Christina Koop

MALDI mass spectrometric imaging based identification of clinically relevant signals in prostate cancer using large-scale tissue microarrays.

To identify molecular features associated with clinico‐pathological parameters and TMPRSS2‐ERG fusion status in prostate cancer, we employed MALDI mass spectrometric imaging (MSI) to a prostate cancer tissue microarray (TMA) containing formalin‐fixed, paraffin‐embedded tissues samples from 1,044 patients for which clinical follow‐up data were available. MSI analysis revealed 15 distinct mass per charge (m/z)‐signals associated to epithelial structures. A comparison of these signals with clinico‐pathological features revealed statistical association with favorable tumor phenotype such as low Gleason grade, early pT stage or low Ki67 labeling Index (LI) for four signals (m/z 700, m/z 1,502, m/z 1,199 and m/z 3,577), a link between high Ki67LI for one signal (m/z 1,013) and a relationship with prolonged time to PSA recurrence for one signal (m/z 1,502; p = 0.0145). Multiple signals were associated with the ERG‐fusion status of our cancers. Two of 15 epithelium‐associated signals including m/z 1,013 and m/z 1,502 were associated with detectable ERG expression and five signals (m/z 644, 678, 1,044, 3,086 and 3,577) were associated with ERG negativity. These observations are in line with substantial molecular differences between fusion‐type and non‐fusion type prostate cancer. The signals observed in this study may characterize molecules that play a role in the development of TMPRSS2‐ERG fusions, or alternatively reflect pathways that are activated as a consequence of ERG‐activation. The combination of MSI and large‐scale TMAs reflects a powerful approach enabling immediate prioritization of MSI signals based on associations with clinico‐pathological and molecular data.

Clinical significance of different types of p53 gene alteration in surgically treated prostate cancer

Despite a multitude of p53 immunohistochemistry (IHC) studies, data on the combined effect of nuclear p53 protein accumulation and TP53 genomic inactivation are lacking for prostate cancer. A tissue microarray including 11,152 prostate cancer samples was analyzed by p53 IHC and fluorescence in situ hybridization. Nuclear p53 accumulation was found in 10.1% of patients including 1.4% with high‐level and 8.7% with low‐level immunostaining. TP53 sequencing revealed that 17 of 22 (77%) cases with high‐level p53 immunostaining, but only 3% (1 of 31) low‐level p53 cases carried putative dominant‐negative mutations. TP53 deletions occurred in 14.8% of cancers. Both deletions and protein accumulation were linked to unfavorable tumor phenotype and prostate specific antigen (PSA) recurrence (p < 0.0001 each). The combination of both methods revealed subgroups with remarkable differences in their clinical course. Tumors with either TP53 deletion (14%) or low‐level p53 positivity (8.7%) had identical risks of PSA recurrence, which were markedly higher than in cancers without p53 alterations (p < 0.0001). Tumors with both p53 deletion and low‐level p53 positivity (1.5%) had a worse prognosis than patients with only one of these alterations (p < 0.0001). Tumors with strong p53 immunostaining or homozygous inactivation through deletion of one allele and disrupting translocation involving the second allele had the worst outcome, independent from clinical and pathological parameters. These data demonstrate a differential clinical impact of various TP53 alterations in prostate cancer. Strong p53 immunostaining—most likely accompanying dominant negative or oncogenic p53 mutation—has independent prognostic relevance and may thus represent a clinical useful molecular feature of prostate cancer.

TMPRSS2-ERG Fusions Are Strongly Linked to Young Patient Age in Low-grade Prostate Cancer

Based on next-generation sequencing of early-onset prostate cancer (PCa), we earlier demonstrated that PCa in young patients is prone to rearrangements involving androgen-regulated genes-such as transmembrane protease, serine 2 (TMPRSS2)-v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) fusion-and provided data suggesting that this situation might be caused by increased androgen signaling in younger men. In the same study, an accumulation of chromosomal deletions was found in cancers of elderly patients. To determine how age-dependent molecular features relate to cancer phenotype, an existing data set of 11,152 PCas was expanded by additional fluorescence in situ hybridization analyses of phosphatase and tensin homolog (PTEN), 6q15 and 5q21. The results demonstrate that the decrease in TMPRSS2-ERG fusions with increasing patient age is limited to low-grade cancers (Gleason ≤3+4) and that the significant increase in the deletion frequency with age was strictly limited to ERG-negative cancers for 6q15 and 5q21 but to ERG-positive cancers for PTEN. These data suggest that the accumulation of non-androgen-linked genomic alterations with advanced patient age may require an appropriate microenvironment, such as a positive or negative ERG status. The strong link of ERG activation to young patient age and low-grade cancers may help to explain a slight predominance of low-grade cancers in young patients.

