Current and Future Role of Tyrosine Kinases Inhibition in Thyroid Cancer: From Biology to Therapy.

Thyroid cancer represents a heterogenous disease whose incidence has increased in the last decades. Although three main different subtypes have been described, molecular characterization is progressively being included in the diagnostic and therapeutic algorithm of these patients. In fact, thyroid cancer is a landmark in the oncological approach to solid tumors as it harbors key genetic alterations driving tumor progression that have been demonstrated to be potential actionable targets. Within this promising and rapid changing scenario, current efforts are directed to improve tumor characterization for an accurate guidance in the therapeutic management. In this sense, it is strongly recommended to perform tissue genotyping to patients that are going to be considered for systemic therapy in order to select the adequate treatment, according to recent clinical trials data. Overall, the aim of this article is to provide a comprehensive review on the molecular biology of thyroid cancer focusing on the key role of tyrosine kinases. Additionally, from a clinical point of view, we provide a thorough perspective, current and future, in the treatment landscape of this tumor.

Cystathione ß-Synthase Is Increased in Thyroid Malignancies.

BACKGROUND: Cystathione ß-synthase (CBS) catalyzes the conversion of homocysteine and cysteine to hydrogen sulfide (H2S) and cystathione, via the trans-sulfuration pathway. CBS protein expression levels are increased in several different human malignancies, with increased protein expression correlating with parameters such as tumor stage, anaplasia, metastases, and chemotherapy resistance. MATERIALS AND METHODS: This study employed tissue microarrays to examine CBS expression in benign thyroid tissue, thyroid oncocytomas, thyroid follicular adenomas, and in follicular, papillary, anaplastic, and medullary thyroid carcinomas. RESULTS: CBS expression was increased in all thyroid carcinomas types compared to benign thyroid tissue, but not in thyroid follicular adenomas or oncocytomas. A similar pattern was observed for nicotinamide phosphoribosyltransferase (NAMPT) tissue microarray analysis comparing thyroid adenomas and follicular carcinomas. CONCLUSION: For the first time, we showed that an H2S-syntheszing enzyme plays a role in thyroid malignancies. Additionally, our data suggest that CBS and NAMPT immunohistochemistry may be useful in differentiating follicular adenomas from follicular carcinomas.

Enhancement of mitochondrial biogenesis and paradoxical inhibition of lactate dehydrogenase mediated by 14-3-3η in oncocytomas.

Oncocytomas represent a subset of benign pituitary adenomas that are characterized by significant mitochondrial hyperplasia. Mitochondria are key organelles for energy generation and metabolic intermediate production for biosynthesis in tumour cells, so understanding the mechanism underlying mitochondrial biogenesis and its impact on cellular metabolism in oncocytoma is vital. Here, we studied surgically resected pituitary oncocytomas by using multi-omic analyses. Whole-exome sequencing did not reveal any nuclear mutations, but identified several somatic mutations of mitochondrial DNA, and dysfunctional respiratory complex I. Metabolomic analysis suggested that oxidative phosphorylation was reduced within individual mitochondria, and that there was no reciprocal increase in glycolytic activity. Interestingly, we found a reduction in the cellular lactate level and reduced expression of lactate dehydrogenase A (LDHA), which contributed to mitochondrial biogenesis in an in vitro cell model. It is of note that the hypoxia-response signalling pathway was not upregulated in pituitary oncocytomas, thereby failing to enhance glycolysis. Proteomic analysis showed that 14-3-3η was exclusively overexpressed in oncocytomas, and that 14-3-3η was capable of inhibiting glycolysis, leading to mitochondrial biogenesis in the presence of rotenone. In particular, 14-3-3η inhibited LDHA by direct interaction in the setting of complex I dysfunction, highlighting the role of 14-3-3η overexpression and inefficient oxidative phosphorylation in oncocytoma mitochondrial biogenesis. These findings deepen our understanding of the metabolic changes that occur within oncocytomas, and shine a light on the mechanism of mitochondrial biogenesis, providing a novel perspective on metabolic adaptation in tumour cells. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Virilizing oncocytic adrenocortical carcinoma: clinical and immunohistochemical studies.

