Metastatic Biopsies Research Articles

Leveraging large-scale PDO-based assays to optimize antibody-drug conjugate efficacy in CRC.

261 Background: Clinical trials are one of the major barriers in contemporary drug development. Functional assays based on patient-derived organoids (PDO) are a promising new tool for derisking clinical development of new therapies, but their use has been limited due to small cohort sizes and the absence of systematic validation studies. Using the largest cohort of matched PDOs, longitudinal clinical data, transcriptomic and WES data in CRC, we explore the use of large PDOs collections to characterize ADC efficacy and affinity through systematic mapping of the relationship between target antigen affinity and payload efficacy. Methods: We have assembled a collection of 85 PDOs from colorectal cancer (CRC), generated from primary tumour samples and metastatic biopsies. Patients ranged from 32 to 89 years old and had experienced an average of 1.54 lines of treatment prior to the PDO-generating tissue sampling. Using gene expression levels of well known target antigens such as CEACAM5, ERBB2, MSLN and TROP2 as a proxy for protein concentration, we explore the relationship between the target antigen affinity and the efficacy of a subset of common payloads including topoisomerase and microtubule inhibitors. This allows us to identify the relationship between two of the three main vectors of ADC efficacy in CRC. Results: We show that topoismerase inhibitor and microtubule inhibitor efficacy relates to the RNA expression level of multiple target antigens commonly used in active clinical trials in CRC. These data match the target antigen and payload couples currently under development or approved in the clinic. We are developing a tool to optimize clinical trial design. This approach will allow us to match patients more accurately with the appropriate payload, ensuring a higher likelihood of response. Conclusions: We report that large-scale PDO-based assays can be a useful tool to anticipate clinical readouts. This work suggests that well-characterized PDO collections could also help redefine patient populations, increasing the likelihood of identifying those more likely to respond to a given ADC, and thus improving the success rates of clinical studies for new treatments in CRC.

P-21 POSSIBLY ECTOPIC PTH SECRETION IN A WOMAN WITH METASTATIC BREAST CANCER

Abstract Introduction Hypercalcemia is a common complication in malignancies, with various causes such as tumor secretion of parathyroid hormone-related peptide (PTHrP), osteolytic metastases or tumor-induced calcitriol production. Ectopic production of parathyroid hormone (PTH) is a rare cause of hypercalcemia. This report presents a rare case of metastatic breast cancer possibly associated with ectopic PTH secretion. Clinical Case A 66-year-old female with a history of invasive ductal carcinoma and metastases of bone and liver was referred to our endocrinology clinic due to persistent hypercalcemia. She had been presented with atypical-pathological femur fracture 2 years ago and metastatic breast cancer had been detected. Her primary cancer treatment included conventional chemotherapy and immunotherapy over the past 2 years. She had been already treated with zoledronic acid from the beginining of the chemotherapy for her bone metastases. During her follow up hypercalcemia had been detected at the 30 th month of the therapy; her calcium was around 9-10 mg/dl and increased to 12 mg/dl abruptly while shewas on zoledronic acid. Laboratory tests showed elevated PTH levels accompanying hypercalcemia. Neck ultrasound and parathyroid scintigraphy were negative for any parathyroid pathology. A biopsy of the liver metastasis was performed during the transarterial chemoembolization procedure to make an immunohistochemical staining for PTH but it was negative. The patient was given cinacalcet with changing dosages according to the calcium levels besides zoledronic acid. Under these medications both calcium and PTH levels continued to rise;during a short period which she could not take cinacalcet because of side effects, her calcium was measured 17.5mg/dl,and didn't come to normal levels despite glucocorticoid and furosemide treatment which she subsequently required hemodialysis. Zoledronic acid was replaced with denosumab. Although calcium levels were controlled with denosumab and low dosage cinacalcet PTH levels remained elevated, suggesting ongoing ectopic PTH secretion. With no identifiable parathyroid hormone source and persistent high PTH levels despite normalized calcium, the diagnosis of ectopic PTH production was assumed. Besides the high levels of PTH we couldn't visualize a lesion secreting PTH on the contrary of direct relationship between the volume of parathyroid lesions and PTH levels. Conclusion This case represents a rare complication of metastatic breast cancer possibly associated with ectopic PTH production causing hypercalcemia. Despite extensive imaging,no parathyroid adenoma or alternative explanation for the elevated PTH was found, reinforcing the need for awareness of ectopic hormone production in the management of hypercalcemia in cancer patients. Early recognition and appropriate management of such atypical cases are crucial to avoid unnecessary interventions and to tailor treatment strategies effectively.

Utilizing Patient-Derived Xenografts to Model Precision Oncology for Biliary Tract Cancer.

