Methylation profiling identified novel differentially methylated markers including <i>OPCML</i> and <i>FLRT2</i> in prostate cancer

<p>To develop new methods to distinguish indolent from aggressive prostate cancers (PCa), we utilized comprehensive high-throughput array-based relative methylation (CHARM) assay to identify differentially methylated regions (DMRs) throughout the genome, including both CpG island (CGI) and non-CGI regions in PCa patients based on Gleason grade. Initially, 26 samples, including 8 each of low [Gleason score (GS) 6] and high (GS ≥7) grade PCa samples and 10 matched normal prostate tissues, were analyzed as a discovery cohort. We identified 3,567 DMRs between normal and cancer tissues, and 913 DMRs distinguishing low from high-grade cancers. Most of these DMRs were located at CGI shores. The top 5 candidate DMRs from the low vs. high Gleason comparison, including <i>OPCML, ELAVL2, EXT1, IRX5</i>, and <i>FLRT2</i>, were validated by pyrosequencing using the discovery cohort. <i>OPCML</i> and <i>FLRT2</i> were further validated in an independent cohort consisting of 20 low-Gleason and 33 high-Gleason tissues. We then compared patients with biochemical recurrence (n=70) vs. those without (n=86) in a third cohort, and they showed no difference in methylation at these DMR loci. When GS 3+4 cases and GS 4+3 cases were compared, <i>OPCML</i>-DMR methylation showed a trend of lower methylation in the recurrence group (n=30) than in the no-recurrence (n=52) group. We conclude that whole-genome methylation profiling with CHARM revealed distinct patterns of differential DNA methylation between normal prostate and PCa tissues, as well as between different risk groups of PCa as defined by Gleason scores. A panel of selected DMRs may serve as novel surrogate biomarkers for Gleason score in PCa.</p

Identification of differentially methylated markers among cytogenetic risk groups of acute myeloid leukemia

<div><p>Aberrant DNA methylation is known to occur in cancer, including hematological malignancies such as acute myeloid leukemia (AML). However, less is known about whether specific methylation profiles characterize specific subcategories of AML. We examined this issue by using comprehensive high-throughput array-based relative methylation analysis (CHARM) to compare methylation profiles among patients in different AML cytogenetic risk groups. We found distinct profiles in each group, with the high-risk group showing overall increased methylation compared with low- and mid-risk groups. The differentially methylated regions (DMRs) distinguishing cytogenetic risk groups of AML were enriched in the CpG island shores. Specific risk-group associated DMRs were located near genes previously known to play a role in AML or other malignancies, such as <i>MN1, UHRF1, HOXB3</i>, and <i>HOXB4</i>, as well as <i>TRIM71</i>, the function of which in cancer is not well characterized. These findings were verified by quantitative bisulfite pyrosequencing and by comparison with results available at the TCGA cancer genome browser. To explore the potential biological significance of the observed methylation changes, we correlated our findings with gene expression data available through the TCGA database. The results showed that decreased methylation at <i>HOXB3</i> and <i>HOXB4</i> was associated with increased gene expression of both <i>HOXB</i> genes specific to the mid-risk AML, while increased DNA methylation at <i>DCC</i> distinctive to the high-risk AML was associated with increased gene expression. Our results suggest that the differential impact of cytogenetic changes on AML prognosis may, in part, be mediated by changes in methylation.</p></div

A Three-Marker FISH Panel Detects More Genetic Aberrations of <i>AR</i>, <i>PTEN</i> and <i>TMPRSS2/ERG</i> in Castration-Resistant or Metastatic Prostate Cancers than in Primary Prostate Tumors

