UNASSIGNED

Acute kidney injury (AKI) is a risk factor for progression to chronic kidney disease, with even subclinical AKI episodes progressing to chronic kidney disease. Several risk factors such as preexisting kidney disease, hyperglycemia, and hypertension may aggravate renal disease after AKI. However, mechanisms underlying the progression of AKI are still unclear. This study identified the effect of human cluster of differentiation 36 (CD36) overexpression on the progression of folic acid-induced AKI.

UNASSIGNED

Pax8-rtTA/tetracycline response element-human CD36 transgenic mice were used to elucidate the effect of human CD36 overexpression in the proximal tubules on folic acid-induced AKI.

UNASSIGNED

Results of histological analysis showed severely dilated tubules with casts and albuminuria in folic acid-treated transgenic mice overexpressing human CD36 compared with folic acid-treated wild-type mice. In addition, analysis of mRNA expression showed a significant increase in the collagen 3a1 gene in folic acid-treated transgenic mice overexpressing human CD 36 compared with folic acid-treated wild type mice.

UNASSIGNED

Human CD36-overexpressing transgenic mice showed severe pathological changes and albuminuria compared with wild-type mice. Moreover, mRNA expression of the collagen 3a1 gene increased in folic acid-treated transgenic mice. These results suggest that human CD36 overexpression is a risk factor of AKI and its progression to chronic kidney disease.

Paired-box gene 8 (PAX8)-peroxisome proliferator-activated receptor-γ (PPARγ) gene fusion has been identified at significant frequency in follicular thyroid carcinomas (FTCs) with cytogenetically detectable translocation t(2;3)(q13;p25). This represents a possible specific molecular marker for follicular carcinoma. In this study, we examined PAX8-PPARγ rearrangement in 24 FTC samples from Japanese patients by reverse transcribed-polymerase chain reaction (RT-PCR) using two upstream PAX8 primers located in exons 7 and 8 and a downstream primer in exon 1 of PPARγ. The fusion gene was detected in only one of 24 FTCs (4%). The FTC with PAX8-PPARγ rearrangement from a 56-year-old man showed a product consistent with fusion between exon 8 of PAX8 and exon 1 of PPARγ. It was confirmed by direct sequencing. This FTC was histologically encapsulated, composed of trabeculae and small follicles and had complete penetration of the capsule by tumor tissues (minimally invasive type). The frequency of the fusion gene in this study was much lower than the 29-63% noted in reports from other countries suggesting that FTCs in Japanese patients may have a special genetic background, and that the high iodine intake from a typical Japanese diet might influence the frequency of the fusion gene in FTCs.

APE/Ref-1 is a multifunctional protein possessing both redox and DNA repair functions. Through its redox activity, APE/Ref-1 controls the DNA-binding function of several transcriptional regulators (AP1, NF-kappaB, p53, Pax proteins). We have previously shown that APE/Ref-1 upregulates the transcriptional activity of the thyroid-specific transcription factor Pax8. In thyroid cells, APE/Ref-1 can be detected both in the nuclear and cytoplasmatic compartments. In this study regulatory mechanisms acting on APE/Ref-1 were revealed using the FRTL-5 cell line. TSH induces both cytoplasm-to-nucleus translocation and neosynthesis of APE/Ref-1 protein. Interestingly, only neosynthesis is dependent on cAMP signalling. In contrast, the cytoplasm-to-nucleus translocation is dependent on redox-mediated mechanisms. Based upon the data shown in this study and in others, a bimodal control of APE/Ref-1 by TSH can be delineated.

Somatic cell hybrid (SCH) panels and radiation hybrid (RH) panels are powerful resources for comparative gene mapping because gene assignments are made without the detection of genetic polymorphism as needed for linkage mapping. A frequently encountered problem, however, is that the gene specific primers may amplify homologous PCR products of equal length from the donor and recipient species of the panel. Here, we describe a simple solution to this problem in which we utilize the formation of interspecies heteroduplexes that can be easily distinguished from the corresponding homoduplexes by native polyacrylamide gel electrophoresis. We denote these DNA-DNA interspecies hybrids, xenoduplexes (xeno = Gr. Xenos, foreigner). A merit of the method is that the formation of xenoduplexes strongly suggests that the PCR products from the two species represent homologous sequences. The method is thus particularly useful for comparative gene mapping when the PCR primers have been designed by use of sequence information from other species. In this study we have successfully used xenoduplex analysis and a pig-rodent SCH panel to map seven porcine genes (ACADM, AT3, HOXD, IL8RB, LEPR, PAX8, PKLR) for which no previous sequence information was available. The assignment of the leptin receptor gene (LEPR) to pig chromosome 6q32-35 excluded LEPR as a candidate gene for a QTL on pig chromosome 4 with a major effect on fatness.

