Expression of miRNA-371a-3p in seminal plasma and ejaculate is associated with sperm concentration.

BACKGROUND: The microRNAs of the miR-371-3 cluster are novel serum markers for testicular germ cell tumors. Sporadic reports suggested the expression of this miRNA in semen. OBJECTIVES: To verify the expression of miR-371a-3p in seminal plasma and unprocessed ejaculate; to compare seminal plasma miRNA levels in germ cell tumors patients with those of controls; to look for an association of miRNA levels with semen quality. MATERIALS AND METHODS: The miR-371a-3p expression was analyzed with qPCR. The study population consisted of 100 participants: seminal plasma samples from 20 germ cell tumors patients and 30 controls, serum samples from 12 healthy men, ejaculate samples from 38 men undergoing fertility testing. RESULTS: The seminal plasma miR-371a-3p levels of germ cell tumors patients were not different from controls. The miRNA expression was very low in serum but much higher in seminal plasma. In ejaculate samples, the miRNA expression significantly correlated with sperm concentration and the total sperm count. DISCUSSION: miR-371-a-3p is present in sperm-containing fluids. Seminal plasma levels cannot be used to distinguish germ cell tumors from controls. The correlation with sperm concentration in ejaculate samples suggests the spermatozoa as possible source of miR-371a-3p production. CONCLUSION: The miR-371a-3p levels in ejaculate could represent a novel biomarker for the non-invasive evaluation of male infertility.

Can germ cell neoplasia in situ be diagnosed by measuring serum levels of microRNA371a-3p?

PURPOSE: Diagnosing germ cell neoplasia in situ (GCNis) can detect germ cell tumours (GCTs) at the pre-invasive stage. To date, testicular biopsy with the potential of surgical complications is the only way of safely diagnosing GCNis. Recently, microRNAs (miRs) 371-3, and miR 367 were shown to be valuable serum biomarkers of GCTs. We explored the usefulness of these candidate miRs as a marker for GCNis. METHODS: 27 patients with GCNis and no concomitant GCT were enrolled. All patients underwent measuring serum levels of miR-371a-3p and miR-367-3p before treatment, 11 had repeat measurement after treatment, 2 also had testicular vein blood examinations. Serum levels were measured by quantitative PCR. In addition, four orchiectomy specimens of patients with GCT were examined immunohistochemically and by in situ hybridization (ISH) with a probe specific for miR-371a-3p to look for the presence of this miR in GCNis cells. RESULTS: The median serum level of miR-371a-3p was significantly higher in patients with GCNis than in controls, miR-367 levels were not elevated. Overall, 14 patients (51.9%) had elevated serum levels of miR-371a-3p. The highest levels were found in patients with bilateral GCNis. Levels in testicular vein serum were elevated in both of the cases. After treatment, all elevated levels dropped to normal. In two orchiectomy specimens, miR-371a-3p was detected by ISH in GCNis cells. CONCLUSIONS: Measuring miR-371a-3p serum levels can replace control biopsies after treatment of GCNis. In addition, the test can guide clinical decision making regarding the need of testicular biopsy in cases suspicious of GCNis.

MicroRNA miR-371a-3p in serum of patients with germ cell tumours: evaluations for establishing a serum biomarker.

As only 60% of the patients with germ cell tumour (GCT) express the classical markers, new markers as for example microRNAs (miRNAs) are required. One promising candidate is miR-371a-3p, but data are sparse to date. We measured serum levels of miR-371a-3p in GCT patients, in controls, and in cases with other malignancies. We also assessed the expression in other body fluids and we looked to the decline of serum miR-371a-3p levels after treatment. miR-371a-3p levels were measured by quantitative polymerase chain reaction in serum samples of 25 GCT patients, 6 testicular intraepithelial neoplasia (TIN) patients, 20 healthy males and 24 non-testicular malignancies (NTMs). Testicular vein blood (TVB) was examined in five GCT patients and five controls. Five GCT patients had serial daily measurements after orchiectomy. Five seminal plasma samples, three urine specimens and one pleural effusion fluid were processed likewise. GCT patients had significantly higher miR-371a-3p serum levels than controls and NTMs. Serum levels of controls, TINs and NTMs were not significantly different. TVB samples of GCT patients had 65.4-fold higher serum levels than peripheral blood. Malignant pleural effusion fluid had extremely high levels of miR-371a-3p, seminal plasma had strongly elevated levels by comparison with serum levels of controls. In urine of GCT patients, no miR-371a-3p expression was detected. Daily measurements after orchiectomy in stage 1 patients revealed a decline by 95% within 24 h. Serum levels of miR-371a-3p appear to be a promising specific biomarker of GCTs as is suggested by high serum levels in GCT patients, the rapid return of elevated levels to normal range after treatment, the association of serum levels with tumour bulk, the non-expression in NTMs and the much higher levels of miR-371a-3p in TVB. This potential marker deserves further exploration in a large-scale clinical study.

