Clonal evaluation of prostate cancer molecular heterogeneity in biopsy samples by dual immunohistochemistry and dual RNA in situ hybridization

Dedigama-Arachchige, Pavithra; Carskadon, Shannon; Li, Jia; Loveless, Ian; Alhamar, Mohamed; Peabody, James O.; Stricker, Hans; Chitale, Dhananjay; Rogers, Craig G.; Menon, Mani; Gupta, Nilesh; Bismar, Tarek A.; Williamson, Sean R.

doi:10.1038/s41379-020-0525-0

Cited by 6 publications

(6 citation statements)

References 33 publications

(35 reference statements)

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“…Notably, no systematic studies have unbiasedly evaluated ETS gene fusions and SPINK1 expression patterns using whole-mount specimens to understand the genetic underpinnings within each tumor focus. In addition to extensive morphological heterogeneity, studies have confirmed significant molecular heterogeneity, with multiple ETS gene fusions and SPINK1 expression present within a single prostate 2,3,8,17 . Although these markers coexist within the prostate, the influence of their combined presence on clinical outcomes remains poorly understood.…”

Section: Discussionmentioning

confidence: 95%

“…To further understand the involvement of any biological factors that may contribute to the differential clinical outcome, we conducted molecular profiling of a large cohort of wholemount radical prostatectomy cases, including the largest AA cohort. We evaluated three ETS gene fusions and SPINK1 simultaneously on wholemount specimens for the first time using the dual IHC and RNA-ISH approach 2 . The primary objective was to explore the expression patterns of these markers in multifocal prostate cancer and their association with clinical outcomes between AA and CA patients.…”

Section: Discussionmentioning

confidence: 99%

“…We have developed methods for simultaneously evaluating the selected four markers by dual IHC and dual RNAISH methods 3,17,18 . IHC was used to detect ERG and SPINK1 protein expression, while RNA-ISH (RNAscope) was selected to detect ETV1 and ETV4 mRNA expression due to the unavailability of cancer-specific monoclonal antibodies 2 . For RNA-ISH, the whole-mount slides were initially incubated at 60°C for 1 hour, then deparaffinized using xylene twice for 5 minutes each with periodic agitation.…”

Section: Dual Ihc For Erg and Spink1 And Dual Rna-ish For Etv1 And Etv4mentioning

confidence: 99%

“…Although ERG, ETV1, and ETV4 belong to the ETS family transcription factors, they have an independent role in prostate cancer. We have reported the independent clonal evolution of tumors with distinct driver molecular aberrations for these markers using needle biopsy and whole-mount prostatectomy specimens 2,3 . It has been shown that ERG and ETV1 control a common transcriptional network but in opposing fashions.…”

Section: Introductionmentioning

confidence: 99%

“…SPINK1, commonly known for its role in pancreatic function, is also associated with aggressive prostate cancer subtypes, including those resistant to androgen deprivation therapy. SPINK1 is more widely expressed in African American (AA) prostate cancer patients than in Caucasian American (CA) patients 2,3,8 . However, the relationship between SPINK1 expression and biochemical recurrence (BCR) after radical prostatectomy is still uncertain, given conflicting evidence.…”

Section: Introductionmentioning

confidence: 99%

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"Unveiling Prostate Cancer’s Molecular Tapestry: Ethnic Disparities and Prognostic Insights from Whole-Mount Prostatectomy Tissue Analysis"

Zhao,

Li,

Carskadon

et al. 2024

Preprint

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BackgroundProstate cancer is a complex and heterogeneous disease with multiple tumor foci, each potentially harboring distinct driver molecular aberrations. This complexity poses challenges to effective management. We took an innovative approach to gain a comprehensive understanding of the genetic underpinnings of each tumor focus and avoid overlooking more minor yet clinically significant foci. Instead of relying solely on a systematic sampling of dominant foci, we conducted molecular analysis on whole-mount radical prostatectomy specimens. Our study aimed to find distinct molecular subsets of prostate cancer and assess their correlation with clinical outcomes, focusing on Caucasians (CA) and African Americans (AA).MethodWe randomly selected 2201 whole-mount radical prostatectomy cases, with 1207 (54.8%) from CA and 994 (45.1%) from AA patients evaluated for a 5-year biochemical recurrence-free survival rate (BCR). Of these 2201 cases, 834 (463 −56% were from CA and 371 −44% from AA patients) were subjected to molecular analysis using dual immunohistochemistry (IHC) for ERG and SPINK1, along with dual RNAin-situhybridization (RNA-ISH) for ETV1 and ETV4 to evaluate tumor molecular heterogeneity on whole-mount specimens. The Chi-squared test examined racial disparities in aberrant oncogene expression. To assess BCR-free survival, we employed the Kaplan-Meier method and Cox-PH models for patients with distinct molecular subsets of prostate cancer. Additionally, Gleason Grade groups of prostate biopsies were summarized using a spaghetti plot and compared using linear mixed models.ResultsAnalysis of the 2201 cases revealed that AA with localized prostate cancer behaved differently with better 5-year BCR-free survival than CA after radical prostatectomy (AA: 0.82, 95% CI 0.80-0.85; CA: 0.71, 95% CI 0.68-0.75; p<.001). Molecular profiling of whole-mount specimens from 834 cases revealed that 16.4%, 58.4%, 21.7%, and 3.5% of patients with localized prostate cancer expressed none, one, two, and three of the four oncogenes, respectively. This finding identified new molecular subsets of prostate cancer with more than one driver mutation in a mutually exclusive manner within the multifocal disease. ERG and SPINK1 expression showed a negative correlation (p<.001). Notably, AA patients exhibited a lower incidence of ERG (38.8% vs. 60.3%) but a higher incidence of SPINK1 (63.3% vs. 35.6%) than CA patients. The incidences of ETV1 (9.4% vs. 9.3%) and ETV4 (4.6% vs. 3.9%) were not statistically significant between the two racial groups. However, significantly, ETV1 expression was associated with worse BCR-free survival in CA patients (hazard ratio [HR]=2.36, 95% CI 1.22-4.57, p=.02), while ETV4 expression was linked to worse BCR-free survival in AA patients (HR=2.65, 95% CI 1.15-6.09, p=.02). Moreover, ETV4 expression was associated with regional lymph node metastasis in AA patients (odds ratio [OR]=5.14, 95% CI 1.3-17.4, p=.01) but not in CA patients (OR=0.60, 95% CI 0.03-3.17, p=.63) at the time of radical prostatectomy. Additionally, in patients who underwent multiple biopsies before radical prostatectomy, the Gleason Grade group increased over time in AA patients (0.25 per year, p<.001) but remained unchanged in CA patients. ERG expression was associated with a lower Gleason Grade group (−0.20, p=.03), while ETV4 expression was linked to a higher Gleason Grade (0.54, p=.01).ConclusionsOur study reveals that AA with localized prostate cancer behaves differently and has better BCR-free survival than CA after radical prostatectomy, even after adjusting for known prognostic factors. Identification of new molecular subsets of prostate cancer with more than one ETS gene fusion within a multifocal prostate shows significant molecular heterogeneity between localized prostate cancer in CA and AA patients. Importantly, given the association of ETV1 and ETV4 expression with worse BCR-free survival in CA and AA, respectively, ETV1 and ETV4 emerge as potential prognostic markers, offering insights for clinical practice to predict prostate cancer recurrence after radical prostatectomy. Identification of new molecular subsets of prostate cancer with more than one ETS gene fusion and SPINK1 in a mutually exclusive pattern indicates the clonal origin of independent tumor foci, which is a rare and unique phenomenon in prostate cancer hitherto unidentified.

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Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%