Advances in Detecting Low Prevalence Somatic TERT Promoter Mutations in Papillary Thyroid Carcinoma.

Background: Two recurrent TERT (telomerase reverse transcriptase) promoter mutations, C228T and C250T, have been reported in thyroid carcinomas and were correlated with high-risk clinicopathological features and a worse prognosis. Although far more frequent in the poorly differentiated and undifferentiated thyroid cancer, the TERT promoter mutations play a significant role on PTC recurrence and disease-specific mortality. However, the prevalence varies considerably through studies and it is uncertain if these differences are due to population variation or the methodology used to detect TERT mutations. In this study we aim to compare three different strategies to detect TERT promoter mutations in PTC. Methods: DNA was isolated from formalin-fixed paraffin-embedded (FFPE) specimens from 89 PTC and 40 paired lymph node metastases. The prevalence of the hot spot TERT C228T and C250T mutations was assessed in FFPE samples using TaqMan SNP genotyping assays. Random samples were tested by Sanger Sequencing and droplet digital PCR (ddPCR). Results: In general, 16 out of 89 (18%) PTC samples and 14 out of 40 (35%) lymph node metastases harbored TERT promoter mutations by TaqMan assay. Sanger sequencing, performed in random selected samples, failed to detect TERT mutations in four samples that were positive by TaqMan SNP genotyping assay. Remarkably, ddPCR assay allowed detection of TERT promoter mutations in six samples that harbor very low mutant allele frequency (≤ 2%) and were negative by both genotype assay and Sanger Sequencing. Conclusion: This study observed a good concordance among the methodologies used to detect TERT promoter mutations when a high percentage of mutated alleles was present. Sanger analysis demonstrated a limit of detection for mutated alleles. Therefore, the prevalence of TERT promoter mutations in PTC may be higher than previously reported, since most studies have conventionally used Sanger sequencing. The efficient characterization of genetic alterations that are used as preoperative or postoperative diagnostic, risk stratification of the patient and individualized treatment decisions, mainly in highly heterogeneous tumors, require highly sensitive and specific approaches.

Comprehensive Assessment of Copy Number Alterations Uncovers Recurrent AIFM3 and DLK1 Copy Gain in Medullary Thyroid Carcinoma.

Medullary thyroid carcinoma (MTC) is a malignant tumor originating from thyroid C-cells that can occur either in sporadic (70-80%) or hereditary (20-30%) form. In this study we aimed to identify recurrent copy number alterations (CNA) that might be related to the pathogenesis or progression of MTC. We used Affymetrix SNP array 6.0 on MTC and paired-blood samples to identify CNA using PennCNV and Genotyping Console software. The algorithms identified recurrent copy number gains in chromosomes 15q, 10q, 14q and 22q in MTC, whereas 4q cumulated losses. Coding genes were identified within CNA regions. The quantitative PCR analysis performed in an independent series of MTCs (n = 51) confirmed focal recurrent copy number gains encompassing the DLK1 (14q32.2) and AIFM3 (22q11.21) genes. Immunohistochemistry confirmed AIFM3 and DLK1 expression in MTC cases, while no expression was found in normal thyroid tissues and few MTC samples were found with normal copy numbers. The functional relevance of CNA was also assessed by in silico analysis. CNA status correlated with protein expression (DLK1, p = 0.01), tumor size (DLK1, p = 0.04) and AJCC staging (AIFM3p = 0.01 and DLK1p = 0.05). These data provide a novel insight into MTC biology, and suggest a common CNA landscape, regardless of if it is sporadic or hereditary MTC.

Retroposed copies of RET gene: a somatically acquired event in medullary thyroid carcinoma.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

LIMD2 Is Overexpressed in BRAF V600E-Positive Papillary Thyroid Carcinomas and Matched Lymph Node Metastases.