CONTEXT: Oncocytic tumors of the adrenal cortex are rare, mostly nonfunctioning and benign. SETTING: Report virilizing oncocytic adrenocortical carcinoma in a 50-year-old woman. PATIENT: She presented a recent and progressive virilization syndrome, associated with high blood pressure. Hormonal evaluation showed elevated serum testosterone and delta-4-androstenedione levels, normal urinary free cortisol level and incomplete suppression of cortisol at the 1 mg dexamethasone suppression test. CT scan of the abdomen revealed a 35 mm left adrenal mass. INTERVENTION: The patient underwent a left adrenalectomy, and the histological study showed a 3 cm oncocytic adrenocortical carcinoma with signs of malignancy. RESULTS: Immunohistochemical study revealed that tumor cells expressed the steroidogenic enzymes involved into androgen synthesis (3ßHSD and P450c17α), P450 aromatase and luteinizing hormone (LH) receptors. Post-operatively, signs of virilization improved rapidly, serum testosterone and delta-4-androstenedione levels returned to normal, as did the dexamethasone suppression test. During follow-up CT-scan and 18-FDG PET/CT showed a right ovary mass, corresponding to a follicular cyst associated with hyperthecosis. The patient is alive with no recurrence 48 months after adrenal surgery. CONCLUSION: Oncocytic adrenocortical carcinomas, although extremely rare, should be considered in women with a virilization syndrome. In this woman immunohistochimical studies revealed the presence of steroidogenic enzymes involved into androgen synthesis and aromatization, and LH receptors could be implicated in this pathology.

Safety and tolerability of sorafenib in patients with radioiodine-refractory thyroid cancer.

Effective adverse event (AE) management is critical to maintaining patients on anticancer therapies. The DECISION trial was a multicenter, randomized, double-blind, placebo-controlled, Phase 3 trial which investigated sorafenib for treatment of progressive, advanced, or metastatic radioactive iodine-refractory, differentiated thyroid carcinoma. Four hundred and seventeen adult patients were randomized (1:1) to receive oral sorafenib (400 mg, twice daily) or placebo, until progression, unacceptable toxicity, noncompliance, or withdrawal. Progression-free survival, the primary endpoint of DECISION, was reported previously. To elucidate the patterns and management of AEs in sorafenib-treated patients in the DECISION trial, this report describes detailed, by-treatment-cycle analyses of the incidence, prevalence, and severity of hand-foot skin reaction (HFSR), rash/desquamation, hypertension, diarrhea, fatigue, weight loss, increased serum thyroid stimulating hormone, and hypocalcemia, as well as the interventions used to manage these AEs. By-cycle incidence of the above-selected AEs with sorafenib was generally highest in cycle 1 or 2 then decreased. AE prevalence generally increased over cycles 2-6 then stabilized or declined. Among these AEs, only weight loss tended to increase in severity (from grade 1 to 2) over time; severity of HFSR and rash/desquamation declined over time. AEs were mostly grade 1 or 2, and were generally managed with dose interruptions/reductions, and concomitant medications (e.g. antidiarrheals, antihypertensives, dermatologic preparations). Most dose interruptions/reductions occurred in early cycles. In conclusion, AEs with sorafenib in DECISION were typically grade 1 or 2, occurred early during the treatment course, and were manageable over time.

Occult oncocytic papillary thyroid carcinoma with lymphoid stroma (Warthin-like tumor): report of a case with concomitant mutations of BRAF V600E and V600K.

Warthin-Like tumor of the thyroid is a recently described rare variant of papillary thyroid cancer. The distinct histological feature of this variant is papillary architecture lining oncocytic epithelial cells with nuclear characteristics of papillary carcinoma, accompanied by prominent lymphocytic infiltration in the papillary stalks. Here, we present a case of occult Warthin-like papillary thyroid carcinoma, 0.5-cm in maximum dimension, underwent left thyroid lobectomy in a 65 years old Chinese woman. In this case, there was no extrathyroid extension, vascular invasion and lymphatic metastasis, as well as no complication of lymphocytic thyroiditis. Immunohistochemistry staining revealed that the tumor cells were positive for Leu-M1, HBME-1, 34ßE12, and MIB-1 labeling index was low. RET/PTC expression was absent in tumor cells. Furthermore, activated point mutations of BRAF V600E and V600K were concurrently detected by DNA sequencing. Further studies are needed to elucidate the prevalence and role of BRAF(V600K) mutation in papillary thyroid carcinoma, and long-term follow-up for the patient is needed to clarify the biological behavior of this variant with dual BRAF mutations.