High lysophosphatidylcholine acyltransferase 1 expression independently predicts high risk for biochemical recurrence in prostate cancers

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) has been suggested to play a role in cancer. To assess its role in prostate cancer, LPCAT1 expression was analyzed on a tissue microarray containing samples from 11,152 prostate cancer patients. In benign prostate glands, LPCAT1 immunostaining was absent or weak. In prostate cancer, LPCAT1 positivity was found in 73.8% of 8786 interpretable tumors including 29.2% with strong expression. Increased LPCAT1 expression was associated with advanced tumor stage (pT3b/T4) (p < 0.0001), high Gleason score (≥4 + 4) (p < 0.0001), positive nodal involvement (p = 0.0002), positive surgical margin (p = 0.0005), and early PSA recurrence (p < 0.0001). High LPCAT1 expression was strongly linked to ERG‐fusion type prostate cancer. Strong LPCAT1 staining was detected in 45.3% of ERG positive but in only 16.7% of ERG negative tumors (p < 0.0001). Within ERG negative cancers, LPCAT1 staining was strongly increased within the subgroup of PTEN deleted cancers (p < 0.0001). Further subgroup analyses revealed that associations of high LPCAT1 expression with PSA recurrence and unfavorable tumor phenotype were largely driven by ERG negative cancers (p < 0.0001) while these effects were substantially mitigated in ERG positive cancers (p = 0.0073). The prognostic impact of LPCAT1 expression was independent of histological and clinical parameters. It is concluded, that LPCAT1 measurement, either alone or in combination, may be utilized for better clinical decision‐making. These data also highlight the potentially important role of lipid metabolism in prostate cancer biology.

The prognostic impact of high Nijmegen breakage syndrome (NBS1) gene expression in ERG-negative prostate cancers lacking PTEN deletion is driven by KPNA2 expression

The Nijmegen breakage syndrome (NBS1) gene was suggested as a prostate cancer susceptibility gene. This study was undertaken to determine, whether NBS1 expression is linked to clinically or molecularly relevant subgroups of prostate cancer. NBS1 expression was analyzed by immunohistochemistry on a tissue microarray containing 11,152 prostate cancer specimens. NBS1 expression was absent or only weakly detectable in benign prostate. In prostate cancers, NBS1 expression was found in 81.3% of interpretable tumors and was considered strong in 41.3% of cases. NBS1 upregulation was tightly linked to ERG‐positive cancers (p < 0.0001). Within ERG‐negative cancers, strong NBS1 immunostaining was linked to advanced pathological tumor stage, high Gleason grade, and positive nodal status (p < 0.0001 each), while high NBS1 immunostaining was only weakly associated with advanced pathological tumor stage in ERG‐positive cancers (p = 0.0099). A comparison with chromosomal deletions revealed a strong NBS1 upregulation in PTEN‐deleted cancers, while deletions of 3p13, 5q21 and 6q15 did not affect NBS1 expression. High NBS1 expression was linked to biochemical recurrence in ERG‐negative and PTEN non‐deleted cancers (p < 0.0001), which was largely driven by high KPNA2 karyopherin alpha 2 expression. In conclusion, our study identifies an association of NBS1 expression with surrogates of genomic instability in prostate cancer including TMPRSS2‐ERG rearrangements and PTEN deletion. The prognostic impact of NBS1 expression in ERG‐negative, PTEN non‐deleted cancers was dependent of the expression status of its interaction partner KPNA2.

