Extracellular vesicles (EVs) represent a promising liquid biopsy platform in multiple myeloma (MM). We developed an MM EV Surface Protein Assay to quantify and dynamically monitor four MM EV subpopulations defined by targetable MM surface proteins (BCMA, CD38, GPRC5D, and CD319) across 336 serial blood samples from 45 relapsed/refractory MM (RRMM) patients treated with anti-BCMA chimeric antigen receptor (CAR) T-cell therapy. All four MM EV subpopulations significantly decreased in 43 patients with initial response, while BCMA+, GPRC5D+, and CD319+ MM EVs increased in 19 patients with progression, and antigen escape was detected by BCMA+ MM EVs. MM EV subpopulations differentiated minimal residual disease (MRD) status and complemented MRD for detecting early relapse before clinical progression. Notably, CD319+ MM EVs were early predictors of progression-free and overall survival in MRD-negative patients. This assay enables noninvasive monitoring of deep response, progression, and antigen escape, and stratifies survival in MRD-negative patients with RRMM.

LBA21 Background: Up to 1/3 of pts develop biochemical relapse following primary therapy. Many are not cured with salvage local therapy, likely because of undetectable distant disease. PSMA is expressed on most PC and can be targeted by radiolabeled J591. 177Lu is a predominantly β-emitting radionuclide and also has γ emission which allows imaging. 111In is predominantly a γ emitter, also with some auger emission for therapy. Hormonal therapy is effective and may increase PSMA expression and radiosensitize. In this DOD-funded study initiated in the pre-PSMA PET and AR signaling inhibitor era, we hypothesized that 177Lu prolongs 18-month (mo) met-free survival (MFS) more than 111In in pts with high risk, M0 CRPC when targeting PSMA via J591 in combo with keto and HC. Methods: Pts with high-risk M0 CRPC defined by PSA DT < 8 mo and/or absolute PSA > 20 ng/mL and serum testosterone < 50 ng/mL with no evidence of metastatic disease on CT/MRI and bone scan were eligible. Treatment included a minimum 4 week lead-in with keto 400 mg TID and HC 20 mg AM, 10 mg PM (both of which could be continued until unacceptable toxicity or development of mets) and a single infusion of J591 with 2:1 randomization to 177Lu (70 mCi/m2) or 111In (5 mCi) in double-blinded fashion. The final version of the protocol was designed to randomize 55 pts for 80% power to detect a difference in 18-mo MFS with one-sided alpha of 10%. Secondary endpoints include median MFS, PSA response, overall survival, and toxicity. Results: 55 pts with median age 68 (range 52 - 88), 75% prostatectomy, 23% primary radiation, 2% primary ADT; 19% local salvage therapy. Median PSA doubling time 3 mo (range 0.87 – 7.85), median baseline PSA 8.0 (range 1-78). In intent to treat analysis (5 without imaging and 4 lost to follow up by 18 mo), 50% developed mets by 18 mo with 177Lu vs 76% with 111In (p=0.066). Median MFS was 23.8 mo vs 20.8 mo, and biochemical PFS was 18.67 vs 8.87 mo, favoring 177Lu in analyses censoring start of new treatment. Confirmed >50% PSA decline occurred in 82% with 177Lu and 71% with 111In. Grade >3 heme AEs were more common with 177Lu vs 111In, including neutropenia (57% vs 11%, with 1 febrile neutropenia) and thrombocytopenia (77% vs 11%, with 25% vs 6% platelet transfusions), whereas Gr >3 non-heme AEs were less common with 177Lu vs 111In, including abdominal pain (0 vs 11%), ALT increase (3.3% vs 22%), and diarrhea (0 vs 22%). Conclusions: Anti-PSMA mAb J591 with keto/HC when radiolabeled with 177Lu leads to improved 18-month met-free survival vs 111In. Most pts had significant PSA decline with either version of radiolabeled J591 with keto/HC. Hematologic toxicity is more common with 177Lu. This supports the development of anti-PSMA radioimmunotherapy for low volume advanced PC, though the optimal radionuclide and targeting agent is unknown. Clinical trial information: NCT00859781 .