Immunoexpression of napsin A in renal neoplasms.

BACKGROUND: Immunohistochemistry (IHC) for napsin A has been widely used to support a diagnosis of lung adenocarcinoma with high sensitivity. In this study, we evaluated immunoreactivity for napsin A in a broad spectrum of renal neoplasms by using tissue microarrays (TMA). METHODS: Duplicate TMA of 159 surgically excised renal neoplasms of various types were constructed. IHC for napsin A was performed on TMAs with appropriate positive and negative controls. RESULTS: Napsin A was expressed in Acquired cystic disease associated renal cell carcinoma (RCC) (2/2, 100.0%), chromophobe RCC (5/45, 11.1%), clear cell RCC (10/23, 43.5%), clear cell papillary RCC (9/19, 47.4%), metanephric adenoma (3/3, 100.0%), oncocytoma (13/23, 56.5%), and papillary RCC (31/37, 83.8%). Expression of napsin A was not seen in mucinous tubular and spindle cell carcinoma (0/1, 0.0%), TFE/MITF RCC 0/1, 0.0%), and urothelial carcinoma (0/6, 0.0%). CONCLUSIONS: Napsin A is expressed in both common and rare sub-types of renal neoplasms with variable sensitivity. Based on our results, napsin A is not specific for lung adenocarcinoma. When a metastatic carcinoma of unknown primary is positive for napsin A, the differential diagnosis should include tumors of both renal and lung origin. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9558727831304717 .

Succinate dehydrogenase B: a new prognostic biomarker in clear cell renal cell carcinoma.

Succinate dehydrogenase B (SDHB) is a mitochondrial enzyme complex subunit. Loss of SDHB protein expression has been found to correlate with SDHx gene mutations. Little is known about its expression in subtypes of renal cell carcinoma (RCC) and whether it is a prognostic indicator. Four hundred fifty renal epithelial neoplasms were analyzed for SDHB, comprising clear cell RCC (CCRCC) (n = 240), papillary RCC (n = 84), chromophobe RCC (n = 49), renal oncocytoma (n = 47), clear cell papillary RCC (CCPRCC) (n = 19), and von Hippel-Lindau (VHL)-associated CCPRCC-like tumors (n = 11). Succinate dehydrogenase B expression was graded based upon staining intensity using a 4-tiered system (0-3+), in which 3+ was strongest and complete absence was 0. Neoplasms were further categorized based upon staining extent into SDHB weak (1+-2+) and strong (3+). Succinate dehydrogenase B was strongly preserved in 131 (55%) of 240 CCRCCs, 84 (100%) of 84 papillary RCCs, 49 (100%) of 49 chromophobe RCCs, 1 (5%) of 19 CCPRCC, 5 (45%) of 11 VHL-associated CCPRCC-like tumors, and 47 (100%) of 47 renal oncocytomas. The remaining 109 CCRCCs, 18 CCPRCCs, and 6 VHL-associated CCPRCC-like tumors had weak but preserved SDHB. Succinate dehydrogenase B expression in CCRCCs with high International Society of Urological Pathology nucleolar grade (G3-G4) correlated significantly with survival (log-rank, P = .0004). Succinate dehydrogenase B is variably expressed in RCCs with clear cell morphology and strongly preserved in most other neoplasms. Therefore, weak staining, particularly in clear neoplasms, should not be misinterpreted as negative. Finally, SDHB expression in CCRCCs with high nucleolar grade (G3-G4) is significantly associated with survival, indicating it may be both a diagnostic and prognostic marker in RCC.

Adrenocortical oncocytoma presenting as Cushing's syndrome: an additional report of a paediatric case.