Biliary tract cancers, which are rare and aggressive malignancies, are rich in clinically actionable molecular alterations. A major challenge in the field is the paucity of clinically relevant biliary tract cancer models that recapitulate the diverse molecular profiles of these tumors. The purpose of this study was to curate a collection of patient-derived xenograft (PDX) models that reflect the spectrum of genomic alterations present in biliary tract cancers to create a resource for modeling precision oncology. PDXs were derived from biliary tract cancer samples collected from surgical resections or metastatic biopsies. Alterations present in the PDXs were identified by whole-exome sequencing and RNA sequencing. PDXs were treated with approved and investigational agents. Efficacy was assessed by change in tumor volume from baseline. Event-free survival was defined as the time to tumor doubling from baseline. Responses were categorized at day 21: >30% decrease in tumor volume = partial response, >20% increase in tumor volume = progressive disease, and any non-partial response/progressive disease was considered stable disease. Genomic sequencing demonstrated key actionable alterations across this cohort, including alterations in FGFR2, isocitrate dehydrogenase I, ERRB2, PIK3CA, PTEN, and KRAS. RNA sequencing demonstrated fusions and expression of antibody-drug conjugate targets, including TROP2, HER2, and Nectin4. Therapeutic matching revealed objective responses to approved and investigational agents that have been shown to have antitumor activity clinically. In this study, we developed a catalog of biliary tract cancer PDXs that underwent comprehensive molecular profiling and therapeutic modeling. To date, this is one of the largest collections of biliary tract cancer PDX models and will facilitate the development of personalized treatments for patients with these aggressive malignancies.

Germline and Somatic Genomic Testing for Metastatic Prostate Cancer: ASCO Guideline.

To evaluate evidence on germline and somatic genomic testing for patients with metastatic prostate cancer and provide recommendations. A systematic review by a multidisciplinary panel with patient representation was conducted. The PubMed database was searched from January 2018 to May 2024. Articles were selected for inclusion if they reported on patients with metastatic prostate cancer who received a germline or somatic genomic test and/or made comparisons between those tests, reported detection rates, prognostic information, or treatment implications. A total of 1,713 papers were identified in the literature search. After applying the eligibility criteria, 14 remained: eight systematic reviews and six clinical trials. Patients with metastatic prostate cancer should undergo both germline and somatic DNA sequencing using panel-based assays. These tests can guide the use of poly(ADP-ribose) polymerase inhibitors, which have a survival benefit in metastatic castration-resistant prostate cancer. In addition, germline testing may have screening implications for additional cancers for patients and cascade testing implications for family members. The data supporting when to perform repeat testing and optimal tissue type to use (eg, primary tumor v metastatic biopsy versus circulating tumor DNA [ctDNA] testing) are more limited, but this panel recommends considering retesting in patients whose results were previously negative or uninformative, and to consider either a metastatic biopsy or ctDNA when a significant change in clinical status occurs. Next-generation genomic sequencing findings that are associated with prognostic only (and not predictive) value should not be used to guide treatment outside of a clinical trial.Additional information is available at www.asco.org/genitourinary-cancer-guidelines.

Efficacy outcomes of CDK4/6 inhibitors in combination with endocrine therapy treatment in hormone receptor-positive/HER2-negative advanced breast cancer according to PAM50 intrinsic subtype: Primary results of SOLTI-1801 CDK-PREDICT study.

The prognostic value of PAM50 intrinsic subtypes (IS), cell cycle, and immune-related gene expression in HR+ /HER2- advanced breast cancer (BC) treated with CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) in a first-line metastatic setting is unclear. This study evaluates these biomarkers in metastatic biopsies from patients diagnosed with HR+ /HER2- advanced BC. CDK-PREDICT study is a multicentric, ambispective observational cohort study conducted in six Spanish hospitals. It included patients diagnosed with HR+ /HER2- advanced BC treated in the first-line setting with CDK4/6i and ET. Baseline biopsies were obtained prior to treatment to determine research-based PAM50 IS, cell cycle and immune-related gene expression. The primary objective was to evaluate progression-free survival (PFS) differences among PAM50 IS using uni- and multivariable Cox regression models. Secondary objectives included overall survival (OS), overall response rate (ORR), and correlating cell cycle and immune response gene expression with PFS. A total of 185 patients were included, with a median follow-up of 38.5 months. PAM50 luminal subtypes were predominant (82.7 %). Non-luminal subtypes showed significantly shorter median PFS (10.2 vs. 25.7 months; HR, 2.50; p < 0.001) and OS (32.3 vs. 58.1 months; HR, 2.54; p < 0.001) than luminal subtypes. Higher cell cycle and immune-related genes expression, such as CCNE1 and PDCD1, as well as tumor infiltrating lymphocytes were associated with poorer outcomes. This study confirms the independent prognostic value of PAM50 IS in HR+ /HER2- advanced BC treated with CDK4/6i and ET. Non-luminal subtypes exhibited the worst prognosis, underscoring the need for novel therapeutic strategies in this population.