BACKGROUND

Paired box 8 (PAX8) has been documented to be downregulated in gastric cancer. However, its biological function in this malignancy is poorly understood.

METHODS

In the present work, we investigated the effects of PAX8 overexpression and knockdown on the aggressive phenotype of gastric cancer cells. We further checked the involvement of forkhead box M1 (FOXM1), a ubiquitously expressed oncogene that can facilitate gastric cancer progression, in the action of PAX8.

RESULTS

Ectopic expression of PAX8 blocked the migration and invasion of both AGS and SGC-7901 cells, but had no effect on cell proliferation. Conversely, knockdown of PAX8 enhanced gastric cancer cell migration and invasion. PAX8 overexpression inhibited epithelial-mesenchymal transition (EMT) and pro-angiogenic activity of gastric cancer cells. Mechanistically, PAX8 overexpression downregulated FOXM1 by stimulating microRNA (miR)-612 expression. Ectopic expression of miR-612 recapitulated the effect of PAX8 overexpression on gastric cancer cells, causing an inhibition of migration, invasion, EMT, and angiogenesis. Knockdown of miR-612 or overexpression of FOXM1 significantly reversed the tumor-suppressive activity of PAX8. In vivo studies further demonstrated that PAX8 overexpression restrained tumor angiogenesis and metastasis in nude mice, which was accompanied by increased expression of miR-612 and decreased expression of FOXM1.

CONCLUSIONS

PAX8 exerts a tumor-suppressive effect against gastric cancer cells, largely through induction of miR-612 and repression of FOXM1. Therefore, restoration of PAX8 expression may offer therapeutic benefits in the treatment of gastric cancer.

BACKGROUND

Congenital hypothyroidism (CH) is a common endocrine disorder in newborns. The cause of CH is thyroid dysgenesis in 80-85% of patients. Paired box gene 8 (PAX8) is a thyroid transcription factor that plays an important role in thyroid organogenesis and development. To date, 22 different PAX8 gene mutations have been reported.

METHODS

Four generations of a Hungarian Jewish family were affected, and in the 3 generations studied, 9 males and 4 females were affected and 3 first-degree relatives were unaffected. Six were diagnosed at birth [thyroid-stimulating hormone (TSH) level 59-442 mU/l] and 7 at 2-48 years of age (TSH level 6-223 mU/l). One affected patient had thyroid hemiagenesis on ultrasound.

RESULTS

Direct sequencing of the PAX8 gene revealed a novel single nucleotide substitution (c.162 A>T) in exon 2 that resulted in the substitution of the normal serine 54 with a cysteine (S54C), which segregated with elevated serum TSH levels. Other mutations of the same amino acid (S54G and S54R) have also been shown to produce functional impairment.

CONCLUSIONS

We report a large family with a novel mutation in the PAX8 gene presenting with variable phenotype and with a high proportion of affected family members.

Specific genotype-phenotype correlations have been identified in conventional-type papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC). In contrast, the genetic alterations underlying the pathogenesis of the follicular variant of PTC (FV-PTC), which shares some clinicopathologic and molecular features with both PTC and FTC, remain to be clarified. This entity shows a PAX8-PPARg fusion gene (associated with FTC), more frequently than BRAF or RET-PTC alterations (associated with PTC). Herein, we report, for the first time, an FV-PTC with the simultaneous occurrence of both RET-PTC and PAX8-PPARg alterations. Neoplastic cells were of the wild type for BRAF and H,K,N-RAS, had an apparently normal karyotype by conventional cytogenetics, and had a balanced genome by array comparative genomic hybridization analysis. In fact, submicroscopic chromosome rearrangements producing RET-PTC3 and PAX8-PPARg chimeric genes were found by interphase fluorescence in situ hybridization. We demonstrated that these 2 genetic alterations coexisted in the same tumor and were confined to 2 different clones. Our findings indicate that molecular heterogeneity, although an uncommon phenomenon, may occur in thyroid carcinoma and demonstrate the coexistence in a case of FV-PTC not only of the histologic but also of the molecular features of both PTC (RET-PTC) and FTC (PAX8-PPARg).

