Molecular testing in anatomic pathology is going to become more and more important during the next decade as we develop assays that can aid in diagnosis, prognosis, and predicting response to therapy. The anatomic pathologist needs to be familiar with the different assays available but also needs to be able to discern which are going to become standard of care and which will not. Three different types of tumors are reviewed: thyroid cancer, oligodendroglioma, and lung carcinoma. Molecular assays that are currently in use or on the near horizon, including translocation analyses for RET-PTC and PPARgamma-PAX8, point mutation analysis for BRAF and epidermal growth factor receptor, and genetic loss for 1p and 19q, are discussed.

Clear cell adenocarcinoma (CCAC) of the urethra is a rare neoplasm, morphologically identical to its homologue arising in the female genital tract. The histogenesis of this neoplasm is uncertain. We present clinical, histopathologic, and immunohistochemical findings of four CCAC of the urethra and discuss the histogenesis and difficulties in diagnosis and differential diagnosis. CCAC of the urethra occurred in females (4/4). Two neoplasms were identified in urethral diverticulum; one of the two cases, in close proximity to a nephrogenic adenoma. CCAC exhibited tubulocystic, papillary, and diffuse/solid growth patterns. The neoplastic cells were cuboidal or columnar with eosinophilic or clear cytoplasm, and nuclear pleomorphism of at least moderate degree. Hobnail features and tumor necrosis were also observed. CCAC expressed p53 (4/4), AMACR (3/4), vimentin (3/4), PAX8 (2/4), CK7 (2/4), cytokeratin 34betaE12 (2/4), RCC (1/4), and CK20 (1/4) and were negative for PSA, WT1, ER, CA 125, uroplakin III, p16, and p63. The immunohistochemical profile supports a possible renal tubular cell differentiation/mesonephric origin for some urethral CCAC. Nephrogenic adenoma and metastatic clear cell carcinoma are the most important differential diagnostic considerations. Multicenter studies on more cases may improve our understanding of this malignancy.

OBJECTIVE

Neuroendocrine tumors, particularly small intestinal tumors, also grouped as 'carcinoids', are defined by an increasing incidence and prevalence, a poor response to current therapies, and confusion regarding appropriate models for drug development. Despite these issues, approximately 350 studies were published in the last year.

RESULTS

Two sources of confusion are clearly apparent. First, pharmacotherapeutic studies using pancreatic tumor cell lines as models for small intestinal or 'carcinoid' tumor biology are considered appropriate. Second, there is continued inclusion and analysis of pancreatic endocrine tumors with small intestinal neuroendocrine tumors in clinical studies. One highlight of this year is additional data confirming the significant differences between pancreatic tumor cell lines and small intestinal cell lines, the different gene expressions, for example, PAX8, between these two tumor types, and the observations that these two tumors respond differently in clinical trials, for example, to mammalian target of rapamycin (mTOR) inhibitors. Other highlights include delineating the role of the tumor microenvironment in the development of fibrosis and developing a minimum pathology dataset and a prognostic nomogram that may have utility in stratifying patients for clinical studies.

CONCLUSIONS

A number of interesting studies have been published during 2009-2010, but critical areas remain that require resolution. Current data, for the most part, reflect amplification of previously held concepts with modest advances in novel information.

A nonsmoker 45-year-old woman, presented with a solid right ovarian mass. Microscopic examination revealed heterogeneous histology with tubular formations and extensive signet ring cell component that resembled the usual appearance of metastatic gastric carcinoma to the ovary. Moreover, the histology also showed solid nests of cells with a microvacuolated basophilic cytoplasm similar to those found in adenosquamous cervical carcinoma of glassy cell type. However, analysis of the patient's past history revealed a lung adenocarcinoma, diagnosed 4 years before, which prompted an immunohistochemical differential diagnosis, showing a strong expression for TTF-1 and Napsin A. A cervical primary was excluded taking into account both macroscopic findings and the negative expression of PAX8 and absence of human papillomavirus-related marker p16. This confirmed the pulmonary origin of ovarian tumor despite its heterogeneous morphology. This is the first reported case of ovarian metastatic lung adenocarcinoma, with a signet ring cell component and solid nests, mimicking both metastatic gastric carcinoma and adenosquamous carcinoma of glassy cell type.