Oncocytomas are tumours predominantly or exclusively composed of oncocytes, cells with granular and eosinophilic cytoplasm filled with mitochondria. Although they can occur in every organ, they are rare in adrenal glands, and in paediatric patients they are even rarer, with only three case reports previously published. We present a preschool child developing Cushing's syndrome due to an adrenocortical oncocytoma, which was confirmed immunohistochemically with antibodies to the mitochondrial electron complex 2. A 5.8-year-old girl presented with clinical features of Cushing's syndrome. ACTH-independent hypercortisolism was confirmed biochemically and a left adrenal mass was detected by imaging and removed by laparotomy. Histopathological analysis revealed a tumour composed of more than 95 % of oncocytes, confirmed immunohistochemically with antibodies to subunits A and B of the mitochondrial enzyme succinate dehydrogenase. Using the Lin-Weiss-Bisceglia score system and the reticulin algorithm, this tumour was categorized as a benign adrenocortical oncocytoma. The patient currently has 64 months of follow-up, without any evidence of relapse of symptoms. To our knowledge, we herein present the youngest patient developing an adrenocortical oncocytoma and the first manifestation of Cushing's syndrome due to this rare neoplasm in paediatric patients. We also emphasize the clinical usefulness of immunohistochemistry to the mitochondrial enzyme succinate dehydrogenase to confirm the oxyphilic nature of adrenocortical oncocytomas.

Matrix metalloproteinase-2 and -9 in the sera and in the urine of human oncocytoma and renal cell carcinoma.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, capable of degrading all the molecular components of extracellular matrix. MMPs have been shown to play critical roles in tumor cell invasion and metastasis. We verified the activity of MMPs in the sera and in the urine of patients with kidney carcinoma by gelatin zymography. Of these patients, 16 had clear cell renal carcinoma (ccRCC) and 4 patients had oncocytoma. The sera and the urine of 16 healthy subjects were used as controls. In the sera, zymography analysis showed gelatinolytic bands at 72 kDa (gelatinase A) at 92, 130 and 240 kDa (gelatinase B). MMP-9 activity was slightly enhanced in sera from ccRCC compared with oncocytoma patients. Serum MMP-2 activity was similar in ccRCC and in oncocytoma patients. In the urine, 2 oncocytoma patients and 3 (33%) of the ccRCC patients showed gelatinolytic activity, whereas MMPs could not be detected in the concentrated urine of healthy subjects. The most abundant lytic activity was at 92 kDa, whereas MMP-2 was present in lesser quantities. However, there was broad overlap of the data and we did not find any correlation to type, stage or grade. Therefore, despite previous evidence, MMP-2 and -9 activity in serum and urine may not be useful biomarker for kidney carcinomas.

Cathepsin K expression in the spectrum of perivascular epithelioid cell (PEC) lesions of the kidney.

The perivascular epithelioid cell (PEC) is a unique cell type coexpressing contractile proteins (mainly α-smooth muscle actin), melanocytic markers, including microphthalmia-associated transcription factor (MITF), and estrogen and progesterone receptors. It is constantly present in a group of tumors called PEComas. Renal PEComas include the common angiomyolipoma as well as less common lesions such as microscopic angiomyolipoma, intraglomerular lesions, angiomyolipoma with epithelial cysts, epithelioid angiomyolipoma, oncocytoma-like angiomyolipoma and lymphangioleiomyomatosis of the renal sinus. It has been demonstrated that most of these lesions are determined by mutations affecting genes of the tuberous sclerosis complex, tuberous sclerosis 1 (TSC1) and tuberous sclerosis 2 (TSC2), with eventual deregulation of the RHEB/MTOR/RPS6KB2 pathway, and it has been observed that some PEComas regressed during sirolimus therapy, an MTOR inhibitor. Recently, overexpression of MITF has been related to the expression of the papain-like cysteine protease cathepsin K in osteoclasts where it has inhibited MTOR. The aim of this study is to evaluate cathepsin K immunohistochemically in the entire spectrum of PEComa lesions in the kidney. The study population consisted of 84 renal PEComa lesions, including 5 composed predominantly of fat (lipoma-like angiomyolipoma), 15 almost exclusively composed of spindle-shaped smooth muscle cells (leiomyoma-like angiomyolipoma) and 31 common angiomyolipomas composed of a mixture of fat, spindle and epithelioid smooth muscle cells, and abnormal thick-walled blood vessels, 15 microscopic angiomyolipomas, 5 intraglomerular lesions, 2 oncocytoma-like angiomyolipomas, 8 epithelioid angiomyolipomas, 2 angiomyolipomas with epithelial cysts and 1 example of lymphangioleiomyomatosis of the renal sinus. In all of the renal PEComas, cathepsin K was found to be constantly and strongly expressed and seems to be a more powerful marker than other commonly used markers for their identification, especially to confirm the diagnosis on needle biopsies.

Carbonic anhydrase IX expression in renal neoplasms: correlation with tumor type and grade.