We previously described that LIM domain containing 2 (LIMD2) overexpression was closely correlated with metastatic process in papillary thyroid carcinoma (PTC). We here evaluated the expression of LIMD2 in a series of non-metastatic and metastatic PTC and their matched lymph node metastases via immunohistochemistry. LIMD2 was expressed in 74 (81%) of primary PTC and 35 (95%) of lymph node metastases. Sub-analysis performed in 37 matched samples demonstrated that in four cases, LIMD2 is expressed in lymph node metastases, while it is not expressed in primary tumors. Moreover, in eight cases, the staining intensity of LIMD2 was stronger in the patient-matched lymph node metastases than in the primary tumors. Next, the expression of LIMD2 was correlated with clinical pathological parameters and BRAF V600E and RET/PTC mutational status. The expression of LIMD2 in primary tumors was correlated with the presence of BRAF V600E mutation (P = 0.0338). Western blot analysis in thyroid cell lines demonstrated that LIMD2 is expressed in two PTC cell lines, while it is not expressed in normal thyroid and follicular thyroid carcinoma cell lines. Importantly, its expression was higher in a PTC cell line that harbors BRAF V600E mutation than in a PTC cell line that harbors RET/PTC1. The available genomic profiling data generated by The Cancer Genome Atlas Research Network confirmed that LIMD2 expression is higher in BRAF-like PTC samples. Our data suggest that LIMD2 may play an important role in the metastatic process of PTC, predominantly in BRAF V600E-positive tumors.

Fusion Oncogenes Are the Main Genetic Events Found in Sporadic Papillary Thyroid Carcinomas from Children.

BACKGROUND: Previous studies reported significant differences in the clinical presentation and outcomes of papillary thyroid carcinoma (PTC) in pediatric patients compared with adults. Previous studies have suggested that the clinicopathological differences observed between pediatric and adult PTCs may be due the existence of distinct genetic alterations. However, the knowledge of genetic events in pediatric PTCs is based primarily on studies in radiation-exposed PTCs or in the few studies that enrolled predominantly adolescent patients. The aim of this study was to characterize the known oncogenic alterations of the MAPK pathway found in adult and radiation-exposed PTCs in a cohort of predominantly sporadic pediatric PTC patients. METHODS: Thirty-five pediatric PTCs were screened for the most prevalent fusions (RET/PTC1, RET/PTC2, RET/PTC3, ETV6-NTRK3, and AGK-BRAF) and point mutations (BRAFV600E and NRASQ61) described in sporadic pediatric PTCs. The mutational status was correlated with clinicopathological data. RESULTS: Mutations were found in 20 out of 35 (57%) PTC cases. Fusion oncogenes were the main genetic alterations found. RET/PTC1-3 rearrangements were found in 13 (37%), ETV6-NTRK3 in 3 (9%), AGK-BRAF in 4 (11%), and BRAFV600E in 3 (9%). No mutation was found in NRASQ61. BRAFV600E was associated with older age and larger tumor size (p < 0.05), and RET/PTC3 was associated with a larger tumor size and multifocality (p < 0.05). CONCLUSIONS: The genetic signature in this cohort was remarkably different than that observed in adults. Although observed at a lower prevalence, the spectrum of mutations was quite similar to that described in radiation-exposed pediatric PTCs. As mutations were unidentifiable in over 40% of the PTC cases, more comprehensive studies conducted in these patients will help to decipher the genetic landscape of sporadic pediatric PTCs.

PVALB diminishes [Ca2+] and alters mitochondrial features in follicular thyroid carcinoma cells through AKT/GSK3ß pathway.

We have identified previously a panel of markers (C1orf24, ITM1 and PVALB) that can help to discriminate benign from malignant thyroid lesions. C1orf24 and ITM1 are specifically helpful for detecting a wide range of thyroid carcinomas, and PVALB is particularly valuable for detecting the benign Hürthle cell adenoma. Although these markers may ultimately help patient care, the current understanding of their biological functions remains largely unknown. In this article, we investigated whether PVALB is critical for the acquisition of Hürthle cell features and explored the molecular mechanism underlying the phenotypic changes. Through ectopic expression of PVALB in thyroid carcinoma cell lines (FTC-133 and WRO), we demonstrated that PVALB sequesters free cytoplasmic Ca(2+), which ultimately lowers calcium levels and precludes endoplasmic reticulum (ER) Ca(2+) refilling. These results were accompanied by induced expression of PERK, an ER stress marker. Additionally, forced expression of PVALB reduces Ca(2+) inflow in the mitochondria, which can in turn cause changes in mitochondria morphology, increase mitochondria number and alter subcellular localization. These findings share striking similarity to those observed in Hürthle cell tumors. Moreover, PVALB inhibits cell growth and induces cell death, most likely through the AKT/GSK-3ß. Finally, PVALB expression coincides with Ca(2+) deposits in HCA tissues. Our data support the hypothesis that the loss of PVALB plays a role in the pathogenesis of thyroid tumors.

