Magi‐Galluzzi, Cristina

Juxtaglomerular cell tumor (JxGCT) is a rare type of renal neoplasm demonstrating morphologic overlap with some mesenchymal tumors such as glomus tumor (GT) and solitary fibrous tumor (SFT). Its oncogenic drivers remain elusive, and only a few cases have been analyzed with modern molecular techniques. In prior studies, loss of chromosomes 9 and 11 appeared to be recurrent. Recently, whole-genome analysis identified alterations involving genes of MAPK-RAS pathway in a subset, but no major pathogenic alterations have been discovered in prior whole transcriptome analyses. Considering the limited understanding of the molecular features of JxGCTs, we sought to assess a collaborative series with a multiomic approach to further define the molecular characteristics of this entity. Fifteen tumors morphologically compatible with JxGCTs were evaluated using immunohistochemistry for renin, single-nucleotide polymorphism array (SNP), low-pass whole-genome sequencing, and RNA sequencing (fusion assay). In addition, methylation analysis comparing JxGCT, GT, and SFT was performed. All cases tested with renin (n=11) showed positive staining. Multiple chromosomal abnormalities were identified in all cases analyzed (n=8), with gains of chromosomes 1p, 10, 17, and 19 and losses of chromosomes 9, 11, and 21 being recurrent. A pathogenic HRAS mutation was identified in one case as part of the SNP array analysis. Thirteen tumors were analyzed by RNA sequencing, with 2 revealing in-frame gene fusions: TFG::GPR128 (interpreted as stochastic) and NAB2::STAT6. The latter, originally diagnosed as JxGCT, was reclassified as SFT and excluded from the series. No fusions were detected in the remaining 11 cases; of note, no case harbored NOTCH fusions previously described in GT. Genomic methylation analysis showed that JxGCT, GT, and SFT form separate clusters, confirming that JxGCT represents a distinct entity (ie, different from GT). The results of our study show that JxGCTs are a distinct tumor type with a recurrent pattern of chromosomal imbalances that may play a role in oncogenesis, with MAPK-RAS pathway activation being likely a driver in a relatively small subset.

The Genitourinary Pathology Society (GUPS) undertook a critical review of the recent advances in bladder cancer focusing on important topics of high interest for the practicing surgical pathologist and urologist. This review represents the second of 2 manuscripts ensuing from this effort. Herein, we address the effective reporting of bladder cancer, focusing particularly on newly published data since the last 2016 World Health Organization (WHO) classification. In addition, this review focuses on the importance of reporting bladder cancer with divergent differentiation and variant (subtypes of urothelial carcinoma) histologies and the potential impact on patient care. We provide new recommendations for reporting pT1 staging in diagnostic pathology. Furthermore, we explore molecular evolution and classification, emphasizing aspects that impact the understanding of important concepts relevant to reporting and management of patients.

The Genitourinary Pathology Society (GUPS) undertook a critical review of the recent advances in bladder neoplasia with a focus on issues relevant to the practicing surgical pathologist for the understanding and effective reporting of bladder cancer, emphasizing particularly on the newly accumulated evidence post-2016 World Health Organization (WHO) classification. The work is presented in 2 manuscripts. Here, in the first, we revisit the nomenclature and classification system used for grading flat and papillary urothelial lesions centering on clinical relevance, and on dilemmas related to application in routine reporting. As patients of noninvasive bladder cancer frequently undergo cystoscopy and biopsy in their typically prolonged clinical course and for surveillance of disease, we discuss morphologies presented in these scenarios which may not have readily applicable diagnostic terms in the WHO classification. The topic of inverted patterns in urothelial neoplasia, particularly when prominent or exclusive, and beyond inverted papilloma has not been addressed formally in the WHO classification. Herein we provide a through review and suggest guidelines for when and how to report such lesions. In promulgating these GUPS recommendations, we aim to provide clarity on the clinical application of these not so uncommon diagnostically challenging situations encountered in routine practice, while also importantly advocating consistent terminology which would inform future work.

