Showing papers by "Lawrence D. True published in 2023"

Reversible epigenetic alterations mediate PSMA expression heterogeneity in advanced metastatic prostate cancer

Abstract: Prostate-specific membrane antigen (PSMA) is an important cell surface target in prostate cancer. There are limited data on the heterogeneity of PSMA tissue expression in metastatic castration-resistant prostate cancer (mCRPC). Furthermore, the mechanisms regulating PSMA expression (encoded by the FOLH1 gene) are not well understood. Here, we demonstrate that PSMA expression is heterogeneous across different metastatic sites and molecular subtypes of mCRPC. In a rapid autopsy cohort in which multiple metastatic sites per patient were sampled, we found that 13 of 52 (25%) cases had no detectable PSMA and 23 of 52 (44%) cases showed heterogeneous PSMA expression across individual metastases, with 33 (63%) cases harboring at least 1 PSMA-negative site. PSMA-negative tumors displayed distinct transcriptional profiles with expression of druggable targets such as MUC1. Loss of PSMA was associated with epigenetic changes of the FOLH1 locus, including gain of CpG methylation and loss of histone 3 lysine 27 (H3K27) acetylation. Treatment with histone deacetylase (HDAC) inhibitors reversed this epigenetic repression and restored PSMA expression in vitro and in vivo. Collectively, these data provide insights into the expression patterns and regulation of PSMA in mCRPC and suggest that epigenetic therapies — in particular, HDAC inhibitors — can be used to augment PSMA levels.

Targeting advanced prostate cancer with STEAP1 chimeric antigen receptor T cell and tumor-localized IL-12 immunotherapy

Abstract: Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is a cell surface antigen for therapeutic targeting in prostate cancer. Here, we report broad expression of STEAP1 relative to prostate-specific membrane antigen (PSMA) in lethal metastatic prostate cancers and the development of a STEAP1-directed chimeric antigen receptor (CAR) T cell therapy. STEAP1 CAR T cells demonstrate reactivity in low antigen density, antitumor activity across metastatic prostate cancer models, and safety in a human STEAP1 knock-in mouse model. STEAP1 antigen escape is a recurrent mechanism of treatment resistance and is associated with diminished tumor antigen processing and presentation. The application of tumor-localized interleukin-12 (IL-12) therapy in the form of a collagen binding domain (CBD)-IL-12 fusion protein combined with STEAP1 CAR T cell therapy enhances antitumor efficacy by remodeling the immunologically cold tumor microenvironment of prostate cancer and combating STEAP1 antigen escape through the engagement of host immunity and epitope spreading.

Tumor-derived biomarkers beyond antigen expression enhance efficacy of CD276/B7H3 antibody-drug conjugate in metastatic prostate cancer

Abstract: Antibody-drug conjugates (ADCs) are promising targeted cancer therapy; however, patient selection based solely on target antigen expression without consideration for cytotoxic payload vulnerabilities has plateaued clinical benefits. Biomarkers to capture patients who might benefit from specific ADCs have not been systematically determined for any cancer. We present a comprehensive therapeutic and biomarker analysis of a B7H3-ADC with pyrrolobenzodiazepine(PBD) payload in 26 treatment-resistant, metastatic prostate cancer(mPC) models. B7H3 is a tumor-specific surface protein widely expressed in mPC, and PBD is a DNA cross-linking agent. B7H3 expression was necessary but not sufficient for B7H3-PBD-ADC responsiveness. RB1 deficiency and/or replication stress, characteristics of poor prognosis, conferred sensitivity and were associated with complete tumor regression in both neuroendocrine (NEPC) and androgen receptor positive(ARPC) prostate cancer models, even with low B7H3 levels. Non-ARPC models, which are currently lacking efficacious treatment, demonstrated the highest replication stress and were most sensitive to treatment. In RB1 wild-type ARPC tumors, SLFN11 expression or select DNA repair mutations in SLFN11 non-expressors governed response. Significantly, wild-type TP53 predicted non-responsiveness (7/8 models). Overall, biomarker-focused selection of models led to high efficacy of in vivo treatment. These data enable a paradigm shift to biomarker-driven trial designs for maximizing clinical benefit of ADC therapies.