Needle tract seeding of thyroid cancer after biopsy of distant metastasis: a retrospective cohort.

Imaging-guided percutaneous core needle biopsy is currently the most common technique for the investigation of potentially malignant bone lesions. It allows precise needle placement and better visual guidance, leading to improved diagnostic accuracy. Needle tract seeding (NTS) is a rare complication of biopsies in general, and its true incidence remains unknown. This study aimed to assess the risk of NTS in patients with thyroid cancer who underwent bone biopsy. For our cohort, we extracted data from the electronic medical record at the Mayo Clinic in Rochester (Minnesota, USA). Inclusion criteria included patients with a history of thyroid cancer who underwent biopsy for bone metastasis between 1/1/2014 and 10/1/2023. We identified a cohort of 20 patients that fit our inclusion criteria. Of these 20 patients, 2 patients developed NTS after CT-guided bone biopsy. Cases of seeding had a larger tumor size, a more aggressive histopathological presentation, a significantly shorter duration between cancer diagnosis and bone metastasis, and underwent more tumor manipulation procedures, such as biopsy and radiofrequency ablation, in contrast to those without seeding. In conclusion, our study identified NTS to have an incidence of 10% after biopsies of bone metastasis related to thyroid carcinoma. These are likely the result of an interplay of risk factors, including tumor biology, penetrated tissues and procedural technical details. Further studies with larger sample sizes are needed to confirm our findings and identify strategies to mitigate NTS.

Stable Biotherapeutic Penetration Screening in Critically Small Colorectal Metastatic Cancer Biopsies Allows for Oncolytic Virus‐Delivered Chemotherapeutic Response Assessment through 3D Bioprinted Organoid Expansion

AbstractOncolytic viral‐delivered chemotherapeutics have exciting potential for metastatic cancer therapies, including colorectal cancer, but require advanced screening systems for better patient prediction. We optimized primary metastatic colorectal tumor processing and 3D (3‐dimensional) bioprinted tumors to prove efficacy as long‐term screening systems. Normally, this time period would use animals, but we show it is possible to gain useful data in vitro before preclinical stages, to reduce animal modeling and give better clinical trial predictions. Liver tumors were collected from 12 colorectal cancer patients, evaluated for expansion, 3D bioprinted, and tested for ability to create long‐term organoid models with screening of oncolytic viral‐loaded FCU1 enzyme conversion of 5‐fluorocytosine (5‐FC) into the highly toxic 5‐fluorouracil (5‐FU). Donated tumor size was the limiting factor. 75% of patients could be used for screening of viral delivery. Response between patients was overall positive, with good secondary tumor development, outer active cellular content and inner necrotic core. Oncolytic challenge shows good screening potential and cellular targeting, demonstrating an added bystander effect, optimizing the low‐dose. Stable long‐term metastatic organoid models were made, lasting many months, with potential for retesting rather than one‐off analysis. Oncolytic virus‐delivered chemotherapy is promising and warrants further investigation for metastatic colorectal cancers.

Influence of Epithelial-Mesenchymal Transition on Risk of Relapse and Outcome to Eribulin or Cyclin-Dependent Kinase Inhibitors in Metastatic Breast Cancer.

The presence of epithelial-mesenchymal transition (EMT) in breast cancer (BC) cells has been linked to worse prognosis and may influence response to systemic treatment. We explored the effect of EMT in tumor samples of patients with metastatic BC on disease-free interval and overall survival in those patients receiving eribulin or cyclin-dependent kinase 4/6 inhibitors (CDK4/6i). Key inclusion criteria included available archived primary BC tissue and, where available, matched metastatic biopsy. Patients received eribulin and/or a CDK4/6i in the metastatic setting. Specimens were assessed for biomarkers by immunohistochemistry (CDH1, AE1/3, VIM, CDH2, ZEB1, pSMAD2, and SMAD4) and gene expression by droplet digital polymerase chain reaction (CDH1, CDH2, SNAI1 & 2, TWIST1, VIM, PTEN, and ZEB1 & 2). Between 2002 and 2020, 127 patients were included (95 early-stage disease at diagnosis with metastatic relapse, 32 de novo metastatic disease). In metastatic samples, presence of ZEB1 overexpression was associated with shorter time to recurrence (48.1 months shorter; P = .003), with pSMAD2 overexpression suggesting clinical significance of 52.0 months shorter; P = .01. High gene expression levels for SNAIL1, TWIST1, and PTEN in the primary BC were associated with significantly longer survival in patients who received eribulin (P < .05); high VIM was associated with a clinically relevant trend toward shorter survival after a CDK4/6i (P = .013). We demonstrate in our exploratory study that biomarkers involved in the process of EMT could have a prognostic impact in a cohort of patients with BC uniformly treated and with long-term follow-up. Genes known to be involved in EMT were associated with improved eribulin efficacy, while suggesting a poorer outcome with CDK4/6i.