MicroRNAs (miRNAs or miRs) and the target genes before and after warm acupuncture at the genetic level were assessed, and the cytokines and neurotransmitters related to insomnia were studied. Male Sprague-Dawley rats were used to create PCPA insomnia rat models and randomly divided into the normal, model, warm acupuncture, and drug groups. The Dinghui Acupoint, Heyi Acupoint, and Xin Acupoint were inserted in the Mongolian medicine warm acupuncture group. The differential expression profile of microRNA in the brain tissue of the insomnia rats was determined before and after Mongolian medicine warm acupuncture for establishment of miR-101a mimics and inhibitor. qPCR was used to detect the expression level of miR-101a. Western blotting was used to detect the expression level of PAX8. The rats receiving Mongolian medicine warm acupuncture had 141 miRNAs with differential expression compared with the normal rats. The expression level of miR-101a in the cells of the hippocampus of the insomnia rats transfected with miR-101a mimics increased significantly at 72 h (P<0.05). The activity of the neuronal cells transfected with miR-101a inhibitor increased significantly at 72 h (P<0.05). The western blotting result indicated that the expression of the PAX8 protein in the neuronal cells of the insomnia model rats was inhibited and downregulated significantly at 72 h after addition of miR-101a mimics compared with that in the scramble added group (P<0.01). The levels of the interleukins IL-1, IL-2, and IL-6 and the tumor necrosis factor-α in the hypothalamus, hippocampus, and prefrontal cortex decreased significantly compared with those in the blank control group (P<0.05). The levels of noradrenaline, dopamine, and glutamic decreased significantly following warm acupuncture or western medicine treatment (P<0.05). In conclusion, this study demonstrates that the upregulation of miR-101a in the rats treated with warm acupuncture is directly associated with PAX8 regulation.

Well-differentiated thyroid carcinomas comprise two well-defined histological types: papillary and follicular (PTCs and FTCs, respectively). Despite being derived from the same cell (thyroid follicular cell), these two types of tumour accumulate distinct genetic abnormalities during progression. The molecular pathology of thyroid cancer is now better understood because of our ability to identify RET/PTC rearrangements and BRAF mutations in the aetiopathogenesis of the large majority of PTCs and the high prevalence of RAS mutations and PAX8/PPARgamma rearrangements in follicular patterned carcinomas (FTCs and follicular variant of PTCs). This review summarises most of the molecular alterations currently used as targets for new biological treatments and looks at some of the changes that are already occurring or may occur in the treatment of patients with thyroid cancer. For simplicity, the review is divided up according to the major genetic alterations identified in well-differentiated thyroid carcinomas (RET/PTC rearrangements, BRAF mutations, RAS mutations and mitochondrial DNA deletions and mutations) and their respective treatments.

Transient Pax8 expression was reported in mouse islets during gestation while a genome-wide linkage and admixture mapping study highlighted PAX8 as candidate gene for diabetes mellitus (DM). Herein we sought the significance of PAX8 expression in mouse and human islet biology. Pax8 was induced in gestating mouse islets and in human islets treated with recombinant prolactin. Global gene expression profiling of human and mouse islets overexpressing the corresponding species specific PAX8 revealed the modulation of distinct genetic pathways that converge on cell survival. Accordingly, apoptosis was reduced in PAX8-overexpressing islets. These findings support that PAX8 could be a candidate gene for the study of gestational DM (GDM). PAX8 was genotyped in patients with GDM and gestational thyroid dysfunction (GTD), a pathology commonly found in patients with mutations on PAX8 A novel missense PAX8 mutation (p.T356M, c.1067C>T) was identified in a female diagnosed with GDM and GTD as well as in her type 2 diabetic father while absent in control subjects. The p.T356M variant did not alter protein stability or cellular localization whereas its trans-activation activity was hindered. In parallel, a retrospective clinical analysis uncovered that a pregnant female harboring a second PAX8 mutation (p.P25R, c.74C>G) previously reported to cause congenital hypothyroidism also developed GDM. These data indicate that increased expression of PAX8 impacts islet viability and PAX8 could be considered as a candidate gene for the study of GDM.

