RET fusion genes in pediatric and adult thyroid carcinomas: cohort characteristics and prognosis.

Thyroid cancer is associated with a broad range of different mutations, including RET (rearranged during transfection) fusion genes. The importance of characterizing RET fusion-positive tumors has recently increased due to the possibility of targeted treatment. The aim of this study was to identify RET fusion-positive thyroid tumors, correlate them with clinicopathological features, compare them with other mutated carcinomas, and evaluate long-term follow-up of patients. The cohort consisted of 1564 different thyroid tissue samples (including 1164 thyroid carcinoma samples) from pediatric and adult patients. Samples were analyzed for known driver mutations occurring in thyroid cancer. Negative samples were subjected to extensive RET fusion gene analyses using next-generation sequencing and real-time PCR. RET fusion genes were not detected in any low-risk neoplasm or benign thyroid tissue and were detected only in papillary thyroid carcinomas (PTCs), in 113/993 (11.4%) patients, three times more frequently in pediatric and adolescent patients (29.8%) than in adult patients (8.7%). A total of 20 types of RET fusions were identified. RET fusion-positive carcinomas were associated with aggressive tumor behavior, including high rates of lymph node (75.2%) and distant metastases (18.6%), significantly higher than in NTRK fusion, BRAF V600E and RAS-positive carcinomas. Local and distant metastases were also frequently found in patients with microcarcinomas positive for the RET fusions. 'True recurrences' occurred rarely (2.4%) and only in adult patients. The 2-, 5-, 10-year disease-specific survival rates were 99%, 96%, and 95%, respectively. RET fusion-positive carcinomas were associated with high invasiveness and metastatic activity, but probably due to intensive treatment with low patient mortality.

Changes on chromosome 11p15.5 as specific marker for embryonal rhabdomyosarcoma?

Rhabdomyosarcomas (RMS) constitute a heterogeneous spectrum of tumors with respect to clinical behavior and tumor morphology. The paternal uniparental disomy (pUPD) of 11p15.5 is a molecular change described mainly in embryonal RMS. In addition to LOH, UPD, the MLPA technique (ME030kit) also determines copy number variants and methylation of H19 and KCNQ1OT1 genes, which have not been systematically investigated in RMS. All 127 RMS tumors were divided by histology and PAX status into four groups, pleomorphic histology (n = 2); alveolar RMS PAX fusion-positive (PAX+; n = 39); embryonal RMS (n = 70) and fusion-negative RMS with alveolar pattern (PAX-RMS-AP; n = 16). The following changes were detected; negative (n = 21), pUPD (n = 75), gain of paternal allele (n = 9), loss of maternal allele (n = 9), hypermethylation of H19 (n = 6), hypomethylation of KCNQ1OT1 (n = 6), and deletion of CDKN1C (n = 1). We have shown no difference in the frequency of pUPD 11p15.5 in all groups. Thus, we have proven that changes in the 11p15.5 are not only specific to the embryonal RMS (ERMS), but are often also present in alveolar RMS (ARMS). We have found changes that have not yet been described in RMS. We also demonstrated new potential diagnostic markers for ERMS (paternal duplication and UPD of whole chromosome 11) and for ARMS PAX+ (hypomethylation KCNQ1OT1).

Next generation sequencing - a science tool or routine pathology?

Molecular assays for translocation detection in different tumors have gradually been incorporated into routine diagnostics. However, conventional methods such as fluorescence in situ hybridization (FISH) and reverse transcriptase-PCR come with several drawbacks. Next-generation sequencing (NGS) can provide in-depth detection of numerous gene alterations. The anchored multiplex PCR assay proved to be a fast and easy-to-analyze approach for routine diagnostics laboratories. Next-generation sequencing-based anchored multiplex PCR technique (Archer FusionPlex Panels) is beneficial in both diagnosis for patient care and in identification of a novel fusion breakpoint in tumors. NGS is useful in identifying targetable molecular changes (point mutations, fusion genes, etc.) in tumors that can serve as a rationale for inclusion of patients with advanced disease in ongoing clinical trials and allow for better risk stratification.

