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.

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.

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.

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.

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.

Cytogenetic biclonality corresponding to multiphasic differentiation in an atypical thyroid adenoma.

Cytogenetic aberrations have been described in about 30% of benign thyroid tumors, but their role for tumorigenesis or progression has not yet been elucidated. We describe the cytogenetic analyses in a thyroid adenoma with two different clonal cytogenetic stemlines: 45,XX,der(1)t(1;14)(p13;q11.2-q(13),t(5;12)(q11.2;q24),del(9)(q12),- 10,der(11)t(11;?;19)(p15;q13),der(14)t(14;15)(q11.2-q13;q23),del(15)(q23 ), der(15)t(9;15)(q12;p10),der(19)t(10;19)(q11.2;q13)/46,X,?inv(x),?inv(3) (p21q29),t(3;8)(q26;q12). Histologic examination revealed an atypical follicular thyroid adenoma containing microfollicular, follicular, trabecular-solid, and oncocytic components. There may be a direct relation between the different cytogenetic stemlines and the histologic diversity of the tumor. Thyroid tumors with complex karyotypes involving the 19q13 breakpoint may represent advanced stages of karyotypic evolution and therefore warrant an extensive clinical follow-up.

Aberrations of chromosome 19. Do they characterize a subtype of benign thyroid adenomas?

We describe the cytogenetic findings in two benign thyroid hyperplasias with aberrations of chromosome 19. In the first patient, two of four nodules showed identical translocations involving chromosome 19 and 22: 46,XX,der(19)t(19;?)(q13;?),der(22)t(22;?)(q12;?), the remaining nodules had an apparently normal karyotype. Two nodules from a second patient were karyotyped. One showed a karyotype 46,XX,t(1;19)(p35-36.1;q13) and the other had a normal karyotype. From these results as well as those reported previously, we can conclude that structural changes of chromosome 19 characterize a subgroup of thyroid adenomas, thyroid hyperplasias, or both.

Deletion of part of the long arm of chromosome 13 as the only karyotypic aberration in a follicular thyroid adenoma.

A follicular thyroid adenoma is described showing a del(13)(q14) as the only karyotypicabnormality. The karyotypic similarities to another benign tumor, the lipoma, are discussed.

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.

Cytogenetic investigations on a cell line derived from a carcinoma arising in a salivary gland pleomorphic adenoma.

This article discusses the results of cytogenetic investigations of a cell line derived from a malignant tumor arising in a benign salivary gland pleomorphic adenoma. Initial karyotypic studies became possible with cells of the second in vitro passage and revealed the existence of hypertetraploidy. Furthermore, a marked polysomy 7 and a translocation involving 12q13-15, a breakpoint also frequently seen in benign pleomorphic adenomas, were noteworthy. During long term culture, the number of chromosomes per cell decreased as well as the number of copies of chromosome 7. Cell tumorigenicity was proved by heterotransplantation to nude mice. The resulting tumors were karyotyped again. No significant changes of the chromosome number or of the degree of polysomy 7 were found compared to the cells before heterotransplantation. In contrast, the cells from the nude mice tumors showed a remarkable number of different isochromosomes probably indicating an unknown factor supporting the generation of isochromosomes. The consistent presence of the t(12;?)(q13-15;?) makes the cell line well suited for molecular studies of this breakpoint region.

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.

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.

Mapping of the translocation breakpoints of primary pleomorphic adenomas and lipomas within a common region of chromosome 12.

Recent molecular cytogenetic analysis of uterine leiomyoma cell lines with chromosomal aberrations of 12q14-q15 have indicated that the chromosome 12 breakpoints cluster in a 445-kb region designated ULCR12 (uterine leiomyoma cluster region of the chromosome 12 breakpoints). Here we report the results of FISH studies of five primary pleomorphic adenomas and six primary lipomas and established cell lines of these tumor types characterized by translocations involving the chromosomal segment 12q13-q15. The results reveal that for nearly all tumors and cell lines analyzed, the chromosome 12 breakpoints map within a 350-kb region included in ULCR12, despite the previous cytogenetic assignment of the breakpoints to different bands of that region. In some cases the primary material and additionally analyzed cell lines allowed an even more precise localization of the breakpoints to less than 100 kb. Furthermore, a previously hidden translocation of ULCR12 in one primary tumor could be detected by FISH.

[Ischemic cardiopathies and psychologic pattern in three male cohorts (author's transl)]. / Cardiopathies ischémiques et profil psychologique dans trois cohortes masculines. Données de prévalence.