Shared decision-making is essential to the care of patients with advanced renal carcinoma which can empower patients and help the healthcare team understand the patient’s goals of care. An important topic during the shared-decision making process is identification and management of treatment-related adverse events. A patient author and two healthcare professionals with real-world experience provide insight into the importance of shared decision-making and its utility in the management of treatment-related adverse events in patients with renal cell carcinoma who are receiving axitinib in combination with an immunotherapy agent.

Supplemental Table 4. Univariate and multivariable Cox regression analyses for patients with detectable CTCs adjusted for minimum nuclear size.

PURPOSE:Biochemical recurrence (BCR) of prostate cancer (PCa) after definitive surgery and/or radiation (including salvage strategies) is a burgeoning area of clinical research inspired by ultrasensitive next-generation imaging. Most phase III trials in PCa have focused on metastatic disease, defined by conventional imaging. Despite the emergence of new imaging, clinical trial principles from metastatic studies will not optimize future BCR trials. METHODS:A Working Group convened at the National Cancer Institute on November 13, 2024 (NCI BCR WG). Key areas of discussion included nomenclature, baseline criteria for data capture, imaging considerations, delineation of high-risk populations to be targeted for trial development, requirements of metastasis-directed therapy (MDT) or hormonal therapy, quality-of-life considerations, and potential study end points. RESULTS:The NCI BCR WG defined the novel term "prostate-specific membrane antigen (PSMA)+BCR" to identify the emerging concept of recurrent PCa identifiable only on PSMA positron emission tomography (PET), overlapping with BCR and distinct from metastatic hormone-sensitive PCa as traditionally defined by conventional imaging. The WG suggested defining high-risk BCR with a prostate-specific antigen doubling time of ≤6 months, regardless of PET findings. The WG provided recommendations for baseline data capture and imaging requirements. Neither systemic therapy nor MDT were considered mandatory for control arms. The WG also discussed novel end points and quality-of-life metrics in this disease space. CONCLUSION:These discussions should inform future clinical BCR trials in this distinct disease space relative to metastatic disease defined by conventional imaging. The NCI BCR WG strongly advocates that future trials explore deintensification of treatment to minimize toxicity in this relatively indolent disease state.

BACKGROUND:B7-H3 is a promising target for cancer therapy, notably in prostate cancer (PCa), particularly in metastatic, castration-resistant PCa (mCRPC). With the development of B7-H3-targeted therapies, there is a need for a rapid, reliable, and cost-effective method to detect and monitor B7-H3 expression. Leveraging their abundance and stability, we developed a liquid biopsy assay using extracellular vesicles (EVs) for this purpose. METHODS:B7-H3+ EVs were isolated using a B7-H3 antibody-mediated, click chemistry-based enrichment method. Antibodies were conjugated to methyltetrazine-grafted microbeads. EVs were isolated from 100 µL of plasma from metastatic, castration-sensitive PCa (mCSPC) (n = 43) and mCRPC (n = 103) patients and quantified using RT-qPCR of ACTB. Measurements were compared with the patient's disease status over time. RESULTS:The assay detected higher B7-H3+ EVs in mCRPC than mCSPC and increased when mCSPC transitioned to mCRPC. Elevated B7-H3+ EVs were associated with lower overall survival (Hazard ratio (HR) 2.19, p = 0.01). In patients with serial plasma samples, B7-H3+ EV levels reflected treatment response and disease progression. CONCLUSIONS:This B7-H3+ EV assay represents a significant advancement in utilizing tumor-derived EVs for a non-invasive, quantitative, and consistent real-time measurement of B7-H3. This assay warrants further development as a companion diagnostic for B7-H3 targeted therapies in PCa and other conditions.