BACKGROUND

The distinction of metastatic ovarian carcinoma from other metastatic carcinomas and primary adnexal lesions in the skin is often difficult. PAX8 is a transcription factor that plays a critical role in development of the Müllerian system and has been shown to be a useful discriminatory marker between ovarian and breast carcinomas. Identification of ovarian cutaneous metastases may be of benefit in patients with familial breast-ovarian carcinoma syndrome.

METHODS

PAX8 immunohistochemical analysis was performed on 24 cases of metastatic adenocarcinomas to the skin and compared with 7 cases of primary adnexal lesions and also compared with p63 immunohistochemical staining results. Patients with metastatic adenocarcinomas had clinically documented primary malignancies, and patients with primary adnexal carcinomas had no known history of another adenocarcinoma.

RESULTS

Cutaneous ovarian and renal cell carcinoma metastases (2/2 and 8/8, respectively) showed positive nuclear expression of PAX8. PAX8 immunohistochemical staining in primary adnexal and other cutaneous metastases was completely negative (0/7 and 0/16, respectively). The p63 expression profile supported the distinction between adnexal and metastatic adenocarcinomas.

CONCLUSIONS

Although cutaneous ovarian metastasis is a rare phenomenon, the prognosis is extremely poor. PAX8 expression is a useful marker that effectively discriminated metastatic ovarian carcinomas from metastatic breast carcinomas and primary adnexal tumors.

BACKGROUND

The majority of differentiated thyroid cancers are characterised by one of several point mutations or gene rearrangements. Limited data are available on the prevalence and clinical correlations of these mutations in the Australian population.

OBJECTIVE

The aim of the present study was to characterise the mutation profile of differentiated thyroid tumours in the local population.

METHODS

The study involved 148 patients with differentiated thyroid cancer. The following tumours were examined: 109 papillary carcinomas (PTC), 27 follicular carcinomas (FC) and 12 Hurthle cell carcinomas (HCC). Polymerase chain reaction (PCR) was performed for BRAF and RAS mutations (RNA and DNA) as well as for RET/PTC rearrangements and PAX8-PPARγ translocations (RNA). Clinicopathological parameters and outcome data were analysed according to BRAFV600E status in PTC and RAS mutation status in FC.

RESULTS

BRAFV600E was identified in 74/109 (68%) PTC. BRAFV600E was not significantly correlated with clinicopathological features of aggressive disease. At a median follow up of 48 months, there was no significant difference between BRAFV600E and wild-type BRAF PTC with respect to the rates of nodal recurrence, distant metastases or disease-specific death. In FC, RAS mutations (five NRAS and three HRAS) were present in 8/27 (30%) tumours. RAS mutation was significantly associated with widely invasive histology (P = 0.01) and distant metastases (P = 0.01) on follow up.

CONCLUSIONS

In the present study, BRAF mutation was not associated with negative prognostic indicators or adverse outcomes in PTC. RAS mutation was positively correlated with aggressive features in FC suggesting potential prognostic utility, although confirmation is required from larger studies.

OBJECTIVE

Exposure to ionizing radiation at young age is the strongest risk factor for the occurrence of papillary thyroid carcinoma (PTC). RET/PTC rearrangements are the most frequent genetic alterations associated with radiation-induced PTC, whereas BRAF and RAS mutations and PAX8-PPARG rearrangement have been associated with sporadic PTC. We decided to search for such genetic alterations in PTCs of patients subjected in childhood to scalp irradiation.

METHODS

We studied 67 thyroid tumors from 49 individuals irradiated in childhood for tinea capitis scalp epilation: 36 malignant (12 cases of conventional PTC (cPTC), two cPTC metastases, 20 cases of follicular variant PTC (FVPTC), one oncocytic variant of PTC and one follicular carcinoma) and 31 follicular thyroid adenomas.

METHODS

The lesions were screened for the BRAF(V600E) and NRAS mutations and for RET/PTC and PAX8-PPARG rearrangements.

RESULTS

BRAF(V600E) mutation was detected in seven of 14 (50%) cPTC and two of 20 FVPTC (10%) (P=0.019). NRAS mutation was present in one case of FVPTC (5%). RET/PTC1 rearrangement was found, by RT-PCR, in one of 17 cases (5.9%) and by fluorescence in situ hybridization in two of six cases (33%). PAX8-PPARG rearrangement was not detected in any carcinoma. None of the follicular adenomas presented any of the aforementioned genetic alterations.

CONCLUSIONS

The prevalence of BRAF(V600E) mutation in our series is the highest reported in series of PTCs arising in radiation-exposed individuals. The prevalence of RET/PTC1 rearrangement fits with the values recently described in a similar setting.