Three surveys have been completed on 3.202 male subjects aged 40-59 yr. according to a common protocole; mean ages are 48,3 +/- 10,8 in Brussels-Ghent, 48,5 +/- 8,9 in Marseilles and 43,9 +/- 1,7 in Paris. A higher prevalence of coronary heart disease (CHD) was observed in Brussels (18,2%) as compared to Marseilles (10,5%) and Paris (5,8%). Psychological pattern has been defined according to the answers on 2 questionnaires: a French adaptation of the Bortner Scale and a combination of the questionnaire of Sandler and Hazari with the Eysenck Personality Inventory (S.H.-E.P.I.). The data suggest that the psychological pattern is independent of the other coronary risk factors. In this retrospective study, the score of neuroticism is significantly higher in subjects with CHD (angina, myocardial infarction or coronary insufficiency on the ECG), as compared to normal controls. The score on the Bortner Scale being highly correlated with the score of neuroticism, the former disappears in the discriminant function analysis. The psychological variables do not discriminate normal subjects from those with CHD according to their ECG, but without clinical symptoms of angina. In this study, neuroticism and less so obsessionality has a discriminating power between "coronary free" and "non-free" subjects. This power disappears in the absence of clinical symptoms (angina). As the 3 cohorts are followed during at least 5 years, the predictive power of neuroticism for CHD will be examined in a prospective manner.

Inhibition of caspase-3 differentially affects vascular smooth muscle cell apoptosis in the concave versus convex aortic sites in ascending aneurysms with a bicuspid aortic valve.

Apoptosis of vascular smooth muscle cells (VSMCs) is involved in bicuspid aortic valve (BAV) ascending aorta aneurysms characteristically affecting the convex site. Caspase-3 is a pivotal effector of the apoptosis machinery. The aim of this study was to investigate the impact of an inhibited caspase-3 pathway on apoptosis in convex and concave sites VSMCs of ascending aortic tissue in vitro. Specimens from the convex and concave sites of ascending aortic aneurysm were collected from nine patients with BAV (mean age 58.7+/-14.8). Cultured VSMCs were characterized morphologically and immunohistochemically. Apoptosis activity was measured in VSMCs using Annexin V-APC with propidium iodide nuclear staining in flow cytometry. To investigate apoptotic modulation, caspase-3 was inhibited by N-acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO). Apoptosis was initiated by calcium chloride. Inhibition of caspase-3 with Ac-DEVD-CHO protected VSMCs against calcium chloride apoptosis significantly more in the concave site than in the convex site (25.8+/-9.8 versus 38.5+/-8.0% apoptotic cells, p=0.01). Morphological scanning using light microscopy revealed typical VSMCs. We provide evidence that VSMCs show a different behavior with respect to apoptosis in the concave versus the convex sites in BAV ascending aortic aneurysm. Inhibition of caspase-3 resulted in a significantly increased protection of VSMCs against apoptosis in the concave site compared with the convex site in ascending aortic aneurysm in BAV. These findings may have some implications on understanding aneurysmal formation and its potential modulation.

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.

Quantification of the refrigerants R22 and R134a in mixtures by means of different polymers and reflectometric interference spectroscopy.

The aim of this study was the quantification of vapors of the ozone-depleting refrigerant R22 in the presence of its most important substitute R134a, by the use of the reflectometric interference spectroscopy and polymers as sensitive layers. First, the sorption characteristic of different types of polymers exposed to the vapors of the two analytes was investigated. Then, binary mixtures of the two refrigerants were measured with an array set-up on the basis of six polymer sensors. The measurements were evaluated by the use of neural networks, whereby low limits of detection of 0.45 percentage volume (vol. %)for R22 and 1.45 vol. % for R134a could be established. Additionally, one polar polymer and one microporous polymer were selected for the measurements with a low-cost set-up. The quantification of R22 in the presence of R134a with this low-cost set-up was possible with a limit of detection of 0.44 vol. %, which would enable a fast and economical monitoring at recycling stations.

FISH analyses of a newly established thyroid tumor cell line showing a t(1;19)(p35 or p36.1;q13) reveal that the breakpoint lies between 19q13.3-13.4 and 19q13.4.

Translocations involving the long arm of human chromosome 19 are specific chromosomal alterations found in a subset of benign thyroid tumors. In the present communication we describe the establishment of an SV40-transformed cell line derived from a thyroid adenoma with a t(1;19)(p35 or p36.1;q13). Cytogenetic studies were carried out on this cell line, using a series of cosmids mapping along the long arm of chromosome 19. In situ hybridization with probes for RYR1, ATP1A3, BCKDHA, ERCC1, POLD1, and TRPT revealed that only TRPT mapped distal to the breakpoint of the translocation. The results indicated that the breakpoint is located between POLD1 and TRPT at the boundary between chromosome bands 19q13.3 and 19q13.4.

A characteristic sequence of trisomies starting with trisomy 7 in benign thyroid tumors.

The cytogenetic results from a series of 113 thyroid hyperplasias and adenomas are reported; 15 showed clonal karyotypic alterations. In addition to a group showing translocations involving 19q13, another subset of lesions characterized by polysomies can be found. Based on our own cases belonging to this subset and a review of the cases reported in the literature, we conclude that the characteristic feature of this group is a sequence that always starts with trisomy 7, but that sometimes even leads to chromosome numbers in the hypertriploid range. This subset of thyroid tumors may be an example of a more common genetic pathway in human solid tumors.