We describe an uncommon thyroid tumor in a 56-year-old woman. The widely infiltrating, angioinvasive neoplasm, 5 cm in diameter, exhibited a peculiar architectural growth pattern characterized by follicles with round to oval epithelial tufts growing within, often supported by a fibrovascular core mimicking the renal glomerulus. Colloid-empty follicles, tubular or elongated, were lined by pseudostratified tall, columnar cells with clear cytoplasm. Nuclei were round to oval, with evenly distributed, slightly coarse chromatin. Tumor cells were positive for thyroid transcription factor-1, thyroperoxidase, thyroglobulin, cytokeratin 18, Hector Battifora mesothelial cell, and vimentin. Scattered cells positive for S100, Wilms tumor 1 (WT1), and cytokeratins AE1/AE3 were found, with no reaction detected for cytokeratins 34betaE12, 5/6, 7, 19, or 20. There were PAX8-PPARgamma rearrangement and N-RAS mutation. No mutations were found for APC or BRAF genes, nor were RET/PTC rearrangements detected. Because of the distinctive histologic features, we propose naming this tumor follicular thyroid carcinoma with an unusual glomeruloid pattern of growth.

We have developed an efficient and simple method for extracting and purifying genomic DNA from dried blood stored on filter paper. The quality of the genomic DNA extracted is tested by PCR amplification of a 255-bp fragment of the PAX8 gene sequence and the PCR products are determined for further genetic studies by single strand conformation polymorphism (SSCP) analysis. Larger DNA sequences of the 674-bp of the PAX8 gene and the 1,039-bp of the human beta-globin gene, a housekeeping gene, have also been amplified from the extracted DNA, thus indicating the high quality of the genomic DNA extracted by the developed method for subsequent genetic studies of any gene of interest. The method developed can also be used for the purification of genomic DNA from dried blood specimens stored under different conditions. Moreover, the genomic DNA products can be stored for long-term use due to the highly purified procedure. Therefore, the method is efficient and appropriate for the extraction and purification of genomic DNA from dried blood specimens, which has become an increasingly important tool for genetic and epidemiological studies.

Renal cell carcinoma with TFEB rearrangement (t[6;11][p21;q13]) was initially recognized to be composed of dual populations of large cells with clear cytoplasm and small cells forming rosettes around hyaline material. With increasing awareness, however, the spectrum of described morphology has been found to be more heterogeneous. We report a 54-year-old woman who underwent partial nephrectomy for a 2.4-cm renal mass, composed of fibrosis, hyalinization, calcification, and ossification and a smaller component of epithelioid cells. Immunohistochemical staining revealed diffuse positivity for cytokeratin AE1/AE3 and PAX8, patchy labeling for melan-A, human melanosome, and cathepsin K, and negative caldesmon, smooth muscle actin, TFE3 protein, carbonic anhydrase IX, CD10, cytokeratin 7, epithelial membrane antigen, and inhibin. Fluorescence in situ hybridization confirmed rearrangement of TFEB and not TFE3. Together with one recent case in another report, our findings suggest that extensive sclerosis and ossification may be a less common recurring histology of TFEB-rearrangement renal cell carcinoma.

