Diana Taheri

Renal carcinoma associated with a novel succinate dehydrogenase A mutation: a case report and review of literature of a rare subtype of renal carcinoma ☆

Renal cell carcinoma (RCC) linked to germline mutation of succinate dehydrogenase subunits A, B, C, and D (SDHA, SDHB, SDHC, and SDHD, respectively) has been recently included as a provisional entity in the 2013 International Society of Urological Pathology Vancouver classification. Most SDH-deficient tumors show SDHB mutation, with only a small number of RCC with SDHC or SDHD having been reported to date. Only one case of SDH-deficient renal carcinoma known to be SDHA mutated has been previously reported. Here we report an additional RCC harboring an SDHA mutation occurring in a 62-year-old man with right flank pain and nodal metastasis. The tumor was characterized by an infiltrative pattern with solid, acinar, and papillary components. Loss of SDHA and SDHB protein by immunohistochemistry confirmed the diagnosis. Hybrid capture-based comprehensive genomic profiling identified 3 genomic alterations in tumor tissue: (i) a novel single-nucleotide splice site deletion in SDHA gene, (ii) single-nucleotide deletion in NF2 gene, and (iii) EGFR gene amplification of 19 copies. This is the second report of SDHA-mutated RCC. With increased awareness, this rare tumor can be recognized on the basis of distinctive morphology and confirmation by immunohistochemistry and genomic profiling.

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

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.

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.

Immune-checkpoint status in penile squamous cell carcinoma: a North American cohort

Penile squamous cell carcinoma (SCC) is primarily treated by surgical resection. Locally advanced and metastatic diseases require a multidisciplinary treatment approach. However, mortality and morbidity remain high, and novel molecular and immunotherapeutic targets are actively being sought. We investigated the expression of immune-checkpoint markers in penile cancers. Fifty-three invasive penile SCCs diagnosed between 1985 and 2013 were retrieved from our surgical pathology archives. Representative formalin-fixed, paraffin-embedded archival blocks were used for the construction of 2 high-density tissue microarrays. Tissue microarrays were stained with immunohistochemistry for PD-L1, FOXP3, CD8, and Ki-67. PD-L1 was investigated using rabbit monoclonal anti-PD-L1 antibody (Cell Signaling, Boston, MA; E1L3N, 1:100). Overall, 21 (40%) of 53 penile SCCs had positive PD-L1 expression. PD-L1 was expressed by a significant proportion of advanced penile SCC. Forty-four percent (15/34) of stage pT2 or more SCC and 38% (6/16) of tumors with lymph node metastasis were positive for PD-L1. PD-L1 expression did not correlate with patient age, tumor location, histologic subtype, tumor stage, anatomic depth of invasion, or tumor grade. FOXP3 expression in tumoral immune cells was found in 26 (49%) of 53 cases. FOXP3 expression in stromal immune cells correlated with tumor thickness (P = .0086). The ratio of CD8/FOXP3 was greater than 1 in 62% of cases in tumor-infiltrating immune cells and 34% of cases in stromal immune cells. Our current study is the largest to assess expression of PD-L1 in a clinically well-annotated North American cohort of penile SCC. Our findings support a rationale for targeting immune-checkpoint inhibitor pathways in advanced penile SCC.

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.

Non-invasive detection of urothelial cancer through the analysis of driver gene mutations and aneuploidy

Current non-invasive approaches for detection of urothelial cancers are suboptimal. We developed a test to detect urothelial neoplasms using DNA recovered from cells shed into urine. UroSEEK incorporates massive parallel sequencing assays for mutations in 11 genes and copy number changes on 39 chromosome arms. In 570 patients at risk for bladder cancer (BC), UroSEEK was positive in 83% of those who developed BC. Combined with cytology, UroSEEK detected 95% of patients who developed BC. Of 56 patients with upper tract urothelial cancer, 75% tested positive by UroSEEK, including 79% of those with non-invasive tumors. UroSEEK detected genetic abnormalities in 68% of urines obtained from BC patients under surveillance who demonstrated clinical evidence of recurrence. The advantages of UroSEEK over cytology were evident in low-grade BCs; UroSEEK detected 67% of cases whereas cytology detected none. These results establish the foundation for a new non-invasive approach for detection of urothelial cancer.