Framework for the Pathology Workup of Metastatic Castration-Resistant Prostate Cancer Biopsies.

Lineage plasticity and histologic transformation from prostate adenocarcinoma to neuroendocrine prostate cancer (NEPC) occurs in up to 15-20% of patients with castration-resistant prostate cancer (CRPC) as mechanism of treatment resistance and is associated with aggressive disease and poor prognosis. NEPC tumors typically display small cell carcinoma morphology with loss of androgen receptor (AR) expression and gain of neuroendocrine (NE) lineage markers. However, there is a spectrum of phenotypes that are observed during the lineage plasticity process, and the clinical significance of mixed histologies or those that co-express AR and NE markers or lack all markers is not well defined. Translational research studies investigating NEPC have used variable definitions making clinical trial design challenging. Here we discuss the diagnostic workup of metastatic biopsies to help guide the reproducible classification of phenotypic CRPC subtypes. We recommend classifying CRPC tumors based on histomorphology (adenocarcinoma, small cell carcinoma, poorly differentiated carcinoma, other morphologic variant, or mixed morphology) and immunohistochemical markers with a priority for AR, NKX3.1, INSM1, synaptophysin and cell proliferation based on Ki-67 positivity, with additional markers to be considered based on the clinical context. Ultimately, a unified workup of metastatic CRPC biopsies can improve clinical trial design and eventually practice.

Nodular Regenerative Hyperplasia: Report of 82 Patients and Systematic Review of Literature.

Data about the clinical significance and outcome of patients with nodular regenerative hyperplasia are limited. The aim of this study was to describe the clinical and histopathological characteristics of patients with nodular regenerative hyperplasia and compare our findings with the literature. From January 2015 to March 2021, patients with a diagnosis of nodular regenerative hyperplasia were included. They were extracted from the database of the pathology department of Cliniques universitaires Saint-Luc. Clinical and histological data were retrospectively recorded and complications of portal hypertension and mortality were analyzed. We also performed a systematic review of the literature. Eighty-two histology-proven nodular regenerative hyperplasia were included. The mean age at diagnosis was 58±14years. At least one clinical sign of portal hypertension was present in 37 patients (45%), and liver tissue sampling was performed for 29 of them for evaluation of portal hypertension. Conversely, nodular regenerative hyperplasia was an incidental discovery in 27 patients (33%), mostly after liver resection for metastasis (n=15) or protocol biopsy in liver-transplanted patients (n=9). The 5-year liver-related mortality was 5%. The 5-year non-liver-related mortality was 20%. Patients diagnosed by clinical suspicion (n=55) were compared to patients diagnosed incidentally (n=27). Patients with an incidental diagnosis had more frequently a condition associated with nodular regenerative hyperplasia than patients diagnosed clinically (93% vs. 66%, p=0.008) and they developed significantly lower liver-related complications (4% vs. 27%, p=0.01). A systematic review allowed us to compare our patients with 10 case series in the literature. The clinical spectrum of patients with nodular regenerative hyperplasia is heterogeneous, including patients with clinical liver manifestations and patients diagnosed incidentally who could remain free of liver-related complications. This suggests that nodular regenerative hyperplasia could be a histological epiphenomenon as well as a clinical entity.

Abstract C048: Intrapatient heterogeneity and targeting microenvironment protection mechanisms in melanoma metastasis

Abstract Intrapatient tumor heterogeneity contributes to immunotherapy and targeted therapy resistance in cutaneous metastatic melanoma patients. More than 50% of melanoma patients develop brain metastases (MBM) during disease progressionand these patients have a very poor prognosis with a median overall survival of 4 months. To assess how tumors persist through treatments and adapt to and rely on organ-specific microenvironments, we utilized samples from a patient who had received anti-CTLA4, BRAFi/MEKi, and BRAFi/MEKi+CDK4/6i treatments during the LOGIC2 clinical trial. Eight short-term melanoma cultures (STMCs) were established from different metastatic site biopsies at chronological time points throughout the patient’s treatment and subsequent post-mortem resection of the bone, ovary, and brain metastatic sites. While the initial treatment naïve metastatic tumor in the breast was BRAF V600E mutant, NRAS Q61 mutations were acquired after treatment with CDK4/6i in the bone and ovary tumors. We performed single-cell RNA sequencing and identified that each STMC consists of 2-10 different subpopulations. By cell viability assay, we determined all STMCs were resistant to CDK4/6i, however had varying degrees of response to BRAFi and MEKi in vitro. Two STMCs (MM150604, MM150859) were sensitive to both BRAFi and MEKi. We identified HER3 was upregulated in the sensitive STMCs and more specifically, HER3 expression was elevated in the MBM STMC and in other MBM patient tumors in publicly available datasets. In publicly available data of the MBM tumor microenvironment (TME), neurons produce NRG1 and NRG2 while astrocytes and pericytes produce NRG2, exogenous sources of NRGs in the MBM TME that act as ligands for the HER3 receptor. Treatment with recombinant NRG1 partially rescued the growth of MBM cells and restored MAPK and AKT-mTOR signaling in the presence of BRAFi/MEKi suggesting that the brain microenvironment may play a role in drug tolerance. This novel finding demonstrates a unique role of HER3 in MBM and targeting the NRG-HER3 signaling axis could have therapeutic potential in melanoma patients with active brain metastases. Citation Format: Haley P. Wilson, Glenn L. Mersky, Jelan I. Haj, Jessica L.F. Teh, Phil F. Cheng, Signe Caksa, Manal Mustafa, Isabella Trachtenberg, Francis J. Waltrich Jr, Casey D. Stefanski, Vivian Chua, Dan A. Erkes, Mitchell P. Levesque, Reinhard Dummer, Timothy J. Purwin, Claudia Capparelli, Andrew E. Aplin. Intrapatient heterogeneity and targeting microenvironment protection mechanisms in melanoma metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C048.