AGK-BRAF gene fusion is a recurrent event in sporadic pediatric thyroid carcinoma.

Thyroid cancer is the fastest increasing cancer worldwide in all age groups. Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer in both adults and children. PTC genomic landscape has been extensively studied in adults, but information regarding sporadic pediatric patients is lacking. Although BRAF V600E mutation is highly prevalent in adults, this mutation is uncommon in pediatric cases. As adult and pediatric PTC is a mitogen-activated protein kinase-driven cancer, this altered pathway might be activated by different genetic events. The aim of this study was to investigate the occurrence of AGK-BRAF fusion gene, recently described in radiation-exposed pediatric PTC, in a cohort of exclusively sporadic pediatric PTC. The series consisted of 30 pediatric PTC younger than 18 years of age at the time of diagnosis and 15 matched lymph node metastases (LNM). Primary tumors and matched LNM were screened for the presence of the AGK-BRAF fusion transcript by RT-PCR. To confirm the identity of the amplified products, randomly selected samples positive for the presence of the fusion transcripts were sequenced. Moreover, BRAF dual-color, break-apart probes confirmed BRAF rearrangement. Overall, the AGK-BRAF fusion gene was detected in 10% (3/30) of primary tumors. For one of these cases, paired LNM was also available, which also shows the presence of AGK-BRAF fusion gene. This study described, for the first time, the presence of AGK-BRAF in sporadic pediatric PTC. Understanding the molecular events underlying pediatric PTC may improve preoperative diagnosis, allow molecular prognostication and define a therapeutic approach toward sporadic PTC patients.

Thyroid-Specific Genes Expression Uncovered Age-Related Differences in Pediatric Thyroid Carcinomas.

Despite a more advanced stage of disease at presentation, a better response to radioiodine (RAI) therapy and a reduced overall mortality have been reported in pediatric differentiated thyroid cancer (DTC) in comparison to adult DTC. Few studies suggested that the better response to RAI therapy in pediatric patients might be associated with an increased expression of NIS. However, a marked heterogeneity within the pediatric group has been recognized. Children (<10 years old) usually present a more aggressive disease than adolescents (≥10-18 years old). By analyzing the expression of thyroid-specific genes in 38 sporadic pediatric tumors, we show that the expression of NIS, PDS, and TSHR was lower in children than adolescents (P < 0.05). A linear regression confirmed the association between NIS expression and age. Most significantly, NIS was expressed at similar levels in DTC from children and adults, whereas PDS and TSHR expression was even lower in DTC from children, compared to adolescents and adults. Our data suggest that biological behaviors of DTC in adolescents might differ from those in children and adults. Therefore, the premise that the expression of thyroid-specific genes is higher in tumors from pediatric patients than in adults is not entirely true and might be too oversimplified.

Analysis of somatic mutations in BRAF, CDKN2A/p16 and PI3KCA in patients with medullary thyroid carcinoma.

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 paraffin­embedded 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.

A ten-year clinical update of a large RET p.Gly533Cys kindred with medullary thyroid carcinoma emphasizes the need for an individualized assessment of affected relatives.

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.

Keratinized versus non-keratinized preputial flap onlay urethroplasty: does it make any difference in the histological analysis? An experimental study in rabbits

Purpose To compare the histological characteristics of keratinized versus non-keratinized onlay island flaps in an experimental rabbit model. Materials and Methods Sixteen male rabbits were randomly allocated into two experimental groups: keratinized and non-keratinized onlay island flaps. A defect was created in the ventral aspect of the penile urethra. In the keratinized group, a longitudinal island flap was harvested from the external prepuce and rotated to cover the urethral defect. In the non-keratinized group a transverse island flap was harvested from the inner prepuce. The animals were sacrificed after 2, 4, 8 and 12 weeks. Results The flaps were viable in all animals, and no deaths were associated with the procedure. Two urethrocutaneous fistulas were identified, one in each experimental group. A similar pattern of fibrosis was identified in both groups. The keratinized epithelium of the external prepuce kept its histological aspect and keratin production. Both keratinized and non-keratinized groups presented a slight decrease on the epithelial thickness, however without a statistically significant difference between groups. Conclusions In this short-term rabbit model, we observed that the stratified squamous keratinized epithelium from the external prepuce kept its keratin production. There was no statistical influence of the flap type on the mean epithelial thickness. .