OBJECTIVE

The role of thyroid-specific transcription factors in thyroid malignancy is still poorly understood, so we investigate thyroid-specific transcription factors gene expression both in benign and in malignant thyroid nodules, aiming to study a possible clinical utility of these molecules.

METHODS

We quantified TTF-1, FOXE1 and PAX8 mRNA levels, relating their expression to diagnostic and prognostic features of thyroid tumors. RNA was extracted from 4 normal thyroid tissues, 101 malignant [99 papillary thyroid carcinomas (PTC) and 2 anaplastic thyroid carcinomas] and 99 benign thyroid lesion tissues [49 goiter and 50 follicular adenomas (FA)].

RESULTS

Levels of mRNA of both FOXE1 (P < 0.0001) and PAX8 (P < 0.0001) genes, but not TTF-1 (P = 0.7056), were higher in benign than in malignant thyroid lesions. FOXE1 was able to identify malignant nodules with 75.8 % sensitivity, 76.1 % specificity, 75.8 % positive predictive value, 76.1 % negative predictive value and 75.9 % accuracy. PAX8 was able to identify malignancy with 60.6 % sensitivity, 81.1 % specificity, 76.9 % positive predictive value, 66.4 % negative predictive value and 70.6 % accuracy. Both FOXE1 and PAX8 gene expression patterns were also able to differentiate FA from the follicular variant of PTC-FVPTC. However, the investigated gene expression was neither associated with any clinical feature of tumor aggressiveness nor associated with recurrence or survival.

CONCLUSIONS

We suggest that FOXE1 and PAX8 gene expression patterns may help to diagnose thyroid nodules, identifying malignancy and characterizing follicular-patterned thyroid lesions, but are not determinants of thyroid tumor progression.

A bilateral small cell ovarian carcinoma pulmonary-type (SCCOPT), arising in bilateral mature cystic teratomas (MCTs) presented as stage IIIB in a 37-year-old woman. Microscopically, tumor nests were related to the dermoid protuberance and expressed pancytokeratin, EMA, CD56, chromogranin A, NSE, synaptophysin, and SOX2. SALL4 was also focally positive. CDX2, TTF1, PAX8, CK7, CK20, and several neuroendocrine gut hormones were negative. Serum NSE was elevated. This case represents a SCCOPT arising in an MCT in the right ovary with metastasis to the left one also containing a synchronous MCT. Surface implants and lymphovascular invasion suggested metastasis from the right ovarian SCCOPT and excluded a metastatic origin from usual locations of small cell carcinoma (SCC). SCCOPT is morphologically identical to SCC elsewhere, even sharing NSE serum elevation. Although the tumor was closely related to teratomatous mature tissues, a complex immunohistochemical panel failed to provide a tissue of origin.

Serous ovarian carcinoma is now hypothesized to originate from fallopian tube epithelium (FTE). We investigated the FTE abnormalities in the patients with epithelial ovarian tumors. Our study included 55 cases of serous tumors (24 carcinomas, 8 borderline tumors, and 23 adenomas), 14 mucinous carcinomas, 22 endometrioid carcinomas, 5 clear cell carcinomas, and 2 malignant Brenner tumors. FTE was diagnosed by the diagnostic algorithm, which combines the data of morphology, and p53, Ki-67 immunostaining, as serous tubal intraepithelial carcinoma, serous tubal intraepithelial lesion, p53 signature, and normal/reactive. Serous tubal intraepithelial carcinoma, serous tubal intraepithelial lesion, p53 signature, and normal/reactive were observed in 5, 3, 0, and 16 cases in serous carcinoma; 0, 3, 0, and 5 cases in serous borderline tumor; 0, 1, 1, and 21 cases in serous adenoma; 0, 0, 1, and 13 cases in mucinous carcinoma; 0, 0, 3, and 19 cases in endometrioid carcinoma; 0, 0, 0, and 5 cases in clear cell carcinoma; and 0, 1, 0, and 1 case in malignant Brenner tumor. Among tumors of serous histology and between carcinomas, FTE abnormalities differed significantly (P<0.05). Serous tubal intraepithelial carcinomas were only found in serous carcinoma. The incidence of secretory cell proliferation (SCP) was examined by PAX8 expression. The rate of SCP was extremely high in serous carcinoma (96%). Among tumors of serous histology and between carcinomas, an incidence of SCP differed significantly (P<0.05). Patients with SCP were significantly older (P<0.0001). Our observations were concordant with the hypothesis of serous ovarian carcinogenesis. The SCP has a meaningful association with serous ovarian cancer.