Abstract A001: Analytical validation of a novel multiomic metastatic breast cancer liquid biopsy test that combines ctDNA analysis with CTC HER2-ultralow and ER co-expression, as well as single-cell chromosomal instability

Abstract Background: Metastatic breast cancer (MBC) subtype characterization is critical for effective treatment. The DefineMBC™ multiomic blood biopsy clinical diagnostic combines ctDNA and circulating tumor cell (CTC) analyses to characterize MBC patients when a tissue biopsy is not feasible. Innovation in the DefineMBC 2.0 5-channel immunofluorescent (IF) CTC characterization assay now measures ultralow (UL) levels of HER2 protein expression, ER protein expression, and chromosomal instability (CI) as well as ERBB2 amplification via single- cell sequencing. Cell-level HER2/ER co-detection enables monitoring of receptor conversion during a patient’s MBC treatment journey. As trastuzumab-deruxtecan (T-DxD) has become standard of care for HER2-low disease, distinguishing HER2-UL from HER2[-] by DefineMBC’s liquid biopsy approach may identify additional patients who can benefit from such therapy. This addresses an unmet need given the known shortcomings of tissue biopsy IHC in the metastatic setting as recently noted by communications from NCCN, ASCO, and the College of American Pathologists. Methods: Assay development including analytical validation studies utilized biologically relevant cell lines of epithelial origin and known expression levels of HER2 and ER (MDA-MB-453: HER2[+] &amp; ER[-]; MCF-7: HER2-low &amp; ER[+]; and MCF-7/ERBB2 knockout: HER2[-]) that were spiked into healthy donor blood. Additionally, patients with various forms of MBC were characterized with the new assay. As with all samples, following red blood cell lysis, nucleated cells were deposited on glass slides at high density (∼3E6 WBC/slide). Slides underwent IF staining for cytokeratins (CK), CD31, CD45, HER2, ER, and Hoechst to localize nuclear DNA, followed by scanning and machine learning-based rare cell detection. CK[+], CD31[-]/CD45[-] cells were classified as CTC candidates and assessed for HER2/ER expression. Pathologist-selected CTCs were isolated for single-cell whole genome sequencing and analyzed using a proprietary CTC DNA copy number pipeline to detect CI and amplification at the ERBB2 locus. Results: Analytical validation studies demonstrated the limit of detection at 1 CTC per 12E6 WBC (∼1.6 mL of blood) with a linear range of 1- 300 CTCs per slide. Sensitivity, specificity, accuracy, and precision metrics of the HER2 and ER IF assays were 96%, 96%, 96%, 2% CV; 91%, 95%, 93%, 3% CV; respectively. CTC enumeration precision was validated at greater than 80% for enumeration bins of 0, 1-10, 11-50, and&amp;gt;50 CTCs per sample. During patient testing 7 patients previously called HER2[-] are now designated as consistent with HER2-low by the pathologist, suggesting improved test performance in future clinical validation studies is likely. Conclusions: Advancement of our comprehensive cancer profiling platform now includes ER/HER2-UL/CI co-detection on enumerated CTCs in MBC patients. Clinical studies currently in progress will reveal whether HER2-UL/HER2[+] detection on CTCs will identify patients better suited for T-DxD or trastuzumab, respectively. Citation Format: Raj Srikrishnan, Jordan Ritchie, Alessandra Cunsolo, Andrew Kunihiro, Brandon Guillory, Sara Tin, Zhuo Meng, Ernest T Lam, Bharat Annaldas, Evan Schwab, Nilesh Dharajiya, Timothy Pluard, Martin Blankfard, David Bourdon. Analytical validation of a novel multiomic metastatic breast cancer liquid biopsy test that combines ctDNA analysis with CTC HER2-ultralow and ER co-expression, as well as single-cell chromosomal instability [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A001.

