Gunes Guner

18F-DCFBC PET/CT for PSMA-Based Detection and Characterization of Primary Prostate Cancer

We previously demonstrated the ability to detect metastatic prostate cancer using N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-18F-fluorobenzyl-l-cysteine (18F-DCFBC), a low-molecular-weight radiotracer that targets the prostate-specific membrane antigen (PSMA). PSMA has been shown to be associated with higher Gleason grade and more aggressive disease. An imaging biomarker able to detect clinically significant high-grade primary prostate cancer reliably would address an unmet clinical need by allowing for risk-adapted patient management. Methods: We enrolled 13 patients with primary prostate cancer who were imaged with 18F-DCFBC PET before scheduled prostatectomy, with 12 of these patients also undergoing pelvic prostate MR imaging. Prostate 18F-DCFBC PET was correlated with MR imaging and histologic and immunohistochemical analysis on a prostate-segment (12 regions) and dominant-lesion basis. There were no incidental extraprostatic findings on PET suggestive of metastatic disease. Results: MR imaging was more sensitive than 18F-DCFBC PET for detection of primary prostate cancer on a per-segment (sensitivities of up to 0.17 and 0.39 for PET and MR imaging, respectively) and per-dominant-lesion analysis (sensitivities of 0.46 and 0.92 for PET and MR imaging, respectively). However, 18F-DCFBC PET was more specific than MR imaging by per-segment analysis (specificities of 0.96 and 0.89 for PET and MR imaging for corresponding sensitivity, respectively) and specific for detection of high-grade lesions (Gleason 8 and 9) greater than 1.0 mL in size (4/4 of these patients positive by PET). 18F-DCFBC uptake in tumors was positively correlated with Gleason score (ρ = 0.64; PSMA expression, ρ = 0.47; and prostate-specific antigen, ρ = 0.52). There was significantly lower 18F-DCFBC uptake in benign prostatic hypertrophy than primary tumors (median maximum standardized uptake value, 2.2 vs. 3.5; P = 0.004). Conclusion: Although the sensitivity of 18F-DCFBC for primary prostate cancer was less than MR imaging, 18F-DCFBC PET was able to detect the more clinically significant high-grade and larger-volume tumors (Gleason score 8 and 9) with higher specificity than MR imaging. In particular, there was relatively low 18F-DCFBC PET uptake in benign prostatic hypertrophy lesions, compared with cancer in the prostate, which may allow for more specific detection of primary prostate cancer by 18F-DCFBC PET. This study demonstrates the utility of PSMA-based PET, which may be used in conjunction with MR imaging to identify clinically significant prostate cancer.

Utility of uroplakin II expression as a marker of urothelial carcinoma

Uroplakins are markers of terminally differentiated urothelium. Uroplakin II (UPII) is a newly described sensitive marker for urothelial carcinoma (UC). The expression profile of UPII in different types of UC and its utility in the diagnostic setting are needed. We evaluated UPII expression in bladder tissue microarrays, including urothelial neoplasm of low malignant potential (n = 8), low-grade papillary UC (n = 72), noninvasive high-grade papillary UC (n = 77), UC in situ (n = 27), and invasive high-grade UC (INVUC) (n = 122). UPII expression in 52 breast carcinomas and 38 high-grade prostate adenocarcinomas was also assessed. UPII expression was compared with GATA binding protein 3 (GATA3) and estrogen receptor for its role in facilitating the differential diagnosis of the above 3 types of malignancy. UPII labeling was seen in 83.0% of UC overall, including 95.7% of noninvasive UC and 65.6% of INVUC. UPII labeling was not found in any breast and prostate carcinomas. In comparison, GATA3 labeling was seen in 91.6% of all UCs, including 96.4% of noninvasive UCs and 85.1% of INVUC, with stronger intensity and extent compared with UPII (P < .005). GATA3 labeled 2 (5%) of 38 high-grade prostate adenocarcinoma. Estrogen receptor nuclear labeling was seen in 13.0% of UCs and 12.5% of prostate carcinomas. UPII was highly specific (100%) but only moderately sensitive for UC and can therefore be a potentially useful marker to identify urothelial lineage and help distinguish UC from prostate cancer or, in conjunction with GATA3, from metastatic breast cancer.

