An Unusual Two-Domain Thyropin from Tick Saliva: NMR Solution Structure and Highly Selective Inhibition of Cysteine Cathepsins Modulated by Glycosaminoglycans.

The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin (IrThy), which is secreted in the saliva of Ixodes ricinus ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure-function characterization of IrThy-the first investigated thyropin of parasite origin-and suggests its potential role in host-parasite interactions at the tick bite site.

Methylation analysis as an auxiliary tool in cytological diagnostics of infrequent anogenital lesions - a pilot study.

Methylation silencing of certain cellular genes is a sign of carcinogenesis progression and therefore tests that detect methylation could be used in the diagnosis or staging of malignant diseases. In the diagnosis of squamous cell carcinomas of the cervix which are almost 100% caused by long-term infection with highrisk human papillomavirus (HR-HPV), methylation silencing of certain cellular genes is a highly specific marker of advanced dysplastic lesions and appears to result from aberrant activation of the methyltransferase DNMT1 by viral oncoproteins E6 and E7. A methylation test performed on a cervicovaginal cytology specimen allows to increase the diagnostic value of this non-invasive test and to select patients with severe squamous cell lesions for follow-up. Other less frequent anogenital malignancies that are induced by HR-HPV to a lesser extent can also be detected by cytological examination - glandular lesions of various origins, most commonly cervical and endometrial adenocarcinomas and anal carcinoma. The aim of our pilot study was to evaluate the utility of a methylation test for the diagnosis of these malignancies in a cohort of 50 liquid-based cervicovaginal cytologies with glandular lesion and 74 liquid-based anal cytologies from HIV-positive men having sex with men who are at high risk for anal cancer development.

Histone Methyltransferase DOT1L is Involved in Larval Molting and Second Stage Nymphal Feeding in Ornithodoros Moubata.

Epigenetic mechanisms have not been characterized in ticks despite their importance as vectors of human and animal diseases worldwide. Our investigation identifies and functionally characterizes the orthologue of S-adenosylmethionine (SAM) binding methyltransferase enzyme, disruptor of telomeric silencing 1-like (DOT1L) in Ornithodoros moubata (OmDOT1L), a soft tick vector for the relapsing fever pathogen Borrelia duttonii and the African swine fever virus. The OmDOT1L tertiary structure was predicted and compared to the Homo sapiens DOT1L which had been co-crystalized with SGC0946, a DOT1L-specific inhibitor. The amino acid residues crucial for SAM and SGC0946 binding conserved in most DOT1L sequences available, are also conserved in OmDOT1L. Quantitative PCR of Omdot1l during O. moubata life stages showed that transcripts were significantly upregulated in first-stage nymphs. O. moubata larvae exposed to SGC0946 displayed high mortality during molting to first-stage nymphs. Furthermore, a significant decrease in weight was observed in second-stage nymphs fed on recombinant OmDOT1L-immunized rabbits. In contrast, artificial blood feeding supplemented with SGC0946 did not affect survival and reproductive performance of adult female ticks. We concluded that OmDOT1L plays an essential role in the regulation of larval molting and the feeding of O. moubata second-stage nymphs.

Selection of suitable reference genes for gene expression studies in myxosporean (Myxozoa, Cnidaria) parasites.

Myxozoans (Cnidaria: Myxozoa) are an extremely diversified group of endoparasites some of which are causative agents of serious diseases in fish. New methods involving gene expression studies have emerged over the last years to better understand and control myxozoan diseases. Quantitative RT-PCR is the most extensively used approach for gene expression studies. However, the accuracy of the results depends on the normalization of the data to reference genes. We studied the expression of eight commonly used reference genes, adenosylhomocysteinase (AHC1), beta actin (ACTB), eukaryotic translation elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), DNA-directed RNA polymerase II (RPB2), 18S ribosomal RNA (18S), 28S ribosomal RNA (28S) across different developmental stages of three myxozoan species, Sphaerospora molnari, Myxobolus cerebralis and Ceratonova shasta, representing the three major myxozoan linages from the largest class Myxosporea. The stable reference genes were identified using four algorithms: geNorm, NormFinder, Bestkeeper and ΔCq method. Additionally, we analyzed transcriptomic data from S. molnari proliferative and spore-forming stages to compare the relative amount of expressed transcripts with the most stable reference genes suggested by RT-qPCR. Our results revealed that GAPDH and EF2 are the most uniformly expressed genes across the different developmental stages of the studied myxozoan species.