e20009 Background: Osimertinib resistance in patients with EGFR-driven non-small cell lung cancer (NSCLC) contributes to mortality. Targeting bypass alterations have had limited efficacy. Here, we explored the role of CD105 (endoglin) expression, a co-receptor for bone morphogenetic protein (BMP) signaling as a factor of osimertinib treatment. Methods: Sixty-six NSCLC tumors, 44 of which had EGFR mutations were subjected to imaging mass cytometry to identify CD105 expression status as a predictor of clinical outcomes. To study the effects of EGFR and CD105 disruption two osimertinib-resistant EGFR-mutant cell lines were developed for subsequent characterization by single cell RNA sequencing. The promoter chromatin status and glycolytic state of the lines were evaluated by ATAC sequencing and Single Cell ENergetIc metabolism by profiling Translation inHibition analysis (SCENITH), respectively. Results: There was an inverse correlation between the expression CD105 and overall survival in treatment-naïve patients ( P = 0.0071). Modeling osimertinib resistance highlighted the emergence of a persister cell population expressing tumor dormancy features. Further, the persister cells were found to have elevated CD105 levels with responsivity to the neutralizing antibody, carotuximab (ENV105), in restoring epigenetic and metabolic reprogramming to support osimertinib sensitivity. The carotuximab mechanism of action was through the re-engagement of EGFR signaling by potentiating physical CD105/EGFR interaction. Combination of osimertinib and carotuximab significantly reduced tumor growth in two independent NSCLC mouse models ( P = 0.0056 and P = 0.0008), where either single agent had little benefit. Conclusions: CD105 targeting provides a novel mutation-independent strategy to overcome resistance in EGFR-mutant NSCLC. Based on the efficacy of carotuximab capacity to limit tumor dormancy and restored osimertinib sensitivity, a Phase 1 trial (NCT05401110) investigating the combination therapy is currently accruing subjects.

45 Background: PSMA imaging can identify recurrent prostate cancer after definitive surgery/radiation prior to detection on computed tomography (CT) or bone scan. Radiation to PSMA+ findings is common but lacks clear data demonstrating long term benefit. PSMA+ recurrent prostate cancer (PSMArpc) is often defined and treated as metastatic castration sensitive prostate cancer (mCSPC), yet PSMA imaging alone as an eligibility criteria was never studied in the mCSPC trials. PSMArpc requires better understanding to define at-risk patients (pts). Methods: NCT05588128 enrolls pts after definitive and possibly salvage therapies. Pts are required to be 1 year removed from definitive therapy with a PSA≥0.5 ng/ml, testosterone≥100 ng/dL, and negative CT/bone scans. Lymph nodes (LNs) up to 1.5 cm and prior therapies are permitted. At enrollment pts have a baseline PSMA, which is repeated every 6 months (mos) if positive. If negative PSMA is done annually. CT and bone scans are also repeated annually. Pts are allowed to have radiation therapy or systemic therapies for ≤6 mos and remain on-study. Up to 350 pts will be enrolled and followed for up to 5 years. Results: Over 100 pts have enrolled since 3/2023 and 73 pts were evaluable after the 6-month PSMA scan/follow-up. The pts had a median age of 71 years, PSA=2.8, PSA doubling time=11.1 mos (30% less than 6 mos). In an overlapping descriptive analysis 10 pts were PSMA- and 15 pts had only local disease. For PSMA+ LNs, 15 pts had 1 LN, 8 pts had 2-3 LNs+, 4 pts had 4 LNs+, and 16 pts had 5+ LNs. 6 pts had bone findings, but negative bone scan. 3 pts had PSMA+ serosal nodules. After baseline PSMA 3 pts had radiation to solitary LNs, only 1 resolved/PSA declined. 1 pt elected androgen deprivation and 1 pt had salvage radiation. 4 pts enrolled in a clinical study at the NCI without androgen deprivation. At 6 mos PSMA scan only 2 pts had metastatic disease, both with bone scan findings, no pts had LNs beyond eligibility size criteria, and no pts had new visceral findings. Conclusions: These preliminary data from an ongoing study suggest PSMArpc is an indolent disease process and pts are at limited risk for clinically relevant progression within 6 mos. This study continues to accrue at the NCI and will seek to better define high-risk PSMArpc. These preliminary data may better inform the risks/benefits of aggressive treatment of PSMArpc and clinical studies in PSMArpc. Clinical trial information: NCT05588128 .