MicroRNAs miR-371-3 in serum as diagnostic tools in the management of testicular germ cell tumours.

BACKGROUND: miRNAs are small noncoding RNA molecules that can be released into body fluids. Germ cell tumours (GCTs) overexpress miRNAs of the miR-371-3 cluster. Thus, serum levels of these miRNAs may correlate with tumour load. METHODS: miRNAs of the miR-371-3 cluster were quantified in cubital vein blood samples of 20 GCT patients with clinical stage 1, and of 4 patients with advanced stages before and after treatment. In six patients testicular vein blood (TVB) was examined additionally. Seventeen healthy males served as controls. Likewise, expression of miRNAs in 15 matching tumour specimens was measured. RESULTS: In all patients, serum levels of miRNAs 371-3 were much higher than in controls. In stage 1, levels decreased postoperatively 336.7-fold, 7.4-fold, and 7.7-fold for miRNAs 371a-3p, 372, and 373-3p, respectively (P<0.01). Also, in those cases with advanced disease levels dropped to the normal range after completion of treatment. miR-371-3 levels in TVB exceeded those in peripheral blood in all cases. Expression of miR-371a-3p was also documented in tumour tissue. However, no correlation was found regarding the extent of miRNA expression in tissue and the values measured in matching serum. CONCLUSION: Thus, miR-371a-3p serum level appears to be a useful biomarker in GCTs.

Evidence for a 3p25 breakpoint hot spot region in thyroid tumors of follicular origin.

Epithelial tumors of the thyroid are cytogenetically well-investigated tumors. So far, the main cytogenetic subgroups, characterized by trisomy 7 and by rearrangements of either 19q13 or 2p21, respectively, have been described. Recently, we have been able to describe the involvement of a novel gene called THADA in benign thyroid lesions with 2p21 rearrangements. Other fusion genes found in thyroid lesions are RET/PTC and PAX8/PPAR(gamma). The latter occurs in follicular thyroid carcinomas with a t(2;3)(q13;p25). Here we present molecular-cytogenetic and cytogenetic investigations on a follicular thyroid adenoma with a t(2;20;3)(p21;q11.2; p25). In this case, an intronic sequence of PPAR(gamma) is fused to exon 28 of THADA. We used BAC clones containing the genomic sequence of PPARgamma for fluorescence in situ hybridization to confirm the localization of the breakpoint within intron 2 of PPAR(gamma) . Our findings suggest that the close surrounding of PPAR(gamma) is a breakpoint hot spot region, leading to recurrent alterations of this gene in thyroid tumors of follicular origin including carcinomas as well as adenomas with or without involvement of PAX8.

A 3.4-kbp transcript of ZNF331 is solely expressed in follicular thyroid adenomas.

Translocations involving chromosomal region 19q13 are a frequent finding in follicular adenomas of the thyroid and might represent the most frequent type of structural aberration in human epithelial tumors. By positional cloning, a putative candidate gene, ZNF331 (formerly RITA) located close to the breakpoint was identified. Recently, aberrant expression of ZNF331 has been described in two cell lines of follicular thyroid adenomas with aberrations in 19q13 indicating an involvement of ZNF331 in tumorigenesis. Nevertheless, knowledge about structure and expression of ZNF331 is limited. We performed RACE-PCR and genomic sequence analyses to gain a deeper insight into its molecular structure. To elucidate ZNF331 expression patterns we performed Northern blot analyses on various normal tissues as well as on thyroid carcinoma and adenoma cell lines. Herein, unique expression of a 3.4-kbp transcript is described in thyroid adenoma cell lines with 19q13 aberrations, which was not detected either in normal tissues or in thyroid carcinoma cell lines.

Delineation of a 150-kb breakpoint cluster in benign thyroid tumors with 19q13.4 aberrations.