Multiple legumain isoenzymes in ticks.

By searching nucleotide databases for the North American Lyme disease vector, Ixodes scapularis, we have complemented the previously characterized European Ixodes ricinus legumain IrAE1 with a full set of nine analogous genes (isae1-9). Six of these were PCR confirmed as genes present in all tick genomes tested. The absolute mRNA copy number examined by quantitative (q)PCR enabled expression profiling and an absolute comparison of mRNA levels for individual I. scapularis (Is)AEs in tick tissues. Four IsAEs (1, 2, 4, 9) were expressed solely in the gut and thus are proposed to be involved in host blood digestion. Expression qPCR profiling over developmental stages confirmed IsAE1, the direct analogue of previously characterized I. ricinus IrAE1, as the principle legumain transcript in partially engorged females, and demonstrated its strong regulation by on-host feeding in larvae, nymphs and females. In contrast, IsAE2 was the predominant gut legumain in unfed nymphs, unfed females and males. In-silico, IsAE1 and IsAE2 protein three-dimensional structural models displayed minimal differences in overall proenzyme structures, even in comparison with recently resolved crystal structures of mammalian prolegumain. Three functional studies were performed in I. ricinus with IsAE1/IsAE2 analogues: double IrAE1/IrAE2 RNA interference silencing, feeding of ticks on IrAE1+IrAE2 immunized hosts and in vitro membrane tick feeding on blood containing a legumain-specific inhibitor. The latter experiment led to reduced weights of fully engorged ticks and limited oviposition, and indicated the potential of legumain inhibitors for novel anti-tick interventions.

Tick iron and heme metabolism - New target for an anti-tick intervention.

Ticks are blood-feeding parasites and vectors of serious human and animal diseases. Ixodes ricinus is a common tick in Europe, transmitting tick-borne encephalitis, Lyme borreliosis, anaplasmosis, or babesiosis. Immunization of hosts with recombinant tick proteins has, in theory, the potential to interfere with tick feeding and block transmission of pathogens from the tick to the host. However, the efficacy of tick antigens has, to date, not been fully sufficient to achieve this. We have focused on 11 in silico identified genes encoding proteins potentially involved in tick iron and heme metabolism. Quantitative real-time PCR (qRT-PCR) expression profiling was carried out to preferentially target proteins that are up-regulated during the blood meal. RNA interference (RNAi) was then used to score the relative importance of these genes in tick physiology. Finally, we performed vaccination screens to test the suitability of these proteins as vaccine candidates. These newly identified tick antigens have the potential to improve the available anti-tick vaccines.

Nuclease Tudor-SN Is Involved in Tick dsRNA-Mediated RNA Interference and Feeding but Not in Defense against Flaviviral or Anaplasma phagocytophilum Rickettsial Infection.

Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are conserved components of the basic RNA interference (RNAi) machinery with a variety of functions including immune response and gene regulation. The RNAi machinery has been characterized in tick vectors of human and animal diseases but information is not available on the role of Tudor-SN in tick RNAi and other cellular processes. Our hypothesis is that tick Tudor-SN is part of the RNAi machinery and may be involved in innate immune response and other cellular processes. To address this hypothesis, Ixodes scapularis and I. ricinus ticks and/or cell lines were used to annotate and characterize the role of Tudor-SN in dsRNA-mediated RNAi, immune response to infection with the rickettsia Anaplasma phagocytophilum and the flaviviruses TBEV or LGTV and tick feeding. The results showed that Tudor-SN is conserved in ticks and involved in dsRNA-mediated RNAi and tick feeding but not in defense against infection with the examined viral and rickettsial pathogens. The effect of Tudor-SN gene knockdown on tick feeding could be due to down-regulation of genes that are required for protein processing and blood digestion through a mechanism that may involve selective degradation of dsRNAs enriched in G:U pairs that form as a result of adenosine-to-inosine RNA editing. These results demonstrated that Tudor-SN plays a role in tick RNAi pathway and feeding but no strong evidence for a role in innate immune responses to pathogen infection was found.