Endometrial stromal sarcoma (ESS) involving the urinary bladder is very rare, with no prior series reported. We identified 6 cases of low-grade ESS involving the bladder at our institution (1998 to 2013), 5 of them consults. The median age at bladder involvement was 60 years (range, 44 to 77 y). One patient presented with bladder involvement at initial diagnosis of ESS. The remaining 5 cases with bladder involvement presented 7 to 30 years (mean 18 y) after a known diagnosis of ESS (n=2) or after a remote history of hysterectomy with an uncertain diagnosis (n=3). The location of bladder involvement included dome (n=1), trigone (n=2), diffuse (n=1), and unknown (n=2). Two cases demonstrated worm-like infiltrating tumor nests classic of low-grade ESS with little stromal reaction with retraction artifact mimicking vascular invasion. One case originating from the ovary showed focal glandular differentiation in the bladder, resembling endometriosis. Two cases had abundant keloidal collagen formation, arranged haphazardly or in a sunburst pattern. One case showed primitive cells infiltrating entirely hyalinized stroma, after chemotherapy given for a misdiagnosis of urothelial carcinoma. CD31 was negative in all cases, except for 1 case with obvious large vessel invasion. The differential diagnosis included a large nested variant of urothelial carcinoma, carcinoid tumor, synovial sarcoma, solitary fibrous tumor, Ewing sarcoma/primitive neuroectodermal tumors, and endometriosis. CD10 was strongly positive in 5 cases, and 1 case had very focal, moderate staining. Estrogen receptor showed strong and diffuse staining in all 6 cases. Progesterone receptor showed moderate to strong staining in 5 cases and focal staining in 1 case. One case showed PAX8 expression, and 2 cases showed p16 nuclear and cytoplasmic expression. CD56 showed weak to strong staining in 4 cases. Two cases had diffuse synaptophysin, and 1 case had focal p63 positivity. GATA-3, CD34, and CD99 were negative in all cases. The Ki-67 index was 1% to 10% (mean 4%). The mitotic count was 0 to 3/10 HPF (mean <1/10 HPF). Two patients had metastases to pelvic lymph nodes, and 1 had possible lung metastasis. Three patients were treated with Megace and 1 with Arimidex after surgery. Follow-up averaged 19 years (0 to 33 y) after the initial diagnosis of ESS or hysterectomy and 3.5 years (0 to 11 y) after bladder surgery. ESS involving the bladder is extremely rare with a very long interval from onset to bladder involvement. In female patients, low-grade spindle cell lesions involving the bladder should include ESS in the differential diagnosis.

BACKGROUND

Anaplastic thyroid cancer (ATC) is thought to often be transformed from pre-existing differentiated thyroid cancer. It is one of the most aggressive malignancies and has a dismal prognosis due to its resistance to multimodal therapies. Basic exploratory studies using authentic ATC cell lines that retain its clinical features are necessary. We investigated the characteristics of seven ATC cell lines newly established at our institute to confirm their possible utility for basic studies.

METHODS

Seven distinct cell lines from six patients were established. Their molecular characteristics and sensitivities to cytotoxic anti-cancer drugs were investigated and compared with each other, and with the clinical features of the original tumors.

RESULTS

All cells showed extensive chromosomal abnormality and Pax8 expression, indicating human thyroid follicular cell origin. Vascular endothelial growth factor was secreted from all cells, suggesting possible candidacy for targeted therapy. Vimentin was expressed, but E-cadherin expression was lost in all cells but OCUT-1C, which showed different features from those of OCUT-1F derived from the same tumor, suggesting a mixture of cancer cell clones with various degrees of differentiation within a single ATC tumor. Cells were likely to show sensitivity for taxanes, indicating the usefulness of taxanes as the first-line chemotherapy. OCUT-2, a cell line with both B-Raf and PI3 KCA mutation, demonstrated the importance of molecular target-oriented therapy.

CONCLUSIONS

Basic studies using authentic ATC cell lines retaining the clinical features of the original tumor are useful models for investigating the mechanism of anaplastic transformation and exploring novel therapeutic strategies.

BACKGROUND

Comprehension of the regulatory mechanism involved in the sodium iodide symporter (NIS) expression is of great relevance for thyroid cancer. In fact, restoration of NIS expression would be a strategy to treat undifferentiated thyroid cancer. Previous in vitro findings suggest that the cyclic AMP-response element (CRE) modulator (CREM) is involved in control of NIS expression. In this work, we examined the expression of CREM in a series of thyroid cancer tissues and its action on NIS promoter in human thyroid cancer cells.

METHODS

Expression of mRNA levels for CREM, PAX8 and NIS was measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in 6 normal thyroid tissues, 22 papillary, 12 follicular and 4 anaplastic thyroid cancers. The effect of CREM on transcriptional activity of the NIS promoter was investigated by transient transfection of human thyroid cell lines.