Abstract B050: Human interpretation of 3D histopathology image datasets of whole prostate biopsies reveals more cribriform pattern (Gleason pattern 4) carcinoma than is seen in standard 2D histology images

Abstract: Background: Prostate biopsies of patients suspected of having cancer are taken to identify and grade the cancer, if present. These biopsies are currently diagnosed based on thin (up to 4 micron thick) sections of 1 mm thick biopsies. Consequently, only a minor portion of the tissue is examined (a set of 4 micron thick sections from 1 mm thick biopsies is less than 1% of the tissue). We developed a microscope and process that can nondestructively generate 3D image datasets of whole biopsies, allowing complete examination of the tissue specimen as a series of virtual sections. We hypothesize that clinically important grades of the cancer can be found by examining all levels of the biopsies. Methods: This study is based on 161 biopsies of low to intermediate grade cancer taken from radical prostatectomies that had been collected for an active surveillance project of the Canary Foundation. The formalin-fixed biopsy tissue was deparaffinized, stained with fluorescence analogues of hematoxylin and eosin (H&E), optically cleared, imaged in 3D, and false colored to resemble H&E stains. Two sets of images were created. One set was based on current practice – sections at 3 levels with 20 micron spacing between levels. The second set fully imaged the biopsy in 3D with at least 500 virtual levels per biopsy (at a spacing of ~1 micron per level). The images were acquired using a custom-developed 3D open-top light-sheet (OTLS) microscope and were diagnosed and graded by two pathologists using a web-based viewing platform. Results: Biopsies that had been fully imaged in 3D had more foci of intermediate risk grade cancer (Grade Group/GG 2 to 3) than biopsies that had been imaged based on current standard of practice. With 3D pathology, 13% of the biopsies had the cribriform variant of Gleason pattern 4 cancer when 2D pathology did not, while only 4.3% of the biopsies had cribriform carcinoma in 2D but not in 3D. However, when looking at overall GG, a similar number of biopsies were upgraded from low-grade (GG = 1) to higher-grade cancer (GG > 1) when comparing 3D to 2D images, and vice versa. Conclusion: Due to thorough sampling of tissue, viewing 3D datasets of whole prostate biopsies reveals more biopsies with cribriform pattern carcinoma than is seen in standard 2D pathology images. However, the grade of the PCa in the overall set of 161 biopsies is not changed. This is most likely due to poorly formed glands (Gleason pattern 4) being “downgraded” to well formed glands (Gleason pattern 3) when viewed as 3D vs 2D images. Based on these findings, more research is warranted to examine whether human interpretation of 3D pathology datasets can better predict patient prognosis and, thus, lead to treatment more appropriate for the grade of the cancer in a patient than is provided by standard 2D histopathology. Citation Format: Lawrence D. True, Peter A. Humphrey, Vanessa Roybal, Suet-Ling Sarah Chow, Weisi Xie, Jonathan T. C. Liu. Human interpretation of 3D histopathology image datasets of whole prostate biopsies reveals more cribriform pattern (Gleason pattern 4) carcinoma than is seen in standard 2D histology images [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B050.

3D open-top light-sheet microscopy and 3D microdissection of neuoadjuvant-treated primary prostate cancer reveals latent subclonal mutations.

Abstract: e17109 Background: Intratumoral molecular heterogeneity is a well described phenomenon in primary prostate cancer. Increased genomic heterogeneity is associated with more aggressive clinical behavior, including recurrence and metastasis. However, current methods to characterize heterogeneity suffer from tradeoffs between generating high quality morphologic information, correlating morphology with genomic alterations, and workflow considerations. We describe a technology to image prostate samples in 3D, perform 3D microdissection of regions of interest (ROIs), and preserve nucleic acids for DNA sequencing. Methods: Samples (N = 18) were obtained from patients (N = 12) enrolled in an open-label, single-site, Phase II neoadjuvant trial in men with NCCN high to very-high risk PC. Patients received 12 weeks of apalutamide, abiraterone acetate plus prednisone, degarelix, and indomethacin followed by radical prostatectomy (RP). Fresh frozen prostate samples (~2x2x0.4cm) were obtained from RP specimens. Frozen samples were fixed in an ethanol-based fixative, stained with nuclear (TOPRO-3) and general protein (eosin) fluorescent dyes, cleared with ethyl cinnamate, and imaged using 3D open-top light-sheet (OTLS) fluorescence microscopy. Image-guided 3D microdissection was performed on ROIs with distinct morphologies. Microdissected ROIs measured 0.5x0.5x2mm. Microdissected samples were sent for DNA extraction and sequencing using the comprehensive clinically-validated UW-OncoPlex NGS assay. Results: The tissue from neoadjuvant-treated patients contained sparse carcinoma, with cancer volumes ranging from 0.01-0.3 cm2 and cellularity ranging from 2%-30%. Molecular alterations were detected in a sample with only 0.07 cm2 volume of carcinoma with 5% cellularity. The read depth of 3D microdissected samples was 3.9 times greater than the read depth of FFPE samples from the same patients. At least two morphologically distinct ROIs were 3D microdissected from 11 of 12 patients. Four of the 18 samples had molecular alterations that differed between ROIs, i.e. intra-sample molecular heterogeneity. For example, one sample with three microdissected ROIs had a PTEN alteration exclusive to a single ROI. Conclusions: The findings show the potential for 3D microdissection of morphologically distinct subclones in samples from neoadjuvant-treated patient, to enable sequencing of specific cellular populations. The sparsity of carcinoma necessitated a spatially targeted approach for extracting material for sequencing. The increased yield from avoiding FFPE processing and carcinoma enrichment from targeted 3D microdissection of carcinoma ROIs enabled the detection of spatially distinct molecular subclones. Future work will include the analysis of additional samples and implementation of AI-guided ROI detection to streamline workflows.