GOLPH2 is a highly conserved protein. It is upregulated in a number of tumors and is being considered as an emerging biomarker for related diseases. However, the function of GOLPH2 remains unknown. The Xenopus model is used to study the function of human proteins. We describe the isolation and characterization of Xenopus golph2, which dimerizes and localizes to the Golgi in a manner similar to human GOLPH2. Xenopus golph2 is expressed in the pronephros during early development. The morpholino-mediated knockdown of golph2 results in edema formation. Additionally, Nephrin expression is enhanced in the glomus, and the expression of pronephric marker genes, such as atp1b1, ClC-K, NKCC2, and NBC1, is diminished in the tubules and duct. Expression patterns of the transcription factors WT1, Pax2, Pax8, Lim1, GATA3, and HNF1β are also examined in the golph2 knockdown embryos, the expression of WT1 is increased in the glomus and expanded laterally in the pronephric region. We conclude that the deletion of golph2 causes an increase in the expression of WT1, which may promote glomus formation and inhibit pronephric tubule differentiation.

CONCLUSIONS

Jagged1-mediated Notch signaling regulates hair cell (HC) production in a distinct way rather than lateral inhibition mediated by Hes1 and Hes5. Jagged1 may interact with Notch3, probably via candidate downstream mediators Hesr1 and Hesr2, regulating the prosensory formation in the early stage.

OBJECTIVE

To explore the function of the Jagged1-mediated Notch signaling pathway in mammalian inner ear development and its possible mechanism.

METHODS

Using conditional gene targeting, a novel Jagged1 conditional knockout (Jag1-cko), Pax8(cre/+); Jag1(flox/flox), was established. The auditory brainstem response and swim ability test were utilized to identify functional disability. The expression of Jagged1, Notch3, Hes1, Hesr1, and Hesr2 was detected by immunofluorescence and immunohistochemistry.

RESULTS

Our Jag1-cko model was established and survived well. It presented hearing impairment and balance disturbance with 'waltzing' behavior. Cochleae and vestibular apparatus were all found in our Jag1-cko model. Patch deficiency of outer hair cells (OHCs) was found on the apical and middle turns of the auditory epithelium. OHCs were totally missing on the basal turn. The stereociliary bundles were disorientated on the cristae. Unlike Hes1, no expression of Notch3, Hesr1, and Hesr2 was found on embryonic day 13.5 of the Jag1-cko model.

Today there is a better understanding of the events involved in the initiation and progression of thyroid cancer. It is indeed now known that BRAF and RAS mutations and RET/PTC and PAX8/PPARγ rearrangements account for the majority of molecular alterations detected in differentiated thyroid cancers. Abnormal regulation of microRNAs (miRNAs) is also a promising way of research. The diagnostic utility and prognostic value of detecting these molecular events has been analyzed in several recent studies. BRAF mutation analysis improves the performance of fine-needle aspiration diagnosis by increasing specificity in "indeterminate" cytologies and sensitivity in false negatives. Testing for a "panel of mutations" (BRAF, RAS, RET/PTC and PAX8/PPARγ) improves the performance, detecting papillary carcinomas with non-classic histology. The specificity of these analyzes is excellent but their sensitivity is still insufficient. In the future, specific miRNAs expression profiles in thyroid carcinoma and identification of new mutations might provide interesting information. Several studies have found that BRAF mutations are associated with a more aggressive tumor behavior, a higher risk of recurrence and treatment failure. With regard to the other mutations and rearrangements, current data are conflicting and it seems premature to draw practical conclusions applicable in routine practice. Lastly, targeted therapy with tyrosine kinase inhibitors, based on our understanding of the molecular mechanisms of thyroid oncogenesis, has shown promise in metastatic, progressive, and radioactive iodine-refractory differentiated thyroid carcinomas.

Cystic lesions can be occasionally be found in the mediastinum, and typically include bronchogenic cysts, esophageal duplication cysts, and neuroenteric cysts. In 2005, Hattori described the first mediastinal cyst with Mullerian differentiation. Since that time, three other authors have described similar cysts occurring in the posterior mediastinum. Here we present two cases of patients with ciliated cysts with Mullerian differentiation with expression of estrogen receptor, progesterone receptor, PAX8 and Wilm's tumor 1, occurring in the posterior mediastinum and review the related literature.