RESULTS

Compared to normal tissues, NIS and PAX8 mRNA levels were significantly reduced in all types of thyroid cancer. As expected, the maximal decrease was detected in anaplastic thyroid cancer. Conversely, CREM mRNA levels were increased in all types of thyroid cancer, reaching statistical significance for follicular and anaplastic thyroid carcinoma (p=0.0157 and 0.0045, respectively). Transfection experiments showed an inhibitory effect of CREM on NIS promoter activity in various thyroid cancer cell lines.

CONCLUSIONS

These data demonstrate that CREM expression is increased in thyroid cancer tissue and may play a role in the downregulation of NIS expression in thyroid cancer acting at the transcriptional level.

Angiosarcoma and anaplastic carcinoma are the most lethal neoplasms of the thyroid worldwide and share some similarities, which have led to a longstanding controversy on their etiopathological relationship. Thyroid angiosarcomas are characterized by vessel formation and an immunophenotype common to endothelial cells, while anaplastic carcinomas are partially or wholly composed of mesenchymal-like cells that have lost the morphologic and functional features of normal thyroid follicular cells. To investigate whether angiosarcomas represent the endothelial extreme of the differentiation spectrum of carcinomas or they are bona fide vascular neoplasms, we studied the clinico-morphologic and genetic characteristics of a series of 10 angiosarcomas and 22 anaplastic carcinomas. Immunohistochemically, among the endothelial markers, CD31 and ERG were the most consistently expressed in angiosarcomas. Among the markers of thyroid origin, PAX8 was the most reliable in anaplastic carcinomas, while TTF-1 reactivity was found in only 5% of anaplastic carcinomas and thyroglobulin was always negative. Pankeratin reacted with most angiosarcomas and anaplastic carcinomas and is therefore not useful in the differential diagnosis. Interestingly a mutated pattern of p53 immunostaining prompted a diagnosis of anaplastic carcinoma. To compare the genetic profile, we used the NGS approach to sequence hotspot regions within a panel of 57 genes. As a result, only a few mutations were found in angiosarcomas and all of them were single events (no TP53 or TERT mutation). On the other hand, anaplastic carcinomas were characterized by a higher number of mutations, and TP53 and TERT promoter mutations were the most frequent genetic alterations. The lack in angiosarcomas of the common mutations identified in anaplastic carcinomas supports a different genetic origin and strongly suggests that, in spite of a shared sarcomatous morphology and a similar clinical aggressiveness, angiosarcomas and anaplastic carcinomas rely on a completely different set of genetic alterations during their evolution.

The most common thyroid neoplasms are either follicular derived (papillary, follicular and Hürthle cell lesions) or C-cell derived (medullary carcinoma). The diagnosis of these tumors can usually be made at the histologic level, with immunohistochemical stains necessary in some circumstances. Specific molecular mutations have been described that can be diagnostically useful or explain, in part, their pathogenesis, including the well-known Ret/PTC and PPARgamma-PAX8 translocations, point mutations in the Ret, Ras and BRAF genes, and loss of heterozygosity of multiple different tumor suppressor genes. Some unusual tumors of the thyroid gland are more difficult to diagnose. In examining these lesions, the pathologist may use the hematoxylin and eosin-stained morphology, coupled with an analysis of the immunohistochemical staining profiles and possibly analysis of the underlying molecular mutational patterns. These less common thyroid tumors include tall cell and cribriform-morular variants of papillary carcinoma, hyalinizing trabecular tumor, mucoepidermoid and sclerosing mucoepidermoid carcinoma with eosinophilia, poorly differentiated (insular) carcinoma, and undifferentiated (anaplastic) carcinoma. The diagnostic features of these rare tumors, including the histology, immunohistochemical expression profiles and the known molecular mutational profiles of each, are reviewed.

BACKGROUND

Fluorescence in situ hybridization (FISH) to identify specific DNA target sequences in the nuclei of nondividing cells of numerous solid neoplasms has contributed to the introduction of molecular cytogenetics as a useful adjunct to cytology, leading recently to the "marriage" of the 2 disciplines. Numerous cancer molecular markers can now be investigated using different technical approaches, at both the gene and expression levels, in biopsies of various suspected cancers, including differentiated thyroid carcinoma. The limited amount of bioptic material is often insufficient to carry out multiple tests, and optimizing handling of the biopsy is desirable.