Carbonic anhydrase IX (CAIX), a hypoxia-induced protein, is expressed in some renal tumors. We evaluated its immunohistochemical expression in 317 primary and 42 metastatic renal neoplasms (186 clear cell, 52 papillary, 35 chromophobe, 47 unclassified, and 15 Xp11.2 translocation renal cell carcinomas [RCCs]; 26 oncocytomas; 2 metanephric adenomas; 1 urothelial carcinoma; 1 mixed epithelial and stromal tumor; and 1 angiomyolipoma); 7 neoplasms were unknown as to whether they were primary or metastatic. We also correlated expression with tumor type and grade. Variable staining was seen in clear cell, papillary, unclassified, and Xp11.2 translocation carcinomas. One chromophobe carcinoma had focal expression. No staining was seen with other tumors. An association was found between high expression and clear cell vs non-clear cell carcinomas with all cases (P < .01) and primary (P < .01) cases. An association between CAIX expression and grade (P < .01) in primary clear cell carcinomas was found. CAIX expression is more common in clear cell RCC than other renal tumor types and is associated with grade.

Expression and activity profiles of DPP IV/CD26 and NEP/CD10 glycoproteins in the human renal cancer are tumor-type dependent.

BACKGROUND: Cell-surface glycoproteins play critical roles in cell-to-cell recognition, signal transduction and regulation, thus being crucial in cell proliferation and cancer etiogenesis and development. DPP IV and NEP are ubiquitous glycopeptidases closely linked to tumor pathogenesis and development, and they are used as markers in some cancers. In the present study, the activity and protein and mRNA expression of these glycoproteins were analysed in a subset of clear-cell (CCRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytomas (RO). METHODS: Peptidase activities were measured by conventional enzymatic assays with fluorogen-derived substrates. Gene expression was quantitatively determined by qRT-PCR and membrane-bound protein expression and distribution analysis was performed by specific immunostaining. RESULTS: The activity of both glycoproteins was sharply decreased in the three histological types of renal tumors. Protein and mRNA expression was strongly downregulated in tumors from distal nephron (ChRCC and RO). Moreover, soluble DPP IV activity positively correlated with the aggressiveness of CCRCCs (higher activities in high grade tumors). CONCLUSIONS: These results support the pivotal role for DPP IV and NEP in the malignant transformation pathways and point to these peptidases as potential diagnostic markers.

Peroxiredoxin I is overexpressed in oncocytic lesions of salivary glands.

BACKGROUND: Oncocytic lesions, particularly frequent in the salivary glands, are characterized by cells with an atypical accumulation of mitochondria. This accumulation has been recognized as a compensatory mechanism to intrinsic functional defects of these organelles, resulting in energy production impairment and increased generation of reactive oxygen species (ROS), including hydrogen peroxide (H(2)O(2)). Peroxiredoxin I (Prx I) is a H(2)O(2) scavenging protein and the expression of its yeast homolog was reported to be influenced by mitochondrial function. METHODS: In this study, we evaluated Prx I expression in oncocytic lesions of salivary glands by immunohistochemistry. RESULTS: Our results showed that Prx I is overexpressed in oncocytes regardless of the salivary gland lesion where they appear. CONCLUSIONS: These results suggest that Prx I expression in oncocytes is related to its ability to decompose mitochondrial-derived H(2)O(2) and that it could provide to the cells a protective role in an environment that, by continuously producing potential DNA-damaging ROS, predisposes to genome instability and cellular transformation.

Acid, basic, and neutral peptidases present different profiles in chromophobe renal cell carcinoma and in oncocytoma.

Renal cell carcinomas (RCCs) are neoplasias with high prevalence and mortality. We previously reported that several peptidases may be involved in the pathophysiology of clear cell renal cell carcinoma (CCRCC). Now, to gain insight into the reasons that lead the various RCC types to behave very differently with regard to aggressiveness and response to anticancer treatments, we analyzed subsets of chromophobe renal cell carcinoma (ChRCC), and renal oncocytoma (RO), a benign tumor; as well as different grades and stages of CCRCCs. Particulate APN, APB, and APA activities were decreased in both ChRCC and RO (tumor vs. nontumor tissues). Interestingly, activities were downregulated in a tumor-type specific way and the intensities of the decreases were stronger in the benign tumor than in the malignant type. Moreover, when two key histopathological parameters for tumor prognosis (high vs. low stage and grade) were analyzed, increases of activity were also observed in several of these cell surface peptidases (APN, APB). Some soluble activities (APB, Asp-AP) were also downregulated in the RCCs. With respect to genetic expression, PSA and APN were in a positive correlation related to their activities in both ChRCC and RO; but not APB, Asp-AP, APA, and PGI. These results may suggest an involvement of several peptidases in the pathophysiology of renal cancer, since they presented different patterns of activity and expression in tumors with different behaviors.