Keratinized versus non-keratinized preputial flap onlay urethroplasty: does it make any difference in the histological analysis? An experimental study in rabbits.

PURPOSE: To compare the histological characteristics of keratinized versus non-keratinized onlay island flaps in an experimental rabbit model. MATERIALS AND METHODS: Sixteen male rabbits were randomly allocated into two experimental groups: keratinized and non-keratinized onlay island flaps. A defect was created in the ventral aspect of the penile urethra. In the keratinized group, a longitudinal island flap was harvested from the external prepuce and rotated to cover the urethral defect. In the non-keratinized group a transverse island flap was harvested from the inner prepuce. The animals were sacrificed after 2, 4, 8 and 12 weeks. RESULTS: The flaps were viable in all animals, and no deaths were associated with the procedure. Two urethrocutaneous fistulas were identified, one in each experimental group. A similar pattern of fibrosis was identified in both groups. The keratinized epithelium of the external prepuce kept its histological aspect and keratin production. Both keratinized and non-keratinized groups presented a slight decrease on the epithelial thickness, however without a statistically significant difference between groups. CONCLUSIONS: In this short-term rabbit model, we observed that the stratified squamous keratinized epithelium from the external prepuce kept its keratin production. There was no statistical influence of the flap type on the mean epithelial thickness.

The buccal mucosa fenestrated graft for Bracka first stage urethroplasty: experimental study in rabbits

To histologically evaluate, in an experimental study in rabbits, the integration process of the buccal mucosa fenestrated graft applied in the corpora cavernosa for Bracka first stage urethroplasty.

A urethral defect was surgically created in 16 male rabbits of the New Zealand breed through the excision of the penile urethra. The urethral defect was corrected by applying buccal mucosa fenestrated graft through two cruciform incisions in the distal portions of its longitudinal axis. The animals were sacrificed at 2, 4, 8 and 12 weeks post surgery and their genitals were subjected to clinical and histological assessment.

The buccal mucosa fenestrated graft showed complete uptake in all groups, with keratinization squamous metaplasia and mucosal proliferation of the fenestrated areas. The fenestrated graft area represented an increase in length of 25% in length in relation to the original standard graft.

The fenestrated buccal mucosa graft presented total integration to the adjacent epithelia with re-epithelization of the incision areas of the graft (fenestrations) and no significant inflammatory or scarring reactions when compared to other mucosa transplanted areas; therefore its application is viable in cases of extensive urethral defect whenever the donating area might be insufficient. .

Extended RET gene analysis in patients with apparently sporadic medullary thyroid cancer: clinical benefits and cost.

RET sequencing has become an important tool in medullary thyroid cancer (MTC) evaluation and should be performed even in the absence of family history of MTC. The most commonly studied exons in index cases are 8, 10, 11, and 13-16. To address the ATA guidelines regarding the sequencing of the entire coding region of RET, we selected 50 patients with sporadic MTC (sMTC) without mutations in the hot spot regions of RET for extended investigation of exons 1-7, 9, 12, 17, 18, and 19. Twenty-seven of 50 patients presented with one or more features suggesting familial disease. We found only a new RET variant (p.Gly550Glu) in one patient with MTC. Several polymorphisms were observed, and their frequency was histogram scaled by exons and introns. Eight patients were also included for somatic mutation search. We estimated the sequencing cost by stratifying into four investigation approaches: (1) hot spot exons in a new patient, (2) the remaining exons if the hot spots are negative in a patient with suspected familial disease, (3) a relative of a carrier for a known RET mutation, and (4) tumor sequencing. In spite of the increasing number of variants being described in MTC, it appears that there is no direct clinical benefit in extending RET germ line analysis beyond the hot spot regions in sMTC. The cost evaluation in apparent sMTC using a tiered approach may help clinicians make more suitable decisions regarding the benefits of investigating only the hot spots against the entire coding region of RET.

Vaginal reconstruction with two lower abdominal skin flaps in rabbits: histological and macroscopic evaluation.