METHODS

We have developed a home-brew tetracolor break-apart probe able to simultaneously identify the 2 most common genetic alterations in differentiated thyroid carcinoma: RET/PTC variants in papillary thyroid carcinoma and PAX8/PPARg fusion and variants in follicular thyroid carcinoma.

RESULTS

The probe had 100% specificity, 99.5% sensitivity, and ≥ 3% cutoff. The probe was tested on RET/PTC and PAX8/PPARg RT-PCR positive controls, and feasibility was assessed in 368 thyroid nodule fine-needle aspirations (FNA). In the latter analysis, 24 FNAs had split RET signal, and 9 had split PPARg signal. FISH analysis of available surgically removed nodules confirmed the sensitivity of FISH in detecting abnormal clones and oligoclones.

CONCLUSIONS

The home-brew tetracolor probe showed high feasibility, optimizing the use of the biological material in relation to the available molecular tests and maximizing the FISH experimental and slide-scoring times. This probe may be considered an alternative to RT-PCR when recovery and quality of RNA amplification from FNA are insufficient.

Nine cases of ectopic hamartomatous thymoma are described. The patients were 5 men and 4 women aged 34 to 52 years (mean, 43 y). All patients presented with solitary lower neck masses ranging in size from 3.5 to 8.0 cm (mean, 5.4 cm). Grossly, the lesions were circumscribed and lobulated masses with a fleshy white cut surface; cystic changes were identified in 3 cases. Histologically, the tumors were composed of varying proportions of spindle cells arranged in fascicles, mature adipose tissue, and an epithelial component composed of squamoid elements and glandular or ductal structures. In 1 case, duct-like structures dominated the histologic picture. Structures reminiscent of Hassall corpuscles were identified in 2 cases. No overt malignant changes were seen, and the mitotic activity ranged from 0 to 2 mitoses per 10 high-power fields. Immunohistochemically, the spindle cells coexpressed CK5/6, CD34, and smooth muscle actin, whereas the squamous component was positive for CK5/6 only. Bcl-2 was variably expressed in the spindle and epithelial elements, whereas Pax8 and STAT6 were uniformly negative. Clinical follow-up revealed that all patients were alive and well 2 to 5 years after diagnosis. Ectopic hamartomatous thymomas are benign neoplasms with a wide morphologic spectrum. The histologic and immunohistochemical features are reminiscent of thymic derivation and suggest possible origin from remnants of the thymic anlage. Changes in the nomenclature may be advised to more specifically designate tumor differentiation and to avoid confusion with true thymomas as these represent entirely separate clinicopathologic entities.

Neural precursor cell expressed, developmentally downregulated 9 (NEDD9) supports oncogenic signaling in a number of solid and hematologic tumors. Little is known about the role of NEDD9 in ovarian carcinoma (OC), but available data suggest elevated mRNA and protein expression in advanced stage high-grade cancers. We used a transgenic MISIIR-TAg mouse OC model combined with genetic ablation of Nedd9 to investigate its action in the development and progression of OC. A Nedd9-/- genotype delayed tumor growth rate, reduced incidence of ascites, and reduced expression and activation of signaling proteins including SRC, STAT3, E-cadherin, and AURKA. Cell lines established from MISIIR-TAg;Nedd9-/- and MISIIR-TAg;Nedd9+/+ mice exhibited altered migration and invasion. Growth of these cells in a syngeneic allograft model indicated that systemic Nedd9 loss in the microenvironment had little impact on tumor allograft growth, but in a Nedd9 wild-type background Nedd9-/- allografts exhibited significantly reduced growth, dissemination, and oncogenic signaling compared to Nedd9+/+ allografts. Gene expression analysis revealed that Nedd9+/+ tumors exhibited more mesenchymal "stem-like" transcriptional program, including increased expression of Aldh1a1 and Aldh1a2. Conversely, loss of Nedd9 resulted in increased expression of differentiation genes, including fallopian tube markers Foxj1, Ovgp1, and Pax8. Collectively, these data suggest that tumor cell-intrinsic Nedd9 expression promotes OC development and progression by broad induction of oncogenic protein signaling and stem/mesenchymal gene expression.