An unusual fusion gene EML4-ALK in a patient with congenital mesoblastic nephroma.

Congenital mesoblastic nephroma (CMN), the most common renal tumor of infancy, is a mesenchymal neoplasm histologically classified into classic, cellular, or mixed types. Most cellular CMNs harbor a characteristic ETV6-NTRK3 fusion. Here, we report an unusual congenital mesoblastic nephroma presenting in a newborn boy with a novel EML4-ALK gene fusion revealed by Anchored Multiplex RNA Sequencing Assay. The EML4-ALK gene fusion expands the genetic spectrum implicated in the pathogenesis of congenital mesoblastic nephroma, with yet another example of kinase oncogenic activation through chromosomal rearrangement. The methylation profile of the tumor corresponds with infantile fibrosarcoma showing the biological similarity of these two entities.

NTRK Fusion Genes in Thyroid Carcinomas: Clinicopathological Characteristics and Their Impacts on Prognosis.

Chromosomal rearrangements of NTRK genes are oncogenic driver mutations in thyroid cancer (TC). This study aimed to identify NTRK fusion-positive thyroid tumors and to correlate them with clinical and pathological data and determine their prognostic significance. The cohort consisted of 989 different TC samples. Based on the detected mutation, samples were triaged, and those that were positive for a BRAF, HRAS, KRAS, NRAS, RET, RET/PTC or PAX8/PPARγ mutation were excluded from further analyses. NTRK fusion gene testing was performed in 259 cases, including 126 cases using next-generation sequencing. NTRK fusion genes were detected in 57 of 846 (6.7%) papillary thyroid carcinomas and in 2 of 10 (20.0%) poorly differentiated thyroid carcinomas. A total of eight types of NTRK fusions were found, including ETV6/NTRK3, EML4/NTRK3, RBPMS/NTRK3, SQSTM1/NTRK3, TPM3/NTRK1, IRF2BP2/NTRK1, SQSTM1/NTRK1 and TPR/NTRK1.NTRK fusion-positive carcinomas were associated with the follicular growth pattern, chronic lymphocytic thyroiditis and lymph node metastases. NTRK1-rearranged carcinomas showed a higher frequency of multifocality and aggressivity than NTRK3-rearranged carcinomas. Tumor size, presence of metastases, positivity for the NTRK3 or NTRK1 fusion gene and a late mutation event (TERT or TP53 mutation) were determined as factors affecting patient prognosis. NTRK fusion genes are valuable diagnostic and prognostic markers.

RET, NTRK, ALK, BRAF, and MET Fusions in a Large Cohort of Pediatric Papillary Thyroid Carcinomas.

Background: Pediatric papillary thyroid carcinoma (PTC) is a rare malignancy, but with increasing incidence. Pediatric PTCs have distinct clinical and pathological features and even the molecular profile differs from adult PTCs. Somatic point mutations in pediatric PTCs have been previously described and studied, but complex information about fusion genes is lacking. The aim of this study was to identify different fusion genes in a large cohort of pediatric PTCs and to correlate them with clinical and pathological data of patients. Methods: The cohort consisted of 93 pediatric PTC patients (6-20 years old). DNA and RNA were extracted from fresh frozen tissue samples, followed by DNA and RNA-targeted next-generation sequencing analyses. Fusion gene-positive samples were verified by real-time polymerase chain reaction. Results: A genetic alteration was found in 72/93 (77.4%) pediatric PTC cases. In 52/93 (55.9%) pediatric PTC patients, a fusion gene was detected. Twenty different types of RET, NTRK3, ALK, NTRK1, BRAF, and MET fusions were found, of which five novel, TPR/RET, IKBKG/RET, BBIP1/RET, OPTN/BRAF, and EML4/MET, rearrangements were identified and a CUL1/BRAF rearrangement that has not been previously described in thyroid cancer. Fusion gene-positive PTCs were significantly associated with the mixture of classical and follicular variants of PTC, extrathyroidal extension, higher T classification, lymph node and distant metastases, chronic lymphocytic thyroiditis, and frequent occurrence of psammoma bodies compared with fusion gene-negative PTCs. Fusion-positive patients also received more doses of radioiodine therapy. The most common fusion genes were the RET fusions, followed by NTRK3 fusions. RET fusions were associated with more frequent lymph node and distant metastases and psammoma bodies, and NTRK3 fusions were associated with the follicular variant of PTC. Conclusions: Fusion genes were the most common genetic alterations in pediatric PTCs. Fusion gene-positive PTCs were associated with more aggressive disease than fusion gene-negative PTCs.

