Serum and CSF microRNAs in paediatric malignant GCTs

BACKGROUND: The current biomarkers alpha-fetoprotein and human chorionic gonadotropin have limited sensitivity and specificity for diagnosing malignant germ-cell tumours (GCTs). MicroRNAs (miRNAs) from the miR-371-373 and miR-302/367 clusters are overexpressed in all malignant GCTs, and some of these miRNAs show elevated serum levels at diagnosis. Here, we developed a robust technical pipeline to quantify these miRNAs in the serum and cerebrospinal fluid (CSF). The pipeline was used in samples from a cohort of exclusively paediatric patients with gonadal and extragonadal malignant GCTs, compared with appropriate tumour and non-tumour control groups. METHODS: We developed a method for miRNA quantification that enabled sample adequacy assessment and reliable data normalisation. We performed qRT-PCR profiling for miR-371-373 and miR-302/367 cluster miRNAs in a total of 45 serum and CSF samples, obtained from 25 paediatric patients. RESULTS: The exogenous non-human spike-in cel-miR-39-3p and the endogenous housekeeper miR-30b-5p were optimal for obtaining robust serum and CSF qRT-PCR quantification. A four-serum miRNA panel (miR-371a-3p, miR-372-3p, miR-373-3p and miR-367-3p): (i) showed high sensitivity/specificity for diagnosing paediatric extracranial malignant GCT; (ii) allowed early detection of relapse of a testicular mixed malignant GCT; and (iii) distinguished intracranial malignant GCT from intracranial non-GCT tumours at diagnosis, using CSF and serum samples. CONCLUSIONS: The pipeline we have developed is robust, scalable and transferable. It potentially promises to improve clinical management of paediatric (and adult) malignant GCTs.Grant funding was from CwCUK/GOSHCC (M.J. Murray, K.L. Raby, J.C. Nicholson, N. Coleman), SPARKS (M.J. Murray, J.C. Nicholson, N. Coleman), AstraZeneca (E. Bell, H. Brown and B. Destenaves), CRUK (N. Coleman), MRC (M.J. Murray) and an Erasmus MC MRACE grant (M.A. Rijlaarsdam). The authors also thank the Max Williamson Fund and The Perse Preparatory School, Cambridge for supporting this study.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/bjc.2015.42

Comparing genome-scale DNA methylation and CNV marks between adult human cultured ITGA6+ testicular cells and seminomas to assess in vitro genomic stability

Autologous transplantation of spermatogonial stem cells is a promising new avenue to restore fertility in infertile recipients. Expansion of the initial spermatogonial stem cell pool through cell culturing is a necessary step to obtain enough cells for effective repopulation of the testis after transplantation. Since in vitro propagation can lead to (epi-)genetic mutations and possibly malignant transformation of the starting cell population, we set out to investigate genome-wide DNA methylation status in uncultured and cultured primary testicular ITGA6+ sorted cells and compare them with germ cell tumor samples of the seminoma subtype. Seminomas displayed a severely global hypomethylated profile, including loss of genomic imprinting, which we did not detect in cultured primary testicular ITGA6+ cells. Differential methylation analysis revealed altered regulation of gamete formation and meiotic processes in cultured primary testicular ITGA6+ cells but not in seminomas. The pivotal POU5F1 marker was hypomethylated in seminomas but not in uncultured or cultured primary testicular ITGA6+ cells, which is reflected in the POU5F1 mRNA expression levels. Lastly, seminomas displayed a number of characteristic copy number variations that were not detectable in primary testicular ITGA6+ cells, either before or after culture. Together, the data show a distinct DNA methylation patterns in cultured primary testicular ITGA6+ cells that does not resemble the pattern found in seminomas, but also highlight the need for more sensitive methods to fully exclude the presence of malignant cells after culture and to further study the epigenetic events that take place during in vitro culture

A pipeline to quantify serum and cerebrospinal fluid microRNAs for diagnosis and detection of relapse in paediatric malignant germ-cell tumours