Abstract 6617: Gleason score 6 often coexists with Gleason score 7 adenocarcinoma within 3D z-levels of a prostate biopsy

Abstract: Background: Primary prostate cancer is diagnosed by biopsy. However, biopsies sample only a limited amount of the prostate gland. Many studies report that GS6 cancer at biopsy is often upgraded in the radical prostatectomy. Undergrading results from sampling limitations of the biopsy - both of the prostate and of tissue within the biopsy. Since pathological diagnosis is based on 4 micron thick sections of 1 mm thick biopsies, only 0.4% of the biopsy is analyzed. We hypothesize that the evaluation of a biopsy in 3D results in variable Gleason scores or even no cancer in certain z-levels. Here, we examined 3D pathology image datasets of Gleason score 7 (GS7) biopsies using a web-based image viewing platform to determine if they underdiagnose and/or undergrade cancer when examined by conventional 2D slide-based histology. Methods: We used a custom-developed 3D open-top light-sheet (OTLS) microscope, not yet available for clinical use, to study all z-levels of a prostate biopsy. The patient cohort used in this study came from the Canary TMA study. 105 prostate biopsies were non-destructively digitized in 3D with at least 500 z-levels per biopsy (at a spacing of ~1 micron per level). Samples were stained with fluorescence analogs of H&E. The image datasets were false colored to mímic conventional H&E histology. A pathologist reviewed the images using a web-based image platform and gave overall Gleason scores (OGS) based examing all z-levels within a biopsy. Six z-levels (1, 100, 200, 300, 400 and 500) were selected for 2D Gleason scoring. Results: 63 images had a OGS of 3+4 and 42 a OGS 4+3. We found presence of GS6 (Gleason grade 3 glands only) in some z-levels: 32 cases (50.8%) with OGS 3+4 and 4 cases (9.5%) with OGS 4+3. When we only studied the 6 selected z-levels, we found z-levels with no cancer in 4 cases (6.3%) of OGS 3+4 and 2 cases (4.8%) in OGS 4+3. In addition, we found z-levels with limited number of glands. We reported these levels as Atypical Small Acinar Proliferation (ASAP) in 2 cases (3.2%) with OGS 3+4 and 3 cases (7.1%) with OGS 4+3. Examining all z-levels enabled unambiguous grading of biopies containing z-levels with ASAP and tangential sections. Conclusion: We describe the frequent coexistence of GS6 z-levels within a OGS7 3D prostate biopsy. This finding supports prior observations that finding of GS6 prostate cancer in a 2D-based sections of prostate cancer does not rule out a higher Gleason grade areas in other parts of the prostate nor in other levels of each biopsy. We also show that availability of 3D levels eases the interpretation of ASAP glands and tangential sections. Citation Format: Xavier Farré, Vanessa Roybal, Can Koyuncu, Sarah Chow, Hongyi Huang, Alan Aberdeen, Andrew Janowczyk, Anant Madabhushi, Lawrence D. True, Jonathan T. Liu. Gleason score 6 often coexists with Gleason score 7 adenocarcinoma within 3D z-levels of a prostate biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6617.

Nondestructive 3D pathology with analysis of nuclear features for prostate cancer risk assessment

