cell proliferation, energy metabolism, transcription, translation and redox balance. Nuclear levels of the antioxidant enzyme Peroxiredoxin 2 (PRDX2) were found to oscillate rhythmically over two entire 24h long cycles. Donwregulation of PRDX2 resulted in upregulation of the mitochondrion-specific Peroxiredoxin 3 (PRDX3), all other members of the Peroxiredoxin family remained unaltered. Furthermore, PRDX2 knockdown increased intracellular levels of reactive oxygen species (ROS) and impaired cell cycle progression and proliferation. HaCaT cells transduced with a scramble shRNA were used as control. Our work is the first to show that nuclear levels of PRDX2 display circadian oscillation participating in the regulation of human keratinocytes redox balance.

PRDX1 and PRDX6 are repressed in papillary thyroid carcinomas via BRAF V600E-dependent and -independent mechanisms.

Many clinical studies highlight the dichotomous role of PRDXs in human cancers, where they can exhibit strong tumor-suppressive or tumor-promoting functions. Recent evidence suggests that lower expression of PRDXs correlates with cancer progression in colorectal cancer (CRC) or in esophageal squamous carcinoma. In the thyroid, increased levels of PRDX1 has been described in follicular adenomas and carcinomas, as well as in thyroiditis, while reduced levels of PRDX6 has been found in follicular adenomas. We studied the expression of PRDX1 and PRDX6, in a series of thyroid tissue samples, covering different thyroid diseases, including 13 papillary thyroid carcinomas (PTCs). Our results show that PRDX1 and PRDX6 are significantly reduced in all PTCs compared to normal tissues, to non-neoplastic tissue (MNG) or follicular neoplasms. This reduction is strongly evident in PTCs harboring BRAF V600E (31% of our cases). Using TPC-1 and BCPAP and FRTL-5 cell lines, we demonstrate for the first time that the presence of BRAF V600E is responsible of the hypoexpression of PRDX1 and PRDX6 both at mRNA and protein levels. Finally, independently of BRAF status, we observe an interesting correlation between the tumor size, the presence of lymph node metastasis and the lowest PRDX1 and PRDX6 levels. Therefore, these data indicate that PRDX1 and PRDX6 expression not only may play a key role in papillary thyroid carcinogenesis via a BRAF V600E-dependent mechanism, but their determination could be considered as potential tumor marker for indicating tumor progression in PTCs, independently of BRAF status.

Effects of the Smad4 C324Y mutation on thyroid cell proliferation.

Smad4 is a key mediator of the transforming growth factor-ß (TGF-ß) superfamily that is involved in the control of cell proliferation and differentiation. We recently demonstrated that a Smad4 mutation, Smad4 C324Y, isolated from nodal metastases of papillary thyroid carcinoma, causes an increase of TGF-ß signaling, responsible for the acquisition of transformed phenotype and invasive behaviour in thyroid cells stably expressing this mutation. In this paper, we demonstrate that the stable expression of Smad4 C324Y mutation in FRTL-5 cells is responsible for TSH-independent growth ability. Our data show that the Smad4 C324Y mutation interacts with P-Smad3 more strongly than Smad4 wt, already in basal condition; this interaction is responsible for TGF-ß signaling and PKA activation that, in turn, determines an increased phosphorylation of CREB, necessary for the mitogenic actions of TSH. The expression of cyclin D1 also increases in all cells overexpressing the Smad4 C324Y mutation. All together, these data demonstrate that Smad4 C324Y mutation, interacting with the PKA pathway, gives cells the ability to proliferate independently from TSH.

The p85α regulatory subunit of PI3K mediates cAMP-PKA and retinoic acid biological effects on MCF7 cell growth and migration.

Phosphoinositide-3-OH kinase (PI3K) signalling regulates various cellular processes, including cell survival, growth, proliferation and motility, and is among the most frequently mutated pathways in cancer. Although the involvement of p85αPI3K SH2 domain in signal transduction has been extensively studied, the function of the SH3 domain at the N-terminus remains elusive. A serine (at codon 83) adjacent to the N-terminal SH3 domain in the PI3K regulatory subunit p85αPI3K that is phosphorylated by protein kinase A (PKA) in vivo and in vitro has been identified. Virtually all receptors binding p85αPI3K can cooperate with cAMP-PKA signals via phosphorylation of p85αPI3KSer83. To analyse the role of p85αPI3KSer83 in retinoic acid (RA) and cAMP signalling, in MCF7 cells, we used p85αPI3K mutated forms, in which Ser83 has been substituted with alanine (p85A) to prevent phosphorylation or with aspartic acid (p85D) to mimic the phosphorylated residue. We demonstrated that p85αPI3KSer83 is crucial for the synergistic enhancement of RARα/p85αPI3K binding induced by cAMP/RA co-treatment in MCF7 cells. Growth curves, colorimetric MTT assay and cell cycle analysis demonstrated that phosphorylation of p85αPI3KSer83 plays an important role in the control of MCF7 cell proliferation and in RA-induced inhibition of proliferation. Wound healing and transwell experiments demonstrated that p85αPI3KSer83 was also essential both for the control of migratory behaviour and for the reduction of motility induced by RA. This study points to p85αPI3KSer83 as the physical link between different pathways (cAMP-PKA, RA and FAK), and as an important regulator of MCF7 cell proliferation and migration.

A novel human Smad4 mutation is involved in papillary thyroid carcinoma progression.