Background:The current biomarkers alpha-fetoprotein and human chorionic gonadotropin have limited sensitivity and specificity for diagnosing malignant germ-cell tumours (GCTs). MicroRNAs (miRNAs) from the miR-371-373 and miR-302/367 clusters are overexpressed in all malignant GCTs, and some of these miRNAs show elevated serum levels at diagnosis. Here, we developed a robust technical pipeline to quantify these miRNAs in the serum and cerebrospinal fluid (CSF). The pipeline was used in samples from a cohort of exclusively paediatric patients with gonadal and extragonadal malignant GCTs, compared with appropriate tumour and non-tumour control groups.Methods:We developed a method for miRNA quantification that enabled sample adequacy assessment and reliable data normalisation. We performed qRT-PCR profiling for miR-371-373 and miR-302/367 cluster miRNAs in a total of 45 serum and CSF samples, obtained from 25 paediatric patients.Results:The exogenous non-human spike-in cel-miR-39-3p and the endogenous housekeeper miR-30b-5p were optimal for obtaining robust serum and CSF qRT-PCR quantification. A four-serum miRNA panel (miR-371a-3p, miR-372-3p, miR-373-3p and miR-367-3p): (i) showed high sensitivity/specificity for diagnosing paediatric extracranial malignant GCT; (ii) allowed early detection of relapse of a testicular mixed malignant GCT; and (iii) distinguished intracranial malignant GCT from intracranial non-GCT tumours at diagnosis, using CSF and serum samples.Conclusions:The pipeline we have developed is robust, scalable and transferable. It potentially promises to improve clinical management of paediatric (and adult) malignant GCTs

Comparing genome-scale DNA methylation and CNV marks between adult human cultured ITGA6+ testicular cells and seminomas to assess in vitro genomic stability

Autologous transplantation of spermatogonial stem cells is a promising new avenue to restore fertility in infertile recipients. Expansion of the initial spermatogonial stem cell pool through cell culturing is a necessary step to obtain enough cells for effective repopulation of the testis after transplantation. Since in vitro propagation can lead to (epi-)genetic mutations and possibly malignant transformation of the starting cell population, we set out to investigate genome-wide DNA methylation status in uncultured and cultured primary testicular ITGA6+ sorted cells and compare them with germ cell tumor samples of the seminoma subtype. Seminomas displayed a severely global hypomethylated profile, including loss of genomic imprinting, which we did not detect in cultured primary testicular ITGA6+ cells. Differential methylation analysis revealed altered regulation of gamete formation and meiotic processes in cultured primary testicular ITGA6+ cells but not in seminomas. The pivotal POU5F1 marker was hypomethylated in seminomas but not in uncultured or cultured primary testicular ITGA6+ cells, which is reflected in the POU5F1 mRNA expression levels. Lastly, seminomas displayed a number of characteristic copy number variations that were not detectable in primary testicular ITGA6+ cells, either before or after culture. Together, the data show a distinct DNA methylation patterns in cultured primary testicular ITGA6+ cells that does not resemble the pattern found in seminomas, but also highlight the need for more sensitive methods to fully exclude the presence of malignant cells after culture and to further study the epigenetic events that take place during in vitro culture

Functional enrichment of DMPs.

<p>DMPs were classified according to their functional genomic location (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">Fig 1C</a>). Statistical over- and underrepresentation of probes in certain categories provides clues to differences between GCT subtypes in regarding function of methylation. Enrichment was assessed by comparing the number of probes in a functional category in a subset of DMPs with the that in the total dataset (Fisher’s Exact test, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#sec011" target="_blank">Materials & Methods</a> section). Results are shown for four pairwise (A vs B) comparisons of histological subtypes: <b>(A)</b> SE/DG versus EC/mNS; <b>(B)</b> SE/DG vs type I TE; <b>(C)</b> EC/MNS vs type I TE and <b>(D)</b> SE/DG vs SS. <b>(LEFT)</b> The number (n) of DMPs identified in either the DMP[<u><b>A</b></u>-B] (hypermethylated in A, green) or DMP[A-<u><b>B</b></u>] (hypermethylated in B, red) group. <b>(MIDDLE/RIGHT)</b> Functional enrichment in the DMP[<u><b>A</b></u>-B] and DMP[A-<u><b>B</b></u>] group respectively. X-axis: positive numbers indicate a significant overrepresentation of DMPs in a functional category compared to non-DMPs while negative numbers indicate a significant underrepresentation. Depicted is the log2 ratio of (1) the % of either DMP group assigned to a category and (2) the % of non-DMPs assigned to that category. Only significant enrichments are depicted (2-sided Fisher’s Exact test, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#sec011" target="_blank">Methods</a> section for Bonferroni corrected α threshold). DMPs[se/dgvs<u><b>SS</b></u>].IMPR_P1500 showed significant underrepresentation, but could not be plotted on log scale (0 probes in DMP group). Details of calculations and raw counts and percentages are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.s008" target="_blank">S2 Table</a>. Y-axis: functional categories as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">Fig 1C</a>.</p

Methylation patterns in GCT subtypes and cell lines.

