Marzo, Angelo M. De

Abstract Colorectal cancer develops through a normal-adenoma-carcinoma sequence, yet only 5-10% of adenomas progress to malignancy, and the cellular programs governing that sequence remain poorly defined. Here we generate a spatial multi-omics atlas of human colon adenomas, combining Visium CytAssist and protein co-detection across 24 nonadvanced and advanced tubular adenomas with single-cell resolution Xenium Prime 5K profiling of 101 patient-matched normal, adenoma, and carcinoma cores from 16 patients. Integrating whole-transcriptome and 31-plex protein data identifies nine spatial clusters and two dysplastic epithelial populations that co-express stemness, proliferation, and senescence programs. These programs occupy a shared, spatially confined epithelial niche that expands from adenoma to carcinoma. Spatial analysis revealed GDF15, a senescence-associated secretory factor, mediated the coupling between senescence and stemness in advanced adenomas, and that GDF15-high epithelium locally excludes CD8+ T cells in adenoma and, more broadly, in carcinoma. These findings position senescence as a spatially instructive rather than merely tumor-suppressive program during colorectal carcinogenesis and suggest GDF15 might be a potential candidate target for cancer prevention and interception in the colon.

TPS5125 Background: B7-H3 is a member of the B7 superfamily, which includes PD-L1 (B7-H1) and PD-L2 (B7-DC). Enoblituzumab is a humanized Fc-optimized B7-H3–targeting antibody that induces antibody-dependent cellular cytotoxicity (ADCC). A recent investigator-initiated trial demonstrated that enoblituzumab therapy, used in the neoadjuvant prostate cancer setting, is feasible, safe, may enhance short-term clinical outcomes, and appears to stimulate intratumoral immune activation including CD8+ T cell infiltration and peripheral expansion of tumor-infiltrating T-cell clones that correlated with undetectable PSA at 1-year post-prostatectomy. To assess the impact of enoblituzumab on recurrence-free survival following prostatectomy, a phase 2, randomized, neoadjuvant clinical trial in high-risk localized prostate cancer patients has been started. We hypothesize that targeting B7-H3 with enoblituzumab will delay or prevent recurrence following prostatectomy compared to standard of care (SOC) . Methods: The HEAT trial is an investigator-initiated, multi-center, randomized, phase 2 study that is presently enrolling (NCT06014255). Eligible patients will undergo a pre-treatment prostate biopsy and conventional imaging (CT and bone scan) as well as PSMA-PET. Patients who have clinical stage cT1c-T3b, cN0 cM0 disease by conventional imaging (N1 by PSMA allowed with up to 3 LNs each ≤1 cm) will be eligible as long as concurrent hormonal or radiation therapy is not given. Prostate biopsy, within 3 months of enrollment, needs to show at least 3 positive cores containing at least 1 core with at least 50% disease involvement with Gleason ≥4+3=7 disease (with at least 1 additional high-risk feature such as PSA>20 ng/ml or cT3) or a Gleason sum ≥8. Patients will receive enoblituzumab at a dose of 15mg/kg IV biweekly for 6 doses (12 weeks) on the treatment arm, followed by prostatectomy, or will be scheduled for prostatectomy directly within 4-8 weeks on the SOC arm. 219 patients will be randomized (2:1) to treatment versus SOC. Pre-treatment, on-treatment, and post-treatment biomarkers of response and resistance will be collected. Patients will be followed according to standard institutional practices, but will require PSA evaluations every 3 (±1) months during year 1 and every 6 (±2) months during years 2-5. In both arms, salvage or adjuvant therapy will occur after biochemical or radiographic progression. The primary endpoint is recurrence-free survival (RFS) defined as any metastasis events, local pelvic visceral or lymph node recurrence, detectable prostate-specific antigen (PSA), or start of subsequent local or systemic therapy (including salvage or adjuvant therapy), or death for any cause, whichever occurs first. Secondary endpoints include additional clinical and immunologic correlates. Clinical trial information: NCT06014255 .

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.

93 Background: A key eligibility criterion in many active surveillance (AS) programs is that the biopsy exhibit only Gleason pattern 3 (G3) for a Gleason score of 6 or less. However, 25 to 35% of biopsy Gleason 6 is upgraded to Gleason 7 or higher in the prostatectomy (RP) specimen. Thus, there is a great need for biomarkers that, when measured on G3 tissue in a Gleason 6 biopsy, can predict the presence of unsampled higher grade tumor in the whole prostate. We evaluated PTEN loss by immunohistochemistry (IHC), and PTEN deletion, chromosome 8q (MYC) gain and 8p (LPL) loss by fluorescence in situ hybridization (FISH) for their ability to predict unsampled G4 tumor. Methods: A tissue microarray (TMA) was constructed of RP tissue from three groups of patients (n=50 per group) whose prostates exhibited only Gleason 3+3, only 3+4, or only 4+3 tumor, matched on age, year of RP, and race. In each patient, multiple cores sampled only from areas of G3 were evaluated for PTEN deletion by FISH, PTEN loss by IHC, and chromosome 8p/8q alterations by FISH. Biomarker results were compared between Gleason 6 versus 7 tumors using conditional logistic regression. Results: Patients underwent RP in 2001 to 2009, had median age 60, and median prostate-specific antigen 5.2; 63% of tumors were organ confined. In univariate analyses 8q gain (odds ratio OR=8.9, p<.0001), 8p loss (OR=6.9, p<.0001), PTEN loss by IHC (OR=5.7, p=.025), but not PTEN deletion by FISH (OR=1.5, p=.477) were significantly more common in G3 cores from Gleason 7 tumors than G3 cores from Gleason 6 tumors. In multivariable analyses, 8q gain (OR=6.2, p=.002) and 8p loss (OR=5.2, p=.0002) remained highly significant. At least one high risk biomarker (8q gain, 8p loss, PTEN loss, or PTEN deletion) was found in 35.7% of Gleason 3+3 versus 77.1% of Gleason 3+4 versus 91.3% of Gleason 4+3 tumors, p<.0001. Adjustment for confounding factors did not change the results. Conclusions: Chromosomal 8q gains (MYC), 8p loss (LPL), and PTEN loss measured in Gleason G3 TMA cores strongly differentiate whether the core comes from Gleason 6 or Gleason 7 tumor. If validated in biopsy Gleason 6 cores to predict prostatectomy Gleason 7 tumor these biomarkers could facilitate safe selection of men for active surveillance.