Pax proteins are transcriptional regulators that play important roles during embryogenesis. These proteins recognize specific DNA sequences via a conserved element: the paired domain (Prd domain). The low level of organized secondary structure, in the free state, is a general feature of Prd domains; however, these proteins undergo a dramatic gain in alpha-helical content upon interaction with DNA ('induced fit'). Pax8 is expressed in the developing thyroid, kidney and several areas of the central nervous system. In humans, mutations of the Pax8 gene, which are mapped to the coding region of the Prd domain, give rise to congenital hypothyroidism. Here, we have investigated the molecular defects caused by a mutation in which leucine at position 62 is substituted for an arginine. Leu62 is conserved among Prd domains, and contributes towards the packing together of helices 1 and 3. The binding affinity of the Leu62Arg mutant for a specific DNA sequence (the C sequence of thyroglobulin promoter) is decreased 60-fold with respect to the wild-type Pax8 Prd domain. However, the affinities with which the wild-type and the mutant proteins bind to a non-specific DNA sequence are very similar. CD spectra demonstrate that, in the absence of DNA, both wild-type Pax8 and the Leu62Arg mutant possess a low alpha-helical content; however, in the Leu62Arg mutant, the gain in alpha-helical content upon interaction with DNA is greatly reduced with respect to the wild-type protein. Thus the molecular defect of the Leu62Arg mutant causes a reduced capability for induced fit upon DNA interaction.

BACKGROUND

Non-small cell lung cancers (NSCLC) are highly heterogeneous at the molecular level and comprise 75% of all lung tumors. We have previously shown that the receptor tyrosine kinase (RTK) MET frequently suffers gain-of-function mutations that significantly promote lung tumorigenesis. Subsequent studies from our lab also revealed that PAX5 transcription factor is preferentially expressed in small cell lung cancer (SCLC) and promotes MET transcription. PAX8, however, is also expressed in NSCLC cell lines. We therefore investigated the role of PAX8 in NSCLC.

METHODS

Using IHC analysis, PAX8 protein expression was determined in archival NSCLC tumor tissues (n = 254). In order to study the effects of PAX8 knockdown on NSCLC cellular functions such as apoptosis and motility, siRNA against PAX8 was used. Confocal fluorescence microscopy was used to monitor the localization of MET, RON and PAX8. The combinatorial effect of PAX8 knockdown and MET inhibition using SU11274 was investigated in NSCLC cell viability assay.

RESULTS

Relative levels of PAX8 protein were elevated (≥ + 2 on a scale of 0-3) in adenocarcinoma (58/94), large cell carcinoma (50/85), squamous cell carcinoma (28/47), and metastatic NSCLC (17/28; lymph node). Utilizing early progenitors isolated from NSCLC cell lines and fresh tumor tissues, we observed robust overexpression of PAX8, MET, and RON. PAX8 knockdown A549 cells revealed abrogated PAX8 expression with a concomitant loss in MET and the related RON kinase expression. A dramatic colocalization between the active form of MET (also RON) and PAX8 upon challenging A549 cells with HGF was visualized. A similar colocalization of MET and EGL5 (PAX8 ortholog) proteins was found in embryos of C. elegans. Most importantly, knockdown of PAX8 in A549 cells resulted in enhanced apoptosis (~6 fold) and decreased cell motility (~45%), thereby making PAX8 a potential therapeutic target. However, the combinatorial approach of PAX8 knockdown and treatment with MET inhibitor, SU11274, had marginal additive effect on loss of NSCLC cell viability.

CONCLUSIONS

PAX8 provides signals for growth and motility of NSCLC cells and is necessary for MET and RON expression. Further investigations are necessary to investigate the therapeutic potential of PA8 in NSCLC.

BACKGROUND

Thyroid dysfunction is common in newborn infants with Down's syndrome (DS), but defects causing classic thyroid dysgenesis (TD) with permanent congenital hypothyroidism (CH) have not been described.

OBJECTIVE

We studied a girl with DS and CH who had a mutation in the promoter sequence of the PAX8 gene.