A multi-modal single-cell and spatial expression map of metastatic breast cancer biopsies across clinicopathological features

Although metastatic disease is the leading cause of cancer-related deaths, its tumor microenvironment remains poorly characterized due to technical and biospecimen limitations. In this study, we assembled a multi-modal spatial and cellular map of 67 tumor biopsies from 60 patients with metastatic breast cancer across diverse clinicopathological features and nine anatomic sites with detailed clinical annotations. We combined single-cell or single-nucleus RNA sequencing for all biopsies with a panel of four spatial expression assays (Slide-seq, MERFISH, ExSeq and CODEX) and H&E staining of consecutive serial sections from up to 15 of these biopsies. We leveraged the coupled measurements to provide reference points for the utility and integration of different experimental techniques and used them to assess variability in cell type composition and expression as well as emerging spatial expression characteristics across clinicopathological and methodological diversity. Finally, we assessed spatial expression and co-localization features of macrophage populations, characterized three distinct spatial phenotypes of epithelial-to-mesenchymal transition and identified expression programs associated with local T cell infiltration versus exclusion, showcasing the potential of clinically relevant discovery in such maps.

Pre-treatment metastatic biopsy: a step towards precision oncology for urothelial cancer.

Early metastatic spread and clonal expansion of individual mutations result in a heterogeneous tumour landscape in metastatic urothelial cancer (mUC). Substantial molecular heterogeneity of common drug targets, such as membranous NECTIN4, FGFR3 mutations, PDL1 or immune phenotypes, has been documented between primary and metastatic tumours. However, translational and clinical studies frequently do not account for such heterogeneity and often investigate primary tumour samples that might not be representative in patients with mUC. We propose this as a potential factor for why many biomarkers for mUC have failed to be integrated into clinical practice. Fresh pre-treatment metastatic biopsies enable the capturing of prevailing tumour biology in real time. The characterization of metastatic tumour samples can improve response prediction to immunotherapy, the anti-NECTIN4 antibody-drug conjugate enfortumab vedotin and the FGFR inhibitor erdafitinib. Routine metastatic biopsy can thus improve the precision of identifying driver druggable alterations, thus improving treatment selection for patients with mUC.

The lncRNA AFAP1-AS1 is upregulated in metastatic triple-negative breast tumors and controls hypoxia-activated vasculogenic mimicry and angiogenesis

BackgroundVasculogenic mimicry (VM) is an alternative intratumoral microcirculation system that depends on the capacity of tumor cells to reorganize and grow in three-dimensional (3D) channel architectures like the capillaries formed by endothelial cells. Both VM and angiogenesis may coordinately function to feed cancer cells, allowing tumor growth. Long noncoding RNAs (lncRNAs) regulate critical cellular functions in cancer cells, including cell proliferation, apoptosis, angiogenesis, invasion, and metastasis. The lncRNA, known as actin filament-associated protein 1-antisense RNA 1 (AFAP1-AS1), has been described as an oncogene in diverse types of cancers. However, its role in VM and metastasis in triple-negative breast cancer (TNBC) is unknown.MethodsReverse transcription and quantitative polymerase chain reaction (RT‒qPCR) experiments were performed to evaluate the expression of 10 selected lncRNAs from literature in metastatic and nonmetastatic biopsies from TNBC patients. The expression of AFAP1-AS1 was analyzed in Genotype-Tissue Expression Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) datasets. The AFAP1-AS1 expression was knocked in TNBC Hs578T cells by transfection of specific siRNAs. Channel-like formation assays were performed using 3D cultures over Matrigel in hypoxia-treated Hs578T cancer cells with diminished expression of AFAP1-AS1. The angiogenesis tests were conducted using human umbilical vein endothelial cells (HUVECs) and AFAP1-AS1- silenced Hs578T cells on 3D cell cultures. The presence of VM (CD31-/PAS+) in tumor tissues from TNBC patients with and without metastasis was assessed through immunohistochemistry using endothelial marker CD31 antibodies and periodic acid-Schiff (PAS) staining.ResultsCompared with normal mammary tissues, AFAP1-AS1 expression was higher in breast cancer tissues. Moreover, AFAP1-AS1 expression was upregulated in the TNBC subtype compared to receptor-positive breast tumors. In addition, the expression of AFAP1-AS1 was correlated with the expression of the thirteen genes characteristic of a previously reported hypoxia signature. Interestingly, AFAP1-AS1 was upregulated in primary TNBC tumors from patients who developed metastasis compared with the nonmetastatic group. Functional analysis revealed that the knockdown of AFAP1-AS1 in Hs578T cells significantly impaired the hypoxia-induced VM, accompanied by a decrease in the development of 3D channel networks. Similarly, AFAP1-AS1 knockdown counteracts the angiogenic potential of cancer cells, as indicated by a reduction in the number of polygons, sprouting cells, and nodes in HUVEC cells. Remarkably, an increase in CD31-/PAS + staining of 3D channel networks in primary breast tumors from metastatic patients was found compared with the nonmetastatic group. Finally, we found that the number of blood vessels increased in the nonmetastatic group more than in the metastatic cohort.ConclusionsOur data suggested that AFAP1-AS1 controls both VM and angiogenesis in Hs578T breast cancer cells and that increased metastasis is associated with VM in TNBC patients.