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

Abstract Background: Prostate cancer molecular subtypes based on ETS gene fusions and SPINK1 were originally identified through distinct gene expression profiles. 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 and explored clinical associations using global gene expression profiles in a large cohort of PCa. Methods: We analyzed 1,577 patient Affymetrix Human Exon 1.0ST GeneChip expression profiles from 8 radical prostatectomy (RP) cohorts; 5 of them generated as part of the Decipher® platform for the Decipher® discovery or validation. Multi-feature random forest classifiers and outlier analysis were used to define microarray-based molecular subtypes and characterize clinical associations. Results: A random forest (RF) classifier (m-ERG) was trained and validated to predict ERG fusion status using separate subsets of a single-institution RP cohort (total n=407) with known ERG rearrangement status defined by FISH, achieving &gt;95% sensitivity and specificity in the validation subset. The model was then applied to 7 independent RP cohorts to predict ERG rearrangement status. Less frequent rearrangements involving other ETS genes (ETV1, ETV4, ETV5, FLI1) 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+, ERG—ETS+, ERG—SPINK+, and Triple Negative (ERG—/ETS—/SPINK1—), respectively. Global gene expression analysis shows that the four subtypes could be collapsed into three entities (ERG+, ERG—ETS+ and SPINK+/Triple Negative) based on expression patterns and clinical characteristics similarity. Based on multivariable analysis, ERG+ is significantly associated with lower pre-PSA (p&lt;0.001), lower Gleason score (p&lt;0.001), the presence of extraprostatic extension (p&lt;0.001) and European Americans (p&lt;0.001), but not associated with outcome. ERG—ETS+ is significantly associated with SVI (p=0.01) and ERG—SPINK+ is enriched in African Americans (p&lt;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 and provides insights into the development of novel therapeutic approaches. Citation Format: Mohammed Alshalalfa, Scott A. Tomlins, Nicholas Erho, Kasra Yousefi, Shuang Zhao, Robert B. Den, Adam P. Dicker, Bruce Trock, Angelo DeMarzo, Edward M. Schaeffer, Ashley Ross, Eric A. Klein, Cristina Magi-Galluzzi, Jeffery R. Karnes, Robert B Jenkins, Elai Davicioni, Felix Feng. Molecular and clinical characterization of 1,577 primary prostate cancer tumors reveals novel clinical and biological insights into its subtypes. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-64.

Abstract NKX3.1 is a homeobox gene located at chromosome 8p21.2, and one copy is frequently deleted in prostate carcinoma. Prior studies of NKX3.1 mRNA and protein in human prostate cancer and prostatic intraepithelial neoplasia (PIN) have been conflicting, and expression in focal prostate atrophy lesions has not been investigated. Immunohistochemical staining for NKX3.1 on human tissue microarrays was decreased in most focal atrophy and PIN lesions. In carcinoma, staining was inversely correlated with Gleason grade. Fluorescence in situ hybridization showed that no cases of atrophy had loss or gain of 8p, 8 centromere, or 8q24 (C-MYC) and only 12% of high-grade PIN lesions harbored loss of 8p. By contrast, NKX3.1 staining in carcinoma was correlated with 8p loss and allelic loss was inversely related to Gleason pattern. Quantitative reverse transcription-PCR for NKX3.1 mRNA using microdissected atrophy revealed a concordance with protein in five of seven cases. In carcinoma, mRNA levels were decreased in 6 of 12 cases but mRNA levels correlated with protein levels in only 4 of 12 cases, indicating translational or post-translational control. In summary, NKX3.1 protein is reduced in focal atrophy and PIN but is not related to 8p allelic loss in these lesions. Therefore, whereas genetic disruption of NKX3.1 in mice leads to PIN, nongenetic mechanisms reduce NKX3.1 protein levels early in human prostate carcinogenesis, which may facilitate both proliferation and DNA damage in atrophic and PIN cells. Monoallelic deletions on chromosome 8p are associated with more advanced invasive and aggressive disease. (Cancer Res 2006; 66(22): 10683-90)