Renal papillary adenoma--a putative precursor of papillary renal cell carcinoma.

The precursor lesions of renal cell carcinoma (RCC) are unknown. The purpose of this study is to determine the incidence, histomorphological features, and immunohistochemical features of papillary adenoma and elucidate its potential relationship to RCC. We reviewed 542 consecutive nephrectomy specimens over an 8-year period. Immunohistochemistry was carried out with antibodies specific for alpha-methyl-coenzyme A racemase (AMACR) and glutathione S-transferase alpha (clear-cell RCC marker). Thirty-eight (7%) nephrectomy specimens showed histologic evidence of papillary adenoma. Of these 38 cases, 18 (47%) arose in the setting of papillary RCC (PRCC). Seven papillary adenomas (18%) occurred in the setting of acquired polycystic kidney disease (APKD), 6 in clear-cell RCCs, 3 in chromophobe RCCs, 2 in end-stage kidney disease, 1 in oncocytoma, 1 in angiomyolipoma, and 1 in renal schwannoma. Furthermore, papillary adenomas were more commonly found in kidneys removed for PRCC (25%, 18/71) than in kidneys harboring clear-cell RCC (1.9%, 6/318). Histomorphologically, papillary adenomas were characterized by varying proportions of papillae and tubules formed by cuboidal cells with scant basophilic cytoplasm similar to those in type 1 PRCC. Adenomas associated with PRCC tend to be multiple in number (61% [11/18] of cases had >2 adenomas; mean, 5). In contrast, 100% of papillary adenomas arising in other conditions had less than 2 adenomas. Most of the adenomas (82%, 31/38) stained strongly for AMACR in a fashion similar to that of PRCC. The 7 AMACR-negative cases all arose in the setting of APKD. In this study of surgical specimens, the high coincidence, multifocality, and histologic and immunohistochemical similarities between papillary adenoma and PRCC suggest that the 2 are strongly associated and may represent a continuum of 1 biologic process. In contrast, adenomas associated with APKD exhibit distinct morphological and immunohistochemical features and, therefore, may have an entirely different pathogenesis.

Defective oxidative phosphorylation in thyroid oncocytic carcinoma is associated with pathogenic mitochondrial DNA mutations affecting complexes I and III.

Oncocytic tumors are characterized by cells with an aberrant accumulation of mitochondria. To assess mitochondrial function in neoplastic oncocytic cells, we studied the thyroid oncocytic cell line XTC.UC1 and compared it with other thyroid non-oncocytic cell lines. Only XTC.UC1 cells were unable to survive in galactose, a condition forcing cells to rely solely on mitochondria for energy production. The rate of respiration and mitochondrial ATP synthesis driven by complex I substrates was severely reduced in XTC.UC1 cells. Furthermore, the enzymatic activity of complexes I and III was dramatically decreased in these cells compared with controls, in conjunction with a strongly enhanced production of reactive oxygen species. Osteosarcoma-derived transmitochondrial cell hybrids (cybrids) carrying XTC.UC1 mitochondrial DNA (mtDNA) were generated to discriminate whether the energetic failure depended on mitochondrial or nuclear DNA mutations. In galactose medium, XTC.UC1 cybrid clones showed reduced viability and ATP content, similarly to the parental XTC.UC1, clearly pointing to the existence of mtDNA alterations. Sequencing of XTC.UC1 mtDNA identified a frameshift mutation in ND1 and a nonconservative substitution in cytochrome b, two mutations with a clear pathogenic potential. In conclusion, this is the first demonstration that mitochondrial dysfunction of XTC.UC1 is due to a combined complex I/III defect associated with mtDNA mutations, as proven by the transfer of the defective energetic phenotype with the mitochondrial genome into the cybrids.

Alpha-methyl CoA racemase expression in renal cell carcinomas.