The role of fine-needle aspiration biopsy (FNAB) in the diagnostic management of parotid gland masses with emphasis on potential pitfalls.

PURPOSE: The treatment strategy of parotid gland tumours depends mainly on the histopathological type of the lesion. Fine-needle aspiration biopsy (FNAB) is recommended in preoperative diagnostics. The aim of the study was to evaluate the FNAB standing in the diagnostic algorithm of parotid gland lesions and to correlate FNAB results in relation to the definitive histopathological diagnosis. MATERIAL AND METHODS: The retrospective analyses of 651 examined and consequently surgically treated parotid gland lesions at the Department of Otorhinolaryngology and Head and Neck Surgery, 1st Faculty of Medicine, Charles University in Prague between 2006 and 2016 were used. Preoperative cytological results were consequently evaluated in relation to the definitive histopathological diagnosis. RESULTS: The cohort consisted of 367 women and 284 men (average age 58 years). FNAB was diagnostic in 604 (92.8%) patients and non-diagnostic in 47 (7.2%) patients. The result of FNAB was positive (suspicious for malignant tumour) in 89 (14.7%) patients and negative (benign) in 515 (85.3%) patients. Sensitivity of the examination was 80.00%, specificity was 93.82%, PPV 62.92%, NPV 97.28%, and LR + and LR- were 12.95 and 0.21, respectively, with an accuracy of 92.22%. CONCLUSION: Our results confirm the significant role of FNAB in the diagnostic algorithm of parotid gland lesions.

Somatic genetic alterations in a large cohort of pediatric thyroid nodules.

There is a rise in the incidence of thyroid nodules in pediatric patients. Most of them are benign tissues, but part of them can cause papillary thyroid cancer (PTC). The aim of this study was to detect the mutations in commonly investigated genes as well as in novel PTC-causing genes in thyroid nodules and to correlate the found mutations with clinical and pathological data. The cohort of 113 pediatric samples consisted of 30 benign lesions and 83 PTCs. DNA from samples was used for next-generation sequencing to identify mutations in the following genes: HRAS, KRAS, NRAS, BRAF, IDH1, CHEK2, PPM1D, EIF1AX, EZH1 and for capillary sequencing in case of the TERT promoter. RNA was used for real-time PCR to detect RET/PTC1 and RET/PTC3 rearrangements. Total detection rate of mutations was 5/30 in benign tissues and 35/83 in PTCs. Mutations in RAS genes (HRAS G13R, KRAS G12D, KRAS Q61R, NRAS Q61R) were detected in benign lesions and HRAS Q61R and NRAS Q61K mutations in PTCs. The RET/PTC rearrangement was identified in 18/83 of PTCs and was significantly associated with higher frequency of local and distant metastases. The BRAF V600E mutation was identified in 15/83 of PTCs and significantly correlated with higher age of patients and classical variant of PTC. Germline variants in the genes IDH1, CHEK2 and PPM1D were found. In conclusion, RET/PTC rearrangements and BRAF mutations were associated with different clinical and histopathological features of pediatric PTC. RAS mutations were detected with high frequency in patients with benign nodules; thus, our results suggest that these patients should be followed up intensively.

