Targeted sequencing of plasmacytoid urothelial carcinoma reveals frequent TERT promoter mutations.

Activating mutations in the promoter of the telomerase reverse transcriptase (TERT) gene are the most common genetic alterations in urothelial carcinoma (UC) of the bladder and upper urinary tract. Although the cadherin 1 (CDH1) gene is commonly mutated in the clinically aggressive plasmacytoid variant of urothelial carcinoma (PUC), little is known about their TERT promoter mutation status. A retrospective search of our archives for PUC and UC with plasmacytoid and/or signet ring cell features (2007-2014) was performed. Ten specimens from 10 patients had archived material available for DNA analysis and were included in the study. Intratumoral areas of nonplasmacytoid histology were also evaluated when present. Samples were analyzed for TERT promoter mutations with Safe-SeqS, a sequencing error-reduction technology, and sequenced using a targeted panel of the 10 most commonly mutated genes in bladder cancer on the Illumina MiSeq platform. TERT promoter mutations were detected in specimens with pure and focal plasmacytoid features (6/10). Similar to conventional UC, the predominant mutation identified was g.1295228C>T. In heterogeneous tumors with focal variant histology, concordant mutations were found in plasmacytoid and corresponding conventional, glandular, or sarcomatoid areas. Co-occurring mutations in tumor protein p53 (TP53, 2 cases) and kirsten rat sarcoma (KRAS) viral proto-oncogene (1 case) were also detected. TERT promoter mutations are frequently present in PUC, which provides further evidence that TERT promoter mutations are common events in bladder cancer, regardless of histologic subtype, and supports their inclusion in any liquid biopsy 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.

High prevalence of TERT promoter mutations in micropapillary urothelial carcinoma.

Somatic activating mutations in the promoter of the telomerase reverse transcriptase (TERT) gene are the most common genetic alterations in urothelial carcinoma (UC) of the bladder and upper urinary tract. Little is known, however, about TERT-mutation status in the relatively uncommon but clinically aggressive micropapillary (MPC) variant. We evaluated the presence of TERT promoter mutations in MPC of the bladder and upper urinary tract. A retrospective search of our archives for MPC and UC with micropapillary features (2005-2014) was performed. All slides were reviewed to confirm the histologic diagnosis. Thirty-three specimens from 31 patients had FFPE blocks available for DNA analysis and were included in the study. Intratumoral areas of non-micropapillary histology were also evaluated when present. Samples were analyzed with Safe-SeqS, a sequencing error reduction technology, and sequenced using the Illumina MiSeq platform. TERT promoter mutations were detected in all specimens with pure MPC (18 of 18) and UC with focal micropapillary features (15 of 15). Similar to conventional UC, the predominant mutations identified occurred at positions -124 (C228T) (85 %) and -146 (C250T) (12 %) bp upstream of the TERT ATG start site. In heterogeneous tumors with focal variant histology, intratumoral concordant mutations were found in variant (MPC and non-MPC) and corresponding conventional UC. We found TERT promoter mutations, commonly found in conventional UC, to be frequently present in MPC. Our finding of concordant intratumoral mutational alterations in cases with focal variant histology lends support to the common oncogenesis origin of UC and its variant histology.

For staining of ALK protein, the novel D5F3 antibody demonstrates superior overall performance in terms of intensity and extent of staining in comparison to the currently used ALK1 antibody.

Inflammatory myofibroblastic tumor (IMT) is a rare neoplasm. Approximately 50 % of IMTs show an anaplastic lymphoma kinase (ALK) gene fusion resulting in ALK overexpression on immunohistochemistry (IHC). A novel anti-ALK monoclonal antibody (D5F3) has been suggested to be of superior sensitivity to the ALK1 antibody which is currently used. We compared the performance of D5F3 in detecting ALK protein expression in IMTs from various anatomic sites compared to the currently utilized ALK1. We selected 25 IMTs from our surgical pathology files (2005-2015). The novel rabbit monoclonal anti-human CD246 (clone D5F3) and the currently used mouse monoclonal anti-human CD246 (clone ALK1) were used for immunohistochemical staining (IHC) in an automated slide stainer. The percentage of immunoreactive tumor cells (0, <5 %, 5-50 %, >50 %) and cytoplasmic staining intensity (graded 0-3) were assessed and compared between the two antibodies. Fluorescence in situ hybridization (FISH) studies for ALK gene rearrangement were performed on 11 tumors. D5F3 antibody stained 76 % and ALK1 antibody stained 72 % of IMTs (p = 0.747). Compared to staining with ALK1, D5F3 stained a higher proportion of cases extensively (>50 % cells) (76 vs. 28 %, p < 0.001) and with high intensity (grade 3 76 % vs 0; p < 0.001). FISH and IHC findings (for both antibodies) were concordant in 9/10 (90 %) IMTs, in which results were informative. The novel anti-ALK rabbit monoclonal antibody (D5F3 clone) demonstrates superior overall performance in term of intensity and extent of staining of ALK protein in IMT. We found IHC staining with both antibody clones to correlate equally well with FISH results for detection of ALK rearrangement.