Structural rearrangements involving the long arm of chromosome 19 characterize a cytogenetic subgroup of benign thyroid tumors and constitute one of the most frequent specific chromosome abnormalities in epithelial tumors. Recently, we have been able to narrow down the breakpoint region affected in two cell lines to a region covered by a single PAC clone. Close to that region a candidate gene has been identified which we tentatively referred to as RITA (Rearranged In Thyroid Adenomas) now named ZNF331 according to HUGO nomenclature. However, the results had been obtained on two cell lines only making it necessary to extend the studies to a larger number of tumors including primary material. Herein, we have used four further primary tumors showing translocations involving 19q13 for fluorescence in situ hybridization (FISH) mapping studies using a variety of molecular probes from a 470-kbp cosmid/BAC contig. Ten new STSs were characterized and physically mapped within an EcoRI restriction map. The results enabled us to define an approximately 150-kbp breakpoint cluster region of the 19q13 aberrations in benign thyroid tumors flanked by two newly established STS markers.

Cytogenetic tetraclonality in a rare spindle cell variant of an anaplastic carcinoma of the thyroid.

We report a case of an unusual anaplastic carcinoma with spindle cell differentiation in an 85-year-old patient. Although the tumor showed sarcoma-like features its occurrence in the thyroid of an elderly person supported the diagnosis of an anaplastic carcinoma. This diagnosis is also supported by the results of cytogenetic studies that revealed four independent clones. Of these, three clones showed complex chromosomal rearrangements including translocations, deletions and inversions while the remaining clone only showed two balanced translocations. The patient is still alive after 13 months.

Molecular cytogenetic investigations define a subgroup of thyroid adenomas with 2p21 breakpoints clustered to a region of less than 450 kb.

Structural rearrangements involving chromosome band 2p21 characterize a cytogenetic subgroup of benign thyroid tumors. To narrow down the breakpoints of these aberrations, we established two cell lines from benign thyroid tumors showing translocations involving 2p21. These two cell lines and one additional primary tumor were used for FISH-studies with 18 BAC clones. All breakpoints were mapped to a cluster of about 450 kb.

Detection of Epstein-Barr virus in Hodgkin-Reed-Sternberg cells : no evidence for the persistence of integrated viral fragments inLatent membrane protein-1 (LMP-1)-negative classical Hodgkin's disease.

Classical Hodgkin's disease (HD) is associated with Epstein-Barr virus (EBV) infection. Although in developing countries EBV can be demonstrated in Hodgkin-Reed-Sternberg (H-RS) cells in up to 95% of HD cases, in industrialized countries only about 50% of HD cases are associated with EBV. An open question remains whether EBV in the EBV-negative cases has escaped detection by standard screening procedures due to deletions in the viral genome associated with integration of viral fragments into the host cell genome. We, among others, recently described this phenomenon in Burkitt's lymphoma cells. To investigate whether H-RS cells in latent membrane protein-1 (LMP-1)-negative HD cases harbor fragments of the EBV genome, we combined fluorescence in situ hybridization (FISH) using a set of six overlapping DNA probes spanning the whole EBV genome with immunophenotyping of fresh frozen lymphoma sections. Results in the eight cases analyzed were as follows: in three LMP-1-positive cases, FISH analysis yielded specific signals for each EBV DNA probe in H-RS cells, which had been identified by morphology and CD30 staining. In contrast, none of the EBV DNA probes hybridized to the H-RS cells in the five LMP-1-negative cases. Thus, there is no evidence for the presence of fragments of the viral genome integrated into the host cell genome in the LMP-1-negative cases. Furthermore, in the LMP-1-positive cases analyzed, no large deletions in the viral genome were detected. These results show that, in classical HD, LMP-1-negative cases do not harbor EBV DNA within the H-RS cells. Whether, in these cases, a still unknown virus contributes to the transformation and maintenance of the malignant phenotype remains to be established.

Structural abnormalities of chromosome 2 in benign thyroid tumors. Three new cases and review of the literature.

Here we report our cytogenetic findings on three cases of nodular goiter, all showing structural clonal abnormalities of chromosome 2. In the first case, we found a t(2;3)(q21;q27 or q28) in two nodules of the same patient. The second case revealed a t(2;20;3)(p21;q11.2;p25), and the third case showed a t(1;2)(p22;p13). When the data from the literature and the present cases are summarized, the results suggest the existence of at least three breakpoint clusters of chromosome 2 in benign thyroid tumors or hyperplasias.

A KRAB zinc finger protein gene is the potential target of 19q13 translocation in benign thyroid tumors.