IrFC - An Ixodes ricinus injury-responsive molecule related to Limulus Factor C.

Limulus Clotting Factor C is a multi-domain serine protease that triggers horseshoe crab hemolymph clotting in the presence of trace amounts of bacterial lipopolysaccharides. Here we describe and functionally characterize an homologous molecule, designated as IrFC, from the hard tick Ixodes ricinus. Tick Factor C consists of an N-terminal cysteine-rich domain, four complement control protein (sushi) modules, an LCCL domain, a truncated C-lectin domain and a C-terminal trypsin-type domain. Developmental expression profiling by quantitative real-time PCR revealed that the irfc mRNA is expressed in all stages including eggs. In tissues dissected from adult I. ricinus females, the irfc mRNA is present mainly in tick hemocytes and accordingly, indirect immunofluorescence microscopy localized IrFC intracellularly, in tick hemocytes. Irfc mRNA levels were markedly increased upon injection of sterile saline, or different microbes, demonstrating that the irfc gene transcription occurs in response to injury. This indicates a possible role of IrFC in hemolymph clotting and/or wound healing, although these defense mechanisms have not been yet definitely demonstrated in ticks. RNAi silencing of irfc expression resulted in a significant reduction in phagocytic activity of tick hemocytes against the Gram-negative bacteria Chryseobacterium indologenes and Escherichia coli, but not against the yeast, Candida albicans. This result suggests that IrFC plays a role in the tick primordial complement system and as such possibly mediates transmission of tick-borne pathogens.

Hybrid peripheral nerve sheath tumors, including a malignant variant in type 1 neurofibromatosis.

The authors report a small case series of hybrid nerve sheath tumors occurring in the setting of type 1 neurofibromatosis. Four lesions were benign and consisted of plexiform neurofibromas with considerable areas of perineuriomatous differentiation in patients with type 1 neurofibromatosis. In these lesions, biphasic (Schwannian and perineuriomatous) differentiation was apparent on immunohistochemistry, with the perineuriomatous areas staining for epithelial membrane antigen, glut-1, and claudin-1 and being negative for S-100 protein. Three patients were members of a single family, with a history of various malignant neoplasms. Included in the series is 1 hybrid lesion in which neurofibromatous and perineuriomatous areas were clearly visible on hematoxylin- and eosin-stained slides. The lesion was unique in that it manifested malignant change in the S-100 protein-positive component, which was classified as malignant peripheral nerve sheath tumor. The malignant component showed areas with an epithelioid cell morphology.

Cribriform adenocarcinoma of minor salivary gland origin principally affecting the tongue: characterization of new entity.

We present a series of 23 cases of a distinctive, hitherto poorly recognized low-grade adenocarcinoma, with several histologic features reminiscent of papillary carcinoma of the thyroid, and which mostly but not exclusively occurs in the tongue. All the tumors were unencapsulated and were divided into lobules that were composed mainly of cribriform and solid growth patterns. Therefore, we propose the name "cribriform adenocarcinoma of minor salivary gland origin (CAMSG)." All the patients were adults with a mean age at diagnosis of 55.8 years (range, 25 to 85 y). Fourteen of the 23 tumors were localized in the tongue, 3 in the soft palate, 2 in the retromolar buccal mucosa, 3 in the lingual tonsils, and 1 in the upper lip. Fifteen patients of 23 had synchronous metastases in the cervical lymph nodes at the time of diagnosis, bilateral in 3 cases. In 3 patients, the nodal metastasis was the first evidence of disease, later investigation revealing primary neoplasms in the base of tongue and tonsil, respectively. In addition, 1 patient developed a cervical lymph node metastasis 8 years after excision of a primary tumor of the tongue. Data on treatment and follow-up were available in 14 cases. The patients were treated by radical excision with clear margins (12 cases) or by simple excision (2 cases). Neck dissection was performed in 10 patients; 9 received radiotherapy, but none were treated by chemotherapy. Clinical follow-up ranged from 2 months to 13 years (mean, 6 y and 5 mo). Twelve patients are alive with no evidence of recurrent or metastatic disease after treatment, 1 patient died 2 years after surgery without evidence of tumor, and 1 patient is alive with recurrent tumor of the palate.