RESULTS

A female infant was found to have trisomy 21 and CH, with a venous thyrotropin (TSH) of >150 mU/L and a free thyroxine (fT4) of 15.1 pmol/L (day 12). Thyroid peroxidase antibodies and thyroglobulin antibodies were elevated. Scintigraphy showed normal uptake, but ultrasound identified a small gland with heterogenous echotexture and cystic changes. Sequence analysis of the PAX8 gene revealed a new heterozygous maternally inherited mutation (-3C>T) close to the transcription initiation site. Electromobility shift assay studies of the wild type and the mutant PAX8 sequence incubated with nuclear extracts from PCCL3 cells exhibited that the sequence at position -3 is not involved in specific protein binding. However, the mutant PAX8 promoter showed a significantly reduced transcriptional activation of a luciferase reporter gene in vitro tested in HEK, PCCL3, as well as in HeLa cells, indicating that this mutation is very likely to lead to reduced PAX8 expression.

CONCLUSIONS

The persistent CH in this patient with DS is likely to be attributable to the diminished PAX8 expression due to a new heterozygous mutation in the PAX8 promoter sequence. Our case shows that true CH may occur in DS, as in the general population. Furthermore, it is possible that the trisomy 21 itself may have resulted in a more severe phenotypic expression of the PAX8 mutation in the child than the mother.

We reported previously that overexpression of heterogeneous nuclear ribonucleoprotein F (Hnrnpf) in renal proximal tubular cells (RPTCs) suppresses angiotensinogen (Agt) expression, and attenuates systemic hypertension and renal injury in diabetic Hnrnpf-transgenic (Tg) mice. We thus hypothesized that deletion of Hnrnpf in the renal proximal tubules (RPT) of mice would worsen systemic hypertension and kidney injury, perhaps revealing novel mechanism(s). Tubule-specific Hnrnpf knockout (KO) mice were generated by crossbreeding Pax8-Cre mice with floxed Hnrnpf mice on a C57BL/6 background. Both male and female KO mice exhibited elevated systolic blood pressure, increased urinary albumin/creatinine ratio, tubulo-interstitial fibrosis and glycosuria without changes in blood glucose or glomerular filtration rate compared with control littermates. However, glycosuria disappeared in male KO mice at the age of 12 weeks, while female KO mice had persistent glycosuria. Agt expression was elevated, whereas sodium-glucose co-transporter 2 (Sglt2) expression was down-regulated in RPTs of both male and female KO mice as compared to control littermates. In vitro, KO of HNRNPF in human RPTCs (HK-2) by CRISPR gRNA up-regulated AGT and down-regulated SGLT2 expression. The Sglt2 inhibitor canagliflozin treatment had no effect on Agt and Sglt2 expression in HK-2 and in RPTCs of wild-type mice but induced glycosuria. Our results demonstrate that Hnrnpf plays a role in the development of hypertension and glycosuria through modulation of renal Agt and Sglt2 expression in mice, respectively.

BACKGROUND

Here, we demonstrate the successful differentiation of induced pluripotent stem (iPS) cells into functional thyroid cells indicating the therapeutic potential of this approach when applied to individuals with thyroid deficiency.

METHODS

Using embryonic murine fibroblasts, we generated iPS cells with a single lentiviral "stem cell cassette" vector and then differentiated these iPS cells into thyroid cells after transfection with PAX8 and NKX2-1 by Activin A and TSH stimulation.

RESULTS

The generated iPS cells expressed pluripotent stem cell markers as assessed using both reverse transcription quantitative PCRs and immunofluorescence staining with ~0.5% reprograming efficiency. Compared to control cells, the expression of thyroid-specific genes NIS, TSHR, Tg, and TPO were greatly enhanced in PAX8(+)NKX2-1(+) iPS cells after differentiation. On stimulation with TSH, these differentiated iPS cells were also capable of dose-dependent cAMP generation and radioiodine uptake indicative of functional thyroid epithelial cells. Furthermore, the cells formed three-dimensional follicles in culture, and "thyroid organoids" formed after PAX8(+)NKX2-1(+) iPS cells transplanted into nude mice, and all expressed Tg protein as judged immunohistochemically. Taken together, thyroid epithelial cells differentiated from iPS cells, which were themselves derived from murine fibroblasts, exhibited very similar properties to thyroid cells previously developed from traditional murine embryonic stem cells.

CONCLUSIONS

Thyroid cells differentiated from iPS cells offer the opportunity to examine the detailed transcriptional regulation of thyroid cell differentiation and may provide a useful future source for individualized regenerative cell therapy.

Purpose: Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder; however, its molecular etiology remains poorly understood.