In an attempt to identify the target gene of specific translocations involving chromosomal band 19q13 in benign follicular thyroid tumors, we have used two cell lines derived from benign thyroid tumors showing translocations with 19q13 breakpoints for fluorescence in situ hybridization mapping studies with cosmid and PAC clones located in a 400-kbp region. The breakpoints of the chromosome 19 abnormalities mapped within a 140-kb segment covered by a single PAC clone. Sequencing of part of this PAC clone allowed us to establish the cDNA sequence and the genomic structure of a candidate gene located in close vicinity to the breakpoints. The gene that we tentatively refer to as RITA (rearranged in thyroid adenomas) belongs to the KRAB zinc finger protein coding genes. From our results we have concluded that in the two cell lines investigated the breaks have occurred either within the 5' untranslated region of RITA or in its close 5' vicinity. By Northern blot analyses two transcripts of about 4.7 kbp and 5 kbp were detected in normal thyroid tissue as well as in other normal tissues tested. An additional 2.1-kbp transcript was found only in testicular tissue. In contrast to all normal tissues, both cell lines with 19q aberrations expressed larger transcripts of approximately 5.5 kbp and 6.2 kbp. From the close vicinity to the breakpoint region, the expression patterns of the gene, and its type, we consider RITA a strong candidate target gene of the specific 19q aberrations in benign thyroid tumors.

Involvement of the HMGI(Y) gene in a microfollicular adenoma of the thyroid.

Follicular thyroid adenomas are epithelial tumors characterized by subgroups with specific chromosomal aberrations. Here we present a case with translocation t(1;6)(p35;p21). In 1p35 and 6p21, two genes of the high-mobility group of proteins are located. With P1-derived Artificial Chromosome (PACs) derived from the HMG17 gene located at 1p35 and HMGI(Y) located at 6p21, fluorescence in situ hybridization was performed. The breakpoints were located distal to HMG17 and proximal to HMGI(Y), but no rearrangement of the genes was shown by FISH. However, an overexpression of the HMGI(Y) gene was detected by RT-PCR as well as by immunohistochemistry techniques. This suggests a breakpoint in the proximity of the HMGI(Y) deregulating HMGI(Y) gene expression, a situation found in a variety of human benign mesenchymal tumors with involvement of chromosome band 6p21.

Follicular thyroid carcinoma: chromosome analysis of 19 cases.

Short-term cultures of 19 follicular thyroid carcinomas were examined cytogenetically. Clonal chromosomal changes were detected in 12 tumors. Two follicular carcinomas had only numerical alterations: one with a hyperdiploid karyotype with trisomies/polysomies of chromosomes 7 and 12, similar to the karyotypes previously identified in a sub-group of benign thyroid lesions, and the other with monosomy 20. In the remaining ten cases several structural chromosome anomalies were found. Loss of the short arm of chromosome 3 was observed in one tumor. In two widely invasive and metastasizing follicular carcinomas there was a t(7;8)(p15;q24) as the sole abnormality in one case and a der(8)t(7;8)(p15;q24) together with other cytogenetic alterations in the other case. This finding suggests that t(7;8)(p15;q24) may be related to an aggressive behavior of follicular thyroid carcinomas.

Cytogenetic investigations of 340 thyroid hyperplasias and adenomas revealing correlations between cytogenetic findings and histology.

Cytogenetic analyses were performed on 340 follicular thyroid adenomas and goiters after short-term culture. Clonal chromosomal changes were found in 67 cases. Trisomy 7 as the sole abnormality or along with other trisomies was the most frequent type of aberration (19 cases). Other recurrent numerical changes were loss of chromosome 22 (4 cases) and the second X or the Y chromosome (5 cases). Translocations involving 19q13 (12 cases) were frequent structural chromosomal changes. Dicentric chromosomes or telomeric associations were frequent in goiters (12 cases). After a histopathologic classification of all cases, we have correlated the cytogenetic findings with the histology of the tumors. Only 8.4% of the goiters showed clonal abnormalities, whereas 44.9% of the adenomas revealed clonal abnormalities. Furthermore, simple clonal changes were predominantly found in goiters and complex changes in adenomas. The most impressive correlation was found in the group of lesions with trisomy 7. Although all but one lesion with one or two additional trisomies were goiters, those having three or more additional trisomies were all adenomas or adenomatous goiters.

Breakpoints of 19q13 translocations of benign thyroid tumors map within a 400 kilobase region.

Structural rearrangements involving the long arm of chromosome 19 characterize a cytogenetic subgroup of benign thyroid tumors. To localize the breakpoint of the 19q13 aberrations, we have established three cell lines derived from benign thyroid tumors showing translocations in this region. We have used these cell lines and four additional primary tumors with 19q13 abnormalities for fluorescence in situ hybridization (FISH) mapping studies with ten cosmid clones located between the molecular markers POLD1 and TNNT1. The breakpoints of all chromosome 19 abnormalities mapped within a 400 kb region.