[KRAS mutation testing in therapeutic algorithm for treatment of metastatic colorectal carcinoma]. / Vysetrení mutacního statutu genu KRAS jako soucást algoritmu lécby metastatického kolorektálního karcinomu.

Targeted therapy has become an integral part of treatment procedures of malignant tumors. Colorectal carcinomas are frequently targeted with monoclonal anti-EGFR antibodies (cetuximab and panitumumab). Activating somatic mutations in codons 12 and 13 of the exon 2 of KRAS gene are considered negative predictive factors of response to anti-EGFR therapy in patients with metastatic colorectal cancer. In the Czech Republic, evaluation of mutational status of KRAS gene is performed in several referral laboratories. In 2009, these laboratories performed 2580 tests of the KRAS mutational status--out of these, 60.2% cases reported non-mutated, wild-type KRAS. In one of the referral laboratories, we demonstrate the logistics of KRAS testing procedure. Stratification of patients with metastatic colorectal tumors based on their KRAS mutational status has evolved to a standard procedure. Laboratories performing these methods shall therefore adhere to the recommendations of the professional and accredited societies.

Extra nuchal-type fibroma associated with elastosis, traumatic neuroma, a rare APC gene missense mutation, and a very rare MUTYH gene polymorphism: a case report and review of the literature*.

We report a case of an extra nuchal-type fibroma in a 51-year-old male suspected to have attenuated familial adenomatous polyposis (Gardner's syndrome), who presented with a longstanding buttock mass excised due to enlargement and pain. Histopathologically, lobules of haphazard, hypocellular, hyalinized collagen bundles replaced the dermis and subcutis and entrapped nerve bundles, mimicking Morton neuroma. Ramifying nerve twigs found around larger nerve fascicles showed the co-existence of traumatic neuroma. Elastic tissue stain revealed elastosis characterized by large, arborizing fibers lying between and within the hyalinized collagen bundles. Modified Masson's trichrome stain showed light blue staining of collagen bundles producing the hyalinized nodules with irregular, light red staining of collagen bundles at their periphery and within tumor collagen. Compression and/or degeneration of collagen and secondary elastosis with later entrapment by tumor collagen could explain this microscopic phenotype. By immunohistochemistry, tumor spindle cells expressed nuclear ß-catenin and cyclin D1, mostly within regions of fibrosis implicating activation of the adenomatous polyposis coli (APC)-Wnt pathway. Genetic analysis showed a missense mutation in APC gene (c.7504G>A, p.G2502S in exon 15) and a functional homozygous polymorphism in the MUTYH gene (c.36+325G>C, (IVS1+5G/C)). Nuchal-type fibroma has been associated with Gardner's syndrome and trauma. In this patient, genetic predisposition coupled with repetitive, localized trauma and collagen degeneration may have provided the stimulus for the development of extra nuchal-type fibroma.

Borderline papillary serous tumor of the fimbriated end of the fallopian tube with peritoneal implants.

Diagnosis of Borderline papillary serous tumor of the fallopian tube was comprehensively established by Zheng in 1996 supported mostly by a histological similarity to its ovarian counterpart. It is a very rare entity with eight cases published so far and the ninth case described here as a 41-year-old woman presented with non-specific lower abdominal pain, dyspareunia and dysuria. Left adnexal mass was identified and she was operated on. It turned out the tumor was attached exclusively to the left tube, with no connection to any of the surrounding structures and with histology of borderline serous tumor with non-invasive implants in the left and right ovary and visceral peritoneum. Reviewing available data on genetics of these tumors there was diploid status in one examined tumor, and in our case no mutations of KRAS, BRAF and p53 genes were found. Histomorphology remains the mainstay of diagnosis and staging operation is the mainstay of patient management. Prognosis is uncertain with a 6-year survival documented in one case.