<div><p><i>TMPRSS2</i>/<i>ERG</i> rearrangement, <i>PTEN</i> gene deletion, and androgen receptor (<i>AR</i>) gene amplification have been observed in various stages of human prostate cancer. We hypothesized that using these markers as a combined panel would allow better differentiation between low-risk and high-risk prostate cancer. We analyzed 110 primary prostate cancer samples, 70 metastatic tumor samples from 11 patients, and 27 xenograft tissues derived from 22 advanced prostate cancer patients using fluorescence in situ hybridization (FISH) analysis with probes targeting the <i>TMPRSS2</i>/<i>ERG</i>, <i>PTEN</i>, and <i>AR</i> gene loci. Heterogeneity of the aberrations detected was evaluated. Genetic patterns were also correlated with transcript levels. Among samples with complete data available, the three-marker FISH panel detected chromosomal abnormalities in 53% of primary prostate cancers and 87% of metastatic (Met) or castration-resistant (CRPC) tumors. The number of markers with abnormal FISH result had a different distribution between the two groups (<i>P</i><0.001). At the patient level, Met/CRPC tumors are 4.5 times more likely to show abnormalities than primary cancer patients (<i>P</i><0.05). Heterogeneity among Met/CRPC tumors is mostly inter-patient. Intra-patient heterogeneity is primarily due to differences between the primary prostate tumor and the metastases while multiple metastatic sites show consistent abnormalities. Intra-tumor variability is most prominent with the <i>AR</i> copy number in primary tumors. <i>AR</i> copy number correlated well with the <i>AR</i> mRNA expression (rho = 0.52, <i>P</i><0.001). Especially among <i>TMPRSS2:ERG</i> fusion-positive CRPC tumors, <i>AR</i> mRNA and <i>ERG</i> mRNA levels are strongly correlated (rho = 0.64, <i>P</i><0.001). Overall, the three-marker FISH panel may represent a useful tool for risk stratification of prostate cancer patients.</p></div

Summary of intratumoral heterogeneity.

1<p>Number of tumors in sample.</p>2<p>Median for analyzed tissue.</p><p>Predicted values and covariance parameter estimates are from linear mixed models predicting copy number by tumor type, with random patient effects and separate covariance parameter estimates (within-patient heterogeneity and measurement error) for each tumor type.</p

The three-marker FISH panel including <i>TMPRSS2</i>/<i>ERG</i> rearrangements, <i>AR</i> gene amplification, and <i>PTEN</i> gene deletion.

<p>(A) Illustration of the 4-color FISH technique for the detection of rearrangements of <i>TMPRSS2</i> and/or <i>ERG</i>. FISH probes target 5′-<i>TMPRSS2</i> (red, probe I), 3′-<i>TMPRSS2</i> (green, probe II), 5′-<i>ERG</i> (gold, probe III), and 3′-<i>ERG</i> (blue, probe IV) simultaneously, detecting various signal patterns including normal (i), single fusion(ii), dual/complex fusion(iii), alternative rearrangement without fusion (iv), and copy number increase(CNI) without rearrangements. Captured FISH images of (i) and (ii) are shown in the left panel of 1C; images of (iii) – (v) are shown below the corresponding illustration. (B) FISH probes used to detect <i>AR</i> gene amplification and <i>PTEN</i> gene deletion <i>AR</i> gene amplification was analyzed using probes targeting <i>AR</i> (orange) and the X-chromosome centromere (green, CEPX). <i>PTEN</i> gene deletion was detected using probes targeting <i>PTEN</i> (orange) and the chromosome 10 centromere (green, CEP10). (C) Representative interphase FISH images. Top left, normal <i>TMPRSS2</i> and <i>ERG</i> signal pattern demonstrating two sets of the four probes per nucleus; Bottom left, <i>TMPRSS2</i>: <i>ERG</i> fusion shown as juxtaposed red and blue signals concurrent with missing or separation of the interstitial green and gold signals; Top middle, normal <i>AR</i> signal pattern demonstrating one orange <i>AR</i> and one green X signal per nucleus; Bottom middle, <i>AR</i> gene amplification presenting more than twice the number of <i>AR</i> signals than the CEPX signals; Top right, normal <i>PTEN</i> signal pattern demonstrating 2 orange <i>PTEN</i> and 2 green CEP10 signals per nucleus; Bottom right, <i>PTEN</i> deletion showing none or 1 copy of <i>PTEN</i> signals per nucleus.</p

Prevalence of abnormalities detected by each FISH marker among the primary and the metastatic or castration-resistant prostate cancer (Met/CRPC) patients.