Primary angiosarcoma of the femoral artery in patient with kidney and liver polycystosis and multiple arterial aneurysms: report of the case and review of the literature.

The association between kidney and liver polycystosis and arterial aneurysms is well documented. However, it remains unclear whether these patients are at increased risk of malignant transformation. In this article, we describe a case of a primary angiosarcoma of the femoral artery with metastatic spread into the lungs and hilar lymph node arising in a 74-year-old man with kidney and liver polycystosis and multiple arterial aneurysms.

Polymorphisms in selected DNA repair genes and cell cycle regulating genes involved in the risk of papillary thyroid carcinoma.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. In addition to causal somatic mutations in the BRAF gene and RET/PTC rearrangements, the contribution of single nucleotide polymorphisms (SNPs) in low-penetrance genes in the development of PTC has been proposed. METHODS: Four SNPs in the XRCC1 (Arg399Gln, Arg280His, Arg194Trp and T-77C) and one SNP from each of three other genes participating in DNA repair pathways and/or cell cycle regulation (ATM Asp1853Asn, TP53 Arg72Pro, CDKN1B Val109Gly) were selected. The allelic and genotypic distributions of these variants as well as haplotypes of the XRCC1 were examined in 583 individuals comprising well-characterized cohorts of 209 PTC patients and 374 healthy volunteers. Correlations of polymorphism with clinical-pathological data and mutation status were performed. RESULTS: XRCC1 T-77C polymorphism affects the genetic susceptibility for PTC development in men, the specific combination of XRCC1 haplotypes correlates with RET/PTC incidence, CDKN1B Val109Gly significantly influences the risk of developing PTC regardless of gender and in PTC cases, selected genotypes of TP53 Arg72Pro and ATM Asp1853Asn were significantly associated with monitored tumour characteristics. CONCLUSION: It seems that SNPs in studied regulating genes contribute to the development of PTC and modify the tumour behaviour or characteristics.

[Transplantations of lungs in the Czech Republic - from the perspective of the pathologist]. / Transplantace plic v Ceské republice - z pohledu patologa.

Lung transplantation has become a standard therapeutic procedure for patients with end-stage pulmonary diseases in the Czech Republic. There were 246 lung transplantations performed from December 1997 to the end of November 2014 at the 3rd Department of Surgery, 1st Faculty of Medicine, Charles University in Prague and Motol University Hospital. The most common indications for transplantation were chronic obstructive pulmonary disease in 39.4 % of patients, idiopathic pulmonary fibrosis in 28.9 % of patients and cystic fibrosis in 19.1 % of patients. The trans-bronchial biopsy is important for monitoring patients after lung transplantation. The biopsy helps to detect acute cellular rejection, which was found within 63 % of our patients. Patients with the mild and moderate grade of acute cellular rejection got better after the anti-rejection therapy. The severe rejection in three patients led to the shock change in lung and to respiratory failure. Humoral rejection cannot be determined based on biopsy only - the capillaritis and the linear binding of C4d fraction of the complement to the capillaries are inconsistent findings and are not pathognomonic. The classification of chronic rejection, which corresponds to the bronchiolitis obliterans, is limited for the common absence of bronchioli in the biopsy. Therefore, bronchiolitis obliterans in our study group was detected in only 3.7 % of patients.Since the first transplantation, 109 of our patients have survived (44.3 %). After transplantation about 90 % of patients live one year, about 70.9 % of patients live 3 years and 69.1 % live 5 years. An autopsy at our department was performed in 79 cases. The most common causes of death were mycotic infections (aspergillosis, candidiasis), bacterial infections (Klebsiela, Pseudomonas aeruginosa, Burkholderia cepacia) followed by sepsis and viral infection (CMV, varicella zoster). At the autopsy, chronic rejection was found in 13 patients and it led to chronic respiratory failure, which was often complicated by an infection. The tumors as the cause of death were mostly generalized carcinomas.