Renal small cell oncocytoma with pseudorosettes A histomorphologic, immunohistochemical, and molecular genetic study of 10 cases.

A cohort of a heretofore not described rare subtype of renal oncocytoma, small cell oncocytoma with pseudorosettes is presented. Patients were 6 women and 4 men with ages ranging from 51 to 76 years. The tumors displayed areas composed of small cells ("oncoblasts") featuring scant cytoplasm and small, round monomorphic nuclei. The small cell areas constituted 15% to 60% of the total tumor volume (mean, 28.5%; median, 22.5%). No necrosis or mitotic activity was discerned. All tumors also contained areas composed of characteristic oncocytes comprising 40% to 85% of the total tumor volume. In all cases, a varying number of pseudorosettes were identified. The pseudorosettes were composed of small globules of (periodic acid-Schiff-positive) hyaline basal membrane-like material surrounded by small "oncoblastic" cells. The immunohistochemical profile was variable, including at least focal positivity for AE1-3 (10/10), cytokeratin 7 (7/10), epithelial membrane antigen (10/10), c-kit (6/10), antimitochondrial antigen (MIA;10/10), PAX-2 (9/10), AMACR (racemase;6/10), CD10 (5/10), parvalbumin (8/10), vimentin (6/10), claudin 7 (10/10), and claudin 8 (3/10). No immunoreactivity for carbonic anhydrase 9, HMB-45, S-100A1, and TFE3 was documented. We found no differences in the immunophenotype in the small cell oncocytes/oncoblasts that formed pseudorosettes and those that did not. However, there were differences in the immunohistochemical profile of classic oncocytes and small cell oncocytes/oncoblasts. Using array comparative genomic hybridization, no chromosomal changes were identified in any of the cases examined (n = 3). No numerical changes of chromosomes 7 and 17 were revealed on fluorescence in situ hybridization analysis (n = 3). In conclusion, we herein present the first study on small cell renal oncocytomas with formation of pseudorosettes. This is a rare subtype of oncocytoma, which may, especially on a core biopsy, present differential diagnostic difficulties. The immunohistochemical profile of these tumors is variable and differs in significant respects from that of conventional renal oncocytoma. Awareness of this entity and its immunohistochemical variability should help in distinguishing this rare tumor from malignant tumors with similar (small cell) histomorphologic features. All tumors behaved in a benign fashion during follow-up (mean, 3.1 years; median, 1 year).

Lymphocyte-rich renal cell carcinoma: an unusual histomorphologic manifestation of a tumor that is not part of lynch syndrome.

Lynch syndrome (LS) is characterized by familial clustering of early cancer development in various organs. One of the histologic characteristics of carcinomas associated with LS is prominent intratumoral and peritumoral lymphocytes. The relationship between LS and the rare lymphocyte-rich clear cell renal cell carcinoma (CRCC) has been not been studied. We compared lymphocyte-rich CRCC (N=15) with CRCC with no or minimal lymphoid infiltration, occurring in young (<40 y of age) patients (N=15) and in a control group of older (>62 y of age) patients (N=5) with CRCC having conventional histologic features. All cases were analyzed histologically and immunohistochemically using antibodies against the mismatch repair (MMR) proteins MLH1, MSH2, MSH6, and PMS2. The mutational status of the Von Hippel-Lindau (VHL) gene was analyzed successfully in 25 cases. We found no case with complete absence of immunoreactivity for the MMR proteins/ enzymes studied in any of the groups. Hence, none of the RCCs with heavy lymphocytic infiltration displayed complete absence of protein expression of any of the MMR enzymes. We found mutation of the VHL gene in 2 of 8 tumors from group A, in 4 of 12 tumors from group B, and no mutation in the control group C. We conclude that this rare subset of renal neoplasms is not part of the spectrum of tumors seen in LS. The results of the VHL gene analysis are in concordance with published data on sporadic CRCCs.