Methods: We performed genetic analysis of 24 causative genes using next-generation sequencing in 167 CH cases, comprising 57 dyshormonogenesis (DH), 32 dysgenesis (TD) and 78 undiagnosed. The pathogenicity of variants was assessed by the American College of Medical Genetics guidelines, inheritance pattern, and published evidence. Furthermore, we compared the oligogenic groups and monogenic groups to examine the correlation between variant dosage and severity.

Results: We identified variants in 66.5% cases (111/167) and 15 genes, DUOX2, TSHR, PAX8, TG, TPO, DUOXA2, JAG1, GLIS3, DUOX1, IYD, SLC26A4, SLC5A5, SECISBP2, DIO1, and DIO3. Biallelic variants were identified in 12.6% (21/167), oligogenic in 18.0% (30/167), and monogenic in 35.9% (60/167); however, 68.5% of variants were classified as variant of unknown significance (VUS). Further examinations showed that 3 out of 32 cases with TD (9.4%) had pathogenic variants (2 of TSHR and 1 of TPO), and 8 out of 57 cases with DH (14.0%) (7 of DUOX2, 1 of TG) had pathogenic variants. In addition, TSH levels at the first visit were significantly higher in the oligogenic group than in the monogenic group.

Conclusions: The detection rate of pathogenic variants in Japanese CH was similar to that previously reported. Moreover, oligogenic cases were likely to be more severe than monogenic cases, suggesting that CH may exhibit a gene dosage effect. Further analysis of VUS pathogenicity is required to clarify the molecular basis of CH.

Keywords: Congenital hypothyroidism; genes; newborn screening; oligogenic inheritance; targeted next-generation sequencing.

Immunohistochemistry is frequently used to identify ovarian mucinous neoplasms as primary or metastatic; however, there is significant overlap in expression patterns. We compared traditional markers (CK7, CK20, CDX2, PAX8, estrogen receptor, β-catenin, MUC1, MUC2, and MUC5AC) to 2 novel proteins identified through mining of the Human Protein Atlas expression database: SATB2 and POF1B. The study cohort included 49 primary gastrointestinal (GI) mucinous adenocarcinomas (19 colorectal, 15 gastric, 15 pancreatobiliary), 60 primary ovarian mucinous neoplasms (19 cystadenomas, 21 borderline tumors, 20 adenocarcinomas), and 19 metastatic carcinomas to the ovary (14 lower and 5 upper GI primaries). Immunohistochemistry was performed on tissue microarrays, scored and interpreted as negative (absent or focal/weak) or positive. Metastatic tumors were frequently unilateral (42.8% of tumors from lower and 40% of tumors from upper tract) and ≥10 cm (85.7% of tumors from lower and 80% of tumors from upper tract). CK7 was positive in 88.5% upper GI and 88.3% primary ovarian compared with 24.3% lower GI neoplasms. CK20 and CDX2 were positive in 84.8% and 100% of lower GI tumors, respectively; however, expression was also common in upper GI (CK20 42.8%, CDX2 50%) and primary ovarian neoplasms (CK20 65.7%, CDX2 38.3%). Conversely, SATB2 was more specific for lower GI origin, being positive in 78.8% lower GI but only 11.5% upper GI and 1.7% primary ovarian neoplasms. PAX8 expression was common in primary ovarian neoplasms (75% of all neoplasms, 65% of carcinomas); only 1 (1.5%) GI tumor was positive. MUC2 and β-catenin were frequently positive in lower GI tumors (96.9% and 51.5%, respectively). Estrogen receptor expression was only seen in primary ovarian neoplasms (13.3%). Nuclear premature ovarian failure 1B (POF1B) expression was seen in malignant tumors regardless of their origin. A panel including CK7, SATB2, and PAX8 separated primary from secondary GI neoplasms with up to 77.1% sensitivity and 99% specificity, outperforming tumor laterality and size. Second-line markers such as CDX2, MUC2, estrogen receptor, MUC1, and β-catenin increased the sensitivity of immunohistochemistry in excluding lower GI origin. Biomarker search using proteomic databases has a value in diagnostic pathology, as shown with SATB2; however, as seen with POF1B, expression profiles in these databases are not always reproduced in larger cohorts.

OBJECTIVE

The application of molecular tests to thyroid fine needle aspiration (FNA) has been shown to be a valuable tool to better refine the pre-operative malignant risk of patients with indeterminate cytology results. In this study, we investigated the feasibility of using the laser capture microdissection (LCM) technique to obtain DNA and RNA for molecular tests in routine thyroid FNA smears.