Smad proteins are the key effectors of the transforming growth factor ß (TGFß) signaling pathway in mammalian cells. Smad4 plays an important role in human physiology, and its mutations were found with high frequency in wide range of human cancer. In this study, we have functionally characterized Smad4 C324Y mutation, isolated from a nodal metastasis of papillary thyroid carcinoma. We demonstrated that the stable expression of Smad4 C324Y in FRTL-5 cells caused a significant activation of TGFß signaling, responsible for the acquisition of transformed phenotype and invasive behavior. The coexpression of Smad4 C324Y with Smad4 wild-type determined an increase of homo-oligomerization of Smad4 with receptor-regulated Smads and a lengthening of nuclear localization. FRTL-5 clones overexpressing Smad4 C324Y showed a strong reduction of response to antiproliferative action of TGFß1, acquired the ability to grow in anchorage-independent conditions, showed a fibroblast-like appearance and a strong reduction of the level of E-cadherin, one crucial event of the epithelial-mesenchymal transition process. The acquisition of a mesenchymal phenotype gave the characteristics of increased cellular motility and a significant reduction in adhesion to substrates such as fibronectin and laminin. Overall, our results demonstrate that the Smad4 C324Y mutation plays an important role in thyroid carcinogenesis and can be considered as a new prognostic and therapeutic target for thyroid cancer.

EGF and TGF-ß1 Effects on Thyroid Function.

Normal epithelial thyroid cells in culture are inhibited by TGF-ß1. Instead, transformed thyroid cell lines are frequently resistant to its growth inhibitory effect. Loss of TGF-ß responsiveness could be due to a reduced expression of TGF-ß receptors, as shown in transformed rat thyroid cell lines and in human thyroid tumors, or to alterations of other genes controlling TGF-ß signal transduction pathway. However, in thyroid neoplasia, a complex pattern of alterations occurring during transformation and progression has been identified. Functionally, TGF-ß1 acts as a tumor suppressor in the early stage of transformation or as a tumor promoter in advanced cancer. This peculiar pleiotropic behaviour of TGF-ß may result from cross-talk with signalling pathways mediated by other growth factors, among which EGF-like ligands play an important role. This paper reports evidences on TGF-ß1 and EGF systems in thyroid tumors and on the cross-talk between these growth factors in thyroid cancer.

Role of reduced expression of SMAD4 in papillary thyroid carcinoma.

It has been demonstrated that transforming growth factor-ß (TGFß) and other members of TGFß superfamily play an important role in thyroid proliferative diseases. The deficiencies of SMAD4 are responsible to accelerate the malignant progression of neoplastic lesions in several types of tumors. Therefore, the objective of the present study was to determine the functional role of reduced expression of SMAD4 in human papillary thyroid carcinogenesis. For this purpose, we examined the TGFß response in two cell lines, TPC-1 and BCPAP. Our data demonstrated for the first time that these cells showed a strong reduction in the level of SMAD4 protein, which was responsible for an alteration of TGFß signaling and for some of the TGFß-mediated biological effects. The overexpression of SMAD4, restoring TGFß transduction, determined a significant increase of antiproliferative response to TGFß, and reduced the invasive behavior of these cells. Therefore, our data indicated that reduction of SMAD4 may play a significant role in thyroid carcinogenesis.

Reduction of invasive potential in K-ras-transformed thyroid cells by restoring of TGF-beta pathway.

Transforming Growth Factor-beta1 (TGF -beta1) is a multifunctional cytokine that regulates a number of cellular processes such as cell growth, differentiation, plasticity, cell motility, adhesiveness, embryogenesis, development and apoptosis through binding to TGF-beta receptors. We have previously demonstrated that K-ras-transformed rat thyroid cells, K10, are resistant to the growth inhibitory action of TGF-beta1, because they show a decreased expression of type II receptor (TbetaRII). Clones obtained transfecting TbetaRII, partially revert their malignant phenotype, showing a reduction in the anchorage-dependent and -independent cell growth and a statistically significant decrease in tumourigenicity with respect to the highly malignant parental cells, both in spontaneous and artificial metastases, when transplanted in athymic nude mice. The purpose of the present work is to elucidate the molecular events involved in the modulation of the tumourigenic potential of K-ras-transformed rat thyroid cells overexpressing TbetaRII. Our data demonstrate that the TbetaRII overexpressed in K-ras-transformed thyroid cell clones is a functional receptor and is essential to restore in these cells behaviour similar to that of control cells. The TbetaRII overexpression is responsible for a strong reduction of adhesive and migratory behaviour of highly malignant K-ras-transformed thyroid cells. These results suggest that the restore of a functional TGF-beta receptor in these cells may be useful for the limitation of tumour spread and dissemination.

TGF-beta control of rat thyroid follicular cells differentiation.

TGF-beta1 is a potent inhibitor of growth and DNA synthesis in thyroid cells. It has also been shown that TGF-beta1 inhibits thyrocyte function. The functional inhibition is represented by a downregulation of thyroid specific genes, such as Na(+)/I(-) symporter (NIS), thyroglobulin (TG) and thyroperoxidase (TPO). The transcriptional control of these genes is mediated by thyroid-specific transcription factors: thyroid transcription factor-1 (TTF-1) and PAX-8. It has been shown that Smad proteins play a pivotal role in the intracellular signal transduction of the TGF-beta family members. In this paper, the functional relevance of Smad4, in the control of thyroid differentiation genes and thyroid-specific transcription factors, has been investigated. The data obtained provides, for the first time, evidence that D.N. Smad4-100T is capable of blocking TGF-beta1 action in the regulation of thyroid-specific genes expression. Such action is possible by blocking nuclear translocation of Smad4 and Smad2.