High prevalence of TERT promoter mutations in primary squamous cell carcinoma of the urinary bladder

TERT promoter mutations (TERT-mut) are detectable in the majority of urothelial carcinomas. The detection of TERT-mut in urine is under investigation as a potential urine-based molecular-screening assay for bladder cancer. A small but significant number of bladder carcinomas are pure squamous cell carcinoma. We sought to assess the incidence of TERT-mut in squamous cell carcinoma of the urinary bladder. A retrospective search of the institutional pathology archives yielded 15 cystectomy specimens performed for squamous cell carcinoma (2000–2014). Histologic slides were reviewed by a senior urologic pathologist to confirm the diagnosis and select a representative formalin-fixed paraffin-embedded tissue block for mutational analysis. All cases yielded adequate material for DNA analysis. Sequencing for TERT-mut was performed using previously described SafeSeq technique. We detected TERT-mut in 12/15 (80%) of bladder squamous cell carcinomas. TERT promoter mutations, commonly found in conventional urothelial carcinoma, are also highly prevalent in urinary bladder squamous cell carcinoma suggesting a common tumorigenesis and potential utility as a molecular urine-based-screening assay.

Detection of TERT promoter mutations in primary adenocarcinoma of the urinary bladder.

TERT promoter mutations (TERT-mut) have been detected in 60% to 80% of urothelial carcinomas. A molecular urine-based screening assay for the detection of TERT-mut is currently being pursued by our group and others. A small but significant number of bladder carcinomas are adenocarcinoma. The current study assesses the incidence of TERT-mut in primary adenocarcinomas of urinary bladder. A retrospective search of our institutional pathology records identified 23 cystectomy specimens with a diagnosis of adenocarcinoma (2000-2014). All slides were reviewed by a senior urologic pathologist to confirm tumor type and select a representative formalin-fixed, paraffin-embedded block for mutational analysis. Adequate material for DNA testing was available in 14 cases (7 enteric type and 7 not otherwise specified). TERT-mut sequencing analysis was performed using previously described SafeSeq technique. Overall, 28.5% of primary adenocarcinoma harbored TERT-mut. Interestingly, 57% of nonenteric adenocarcinomas were mutation positive, whereas none of the enteric-type tumors harbored mutations. Similar to urothelial carcinoma, we found a relatively higher rate of TERT-mut among nonenteric-type adenocarcinomas further supporting the potential utility of TERT-mut urine-based screening assay for bladder cancer.

Absence of cytomegalovirus in glioblastoma and other high-grade gliomas by real-time PCR, immunohistochemistry, and in situ hybridization

Purpose: Reports of cytomegalovirus (CMV) detection in high-grade gliomas (HGG)/glioblastoma have been conflicting. We undertook a comprehensive approach to determine the presence or absence of CMV in tissue, plasma, and serum of HGG patients. Experimental Design: In a retrospective arm, 25 fresh frozen tissues from glioblastoma patients were tested for CMV by real-time PCR. Tissue microarrays from 70 HGG patients were tested by IHC and 20 formalin-fixed paraffin-embedded (FFPE) glioblastoma tissues by IHC and chromogenic in situ hybridization (CISH), targeting CMV-encoded IE1/2 and pp65. In a prospective arm, 18 patients with newly diagnosed HGG provided tissue and blood samples. Results: All retrospectively collected tissues were negative for CMV by all methods. In the prospective cohort, 18 patients with newly diagnosed HGG provided blood samples at the time of diagnosis and during follow-up. Of 38 plasma specimens, CMV DNA was detected in 3 of 18 samples at baseline and 1 of 20 follow-up samples. Serum CMV IgG was positive in 8 of 15 (53%) of patients. Among the FFPE samples tested in the prospective arm, all were negative for CMV by IHC, CISH, and PCR. Conclusions: Utilizing 6 highly sensitive assays with three orthogonal technologies on multiple specimens and specimen types, no evidence for CMV in glioblastoma tissues was found. Our findings call for multicenter blinded analyses of samples collected from different geographical areas with agreed upon study designs and determination of causality or lack thereof of CMV in HGG/glioblastoma for future guidance on the necessary antiviral and/or CMV-based therapies. Clin Cancer Res; 23(12); 3150–7. ©2016 AACR.