OBJECTIVE: To evaluate the use of two skin flaps of the lower abdominal wall in the creation of a cylindrical conduit in vaginal reconstruction surgery in rabbits, through macroscopic and histological analysis. STUDY DESIGN: An experimental study was performed in 16 female New Zealand rabbits, consisting of the use of two rectangular-shaped skin flaps of the lower abdominal wall measuring 1cm longitudinally and 3 cm transversely anastomosed to each other through continuous suture of the edges of the two flaps to create a tube. Hysterectomy and excision of the vaginal vault were performed, and the skin tube was anastomosed to the remaining vaginal stump with separate points of polyglycolic acid 4.0. Animals were divided into 4 groups according to the euthanasia at 2, 4, 8 and 12 weeks, when after excision of the neovagina, macroscopic and histological evaluation with hematoxylin-eosin and Masson trichrome were performed. RESULTS: Of 16 operated rabbits, only 1 presented partial abdominal wall dehiscence, not compromising the flap viability. The macroscopic analysis of the vaginal conduit showed that it was kept open throughout the experimental steps, with a good patency and gauge, showing a slight retraction in the skin conduit length of no statistical significance. In the histopathological analysis, a local inflammatory process in the anastomosis was observed, which was larger in the early evaluation but decreased in late evaluations, as well as the local fibrosis process. Integration of the vaginal and skin epithelia was made with no alterations in their primary characteristics. CONCLUSION: The use of two skin flaps of the lower abdominal wall as a vaginal conduit presented good integration between skin and vaginal tissue with minimal length retraction, kept the patency during evaluations and did not show strictures, presenting good local healing and a low rate of complications.

The buccal mucosa fenestrated graft for Bracka first stage urethroplasty: experimental study in rabbits.

OBJECTIVE: To histologically evaluate, in an experimental study in rabbits, the integration process of the buccal mucosa fenestrated graft applied in the corpora cavernosa for Bracka first stage urethroplasty. MATERIALS AND METHODS: A urethral defect was surgically created in 16 male rabbits of the New Zealand breed through the excision of the penile urethra. The urethral defect was corrected by applying buccal mucosa fenestrated graft through two cruciform incisions in the distal portions of its longitudinal axis. The animals were sacrificed at 2, 4, 8 and 12 weeks post surgery and their genitals were subjected to clinical and histological assessment. RESULTS: The buccal mucosa fenestrated graft showed complete uptake in all groups, with keratinization squamous metaplasia and mucosal proliferation of the fenestrated areas. The fenestrated graft area represented an increase in length of 25 % in length in relation to the original standard graft. CONCLUSIONS: The fenestrated buccal mucosa graft presented total integration to the adjacent epithelia with re-epithelization of the incision areas of the graft (fenestrations) and no significant inflammatory or scarring reactions when compared to other mucosa transplanted areas; therefore its application is viable in cases of extensive urethral defect whenever the donating area might be insufficient.

Macroscopic and histological evaluation of tunica vaginalis dorsal grafting in the first stage of Bracka's urethroplasty: an experimental study in rabbits.

OBJECTIVE: To evaluate the use of tunica vaginalis as a dorsal free graft in the first stage of Bracka's urethroplasty in a rabbit animal model using macroscopic and histological analysis. MATERIAL AND METHODS: Surgery was performed on 16 rabbits, divided into four groups of four according to the number of weeks after surgery at which they were killed (2, 4, 8 and 12 weeks). The penile urethra was removed and only the ventral aspect of the urethra was reconstructed using a tunica vaginalis free graft dorsally fixed in the corpora cavernosa. Epithelial, inflammatory and fibrotic changes in the graft were evaluated macroscopically then histologically at the point when each rabbit group was killed. RESULTS: Macroscopic evaluation showed good graft uptake with minimal retraction (P > 0.05). Histological evaluation showed a reduction in the inflammatory process during the first 4 weeks, after which inflammation stabilized. The mesothelium was partially healed at 2 and 8 weeks with a metaplasia defined by a stratified squamous, non-keratinized lining. The grafted area was thinner than the surrounding penile skin and similar to urothelial native epithelium. CONCLUSION: Tunica vaginalis used as a free graft placed dorsally on the corpora cavernosa in the first stage of Bracka's urethroplasty shows good tissue integration and develops into a stratified non- keratinized epithelium (metaplasia) without significant retraction, despite exposure to the external environment.

PVALB, a new Hürthle adenoma diagnostic marker identified through gene expression.