A novel RET/PTC variant detected in a pediatric patient with papillary thyroid cancer without ionization history.

Papillary thyroid carcinoma (PTC) is the most frequent type of thyroid cancer. Its development is often caused by the formation of RET/PTC fused genes. RET/PTC1 is the most prevalent form, where exon 1 of CCDC6 gene is fused with the intracellular portion of RET protooncogene starting with exon 12. We have discovered a novel RET/PTC1 variant which we have named RET/PTC1ex9 in metastatic PTC of 8-year-old boy. RET/PTC1ex9 detection was performed by real-time polymerase chain reaction with melting curve analysis and subsequent Sanger and next-generation sequencing. A fusion of exon 1 of CCDC6 with exon 9 of extracellular domain of RET followed by exon 12 of RET was revealed. This is the first RET/PTC variant among PTC cases that contain the extracellular part of RET. This observation could be probably explained by incorrect splicing of RET due to the somatic 32-bp deletion in exon-intron 11 boundary of RET.

Search for new genetic biomarkers in poorly differentiated and anaplastic thyroid carcinomas using next generation sequencing.

BACKGROUND: Poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC) are very rare tumors with extremely aggressive behavior. Their comprehensive genetic background is still unclear. Some of the main genetic changes of differentiated thyroid carcinomas, such as mutations in BRAF and RAS genes, as well as changes in CTNNB1, PIK3CA, TP53, AXIN1, PTEN or APC genes leading to the dedifferentiation of the tumors, are described. MATERIALS AND METHODS: DNAs from fresh frozen thyroid tissues of 3 PDTCs and 5 ATCs were extracted. The next-generation sequencing (NGS) approach was used to target 94 genes involved in cancer. The samples were prepared using a TruSight Cancer panel and sequenced with a MiSeq sequencer. Analysis of variants was performed by the MiSeq Reporter and NextGENe software and stringent criteria for prioritization of the variants were used in the Illumina VariantStudio software. RESULTS: Using NGS, we identified 26 genetic changes in 18 genes, novel variants included. CONCLUSION: NGS is a useful tool for searching for new variants and genes involved in PDTC and ATC. It seems that each of these rare tumor types has its own specific genetic background. These data could be helpful for recognizing new genetic markers and targets for future personalized therapy.

Melanoma cells influence the differentiation pattern of human epidermal keratinocytes.

BACKGROUND: Nodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome. Similarly to other tumors, permissive microenvironment is essential for melanoma progression. Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue. Here, we study the effect of melanoma cells on human primary keratinocytes (HPK). Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC). METHODS: Comparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed. Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively. Differentially expressed candidate genes were verified by RT-qPCR. Biological activity of candidate proteins was assessed on cultured HPK. RESULTS: Epidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases. This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10. We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK. This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies. We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes. CONCLUSION: We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma. The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.

[Soft tissue tumors - the view of the molecular biologist]. / Nádory mekkých tkání ocima molekulárního patologa.

Soft tissue tumors (SSTs) constitute a broad spectrum of neoplasms with diverse biological properties. Rare or unusual types are often difficult to classify. Recent studies show, that a significant subset of SSTs including many types of sarcomas are associated with specific genetic changes such as chromosomal translocations producing chimeric genes, which play a role in the pathogenesis of SSTs. Because SSTs represent a diagnostically challenging group of tumors, molecular-genetic techniques (FISH or PCR) are useful as supplementary and/or confirmatory diagnostic tools. In the present paper we demonstrate the usefulness of a combined diagnostic approach using the tools of classical histopathology and immunohistochemistry together with the molecular diagnostic approach in selected nosologic entites.

Clinical and genetic features of rhabdoid tumors of the heart registered with the European Rhabdoid Registry (EU-RHAB).