The utility of STAT6 and ALDH1 expression in the differential diagnosis of solitary fibrous tumor versus prostate-specific stromal neoplasms.

Solitary fibrous tumor (SFT) diagnosis in prostate can be challenging on small biopsies. Prostatic stromal tumors of unknown malignant potential (STUMP) and SFT have overlapping features. NAB2-STAT6 gene fusions that were recently identified in various SFTs lead to nuclear translocalization of STAT6. Nuclear STAT6 immunostaining is now considered an adjunct for SFT diagnosis. We evaluated STAT6 and an emerging stemness marker, ALDH1, in the differential diagnosis of SFT versus prostatic stromal lesions. Sixteen STUMPs, 12 SFTs, and 4 prostatic stromal sarcomas (12 needle biopsies, 13 radical prostatectomies, 7 transurethral resections) were retrieved (1995-2015). Sections were stained with polyclonal STAT6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA; S20, 1:100) and monoclonal ALDH1 antibody (BD Biosciences, San Jose, CA; clone 44, 1:250). In STAT6 cases, only unequivocal nuclear staining (with/without cytoplasmic staining) was considered positive. Cytoplasmic ALDH1 staining was counted positive. Ten of 11 evaluable SFTs demonstrated strong and diffuse nuclear STAT6 positivity; 4 of 16 STUMPs had nuclear staining that was weak (1/4) or focal (1/4). ALDH1 positivity was seen in 10 of 12 evaluable SFTs and 3 of 15 STUMPs. Prostatic stromal sarcomas were STAT6 negative (4/4); 2 of 4 were ALDH1 positive. The sensitivity and specificity for STAT6 for the diagnosis of SFT were 91% and 75%, respectively. Coexpression of STAT6 and ALDH1 yielded the same sensitivity but improved the specificity (100%) for the diagnosis of SFT. STAT6 is a useful marker in the differential diagnosis of SFT versus STUMP. Using STAT6 and ALDH1 together increases specificity. STUMPs can show STAT6 positivity, and when they do, it is likely to be weak or focal.

Circulating Tumor DNA as a Marker of Therapeutic Response in Patients With Renal Cell Carcinoma: A Pilot Study

Currently, no serum biomarkers are available for renal cell carcinoma (RCC). Circulating tumor DNA (ctDNA) has been shown to correlate with advanced disease and the response to therapy in several solid malignancies; however, little is known about the presence of ctDNA in patients with RCC. We characterized the presence of ctDNA in patients with locally advanced and metastatic RCC. Next-generation sequencing using a panel of 203 candidate genes identified tumor-specific mutations in the 4 queried tumor specimens. One of the identified mutations per patient was then selected and queried in the plasma. Serum polymerase chain reaction detected mutant ctDNA in 1 patient with metastatic disease, with a substitution mutation resulting in a splice site donor of VHL. In this patient, the ctDNA burden decreased after nephrectomy and the initiation of systemic therapy but increased with disease progression, indicating the potential utility of ctDNA as a marker of tumor response in select patients with RCC patients. Future directions will work to optimize the detection of ctDNA in patients with metastatic RCC.