CONTEXT: A better means to accurately identify malignant thyroid nodules and to distinguish them from benign tumors is needed. We previously identified markers for detecting thyroid malignancy, with sensitivity estimated at or close to 100%. One lingering problem with these markers was that false positives occurred with Hürthle cell adenomas (HCA) which lowered test specificity. METHODS: To locate accurate diagnostic markers, we profiled in depth the transcripts of a HCA and a Hürthle cell carcinoma (HCC). From 1146 differentially expressed genes, 18 transcripts specifically expressed in HCA were tested by quantitative PCR in a wide range of thyroid tumors (n = 76). Sensibility and specificity were calculated using receiver operating characteristic (ROC). Selected markers were further validated in an independent set of thyroid tumors (n = 82) by immunohistochemistry. To define the panel that would yield best diagnostic accuracy, these markers were tested in combination with our previous identified markers. RESULTS: Seventeen of the 18 genes showed statistical significance based on a mean relative level of expression (P < 0.05). KLK1 (sensitivity = 0.97) and PVALB (sensitivity = 0.94) were the best candidate markers. The combination of PVALB and C1orf24 increased specificity to >97% and maintained sensitivity for detection of carcinoma. CONCLUSION: We identified tumor markers that can be used in combination for a more accurate preoperative diagnosis of thyroid nodules and for postoperative diagnosis of thyroid carcinoma in tumor sections. This improved test would help physicians rapidly focus treatment on true malignancies and avoid unnecessary treatment of benign tumors, simultaneously improving medical care and reducing costs.

Histological evaluation of an alternative method of neophalloplasty based on two lower abdominal skin flaps and simultaneous buccal mucosa graft in the ventral surface of the neophallus (two-stage urethroplasty): experimental study in rabbits.

OBJECTIVE: To evaluate, in an experimental study in rabbits, a new model of neophalloplasty based on two lower abdominal skin flaps and ventral buccal mucosa graft for planned two-stage urethroplasty procedure. MATERIAL AND METHODS: Sixteen rabbits were operated and divided into four equal groups which were sacrificed at 2, 4, 8 and 12 weeks. The inflammatory pattern, presence of sub-epithelial fibrosis and epithelial changes in the grafted area were evaluated histologically. RESULTS: There were no deaths and no dehiscence of the wound was seen. One animal in the 2-week group developed an ulcer in the grafted area. We found minimal contracture of the neophallus, but this was not statistically significant between groups. Buccal mucosa graft showed good uptake in all groups, with vascular support from subcutaneous tissue of the flaps. The grafted area developed epithelial metaplasia, showing a decrease in cell layers with time, with disappearance of the sub-epithelial papillae and appearance of stratum granulosum and keratinization of the epithelial graft surface. A decrease in sub-epithelial fibrosis with replacement of immature by mature (eosinophilic) collagen was found. In the later groups was also observed an important decrease in inflammatory response, and the chorion of the grafted area presented a dilated capillary network, indicating that the process of neoangiogenesis was effective. CONCLUSION: Buccal mucosa displayed histological integration in the abdominal flaps with epithelial metaplasia in all groups. The surgical aspect of the neophallus was cosmetically acceptable, with minimal contracture.

Dorsal tunica vaginalis graft plus onlay preputial island flap urethroplasty: experimental study in rabbits.

OBJECTIVE: To assess the use of tunica vaginalis graft plus onlay preputial island flap in urethral reconstructive surgery in rabbits through histopathology. MATERIAL AND METHODS: We developed an experimental model of urethroplasty that resembles one-stage complex hypospadias surgery with divided urethral plate. The tunica vaginalis graft is dorsally placed to recreate the urethral plate and the internal preputial island flap is placed onlay to complete the urethroplasty. Sixteen animals were divided into four equal groups and sacrificed at 2, 4, 8 and 12 weeks after surgery, the penis being sent for histopathological evaluation. RESULTS: There were no deaths related to the procedure or wound breakdown; all rabbits voided spontaneously after surgery. Two urethrocutaneous fistulae were found. Microscopically, good tissue integration was observed, the tunica vaginalis mesothelium was gradually replaced by a more stratified epithelial lining, similar to the urothelial lining of the native urethra. The stratified squamous non-keratinized lining of the internal preputial island flap also changed into a thinner epithelial lining with only 4-5 cell layers. Two urethral diverticula were found. CONCLUSION: A tunica vaginalis graft placed dorsally plus an onlay internal preputial island flap was shown to be a successful technique for urethroplasty in an animal model.