Cytoplasmic accumulation of Sequestosome 1 (p62) is a predictor of biochemical recurrence, rapid tumor cell proliferation and genomic instability in prostate cancer

Purpose: Sequestosome 1 (p62) is a multifunctional adapter protein accumulating in autophagy-defective cells. Experimental Design: To evaluate the clinical impact and relationship with key genomic alterations in prostate cancer, p62 protein levels were analyzed by immunohistochemistry on a tissue microarray containing 12,427 prostate cancers. Data on ERG status and deletions of PTEN, 3p13, 5q21, and 6q15 were available from earlier studies. Results: p62 immunostaining was absent in benign prostatic glands but present in 73% of 7,822 interpretable prostate cancers. Strong cytoplasmic p62 staining was tightly linked to high Gleason grade, advanced pathologic tumor (pT) stage, positive nodal status, positive resection margin, and early PSA recurrence (P < 0.0001 each). Increased levels of p62 were significantly linked to TMPRSS2–ERG fusions, both by FISH and immunohistochemical analysis (P < 0.0001 each). For example, moderate or strong p62 immunostaining was seen in 28.5% of cancers with TMPRSS2–ERG fusion detected by FISH and in 23.1% of cancers without such rearrangements (P < 0.0001). Strong p62 staining was significantly linked to the presence of all tested deletions, including PTEN (P < 0.0001), 6q15 (P < 0.0001), 5q21 (P = 0.0002), 3p13 (P = 0.0088), and 6q15 (P < 0.0001), suggesting a link between p62 accumulation and loss of genomic stability. The prognostic role of p62 protein accumulation was striking and independent of Gleason grade, pT stage, pN stage, surgical margin status, and preoperative PSA, regardless of whether preoperative or postoperative parameters were used for modeling. Conclusions: Our study identifies cytoplasmic accumulation of p62 as a strong predictor of an adverse prognostic behavior of prostate cancer independently from established clinicopathologic findings. Clin Cancer Res; 21(15); 3471–9. ©2015 AACR.

High mitochondria content is associated with prostate cancer disease progression

BackgroundMitochondria are suggested to be important organelles for cancer initiation and promotion. This study was designed to evaluate the prognostic value of MTC02, a marker for mitochondrial content, in prostate cancer.MethodsImmunohistochemistry of using an antibody against MTC02 was performed on a tissue microarray (TMA) containing 11,152 prostate cancer specimens. Results were compared to histological phenotype, biochemical recurrence, ERG status and other genomic deletions by using our TMA attached molecular information.ResultsTumor cells showed stronger MTC02 expression than normal prostate epithelium. MTC02 immunostaining was found in 96.5% of 8,412 analyzable prostate cancers, including 15.4% tumors with weak, 34.6% with moderate, and 46.5% with strong expression. MTC02 expression was associated with advanced pathological tumor stage, high Gleason score, nodal metastases (p < 0.0001 each), positive surgical margins (p = 0.0005), and early PSA recurrence (p < 0.0001) if all cancers were jointly analyzed. Tumors harboring ERG fusion showed higher expression levels than those without (p < 0.0001). In ERG negative prostate cancers, strong MTC02 immunostaining was linked to deletions of PTEN, 6q15, 5q21, and early biochemical recurrence (p < 0.0001 each). Moreover, multiple scenarios of multivariate analyses suggested an independent association of MTC02 with prognosis in preoperative settings.ConclusionsOur study demonstrates high-level MTC02 expression in ERG negative prostate cancers harboring deletions of PTEN, 6q15, and 5q21. Additionally, increased MTC02 expression is a strong predictor of poor clinical outcome in ERG negative cancers, highlighting a potentially important role of elevated mitochondrial content for prostate cancer cell biology.

HDAC1 overexpression independently predicts biochemical recurrence and is associated with rapid tumor cell proliferation and genomic instability in prostate cancer.

Histone deacetylases (HDACs) play an important role in tumor development and progression by modifying histone and non-histone proteins. In the current study we analyzed prevalence and prognostic impact of HDAC1 in prostate cancer. HDAC1 expression was analyzed by immunohistochemistry on a tissue microarray containing more than 12,400 prostate cancer specimens. Results were compared to tumor phenotype, biochemical recurrence, and molecular subtypes defined by ERG status as well as genomic deletions of 3p, 5q, 6q and PTEN. HDAC1 immunostaining was detectable in 75.4% of 9744 interpretable cancers and considered strong in 15.4%, moderate in 39.4% and weak in 20.7% of cases. High HDAC1 expression was associated with high Gleason grade (p<0.0001), advanced pathological tumor stage (p<0.0001), positive nodal status (p=0.0010), elevated preoperative PSA-level (p=0.0127), early PSA recurrence (p<0.0001) and increased cell proliferation (p<0.0001). Moreover, high-level HDAC1 staining was associated with TMPRSS2:ERG rearrangement and ERG expression in prostate cancers (p<0.0001) and was linked to deletions of PTEN (p<0.0001), 6q (p<0.0001) and 5q (p=0.0028) in ERG-negative cancers. The prognostic impact of HDAC1 was independent of established clinicopathological parameters and was mostly driven by ERG-negative cancers as revealed by subgroup analyses. HDAC1 has strong prognostic impact in prostate cancer and might contribute to the development of a fraction of genetically instable and particularly aggressive prostate cancers. HDAC1 measurement might therefore be of clinical value for risk stratification of prostate cancer and should be further evaluated in this regard.