Alpha-methyl CoA racemase (AMACR), a new molecular marker for prostate cancer, has been recently reported to be one of the most highly expressed genes in papillary renal cell carcinomas (RCCs). We tested the diagnostic usefulness of AMACR antibody in a series of 110 renal tumors: 53 papillary RCCs (33 type 1, 20 type 2); 25 conventional RCCs; 6 chromophobe RCCs; 9 oncocytomas; 5 mucinous tubular and spindle tumors; 2 urothelial carcinomas; 7 angiomyolipomas; and 2 Bellini carcinomas. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections, with a primary prediluted rabbit monoclonal anti-AMACR antibody. Both type 1 and type 2 papillary RCCs exhibited cytoplasmic immunoreactivity for AMACR, with diffuse strong granular staining in 96.4% (53/55) of tumors, without correlation with type or nuclear grade. The 5 mucinous, tubular, and spindle cell carcinomas strongly expressed AMACR, and only 5 of 25 clear cell RCCs and 1 of 9 oncocytomas were focally reactive. The remaining 6 chromophobe RCCs, 5 urothelial carcinomas, and Bellini duct carcinomas showed no immunoreactivity for AMACR. Because high expression of AMACR is found in papillary RCCs (type 1 and 2) and in mucinous, tubular, and spindle cell carcinomas of the kidney, immunostaining for AMACR should be used in conjunction with other markers when histological typing of a renal tumor is difficult.

Selenoprotein expression in Hürthle cell carcinomas and in the human Hürthle cell carcinoma line XTC.UC1.

Hürthle cell carcinomas (HTC) are characterized by mitochondrial amplification and enhanced oxygen metabolism. To clarify if defects in enzymes scavenging reactive oxygen species are involved in the pathogenesis of HTC, we analyzed selenium (Se)-dependent expression of various detoxifying selenoproteins in the HTC cell line XTC.UC1. Glutathione peroxidase and thioredoxin reductase activity was found both in cell lysates and conditioned media of XTC.UC1 cells and was increased by Na(2)SeO(3). Western blot analysis demonstrated the presence of thioredoxin reductase both in cell lysates and conditioned media and of glutathione peroxidase 3 in conditioned media. Type I 5'-deiodinase, another selenoprotein that catalyzes thyroid hormone metabolism, was detectable only in cell lysates by enzyme assay and Western blot, and responded to stimulation by both Na(2)SeO(3) and retinoic acid. A selenoprotein P signal was detected in conditioned media by Western blot, but was not enhanced by Na(2)SeO(3) treatment. In situ hybridization revealed glutathione peroxidase mRNAs in HTC specimen; glutathione peroxidase 3 mRNA levels were reduced. These data suggest adequate expression and Se-dependent regulation of a couple of selenoproteins involved in antioxidant defense and thyroid hormone metabolism in XTC.UC1 cells, so far giving no evidence of a role of these proteins in the pathogenesis of HTCs.

Peroxiredoxin 5 expression in the human thyroid gland.

Peroxiredoxin 5 (PRDX5) is a newly discovered thioredoxin peroxidase able to reduce peroxides that is implicated in antioxidant protective mechanisms. We report here its expression in the human thyroid gland. Twenty-seven human thyroid specimens were examined by immunohistochemistry. They included six normal thyroid tissues, five multinodular goiters, nine hot nodules, two Hürthle cell adenomas, and five thyroids from patients with Graves' disease. In the control tissue, PRDX5 expression was heterogeneous, being stronger in cubical functionally active follicular cells than in flat quiescent thyrocytes. It was diffuse in the cytoplasm, occasionally localized in inclusions that most likely corresponded to mitochondria. This feature was particularly marked in the Hürthle cell adenoma case. In multinodular goiters, hot nodules, and Graves' thyroids, the cytosolic labeling was enhanced compared to the control tissue and a signal was also detected in few nuclei. To determine whether the level of expression was different between multinodular goiters and hyperthyroid Graves' thyroids, PRDX5 immunoblotting was performed in these two respective tissues. We observed that PRDX5 expression was higher in the thyroid gland of patients with Graves' disease compared to multinodular goiters. In conclusion, our data show that PRDX5 is expressed in the thyroid gland where it could act as antioxidant. The level of expression is directly correlated with the functional status of epithelial cells, being higher in multinodular goiters, and even more pronounced in hyperthyroid tissues, such as Graves' disease.