Novel Assay to Detect RNA Polymerase I Activity In Vivo

This report develops an analytically validated chromogenic in situ hybridization (CISH) assay using branched DNA signal amplification (RNAscope) for detecting the expression of the 5′ external transcribed spacer (ETS) of the 45S ribosomal (r) RNA precursor in formalin-fixed and paraffin-embedded (FFPE) human tissues. 5′ETS/45S CISH was performed on standard clinical specimens and tissue microarrays (TMA) from untreated prostate carcinomas, high-grade prostatic intraepithelial neoplasia (PIN), and matched benign prostatic tissues. Signals were quantified using image analysis software. The 5′ETS rRNA signal was restricted to the nucleolus. The signal was markedly attenuated in cell lines and in prostate tissue slices after pharmacologic inhibition of RNA polymerase I (Pol I) using BMH-21 or actinomycin D, and by RNAi depletion of Pol I, demonstrating validity as a measure of Pol I activity. Clinical human prostate FFPE tissue sections and TMAs showed a marked increase in the signal in the presumptive precursor lesion (high-grade PIN) and invasive adenocarcinoma lesions (P = 0.0001 and P = 0.0001, respectively) compared with non-neoplastic luminal epithelium. The increase in 5′ETS rRNA signal was present throughout all Gleason scores and pathologic stages at radical prostatectomy, with no marked difference among these. This precursor rRNA assay has potential utility for detection of increased rRNA production in various tumor types and as a novel companion diagnostic for clinical trials involving Pol I inhibition. Implications: Increased rRNA production, a possible therapeutic target for multiple cancers, can be detected with a new, validated assay that also serves as a pharmacodynamic marker for Pol I inhibitors. Mol Cancer Res; 15(5); 577–84. ©2017 AACR.

Abstract LB-186: Development and validation of a novel RNA in situ hybridization assay to detect RNA polymerase I activity in vivo

Increased ribosome biogenesis is a critical process requisite for the cancer cell phenotype as it is required to doulbe protein levels for cell replication. Ribosome synthesis is a complex process that is stimulated by a number of well-studied oncogenic pathways (MYC, mTOR, RAS-RAF-ERK) and repressed by tumor suppressors (pRb, p53, PTEN, p14/ARF). A key first step in ribosome biogenesis is the increased synthesis by RNA Polymerase I (Pol I) of the precursor rRNA, referred to as 45S rRNA, that is later processed into the 28S, 5.8S and 18S mature rRNA forms. This process is compartmentalized into the nucleolus. As such, the pharmacological inhibition of RNA Pol I is becoming a potentially important therapeutic strategy in cancer (PMIDs 20055700, 23953479, 24434211). A validated assay that can reflect relative levels of Pol I activity in routinely obtained tissue specimens would be a useful tool. For example it would allow the pretreatment screening (as a predictive biomarker) for those tumors likely to respond to Pol I inhibition, and, could also serve as a pharmacodynamic biomarker of Pol I inhibition. The 5’ external transcribed spacer (5’ETS) of the nascent precursor 45S transcript is rapidly removed and degraded; thus, its levels can be used as a surrogate for new rRNA transcription. We employed branched DNA amplified RNA in situ hybridization using probes developed with Advanced Cellular Diagnostics (ACD RNAscope 2.0). The 5’ ETS/45S CISH signal was restricted to nucleoli. The signal was markedly attenuated in cell lines and in formalin fixed paraffin embedded (FFPE) prostate tissue slices after pharmacological inhibition of RNA Pol I using BMH-21 or actinomycin D, demonstrating validity as a measure of RNA Pol I transcriptional activity. In clinical human prostate FFPE tissue sections and TMAs there was a marked increase in the signal in the presumptive precursor lesion (high grade prostatic intraepithelial neoplasia/HGPIN, n = 25) and in invasive adenocarcinoma lesions (n = 72, Kruskal-Wallis, p = 0.0001 and p = 0.0001, respectively) compared with normal luminal epithelium. These results are consistent with prior work using RT-PCR showing an increase in 5’ ETS/45S rRNA levels in prostate cancer tissues. In adenocarcinomas, the increase in 5’ ETS/45S signal was present throughout all Gleason scores and pathological stages at radical prostatectomy, with no marked difference among these. We developed and analytically validated a chromogenic in situ hybridization (CISH) protocol using RNAscope for detecting the 5’ external transcribed spacer (ETS) of the precursor 45S ribosomal RNA in FFPE human tissues. This novel assay was used to show increased rRNA transcription in human clinical samples of PIN and carcinoma, and should prove useful for detection of increased rRNA production in various tumor types and as a novel pharmacodynamic biomarker in clinical trials of RNA Pol I inhibitors. Citation Format: Gunes Guner, Paul Sirajuddin, Qizhi Zheng, Hester Liu, Ibrahim Kulac, Marikki K. Laiho, Angelo M. De Marzo. Development and validation of a novel RNA in situ hybridization assay to detect RNA polymerase I activity in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-186.