Rhabdoid tumors are rare but highly aggressive malignancies of infancy and early childhood with a generally unfavorable prognosis. Despite a wide variety of anatomic locations rhabdoid tumors share mutational inactivation of the SWI/SNF (SWItch/Sucrose NonFermentable) core component gene SMARCB1 (also known as INI1, hSNF5 or BAF47) in chromosome 22. As this inactivation usually results in loss of SMARCB1 expression, detectable by an antibody against the SMARCB1 protein, the accurate diagnosis of a rhabdoid tumor may be more distinctly and frequently made. Several reports on rhabdoid tumors presenting in various anatomic sites outside the kidneys and CNS are on record. We report two cases of rhabdoid tumors originating in the heart (cardiac tissue), which were entered into the European Rhabdoid Registry (EU-RHAB). The first case presented with intracardial and -cranial lesions as well as malignant ascites, while the second patient demonstrated an isolated cardiac tumor. This induced a different therapeutic approach and subsequently different clinical course (death 7 weeks after diagnosis in patient 1). Patient 2 presented with a bifocal intracardial tumor without metastases and remains in complete remission for 46 months since diagnosis following multimodal therapy. The second case demonstrates that even in a potentially futile clinical situation early and accurate diagnosis followed by prompt and intensive multimodal therapy may offer prolonged survival, potential cure and improved quality of life.

Retinal toxicity after repeated intravitreal carboplatin injection into rabbit eyes.

BACKGROUND: The aim of this study was to assess retinal toxicity in a rabbit model after carboplatin delivered as repeated transcorneal intravitreal injection, in order to determine the highest possible safe dose for use in human retinoblastoma "seeding" tumor chemotherapy. METHODS AND RESULTS: We used six albino rabbits in an in vivo experiment and injected 0.008 mg of carboplatin intravitreally (iv) 4 times at two-week intervals. 0.08 mL saline was injected into the left eye. We recorded electroretinograms (ERGs) before the first carboplatin injection and after the fourth injection. Platinum concentration was measured 1 h after the fifth additional injection. We found reduced dark-adapted b-wave amplitudes and, light-adapted b-wave and a-wave amplitudes. The differences between right and left eyes was significant but we found no histopathologic retinal changes. CONCLUSIONS: 0.008 mg of carboplatin is probably the highest possible safe dose for the treatment of retinoblastoma patients. Questionable is direct extrapolation of retinal toxicity from the rabbit eye model to the human eye.

Topotecan vitreous and plasma levels and retinal toxicity after transcorneal intravitreal delivery in healthy albino rabbits: alternative retinoblastoma treatment.

AIM: To determine intravitreal and plasma concentrations and retinal toxicity after transcorneal intravitreal injection of 1 µg and 2 µg of topotecan (Hycamtin). METHOD: Twelve healthy albino rabbits were included in this in vivo experiment. Six anesthetized albino rabbits received a single transcorneal intravitreal injection of 1 µg (group A) or 2 µg (group B) of topotecan. Vitreous and blood samples were collected until 168 h. Left eyes were treated with the same volume of saline. Plasma and vitreous levels of topotecan were determined by high-performance liquid chromatography. Area under the plasma concentration time curve (AUC) was calculated using trapezoidal rule. Clinical evidence of toxicity was classified into four grades according to anatomical structures. Electroretinograms (ERGs) were recorded. RESULTS: Time to maximum concentration was observed up to 2 h after drug injection in group A whereas up to 1 h in group B. Low levels of topotecan were detected in plasma in both groups and in the vitreous humor of the contralateral eye in group B. Topotecan levels (mean vitreous AUC in group A 2.55 µg/mL.h and in group B 5.338 µg/mL.h) were detectable up to 6 h in both groups. We observed following structural changes in rabbit eyes: corneal vascularization, cataract, hemophthalmus, choroidal edema and focal retinal atrophy. Abnormal ERGs were obtained. CONCLUSION: Our findings proved that transcorneal intravitreal administration of 1 µg and 2 µg of topotecan results in potentially cytotoxic intraocular concentrations. More studies are needed to establish the safety of topotecan for retinoblastoma in children.