[Genetic studies of differential fatty tissue tumor diagnosis]. / Genetische Untersuchungen zur Differentialdiagnose von Fettgewebstumoren.

Adipose tissue tumors are often characterized by typical or even specific chromosomal alterations. In some of the cases the molecular background of these microscopically visible alterations was already elucidated. In myxoid liposarcomas the translocation t(12;16) creates a fusion gene between the CHOP gene and the FUS gene and in lipomas the HMGI-C gene becomes rearranged by structural aberrations involving chromosomal region 12q14-15. Based on examples of a lipoma, a well-differentiated liposarcoma, a myxoid liposarcoma, and an aggressive angiomyxoma it is demonstrated in the present paper how cytogenetic investigation can be used as an additional tool for an improved diagnosis of adipose tissue tumors. Furthermore, the detection of molecular mechanisms underlying the visible cytogenetic alterations will certainly significantly increase our knowledge about the pathogenesis of these diseases.

Integration of Epstein-Barr virus in Burkitt's lymphoma cells leads to a region of enhanced chromosome instability.

BACKGROUND: Several lymphomas are associated with Epstein-Barr virus (EBV) infection. However EBV is not detectable in 100% of cases using standard staining techniques. It still remains an open question whether in these EBV-negative cases EBV has never infected the cell, whether it has infected the cell and escapes conventional screening methods, or whether it has been lost again after initial infection. MATERIALS AND METHODS: The physical status of EBV in the Burkitt's lymphoma cell line BL60-P7 as well as in three somatic cell hybrids between BL60-P7 and its autologous EBV-immortalized lymphoblastoid cell line IARC 277 was analyzed using conventional cytogenetics, Southern blotting, and fluorescence in situ hybridization (FISH). RESULTS: Integration of EBV into the host genome of the lymphoma cell line BL60-P7 leads to an achromatic gap which causes a 'vulnerable site'. In hybrid cells, loss of integrated EBV, together with an adjacent chromosomal fragment, occurs during long-term cultivation. The integrated EBV genome, including genes encoding for LMP and EBER, is partly deleted. CONCLUSION: We assume that integration of EBV into the host cell genome could be a more common event in lymphoma cells. Partially deleted EBV might escape standard detection assays. The integration might constitute a chromosomal region prone to break events akin to the phenomenon of fragile sites, leading to the loss of viral DNA as well as chromosomal DNA. This observation makes it tempting to speculate that under certain conditions EBV can act in lymphomagenesis by a so-called hit-and-run mechanism.

[Cytogenetic changes in benign thyroid gland hyperplasias and adenomas correlated with histology]. / Zytogenetische Veränderungen bei benignen Schilddrüsenhyperplasien und Adenomen korrelieren mit der Histologie.

In order to elucidate cytogenetic changes associated with the development of benign growth of follicular epithelial cells of the thyroid, cytogenetic analyses were performed on 340 follicular thyroid adenomas and goiters after short-term culture. Clonal chromosome changes were found in 67 cases. Trisomy 7 as the sole abnormality or along with other trisomies was the most frequent type of aberration (19 cases). Other recurrent numerical changes were loss of chromosome 22 (4 cases) and the second X or the Y chromosome (5 cases). Translocations involving 19q13 (12 cases) were frequent structural chromosome changes. After a histopathological classification of all cases, we have correlated the cytogenetic findings with the histology of the tumors. Only 8.4% of the goiters showed clonal abnormalities whereas 44.9% of the adenomas revealed clonal abnormalities. Furthermore, simple clonal changes were predominantly found in goiters and complex changes in adenomas. The most impressive correlation was found in the group of lesions with trisomy 7: Whereas all but one lesion with one or two additional trisomies were goiters, those having three or more additional trisomies were all adenomas or adenomatous goiters.

Deletions of the short arm of chromosome 2 characterize a new cytogenetic subgroup of benign thyroid tumors.

Cytogenetic studies of thyroid hyperplasias and adenomas have shown that besides cases with an apparently normal karyotype different groups of cytogenetic abnormalities exist. Herein we describe the cytogenetic analyses of two benign thyroid tumors with deletions of the short arm of chromosome 2. A similar case has been described previously. Besides the previously well-established subgroups, alterations of chromosome 2 may thus characterize a new cytogenetic subgroup of benign thyroid tumors.