Hypomethylation, endogenous retrovirus expression, and interferon signaling in testicular germ cell tumors

Methylation of cytosine residues exerts a critical role in silencing gene transcription. Importantly, DNA methylation patterns are often altered in cancer, with many tumors showing site-specific gain of methylation marks in a background of global hypomethylation (1). In PNAS, Stone et al. (2) show in an ovarian cancer model that treatment with the demethylating agent 5-azacytidine resulted in reexpression of human endogenous retroviruses (HERVs), activation of type I IFN signaling, and increased CD8+ T cells in the tumor microenvironment. These data provide important insights into the role of DNA methylation in suppressing immunogenic pathways and provide additional support for the notion that hypomethylation can resculpt the host antitumor response through derepression of HERV and activation of type I IFN signaling (3, 4). We hypothesized that such elevated HERV expression and type I IFN signaling are also present in tumors that are characterized by constitutive DNA hypomethylation. Seminoma, a type of testicular germ cell tumor (TGCT), provides an opportunity to test this hypothesis. The majority of seminomas are … [↵][1]1To whom correspondence may be addressed. Email: mhaffne4{at}jhmi.edu or syegnasu{at}jhmi.edu. [1]: #xref-corresp-1-1

MP65-14 UROSEEK: A NOVEL NON-INVASIVE MOLECULAR ASSAY FOR SURVEILLANCE OF BLADDER CANCER

Simeon Springer, Baltimore, MD; Maria Del Carmen Rodriguez Pena*, Birmingham, AL; Aline Tregnago, Baltimore, MD; Diana Taheri, Tehran, Islamic Republic of Iran; Stephania Bezerra, Isabela Cunha, S~ ao Paulo, Brazil; Kazutoshi Fujita, Osaka, Japan; Dilek Baydar, Hacettepe, Turkey; Trinity Bivalacqua, Nickolas Papadopoulos, Kenneth W Kinzler, Bert Vogelstein, Baltimore, MD; George Netto, Birmingham, AL

PIK3CA Mutational Analysis in Formalin-Fixed, Paraffin-Embedded Archival Tissues of Urothelial Carcinoma of Urinary Bladder

Objective Urothelial carcinoma of the urinary bladder is the fourth most common cancer in males in the United States. In addition to mutations in FGFR3, TP53, AKT1, TSC1, and PTEN genes, mutations in PIK3CA have been also described in urothelial carcinomas, preferentially in low-grade tumors. Mutations in PIK3CA also has been shown to have implications for prognosis, surveillance and therapeutic response. Thus, determining the PIK3CA status in urothelial carcinomas could potentially improved the clinical management of patients with bladder cancer. Herein, we evaluated the presence of PIK3CA mutations in exons 1, 9, and 20 in 21 urothelial carcinomas of the urinary bladder. Methods Patients were treated by radical cystectomy without neoadjuvant chemotherapy. Representative tissue blocks (1 for each case) were selected. We used a pinpoint DNA extraction technique from formalin-fixed, paraffin-embedded and mutational analysis using the polymerase chain reaction (PCR) assay coupled with sequencing of targeted exons. Patients included 15 men and 6 women, with a median age of 68 years (range, 42 to 76 years), with 3 noninvasive and 18 invasive urothelial carcinomas. Noninvasive carcinomas included 1 case each of low-grade papillary urothelial carcinoma, high-grade papillary urothelial carcinoma, and urothelial carcinoma in situ (CIS). Invasive tumors included 3 pT1, 5 pT2, 6 pT3, and 4 pT4 urothelial carcinomas. Results We did not find mutations in the analyzed exons of the PIK3CA gene, in any of the 21 urothelial carcinomas. The preponderance of invasive high-grade and high-stage tumors could explain the absence of identifiable mutations in our cohort. Conclusions PIK3CA mutations as prognosti-cators of outcome or predictors of therapeutic response await further evaluation.