Abstract 3783: High resolution telomere FISH analysis of testicular germ cell tumors reveals telomere anomalies specific to cancer subtypes and demonstrates telomere shortening as an early event in TGCT carcinogenesis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Testicular germ cell tumor (TGCT) is the most common malignancy of young men between 15 and 40 years of age. TGCT is exceptional, as most men can be completely cured. We evaluated 76 TGCT cases using high resolution (single-cell) telomere-specific fluorescence in situ hybridization (FISH) combined with Oct4 immunofluorescence staining to delineate telomere length patterns in different TGCT subtypes, as well as in the precursor lesion, intratubular germ cell neoplasia unclassified type (ITGCNU). Telomere lengths were qualitatively scored by comparing the intensity of the telomere signals from cancer cells to benign reference cell types and then grouped (short, medium, and long). For isolated tumors and for those part of a mixed germ cell tumor (containing 2 or more TGCT types), we determined the overall trends in telomere length in comparison to reference cell populations. This comparison demonstrated medium telomeres for normal germ cells, short telomeres for ITGCNU and seminomas (p = 0.006 and p = 0.0005, respectively), and a wide range of average telomere lengths in the non-seminomas. Among the non-seminomas, embryonal carcinomas displayed the longest telomeres, on average, and were the only subtype (27% of cases) to display the alternative lengthening of telomeres (ALT) phenotype, as evident from abnormally large telomeric foci and telomere length heterogeneity. ATRX and DAXX protein expression was not absent in a subset of embryonal carcinoma cases. Compared to somatic tissues, germ cells have a high basal telomerase activity and longer telomeres. Hence, short telomeres in ITGCNU are surprising. Telomere attrition does not occur in normal germ cells; therefore, telomere shortening in ITGCNU may play a role in TGCT tumorigenesis. Embryonal carcinoma, the TGCT subtype that is most aggressive and has the worst prognosis, showed very long telomeres. Seminomas, with short telomeres, are exquisitely sensitive to standard platinum based chemotherapy. A recent study implicates telomere damage as a consequence of platinum based chemotherapeutic agents in a male germ cell line. Thus, the knowledge of telomere length differences and that of chemotherapeutic agents’ damage to telomeres together may help shed light on the differential sensitivities of TGCT subtypes to certain chemotherapeutic agents. In summary, TGCT can demonstrate varied telomere lengths depending on lesion subtype and whether the lesion occurs in the context of a mixed tumor. The presence of short telomeres in ITGCNU suggests a major role of telomere shortening in the pathogenesis of TGCT. In the future, telomere length information may also inform point-of-care diagnostic and treatment decisions for clinicians caring for TGCT patients. Citation Format: Mohammed T. Shekhani, John Barber, Christopher M. Heaphy, Leonardo Reis, Gunes Guner, Corinne Joshu, George J. Netto, Alan K. Meeker. High resolution telomere FISH analysis of testicular germ cell tumors reveals telomere anomalies specific to cancer subtypes and demonstrates telomere shortening as an early event in TGCT carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3783. doi:10.1158/1538-7445.AM2015-3783