Context.— Controversies and uncertainty persist in prostate cancer grading. Objective.— To update grading recommendations. Data Sources.— Critical review of the literature along with pathology and clinician surveys. Conclusions.— Percent Gleason pattern 4 (%GP4) is as follows: (1) report %GP4 in needle biopsy with Grade Groups (GrGp) 2 and 3, and in needle biopsy on other parts (jars) of lower grade in cases with at least 1 part showing Gleason score (GS) 4 + 4 = 8; and (2) report %GP4: less than 5% or less than 10% and 10% increments thereafter. Tertiary grade patterns are as follows: (1) replace “tertiary grade pattern” in radical prostatectomy (RP) with “minor tertiary pattern 5 (TP5),” and only use in RP with GrGp 2 or 3 with less than 5% Gleason pattern 5; and (2) minor TP5 is noted along with the GS, with the GrGp based on the GS. Global score and magnetic resonance imaging (MRI)-targeted biopsies are as follows: (1) when multiple undesignated cores are taken from a single MRI-targeted lesion, an overall grade for that lesion is given as if all the involved cores were one long core; and (2) if providing a global score, when different scores are found in the standard and the MRI-targeted biopsy, give a single global score (factoring both the systematic standard and the MRI-targeted positive cores). Grade Groups are as follows: (1) Grade Groups (GrGp) is the terminology adopted by major world organizations; and (2) retain GS 3 + 5 = 8 in GrGp 4. Cribriform carcinoma is as follows: (1) report the presence or absence of cribriform glands in biopsy and RP with Gleason pattern 4 carcinoma. Intraductal carcinoma (IDC-P) is as follows: (1) report IDC-P in biopsy and RP; (2) use criteria based on dense cribriform glands (>50% of the gland is composed of epithelium relative to luminal spaces) and/or solid nests and/or marked pleomorphism/necrosis; (3) it is not necessary to perform basal cell immunostains on biopsy and RP to identify IDC-P if the results would not change the overall (highest) GS/GrGp part per case; (4) do not include IDC-P in determining the final GS/GrGp on biopsy and/or RP; and (5) “atypical intraductal proliferation (AIP)” is preferred for an intraductal proliferation of prostatic secretory cells which shows a greater degree of architectural complexity and/or cytological atypia than typical high-grade prostatic intraepithelial neoplasia, yet falling short of the strict diagnostic threshold for IDC-P. Molecular testing is as follows: (1) Ki67 is not ready for routine clinical use; (2) additional studies of active surveillance cohorts are needed to establish the utility of PTEN in this setting; and (3) dedicated studies of RNA-based assays in active surveillance populations are needed to substantiate the utility of these expensive tests in this setting. Artificial intelligence and novel grading schema are as follows: (1) incorporating reactive stromal grade, percent GP4, minor tertiary GP5, and cribriform/intraductal carcinoma are not ready for adoption in current practice.

Intraductal carcinoma of the prostate (IDC-P) has been recently recognized by the World Health Organization classification of prostatic tumors as a distinct entity, most often occurring concurrently with invasive prostatic adenocarcinoma (PCa). Whether documented admixed with PCa or in its rare pure form, numerous studies associate this entity with clinical aggressiveness. Despite increasing clinical experience and requirement of IDC-P documentation in protocols for synoptic reporting, the specifics of its potential contribution to assessment of grade group (GG) and cancer quantitation of PCa in both needle biopsies (NBx) and radical prostatectomy (RP) specimens remain unclear. Moreover, there are no standard guidelines for incorporating basal cell marker immunohistochemistry (IHC) in the diagnosis of IDC-P, either alone or as part of a cocktail with AMACR/racemase. An online survey containing 26 questions regarding diagnosis, reporting practices, and IHC resource utilization, focusing on IDC-P, was undertaken by 42 genitourinary subspecialists from 9 countries. The degree of agreement or disagreement regarding approaches to individual questions was classified as significant majority (>75%), majority (51% to 75%), minority (26% to 50%) and significant minority (≤25%). IDC-P with or without invasive cancer is considered a contraindication for active surveillance by the significant majority (95%) of respondents, although a majority (66%) also agreed that the clinical significance/behavior of IDC-P on NBx or RP with PCa required further study. The majority do not upgrade PCa based on comedonecrosis seen only in the intraductal component in NBx (62%) or RP (69%) specimens. Similarly, recognizable IDC-P with GG1 PCa was not a factor in upgrading in NBx (78%) or RP (71%) specimens. The majority (60%) of respondents include readily recognizable IDC-P in assessment of linear extent of PCa at NBx. A significant majority (78%) would use IHC to confirm or exclude intraductal carcinoma if other biopsies showed no PCa, while 60% would use it to confirm IDC-P with invasive PCa in NBx if it would change the overall GG assignment. Nearly half (48%, a minority) would use IHC to confirm IDC-P for accurate Gleason pattern 4 quantitation. A majority (57%) report the percentage of IDC-P when present, in RP specimens. When obvious Gleason pattern 4 or 5 PCa is present in RP or NBx, IHC is rarely to almost never used to confirm the presence of IDC-P by the significant majority (88% and 90%, respectively). Most genitourinary pathologists consider IDC-P to be an adverse prognostic feature independent of the PCa grade, although recommendations for standardization are needed to guide reporting of IDC-P vis a vis tumor quantitation and final GG assessment. The use of IHC varies widely and is performed for a multitude of indications, although it is used most frequently in scenarios where confirmation of IDC-P would impact the GG assigned. Further study and best practices recommendations are needed to provide guidance with regards to the most appropriate indications for IHC use in scenarios regarding IDC-P.