The Combination of DNA Ploidy Status and PTEN/6q15 Deletions Provides Strong and Independent Prognostic Information in Prostate Cancer

Purpose: Aberrant DNA content has been discussed as a potential prognostic feature in prostate cancer. Experimental Design: We analyzed the clinical significance of DNA ploidy in combination with prognostic relevant deletions of PTEN and 6q15 in 3,845 prostate cancers. Result: The DNA status was diploid in 67.8%, tetraploid in 25.6%, and aneuploid in 6.8% of tumors, and deletions of PTEN and 6q15 occurred in 17.8% and 20.3% of tumors. Abnormal DNA content and deletions were linked to high Gleason score, advanced tumor stage, and positive nodal stage (P < 0.0001 each). The risk of PSA recurrence increased from diploid to tetraploid and from tetraploid to aneuploid DNA status (P < 0.0001 each). However, 40% of patients with Gleason score ≥4+4 and 55% of patients with PSA recurrence had diploid cancers. This fraction decreased to 21% (Gleason ≥4+4) and 29% (PSA recurrence) if PTEN and/or 6q deletion data were added to ploidy data to identify cancers with an aberrant DNA status. The significance of combining both deletions and ploidy was further demonstrated in a combined recurrence analysis. Presence of deletions increased the risk of PSA recurrence in diploid (P < 0.0001), tetraploid (P < 0.0001), and aneuploid cancers (P = 0.0049), and the combination of ploidy data and deletions provided clinically relevant information beyond the CAPRA-S nomogram. Multivariate modeling including preoperatively and postoperatively available parameters identified the “combined DNA status” as a strong independent predictor of poor patient outcome. Conclusions: The combinatorial DNA content analysis involving general (ploidy) and specific events (deletions) has the potential for clinical utility in prostate cancer. Clin Cancer Res; 22(11); 2802–11. ©2016 AACR.

Overexpression of enhancer of zeste homolog 2 (EZH2) characterizes an aggressive subset of prostate cancers and predicts patient prognosis independently from pre- and postoperatively assessed clinicopathological parameters

Enhancer of zeste homolog 2 (EZH2) plays an important role in tumor development and progression by interacting with histone and nonhistone proteins. In the current study, we analyzed prevalence and prognostic impact of EZH2 in prostate cancer. EZH2 expression was analyzed by immunohistochemistry on a tissue microarray containing more than 12400 prostate cancer specimens. Results were compared to tumor phenotype, biochemical recurrence and molecular subtypes defined by ERG status as well as genomic deletions of 3p, 5q, 6q and PTEN. EZH2 immunostaining was detectable in 56.6% of 10168 interpretable cancers and considered strong in 1.1%, moderate in 12.2% and weak in 43.3% of cases. High EZH2 expression was strongly associated with high Gleason grade (P < 0.0001), advanced pathological tumor stage (P < 0.0001), positive nodal status (P < 0.0001), elevated preoperative PSA level (P = 0.0066), early PSA recurrence (P < 0.0001) and increased cell proliferation P < 0.0001). High-level EZH2 staining was also associated with TMPRSS2:ERG rearrangement and ERG expression in prostate cancers (P < 0.0001) and was linked to deletions of PTEN, 6q15, 5q21 and 3p13 (P < 0.0001 each) particularly in ERG-negative cancers. The prognostic impact of EZH2 was independent of established pre- and postoperatively assessed clinicopathological parameters. EZH2 has strong prognostic impact in prostate cancer and might contribute to the development of a fraction of genetically instable and particularly aggressive prostate cancers. EZH2 analysis might therefore be of clinical value for risk stratification of prostate cancer.