<p>To illustrate differences in methylation status between histological GCT subtypes two (visualization) methods were applied. Firstly, the methylation pattern over the whole genome and specific functional categories (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">Fig 1C</a>) is visualized using the distribution of the methylation percentage β in all samples of a certain GCT subtype. Next, the discriminatory power of the methylation pattern for each individual sample is shown using principal component analysis. <b>(A) Distribution of methylation percentage.</b> Violin plots: grey areas indicate a kernel density plot of the methylation percentage (β) of all probes in all samples in a certain category. The boxplot indicates the interquartile range (black bars) and median (white squares). X-axis labels indicate histological subgroup according to Fig <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">1B</a>. TE indicates type I TE only. <b>(B) Principal Component Analysis.</b> The first two principal components (PC) are plotted to evaluate the discriminative power of the methylation pattern between the subtypes. Abbreviations of histological subtypes are explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">Fig 1A</a>. CL indicates cell lines. Please note that in the legend of the PCA the TE group is subdivided based on gender and localization: I = type I; II = type II/formally part of the mNS group, s = sacrum, t = testis, o = ovary, m = male, f = female. A more detailed visualization of the TE classes is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.s002" target="_blank">S2 Fig</a>, which also includes the full series of 18 functional categories, bootstrap validation of the PCA and an estimation of the variance explained by the first two principal components.</p

Methylation profile at GCT subtype specific differentially methylated regions (DMRs).

<p>Visualization of the methylation percentage at specific loci is used to zoom in on a predefined region and investigate local methylation differences between GCT subtypes. <b>(A) <i>DMRT3</i>, (B) <i>SOX2</i>, (C) <i>POU5F1</i> (<i>OCT3/4</i>), (D) <i>TEX14</i>. (Visualizations)</b> From top to bottom the following is depicted: (1) Four-color heat map indicating methylation % for each individual probe in the depicted region. For the sample groups specified on the left the median methylation % is shown. (2) Position of all probes in the region of interest (ROI) is annotated as black rectangles. (3) HMM segments are displayed as grey boxes spanning the segment’s width and grouped per state. Numbers indicate the state of each (group of) segment(s). (5) GC% was obtained from the UCSC genome browser database (gc5Base table). (6) Transcripts overlapping with the ROI are plotted at the bottom. Plot generated using the Gviz package. Abbreviations of histological subtypes are explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">Fig 1A</a>. Please note that the TE group is subdivided based on gender and localization: I = type I; II = type II/formally part of the mNS group, s = sacrum, t = testis, o = ovary, m = male, f = female. CL indicates cell lines.</p

GCT methylation status in context of methylation during germ cell development.

<p>The top and bottom line charts depict normal germ cell development in female and male respectively (stages specified in the middle black bar). Methylation status during normal germ cell development is depicted for the global genome, ICRs and chromosome X (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#sec010" target="_blank">Discussion</a>). Putative cells of origin of the various types of GCTs are indicated in the brown boxes. ICR_P/M = ICR regulating paternally/maternally expressed genes. Bimodal indicates a methylation pattern peaking 0 and 100% with the exception of SE/DG (between 0 and ≈50). The table (bottom) provides a summary of the results, mainly Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g006" target="_blank">6</a>. Abbreviations: pf = primordial follicle. Type I tumors are indicated with their type (I), sex (m = male, f = female) and location (s = sacral, t = testis, o = ovary). Other GCT subtypes are indicated with their type (I, II, IV) and the abbreviation of each histological class, which are explained in the main text. Gradient bars indicate percentages of methylation (0→100%, green-white-grey-red) analogous to the gradient used in the other figures.</p

Methylation of imprinting control regions and the X chromosome.

<p>Analogous to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g002" target="_blank">Fig 2</a> the differences in methylation status between histological GCT subtypes is illustrated by two methods. Firstly, the methylation pattern is visualized using the distribution of the methylation percentage β. Next, the discriminatory power of the methylation pattern for each individual sample is shown using principal component analysis. <b>(A)</b> All probes associated with paternally expressed genes (ICR_P). <b>(B)</b> All probes associated with maternally expressed genes (ICR_M). <b>(C)</b> All probes located on the X chromosome. <b>(D)</b> Distribution of methylation in individual TE samples ordered by sex and localization. To compare type I and II TE the n = 3 type II pure TEs from the mNS were included in this visualization. Methylation levels of all probes, and probes associated with ICRs (P/M) and probes on the X chromosome are subsequently shown. <b>(Distribution plots of methylation percentage.)</b> Violin plots: grey areas indicate a kernel density plot of the methylation percentage (β) of all probes in all samples in a certain category. The boxplot indicates the interquartile range (black bars) and median (white squares). X-axis labels indicate histological subgroup according to Fig <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">1B</a>. TE indicates type I TE only. (<b>Principal Component Analysis.)</b> The first two principal components (PC) are plotted to evaluate the discriminative power of the methylation pattern between the subtypes. Abbreviations of histological subtypes are explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">Fig 1A</a>. CL indicates cell lines. Please note that in the legend of the PCA the TE group is subdivided based on gender and localization: I = type I; II = type II/formally part of the mNS group, s = sacrum, t = testis, o = ovary, m = male, f = female.</p