Androgen production, uptake, and conversion (APUC) genes define prostate cancer patients with distinct clinical outcomes.

BACKGROUNDProstate cancer (PC) is driven by aberrant signaling of the androgen receptor (AR) or its ligands, and androgen deprivation therapies (ADTs) are a cornerstone of treatment. ADT responsiveness may be associated with germline changes in genes that regulate androgen production, uptake, and conversion (APUC).METHODSWe analyzed whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) data from prostate tissues (SU2C/PCF, TCGA, GETx). We also interrogated the Caris Precision Oncology Alliance (POA) DNA (592-gene/whole exome) and RNA (whole transcriptome) next-generation sequencing databases. Algorithm for Linking Activity Networks (ALAN) was used to quantify all pairwise gene-to-gene associations. Real-world overall survival was determined from insurance claims data using Kaplan-Meier estimates.RESULTSSix APUC genes (HSD3B1, HSD3B2, CYP3A43, CYP11A1, CYP11B1, CYP17A1) exhibited coalescent gene behavior in a cohort of metastatic tumors (n = 208). In the Caris POA dataset, the 6 APUC genes (APUC-6) exhibited robust clustering in primary prostate (n = 4,490) and metastatic (n = 2,593) biopsies. Surprisingly, tumors with elevated APUC-6 expression had statically lower expression of AR, AR-V7, and AR signaling scores, suggesting ligand-driven disease biology. APUC-6 genes instead associated with the expression of alternative steroid hormone receptors, ESR1/2 and PGR. We used RNA expression of AR or APUC-6 genes to define 2 subgroups of tumors with differential association with hallmark pathways and cell surface targets.CONCLUSIONSThe APUC-6-high/AR-low tumors represented a subgroup of patients with good clinical outcomes, in contrast with the AR-high or neuroendocrine PCs. Altogether, measuring the aggregate expression of APUC-6 genes in current genomic tests identifies PCs that are ligand (rather than AR) driven and require distinct therapeutic strategies.FUNDINGNCI/NIH 1R37CA288972-01, NCI Cancer Center Support P30 CA077598, DOD W81XWH-22-2-0025, R01 CA249279.

Desmosome mutations impact the tumor microenvironment to promote melanoma proliferation.

Desmosomes are transmembrane protein complexes that contribute to cell-cell adhesion in epithelia and other tissues. Here, we report the discovery of frequent genetic alterations in the desmosome in human cancers, with the strongest signal seen in cutaneous melanoma where desmosomes are mutated in >70% of cases. In primary but not metastatic melanoma biopsies, the burden of coding mutations in desmosome genes associates with a strong reduction in desmosome gene expression. Analysis by spatial transcriptomics and protein immunofluorescence suggests that these expression decreases occur in keratinocytes in the microenvironment rather than in primary melanoma cells. In further support of a microenvironmental origin, we find that desmosome gene knockdown in keratinocytes yields markedly increased proliferation of adjacent melanoma cells in keratinocyte/melanoma co-cultures. Similar increases in melanoma proliferation are observed in media preconditioned by desmosome-deficient keratinocytes. Thus, gradual accumulation of desmosome mutations in neighboring cells may prime melanoma cells for neoplastic transformation.

Dual PD-L1/SOX10 Immunohistochemistry Combined With Digital Imaging Enhances Stratification Accuracy of Patients With Metastatic Melanoma.

Immune checkpoint inhibitor therapy has demonstrated an overall survival benefit in patients with advanced melanoma. Though the significance of programmed death-ligand 1 (PD-L1) expression on melanoma cells as a predictive biomarker of response remains inconclusive, some reports indicate that a PD-L1 expression of <1% of tumor cells may be associated with better outcomes with dual immunotherapy. Adequate patient selection for combination therapy is critical given the higher frequency of adverse effects compared with monotherapy. Immunohistochemical (IHC) PD-L1 interpretation in tumor cells is challenging when inflammatory cells are present and cutoffs are low. We studied 36 metastatic melanoma biopsies from Immune checkpoint inhibitor-naive patients, previously stained and scored for PD-L1 IHC using the tumor proportion score (TPS). Cases were classified into 3 groups: <1%, 1% to 5%, and >5%. After de-coverslipping, SRY-related HMG-box-10 (SOX10) IHC was performed on PD-L1 IHC slides with a red chromogen, and subsequently scanned and scored by ≥2 dermatopathologists. This assessment determined that 25% of cases (9/36) had a TPS ≥ 1%, in contrast to the single IHC assay (63.8%). The majority of the 1-5% group (11/13, 84.6%) underwent a change of category to <1% TPS. In the >5% group, 60% of cases (6/10) were downgraded to <1% and 1% to 5% (4 and 2 cases, respectively). Our study suggests that PD-L1 IHC evaluation could benefit from dual PD-L1/SOX10 IHC. Dual IHC is expected to decrease the interference caused by PD-L1 expression on inflammatory cells, and digital imaging proves useful for the preservation and analysis of stains. Refining PD-L1 evaluation in metastatic melanoma may improve clinical decisions between single and combination immunotherapy, with potentially profound consequences in response and quality of life.