METHODS

Nine coupled FNA and histological retrospective cases and 31 prospective FNA cases with a follicular neoplasm/suspicious for a follicular neoplasm (FN/SFN) diagnosis were included in this study. Both cytological and histological specimens were investigated by direct sequencing and reverse transcription-polymerase chain reaction (RT-PCR) for BRAF and RAS mutations and for PAX8/PPARG and RET/PTC rearrangements, respectively.

RESULTS

LCM yielded good DNA and RNA quality in all cases (100%) in both series, irrespective of the staining used (Giemsa, Papanicolaou, immunostain for thyroglobulin) and the cytology technique (conventional or liquid-based preparations). Total mutations found in the FNA and in the corresponding histological specimen in both series were: one PAX8/PPARG rearrangement in a follicular carcinoma (FC), four NRAS mutations [in two FCs, one papillary carcinoma and one follicular adenoma (FA)] and one HRAS mutation in one FA. The sensitivity was 67% and the specificity was 91%.

CONCLUSIONS

LCM is a valuable tool to obtain good quality DNA and RNA for molecular tests in cytological material from thyroid FNA, and can be a useful option in the management of patients with an FN/SFN FNA diagnosis.

Collecting duct carcinoma (CDC) and related tumors [ie, renal medullary carcinoma (RMC)] are rare types of highly aggressive renal cell carcinomas (RCC) with poor prognosis. Because of the rarity and diagnostic uncertainty of them, their molecular pathology and significance have not yet been fully elucidated. CDC, RMC, fumarate hydratase-deficient RCC (including hereditary leiomyomatosis and RCC-associated RCC HLRCC-RCC), and recently reported anaplastic lymphoma kinase (ALK)-rearrangement RCC have significant morphologic overlaps, but they are separately distinct entities having different molecular pathway and clinical settings. CDC is more likely to occur in middle to old age population with immunoreactivity for PAX8 and integrase interactor-1 proteins (INI-1). Various chromosomal and genomic alterations have been reported with inconsistent results. In contrast, RMC is more likely to occur in younger patients with sickle cell trait. In RMC, loss of INI-1 expression and OCT3/4 expression are distinguished compared with other RCCs. Finally, ALK-rearrangement RCC seems to have 2 different clinical settings, one with sickle cell trait (VCL-ALK fusion) and the other without (other fusions such as TPM3-ALK, EML4-ALK, and STRN-ALK fusions). Interestingly, VCL-ALK fusion was found in pediatric patients with sickle cell trait, whereas other fusions were detected in adolescent or adult without sickle cell trait. Taken together, CDC and related tumors such as RMC, fumarate hydratase-deficient RCC (including hereditary leiomyomatosis and RCC-associated RCC), and ALK-rearrangement RCC are the distinct entities and their recognition is important for the development of future personalized therapeutic options. This review updates the clinicopathologic features of these tumors with overlapping morphology and outcome.

This study aimed to identify an immunohistochemical panel to aid in the differential diagnosis for tumors with clear cell morphology. Twenty-five clear cell renal cell carcinomas (CCRCCs), 19 clear cell ovarian carcinoma (CCOCs), 20 cases of adrenal cortical carcinomas(ACCs), and 10 chordomas were stained for renal cell carcinoma marker (RCC Ma), Pax8, brachyury, and steroidogenic factor 1 (SF-1). The extent of stains was scored as focal (<25%), nonfocal (25%-50%), and diffuse (>50%). The intensity was scored as weak, moderate, and strong. Twenty-two CCRCCs were positive for RCC Ma (88%) and Pax8 (88%), respectively. The RCC Ma cytoplasmic staining was largely diffuse (76%) and strong (76%). The nuclear Pax8 staining was usually diffuse (76%) and moderate (64%) to strong (8%). All of CCRCCs were negative for brachyury and SF-1. All of 19 CCOCs were positive for Pax8 nuclear staining. The staining was diffuse, moderate (21%) to strong (79%). All of CCOCs were negative for RCC Ma, brachyury, and SF-1. All of 20 ACCs were positive for SF-1 nuclear staining. The staining was largely diffuse (95%), moderate (55%) to strong (15%). All of ACC were negative for RCC Ma, Pax8, and brachyury. All of 10 chordomas were positive for brachyury nuclear staining. The staining was diffuse and strong. All of chordomas were negative for RCC Ma, Pax8, and SF-1. In summary, the panel of RCC Ma, Pax8, brachyury, and SF-1 is useful in the differential diagnosis of tumors with clear morphology.