Clear cell renal cell carcinoma with focal renal angiomyoadenomatous tumor-like area.

Recently, renal angiomyoadenomatous tumor (RAT) has been identified. However, there are no descriptions about clear cell renal cell carcinoma (RCC) with focal RAT-like features. A 33-year-old Japanese man was found to have a tumor in the left kidney. Macroscopically, the tumor extended into the perinephric fat tissue, and the cut surface showed the yellowish color. The histologic examination of the tumor consisted of 2 components of clear cell RCC and RAT-like area. The RAT-like area showed the admixture of epithelial cells with basophilic or clear cytoplasm and stromal component containing leiomyomatous stroma, fine capillary network, and pericytic network. Immunohistochemically, epithelial neoplastic cells in RAT-like area were diffusely positive for CD10 and RCC Ma. G-band karyotype showed the structural abnormality of chromosome 3 and both components of clear cell RCC and RAT-like area revealed the identical VHL gene mutation. Finally, pathologists should pay attention to the presence of clear cell RCC focally resembling RAT.

Brooke-Spiegler syndrome: report of 10 patients from 8 families with novel germline mutations: evidence of diverse somatic mutations in the same patient regardless of tumor type.

Brooke-Spiegler syndrome (BSS) is an inherited autosomal dominant disease characterized by the development of multiple adnexal cutaneous neoplasms including spiradenoma, cylindroma, spiradenocylindroma, and trichoepithelioma (cribriform trichoblastoma). BSS patients have various mutations in the CYLD gene, a tumor suppressor gene located on chromosome 16q. Our search of the literature revealed 51 germline CYLD mutations reported to date. Somatic CYLD mutations have rarely been investigated. We studied 10 patients from 8 families with BSS. Analysis of germline mutations of the CYLD gene was performed using either peripheral blood or nontumorous tissue. In addition, 19 formalin-fixed paraffin-embedded tumor samples were analyzed for somatic mutations, including loss of heterozygosity studies. A total of 38 tumors were available for histopathologic review. We have identified 8 novel germline mutations, all of which consisted of substitutions, deletions, and insertions/duplications and all except one led to premature stop codons. The substitution mutation in a single case was also predicted to disrupt protein function and seems causally implicated in tumor formation. We demonstrate for the first time that somatic events, loss of heterozygosity, or sequence mutations may differ among multiple neoplasms even of the same histologic type, occurring in the same patient.

Mammary analogue secretory carcinoma of salivary glands, containing the ETV6-NTRK3 fusion gene: a hitherto undescribed salivary gland tumor entity.

We present a series of 16 salivary gland tumors with histomorphologic and immunohistochemical features reminiscent of secretory carcinoma of the breast. This is a hitherto undescribed and distinctive salivary gland neoplasm, with features resembling both salivary acinic cell carcinoma (AciCC) and low-grade cystadenocarcinoma, and displaying strong similarities to breast secretory carcinoma. Microscopically, the tumors have a lobulated growth pattern and are composed of microcystic and glandular spaces with abundant eosinophilic homogenous or bubbly secretory material positive for periodic acid-Schiff, mucicarmine, MUC1, MUC4, and mammaglobin. The neoplasms also show strong vimentin, S-100 protein, and STAT5a positivity. For this tumor, we propose a designation mammary analogue secretory carcinoma of salivary glands (MASC). The 16 patients comprised 9 men and 7 women, with a mean age of 46 years (range 21 to 75). Thirteen cases occurred in the parotid gland, and one each in the minor salivary glands of the buccal mucosa, upper lip, and palate. The mean size of the tumors was 2.1 cm (range 0.7 to 5.5 cm). The duration of symptoms was recorded in 11 cases and ranged from 2 months to 30 years. Clinical follow-up was available in 13 cases, and ranged from 3 months to 10 years. Four patients suffered local recurrences. Two patients died, 1 of them owing to multiple local recurrences with extension to the temporal bone, and another owing to metastatic dissemination to cervical lymph nodes, pleura, pericardium, and lungs. We have shown a t(12;15) (p13;q25) ETV6-NTRK3 translocation in all but one case of MASC suitable for analysis. One case was not analyzable and another was not available for testing. This translocation was not found in any conventional salivary AciCC (12 cases), nor in other tumor types including pleomorphic adenoma (1 case) and low-grade cribriform cystadenocarcinoma (1 case), whereas ETV6-NTRK3 gene rearrangements were proven in all 3 tested cases of mammary secretory carcinoma. Thus, our results strongly support the concept that MASC and AciCC are different entities.