6634 Dual-Mediated PTH And Osteolytic Hypercalcemia In Metastatic Endometrial Carcinoma

Abstract Disclosure: C.K. Persaud: None. D.J. Beckman: None. Background: Hypercalcemia of malignancy occurs in 20-30% of advanced stage cancer patients. Etiologies include parathyroid hormone-related peptides (PTHrP)-mediated, osteolytic metastases-mediated, overproduction of 1,25-dihydroxycholecalciferol, and rarely parathyroid hormone (PTH)-mediated. PTH-mediated hypercalcemia of malignancy is rare and occurs through ectopic PTH production or primary hyperparathyroidism. We report a rare case of both PTH-mediated and osteolytic-mediated hypercalcemia in the setting of malignancy. Clinical Case: A 57-year-old female with history of endometrial cancer and multiple solid organ metastasis, presented with two weeks of progressive poor oral intake, nausea, emesis, and right flank pain. On hospital admission, she had a serum calcium of &amp;gt;20.1 mg/dL (8.6-10.2), PTH of 203 pg/mL (15.0-65.0), and creatinine of 1.7 mg/dL (0.7-1.2, baseline 1.3). Non-contrasted CT chest/abdomen/pelvis noted increased size of known right adrenal metastasis, new left adrenal metastasis, periaortic lymphadenopathy, and scattered nodules in the subcutaneous tissues of the abdomen and chest, however bone metastasis were not noted. Patient’s hypercalcemia was treated with zoledronic acid, calcitonin, cinacalcet, and eventually denosumab due to difficulty controlling hypercalcemia. Additional labs obtained during evaluation included ionized calcium &amp;gt;2.5 nmol/L (1.12-1.32), PTHrP &amp;lt;2.0 pmol/L (&amp;lt;2.0), 1,25-dihydroxycholecalciferol &amp;lt;10 pg/mL (21-65). Patient’s right adrenal metastasis biopsy one month prior to admission was subsequently stained for PTH and was negative. Technetium-99m sestamibi scan was obtained for localization of parathyroid adenoma or carcinoma. The scan did not localize, however did show numerous new lytic lesions in the skull, spine, sternum, rib cage, and right humerus. Patient’s hospital course was complicated by progressively worsening renal function, anemia, and thrombocytopenia. Patient was discharged to a long-term acute rehab facility off hypercalcemia treatment with a corrected calcium of 10.0 mg/dL. Conclusion: PTH-mediated hypercalcemia is a rare cause of hypercalcemia of malignancy. PTH levels are typically low or low-normal in osteolytic mediated hypercalcemia. Generally, if PTH is elevated in hypercalcemia, an alternate etiology is not evaluated. This case highlights a rare dual-mediated hypercalcemia of malignancy. The patient’s PTH level elevation was discordant with her high degree of hypercalcemia and prolonged treatment course. She was subsequently found to have widespread bone metastases contributing to hypercalcemia. While multiple etiologies of hypercalcemia can be rare, it is important to evaluate for all contributing causes, especially with additional clinical conditions, such as malignancy. Presentation: 6/3/2024

The genomic and transcriptomic landscape of metastastic urothelial cancer

Metastatic urothelial carcinoma (mUC) is a lethal cancer, with limited therapeutic options. Large-scale studies in early settings provided critical insights into the genomic and transcriptomic characteristics of non-metastatic UC. The genomic landscape of mUC remains however unclear. Using Whole Exome (WES) and mRNA sequencing (RNA-seq) performed on metastatic biopsies from 111 patients, we show that driver genomic alterations from mUC were comparable to primary UC (TCGA data). APOBEC, platin, and HRD mutational signatures are the most prevalent in mUC, identified in 56%, 14%, and 9% of mUC samples, respectively. Molecular subtyping using consensus transcriptomic classification in mUC shows enrichment in neuroendocrine subtype. Paired samples analysis reveals subtype heterogeneity and temporal evolution. We identify potential therapeutic targets in 73% of mUC patients, of which FGFR3 (26%), ERBB2 (7%), TSC1 (7%), and PIK3CA (13%) are the most common. NECTIN4 and TACSTD2 are highly expressed regardless of molecular subtypes, FGFR3 alterations and sites of metastases.