1<p>Average <i>AR</i> per nucleus ≥1.5 but <2.</p>2<p>Average <i>AR</i> per nucleus ≥2 but average AR/X ratio<2.</p>3<p>Average <i>AR/</i>X ratio≥2.</p>4<p>Average <i>PTEN</i>/CEP10 ratio≤0.75 but >0.2.</p>5<p>Average <i>PTEN</i>/CEP10 ratio≤0.2.</p>6<p>Wald tests of abnormal vs. normal for primary vs. CRPC, generalized estimating equations (GEE) with independence autocorrelation, adjusting for rapid autopsy vs xenograft sample for CRPC. Likelihood ratio test for AR (without adjustment for autocorrelation), since no primary samples had abnormal AR.</p>7<p>Wald tests of abnormal vs. normal for primary vs. CRPC, logistic regression adjusting for rapid autopsy vs xenograft sample for CRPC. Likelihood ratio test for AR, since no primary samples had abnormal AR.</p

Within-patient heterogeneity of <i>AR</i> and <i>PTEN</i>/CEP10 for rapid autopsy patients (n = 11).

<p>Each tumor’s FISH result is represented by a plotting character (grey for metastatic lesions, red for prostate) with multiple lesions in the same patient at the same X coordinate. Confidence intervals for subject-specific average copy number values are shown in black. Thresholds for abnormal signals are marked as horizontal dashed lines on each plot. (A) Average number of <i>AR</i> per nucleus. (B) Average <i>PTEN</i>/<i>CEP10</i> ratio.</p

FISH data of individual castration resistant metastatic patient tumors.

<p>Only samples successfully hybridized with at least one marker were presented in the table, including 56 tumors with <i>TMPRSS2</i>/<i>ERG</i> FISH, 65 tumors with <i>AR</i> FISH, and 62 tumors with <i>PTEN</i> FISH results.</p

FISH data of individual xenograft tumors.

†<p>Xenograft discontinued.</p>*<p>Xenograft derived from patient #9 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074671#pone-0074671-t002" target="_blank">Table 2</a>.</p>**<p>Xenograft derived from a patient with localized prostate cancer.</p><p>Only samples successfully hybridized with at least one marker were presented in the table, including 23 with <i>TMPRSS2</i>/<i>ERG</i> FISH, 26 with <i>AR</i> FISH, and 25 xenografts with <i>PTEN</i> FISH results.</p

The prevalence of genetic aberrations detected by the panel.

<p>A patient with multiple tumors was considered abnormal by a given marker if the aberration was seen in at least one tumor. (A) Pie charts demonstrate the percentage of individuals with no (white), one (light grey), two (dark grey), and three (black) abnormalities detected by the panel among the primary prostate cancer (n = 34) and the metastatic or castration resistant prostate cancer (Met/CRPC) cohort (n = 30), respectively. Among primary patients, an asterisk was used to highlight moderate AR gain (average <i>AR</i> per nucleus > = 1.5 but <2). (B-D) Prevalence of each subtype of abnormalities detected by individual FISH marker among the primary patients (one tumor per patient, n = 34), Met/CRPC tumors (n = 81), and Met/CRPC patients/xenografts (n = 30), respectively. <i>TMPRSS2</i>/<i>ERG</i> abnormalities are categorized as single fusion (light blue), dual/complex fusion (dark blue), alternative rearrangements (green), and copy number increase (CNI) of the normal gene alleles (yellow). <i>AR</i> FISH detected moderate <i>AR</i> gain (light blue), gain of X (dark blue) and <i>AR</i> gene amplification (green). <i>PTEN</i> FISH abnormalities includes heterozygous (light blue) and homozygous (green) <i>PTEN</i> deletions. (E) The co-occurrence of abnormalities in the three markers shown as 3D sphere plots for the primary cancer cohort (left) and the Met/CRPC cohort (right). <i>TMPRSS2</i>/<i>ERG</i>, <i>PTEN</i>, and <i>AR</i> results are presented on X, Y, and Z axes, respectively. The value presented on each axis ranges from 0 to 1. “0” denotes normal for a given marker. For <i>TMPRSS2</i>/<i>ERG</i>, “0.5” indicates rearrangements, including fusion and alternative rearrangements; “1” means CNI of the normal alleles without any rearrangement. For <i>PTEN</i> FISH, both heterozygous and homozygous deletions are presented as “1”. For <i>AR</i> FISH, “1” indicates <i>AR</i> copy number gain (> = 2.0). Patients with the same combination of abnormalities are clustered into a sphere, the volume of which is proportional to the percentage of patients in the respective cohort. Only patients with available data from all three markers are included. The green sphere in the primary patient plot denotes moderate <i>AR</i> gain (>1.5 but <2.0).</p