Large cell calcifying Sertoli cell tumor: a clinicopathologic study of 1 malignant and 3 benign tumors using histomorphology, immunohistochemistry, ultrastructure, comparative genomic hybridization, and polymerase chain reaction analysis of the PRKAR1A gene.

Four cases of large cell calcifying Sertoli cell tumor, 3 benign and 1 malignant, with no clinical signs of Carney complex or Peutz-Jeghers syndrome are reported with results of histologic, immunohistochemical, ultrastructural, and comparative genomic hybridization studies. Analysis of PRKAR1A gene was performed on 2 cases. The age range of the patients was 19 to 54 years. The patient with a malignant large cell calcifying Sertoli cell tumor died of disease 4 years after surgery. Patients with benign tumors have had an uneventful follow-up for 1 and 3 years. All tumors were well circumscribed, unencapsulated, and composed of solid sheets, irregular cords, tubular structures, and nests in a fibrous and/or myxoid stroma with cellular atypia in the malignant case. All tumors showed diffuse immunoreactivity for inhibin, vimentin, calretinin, and S100 protein. Focal positivity for cytokeratin (AE1/AE3) was noticed in 1 case. Tumors were negative for CAM 5.2, Mic-2, Melan-A laminin, placental alkaline phosphatase, and alpha-fetoprotein. The proliferation index was 5% and 10% for 2 of the benign tumors and 30% for the malignant tumor. Comparative genomic hybridization was performed in 2 cases. There was no evidence of any major chromosomal changes. In one case, no PRKAR1A gene mutation was found. In the other case, a heterozygous shift mutation c.65_84dup was found, despite the absence of other clinical signs of Carney complex or Peutz-Jeghers syndrome. Although the combination of large cell calcifying Sertoli cell tumor and PRKAR1A mutation fulfills the criteria for establishing a diagnosis of Carney complex, the clinical relevance of finding a PRKAR1A gene mutation in a patient without any clinical signs of Carney complex or Peutz-Jeghers syndrome remains to be established.

Imprint cytologic features of chromophobe renal cell carcinoma morphologically resembling renal oncocytoma: is this an oncocytic variant of chromophobe renal cell carcinoma?

In this article, we report a case of 76-year-old woman with a rare variant of chromophobe renal cell carcinoma (CRCC). Cytologically, renal tumor cells obtained from imprint cytology were isolated or arranged in small or monotonous population cells with abundant granular cytoplasm. Neoplastic cells showed regular and uniformly shaped small round to oval nuclei with smooth margin. Binucleation was occasionally seen. Immunocytochemically, the cytoplasm of almost all tumor cells was diffusely positive for vimentin and CK 7. Histologically, the cytoplasm was abundant granular eosinophilic and composed of solid cell sheets or pseudoacinar structures. Additionally, tumor cells showed infiltration into some small renal veins covered by a single layer of endothelial cells. These cytological and histological features entirely resembled those of renal oncocytoma. We performed the analysis of von Hippel-Lindau (VHL) gene mutation, 3p loss of heterozygosity (LOH), and fluorescence in situ hybridization (FISH) on chromosomes 7, 10, 13, 17, and 21. As a result, we confirmed monosomy of chromosomes 7, 10, 13, and 17, and these findings corresponded to the diagnosis of CRCC. Finally, we present a case of renal tumor morphologically resembling renal oncocytoma but genetically showing CRCC. We suggest that oncocytic variant of CRCC may actually exist.