AimsDespite efforts to standardise grading of prostate cancer, even among experts there is still a considerable variation in grading practices. In this study we describe the use of Pathology Imagebase, a novel reference image library, for setting an international standard in prostate cancer grading.Methods and resultsThe International Society of Urological Pathology (ISUP) recently launched a reference image database supervised by experts. A panel of 24 international experts in prostate pathology reviewed independently microphotographs of 90 cases of prostate needle biopsies with cancer. A linear weighted kappa of 0.67 (95% confidence interval = 0.62–0.72) and consensus was reached in 50 cases. The interobserver weighted kappa varied from 0.48 to 0.89. The highest level of agreement was seen for Gleason score (GS) 3 + 3 = 6 (ISUP grade 1), while higher grades and particularly GS 4 + 3 = 7 (ISUP grade 3) showed considerable disagreement. Once a two‐thirds majority was reached, images were moved automatically into a public database available for all ISUP members at www.isupweb.org. Non‐members are able to access a limited number of cases.ConclusionsIt is anticipated that the database will assist pathologists to calibrate their grading and, hence, decrease interobserver variability. It will also help to identify instances where definitions of grades need to be clarified.

Abstract Expression of DLL3 was examined in additional tumor types, as it was found to be highly expressed in tumor-initiating cells (TIC) in small cell lung cancer (SCLC), where a DLL3-targeted antibody drug conjugate (ADC), rovalpituzumab tesirine (Rova-T; SC16LD6.5; Saunders et al. 2015 Sci Transl Med 7:302ra136)) exerted clinically meaningful anti-tumor effects in a phase I trial (Spigel et al. 2016 Lancet Oncology; In Press). DLL3 expression was profiled by qRT-PCR, ELISA and immunohistochemistry in multiple tumor types. Patient-derived xenografts (PDX) from melanoma and ovarian small cell carcinoma were established and used for efficacy studies to determine the ability of Rova-T to impact tumor growth and TIC frequency. DLL3 expression was seen in metastatic melanoma (55%), low grade gliomas (90%), glioblastoma (70%), medullary thyroid cancer (65%), carcinoids (33%), dispersed neuroendocrine tumors in the pancreas (9%), bladder (57%) and prostate (24%), testicular cancer (90%), and lung adenocarcinomas with neuroendocrine features (80%). Unlike SCLC, where DLL3 does not predict clinical outcome on standard therapies, DLL3 expression negatively correlates with overall survival in melanoma and small cell bladder cancer. In mice bearing DLL3 positive melanoma PDX, treatment with a single dose of Rova-T resulted in effective and durable responses (>100 days), which correlated with a significant impact on TIC frequency. Similarly, in mice bearing DLL3-positive ovarian small cell PDX, a single dose of Rova-T resulted in effective and durable responses (>100 days). Our results show that DLL3 is expressed in many neuroendocrine tumors (lung, ovarian, prostate, bladder, etc), metastatic melanoma, medullary thyroid cancer, low-grade gliomas and glioblastoma. Given pre-clinical results showing efficacy of Rova-T in melanoma and ovarian small cell carcinoma, as well as encouraging clinical data with Rova-T in patients with recurrent/refractory SCLC, clinical evaluation of Rova-T in DLL3-positive melanoma, glioblastoma, medullary thyroid cancer and other high-grade neuroendocrine carcinomas is warranted. A “basket” trial enrolling patients with DLL3-positive solid tumors is now recruiting patients (NCT02709889). Citation Format: Laura R. Saunders, Samuel A. Williams, Sheila Bheddah, Kumiko Isse, Sarah Fong, Marybeth A. Pysz, Himisha Beltran, Loredana Puca, Verena Sailer, Juan M. Mosquera, Yu Yin, Jiaoti Huang, Andrew J. Armstrong, Jorge Garcia, Cristina Magi-Galluzzi, Vadim Koshkin, Petros Grivas, Farhad Kosari, John Cheville, Justin C. Moser, Thomas J. Flotte, Thorvardur Halfdanarson, Aaron Mansfield, Konstantinos N. Leventakos, Julian R. Molina, Douglas W. Ball, Barry D. Nelkin, Jill E. Shea, Courtney L. Scaife, Scott J. Dylla. Expression of DLL3 in metastatic melanoma, glioblastoma and high-grade extrapulmonary neuroendocrine carcinomas as potential indications for rovalpituzumab tesirine (Rova-T; SC16LD6.5), a delta-like protein 3 (DLL3)-targeted antibody drug conjugate (ADC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3093. doi:10.1158/1538-7445.AM2017-3093