Abstract: Prostate cancer treatment decisions rely heavily on subjective visual interpretation [assigning Gleason patterns or International Society of Urological Pathology (ISUP) grade groups] of limited numbers of two‐dimensional (2D) histology sections. Under this paradigm, interobserver variance is high, with ISUP grades not correlating well with outcome for individual patients, and this contributes to the over‐ and undertreatment of patients. Recent studies have demonstrated improved prognostication of prostate cancer outcomes based on computational analyses of glands and nuclei within 2D whole slide images. Our group has also shown that the computational analysis of three‐dimensional (3D) glandular features, extracted from 3D pathology datasets of whole intact biopsies, can allow for improved recurrence prediction compared to corresponding 2D features. Here we seek to expand on these prior studies by exploring the prognostic value of 3D shape‐based nuclear features in prostate cancer (e.g. nuclear size, sphericity). 3D pathology datasets were generated using open‐top light‐sheet (OTLS) microscopy of 102 cancer‐containing biopsies extracted ex vivo from the prostatectomy specimens of 46 patients. A deep learning‐based workflow was developed for 3D nuclear segmentation within the glandular epithelium versus stromal regions of the biopsies. 3D shape‐based nuclear features were extracted, and a nested cross‐validation scheme was used to train a supervised machine classifier based on 5‐year biochemical recurrence (BCR) outcomes. Nuclear features of the glandular epithelium were found to be more prognostic than stromal cell nuclear features (area under the ROC curve [AUC] = 0.72 versus 0.63). 3D shape‐based nuclear features of the glandular epithelium were also more strongly associated with the risk of BCR than analogous 2D features (AUC = 0.72 versus 0.62). The results of this preliminary investigation suggest that 3D shape‐based nuclear features are associated with prostate cancer aggressiveness and could be of value for the development of decision‐support tools. © 2023 The Pathological Society of Great Britain and Ireland.

Abstract PR005: Multi-level functional genomics reveals molecular and cellular oncogenicity of patient-based 3’ untranslated region mutations

Abstract: It is clear from the high mortality rate in advanced cancers that we need a more complete understanding of the many sources of oncogenic dysregulation in tumors. The importance of the non-coding genome in disease has recently begun to be revealed through expanded use of whole genome sequencing. In particular, 3’ untranslated region (3’UTR) somatic mutations represent an important but largely unexplored avenue of alternative oncogenic gene dysregulation. Individual instances of 3’UTR-mediated oncogenicity are known, including oncogenic RNA binding proteins and microRNAs. However, a comprehensive, high-throughput study of patient-based 3’UTR mutations and their effect on post-transcriptional gene regulation in cancer has yet to be undertaken. To determine the significance of 3’UTR mutations in advanced disease, we identify 3’UTR somatic variants across 185 metastatic castration-resistant prostate tumors, discovering 14,497 single-nucleotide mutations, which are enriched in oncogenic pathways and 3’UTR regulatory elements. We develop two complementary massively parallel reporter assays (MPRAs) to measure how thousands of these patient-based mutations affect two distinct levels of post-transcriptional gene regulation: mRNA translation and stability. These MPRAs identify hundreds of functional variants that allow us to define three determinants of mutation significance: sequence conservation, known regulatory elements, and RNA structure. Furthermore, we demonstrate the clinical relevance of these mutations, observing that CRISPR-Cas9 base editing of distinct patient 3’UTR mutations into endogenous cellular loci increases oncogenic mRNA translation and cellular stress resistance. Finally, we go back to patients, illustrating that those harboring oncogenic 3’UTR mutations discovered by our methods display particularly poor prognosis. This work represents an unprecedented view of the extent to which disease-relevant 3’UTR mutations affect mRNA stability, translation, and cancer progression, uncovering principles of regulatory functionality and potential therapeutic targets in previously unexplored regulatory regions. Citation Format: Samantha L. Schuster, Sonali Arora, Cynthia L. Wladyka, Lukas Corey, Bethany L. Stackhouse, Lori Kollath, Eva Corey, Lawrence D. True, Dave Young, Patrick J. Paddison, Andrew C. Hsieh. Multi-level functional genomics reveals molecular and cellular oncogenicity of patient-based 3’ untranslated region mutations [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR005.

Comprehensive Surface Histology of Fresh Resection Margins With Rapid Open-Top Light-Sheet (OTLS) Microscopy

Abstract: Objective: For tumor resections, margin status typically correlates with patient survival but positive margin rates are generally high (up to 45% for head and neck cancer). Frozen section analysis (FSA) is often used to intraoperatively assess the margins of excised tissue, but suffers from severe under-sampling of the actual margin surface, inferior image quality, slow turnaround, and tissue destructiveness. Methods: Here, we have developed an imaging workflow to generate en face histologic images of freshly excised surgical margin surfaces based on open-top light-sheet (OTLS) microscopy. Key innovations include (1) the ability to generate false-colored H&E-mimicking images of tissue surfaces stained for < 1 min with a single fluorophore, (2) rapid OTLS surface imaging at a rate of 15 min/cm2 followed by real-time post-processing of datasets within RAM at a rate of 5 min/cm2, and (3) rapid digital surface extraction to account for topological irregularities at the tissue surface. Results: In addition to the performance metrics listed above, we show that the image quality generated by our rapid surface-histology method approaches that of gold-standard archival histology. Conclusion: OTLS microscopy has the feasibility to provide intraoperative guidance of surgical oncology procedures. Significance: The reported methods can potentially improve tumor-resection procedures, thereby improving patient outcomes and quality of life.