Primary mediastinal seminomas are unusual tumors that can present in a pure form or as part of a mixed germ cell tumor. Contrary to testicular seminomas, little is known about the expression of novel immunohistochemical markers in mediastinal seminomas. This study investigates the immunohistochemical features of these tumors with a focus on novel markers. Thirty-two cases of primary mediastinal seminomas were reviewed; and representative whole-tissue sections were selected for immunohistochemical studies using antibodies directed against high molecular weight cytokeratin 5/6 (CK5/6), low molecular weight cytokeratin (CAM5.2), octamer-binding transcription factor 3/4 (OCT3/4), spalt-like transcription factor 4 (SALL4), GATA binding protein 3 (GATA-3), sry-related HMG box 2 (SOX2), SOX17, human T cell leukemia/lymphoma 1 (TCL1), glypican 3, melanoma associated antigen C2 (MAGEC2), and paired box gene 8 (Pax8). The percentage of positive tumor cells as well as the intensity of staining was evaluated and scored. Thirty-one cases (97%) expressed SOX17, whereas 29 cases (91%) were positive for OCT3/4 and SALL4, respectively. Twenty-eight cases (88%) expressed MAGEC2 and CAM5.2, respectively. Two cases (6%) were positive for Pax8, and a single case (3%) was positive for TCL1. None of the cases stained with CK5/6, GATA-3, SOX2, or glypican 3. Similar to testicular seminomas, mediastinal seminomas show consistent expression of OCT3/4, SALL4, SOX17, and MAGEC2 and are negative for SOX2, glypican 3, GATA-3, and CK5/6. Pax8 positivity is only inconsistently identified in mediastinal seminomas. Contrary to their testicular counterparts, mediastinal tumors show diffuse expression of low-molecular-weight cytokeratin in up to 90% of cases and are commonly negative for TCL1. Although there is some immunohistochemical overlap between testicular and mediastinal seminomas, considerable differences also exist and should be acknowledged when dealing with these tumors.

PAX8 is expressed in a high percentage of renal cell and ovarian cancers; however, the current existing anti-PAX8 rabbit polyclonal (P) antibodies also recognize B cells, pancreatic cancers, carcinoids, and some soft tissue tumors. Cross-reactivity with B cells can be especially troublesome in lymph nodes when identifying tumors of unknown origin. A new mouse monoclonal (M) anti-PAX8 antibody (Clone BC12) has been developed that recognizes PAX8 expression in a high percentage of renal cell and ovarian carcinomas, whereas exhibiting no staining of B cells. PAX8 (M) was tested for specificity and sensitivity in over 1300 cases of both normal and neoplastic tissues. PAX8 (M) demonstrated superior staining sensitivity in clear cell and papillary renal cell carcinomas (88.8% vs. 84.4%) and in serous and endometrioid ovarian carcinomas (87% vs. 83%), when compared with PAX8 (P). PAX8 (M) also stained a high percentage of endometrial and thyroid cancers, 67.5% and 60.7%, respectively. PAX8 (M) demonstrated low sensitivity in cervical and bladder cancers, 2.5% and 1.4%, respectively. All other cancers including lung, breast, prostate, stomach, liver, soft tissue, pancreas, testis, brain, colon, melanoma, lymphoma, adrenal, pituitary, and rectal were negative. In normal tissue, PAX8 (P) stained lymph nodes, pancreas, and neuroendocrine cells of stomach and colon. In contrast, PAX8 (M) was negative in each of these tissues. These results demonstrate that mouse monoclonal PAX8 [BC12] stains nuclei exclusively and performs well in formalin-fixed paraffin-embedded tissues. PAX8 (M) is a highly sensitive marker for thyroid, renal, and ovarian cancers. Importantly, PAX8 (M) does not stain B cells and does not seem to recognize epitopes of pancreatic origin and neuroendocrine cells in stomach and colon; thus, providing superior specificity and making PAX8 [BC12] an excellent marker for confirming primary tumor site and for differential diagnosis.