AimsDespite efforts to standardize histopathology practice through the development of guidelines, the interpretation of morphology is still hampered by subjectivity. We here describe Pathology Imagebase, a novel mechanism for establishing an international standard for the interpretation of pathology specimens.Methods and resultsThe International Society of Urological Pathology (ISUP) established a reference image database through the input of experts in the field. Three panels were formed, one each for prostate, urinary bladder and renal pathology, consisting of 24 international experts. Each of the panel members uploaded microphotographs of cases into a non‐public database. The remaining 23 experts were asked to vote from a multiple‐choice menu. Prior to and while voting, panel members were unable to access the results of voting by the other experts. When a consensus level of at least two‐thirds or 16 votes was reached, cases were automatically transferred to the main database. Consensus was reached in a total of 287 cases across five projects on the grading of prostate, bladder and renal cancer and the classification of renal tumours and flat lesions of the bladder. The full database is available to all ISUP members at www.isupweb.org. Non‐members may access a selected number of cases.ConclusionsIt is anticipated that the database will assist pathologists in calibrating their grading, and will also promote consistency in the diagnosis of difficult cases.

9 Background: Prostate cancer molecular subtypes based on ETS gene fusions and SPINK1 were originally identified through outlier gene expression profiling analysis. Such molecular subtypes may have utility in disease stratification and clonality assessment, complementing available purely prognostic tests. Hence, we determined the analytical validity of molecular subtyping in a large sample of PCa treated with radical prostatectomy. Methods: We analyzed Affymetrix Human Exon 1.0ST GeneChip expression profiles for 1,577 patients from 8 radical prostatectomy (RP) cohorts. Multi-feature random forest classifiers and outlier analysis were used to define microarray-based molecular subtypes. Results: A random forest (RF) classifier was trained and validated to predict ERG fusion status using a subset with known ERG rearrangement status defined by FISH, achieving >95% sensitivity and specificity in the validation subset. Less frequent rearrangements involving other ETS genes or SPINK1 over-expression were predicted based on gene expression outlier analysis. Across cohorts, 45%, 9% 8% and 38% of PCa were classified as ERG+, ETS+, SPINK+, and Triple Negative, respectively. Global gene expression analysis shows that the four subtypes could be collapsed into three entities (ERG+, ETS+ and SPINK+/Triple Negative) based on expression patterns and clinical characteristics similarity. A series of multivariable analyses further revealed, ERG+ to be associated with lower pre PSA and Gleason scores but more likely to have EPE and occur in patients with European American ancestry compared to the ETS+, SPINK+/Triple Negative tumors (p<0.001). In contrast, patients with ETS+ were more likely to have SVI compared to both ERG+ and SPINK/Triple Negative (p=0.01), while SPINK+/Triple Negative had higher Gleason scores and were more likely to occur in African Americans (p<0.001). Conclusions: The Decipher platform can accurately determine ERG rearrangement status and PCa molecular subtypes. Inclusion of molecular subtyping, such as m-ERG status, may enable additional precision medicine opportunities in prognostic tests