Global 5-Hydroxymethylcytosine Levels Are Profoundly Reduced in Multiple Genitourinary Malignancies

<div><p>Solid tumors are characterized by a plethora of epigenetic changes. In particular, patterns methylation of cytosines at the 5-position (5mC) in the context of CpGs are frequently altered in tumors. Recent evidence suggests that 5mC can get converted to 5-hydroxylmethylcytosine (5hmC) in an enzymatic process involving ten eleven translocation (TET) protein family members, and this process appears to be important in facilitating plasticity of cytosine methylation. Here we evaluated the global levels of 5hmC using a validated immunohistochemical staining method in a large series of clear cell renal cell carcinoma (n = 111), urothelial cell carcinoma (n = 55) and testicular germ cell tumors (n = 84) and matched adjacent benign tissues. Whereas tumor-adjacent benign tissues were mostly characterized by high levels of 5hmC, renal cell carcinoma and urothelial cell carcinoma showed dramatically reduced staining for 5hmC. 5hmC levels were low in both primary tumors and metastases of clear cell renal cell carcinoma and showed no association with disease outcomes. In normal testis, robust 5hmC staining was only observed in stroma and Sertoli cells. Seminoma showed greatly reduced 5hmC immunolabeling, whereas differentiated teratoma, embryonal and yolk sack tumors exhibited high 5hmC levels. The substantial tumor specific loss of 5hmC, particularly in clear cell renal cell carcinoma and urothelial cell carcinoma, suggests that alterations in pathways involved in establishing and maintaining 5hmC levels might be very common in cancer and could potentially be exploited for diagnosis and treatment.</p></div

Global loss of 5hmC in primary renal cell carcinoma and metastases.

<p>(A) Normal kidney tissue shows uniformly strong staining for 5hmC in tubules and glomerular cells. (B) Clear cell renal cell carcinoma exhibit greatly reduced 5hmC levels (arrows). Note that stromal and endothelial cells show labeling with 5hmC antibodies. (C) Boxplots illustrating H-score distribution in normal kidney tissue, primary renal cell carcinoma and metastases. All images were taken at original magnification of 200x. Scale bars indicate 50 μm.</p

Global 5hmC levels in normal testis and testicular neoplasms.

<p>(A) Sertoli cells (arrowheads) show strong immunoreactivity in normal testicular tubules. (B) Low 5hmC staining in intratubular germ cell neoplasia (ITGCN) of the testis (arrows). (C) Representative seminoma case with low nuclear 5hmC staining. (D) Representative mature teratoma exhibiting robust nuclear staining. Representative embryonal carcinoma (E) and yolk sack tumor (F) exhibiting variable degrees of 5hmC staining levels in neoplastic cell nuclei. (G) Boxplots summarizing staining distribution in testicular germ cell tumors (seminoma n = 48; embryonal carcinoma; n = 24, yolk sac tumor n = 16; teratoma n = 16) (TGCT) lesions. Note that in tumors with mixed morphologies, components were scored separately. All images were taken at original magnification of 200x. Scale bars indicate 50 μm.</p

Urothelial carcinoma of the bladder shows low 5hmC levels.

<p>(A) Normal transitional cell epithelium of the bladder shows strong nuclear 5hmC staining in apical cell layers; basal cell layers exhibit reduced staining intensities (arrowhead). (B) Urothelial carcinoma shows reduced nuclear 5hmC (arrow). Note that tumor-associated stromal cells show robust strong staining and function as an internal staining control. (C) Boxplots depicting H-score distribution in normal urothelium and urothelial carcinoma. All images were taken at original magnification of 200x. Scale bars indicate 50 μm.</p

Cumulative incidence of metastasis at 5 years post radical prostatectomy.

<p>A) Difference in re-graded 2005 ISUP modified and original Gleason score; B) Difference in primary re-graded 2005 ISUP modified and original Gleason score; and C) Difference in secondary re-graded 2005 ISUP modified and original Gleason score.</p

(A) Fused glands (400x). In the original Gleason grading this pattern was considered pattern 3. According to the 2005 ISUP Gleason system it would be graded as pattern 4; B) Adenocarcinoma with glomeruloid features currently assigned a Gleason pattern 4 (100x); C) Ill-defined glands (100x). This pattern would be graded as Gleason pattern 4 by the ISUP 2005 Gleason grading; and D) Individual cells (100x).

<p>This pattern was originally accepted under Gleason pattern 3 and would be assigned a Gleason pattern 5 according to the 2005 ISUP Gleason system.</p

Cumulative incidence curves of A) metastasis by original Gleason score, B) metastasis by reviewed Gleason score.

<p>The number at risk in each group is shown in the footnote lines. Patients in the sub-cohort who do not experience the event are weighted by the inverse of the sampling fraction in following the case-cohort design.</p

Study diagram and patient selection criteria.

<p>Study diagram and patient selection criteria.</p

A) Survival concordance index. Reviewed Gleason score by the 2005 ISUP modified Gleason system has the highest c-index compared to original Gleason score. B) Decision curve analysis at 5 years post radical prostatectomy shows the net benefit of original and reviewed Gleason score across probability thresholds.

<p>The reviewed Gleason score shows the highest net benefit.</p