e17090 Background: ADT, a standard treatment for prostate cancer (PC), is associated with significant side effects such as fatigue, muscle loss, weight gain, and mood changes, which can lead to physical function decline and poor quality of life. Wearable technology for continuous activity monitoring combined with routine patient-reported outcome (PRO) collection may provide a more complete and accurate picture of a patient’s treatment experience, offering valuable information for guiding symptom management. This study aimed to evaluate the feasibility and acceptability of remote patient monitoring with wearable technology and PROs in men diagnosed with PC undergoing ADT. Methods: Men diagnosed with PC undergoing ADT, recruited from two healthcare facilities as part of the DigiPRO trial (NCT04575402), wore an activity monitor (Fitbit Charge HR) continuously to monitor daily activity and sleep and completed monthly electronic PROs (NIH PROMIS and PRO-CTCAE) over a 12-week study period. Feasibility was defined as >60% of patients adhering to wearing the device and completing PROs at the 12-week primary study endpoint. Acceptability was also assessed through qualitative interviews among a subset of patients, gathering feedback on the usability and impact of the wearable devices. Additionally, a survey was conducted among clinicians with experience treating PC patients to assess their interest in accessing and using the wearable and PRO data. The proportion of patients adhering to wearing the devices and completing PROs at 12 weeks was calculated. Descriptive statistics were computed to summarize clinician survey responses, and inductive thematic analysis was applied to patient interview data. Results: All 40 PC participants (100%) (Median age: 70 years, range 51-87, 60% White/37% Black) consistently used the monitoring devices and 38 participants (95%) completed monthly PROs over the 12-week period. From the interviews, patients reported that the devices were easy to use and enjoyed wearing them to monitor their PA and sleep. Clinicians expressed overall support for accessing the device data, where 7/10 clinicians responded that they would access patient wearable activity data in the medical chart and believe EMR-integrated wearable activity data would help them better take care of patients. Conclusions: Our findings suggest that remote activity and PRO monitoring are feasible and acceptable for PC patients, with high device adherence, PRO completion, and perceived benefits of use observed by clinicians and patients. Clinicians and researchers should consider incorporating remote activity and PRO monitoring into future research studies. Clinical trial information: NCT04575402 .

5098 Background: PSMA imaging can identify recurrent prostate cancer after definitive surgery/radiation prior to detection on computed tomography (CT) or bone scan. Radiation to PSMA+ findings is common but lacks clear data demonstrating long term benefit. PSMA+ biochemically recurrent prostate cancer (PSMA+ BCR) is often defined and treated as metastatic castration sensitive prostate cancer (mCSPC), yet PSMA imaging alone as an eligibility criteria was never studied in the mCSPC trials. PSA+ BCR requires better understanding to define at-risk patients (pts). Methods: NCT05588128 enrolls pts after definitive and possibly salvage therapies. Pts are required to be 1 year removed from definitive therapy with a PSA>0.5 ng/ml, testosterone>100 ng/dL, and negative CT/bone scans. Lymph nodes (LNs) up to 1.5 cm and prior therapies are permitted. At enrollment pts have a baseline PSMA, which is repeated every 6 months (mos) if positive. If negative PSMA is done annually. CT and bone scan are also repeated annually. Pts are allowed to have radiation therapy or systemic therapies for ≤6 mos and remain on-study. Up to 350 pts will be enrolled and followed for up to 5 years. Results: Over 120 pts have enrolled since 3/2023 and 86 pts are evaluable after the 6-month PSMA scan/follow-up. The pts have a median age of 71 years, PSA=3.05, PSA doubling time=11.1 mos (29% less than 6 mos). In an overlapping descriptive analysis 10 pts were PSMA- and 17 pts had only local disease. For PSMA+ LNs, 17 pts had 1 LN, 9 pts had 2-3 LNs+, 5 pts had 4 LNs+, and 18 pts had 5+ LNs. 7 pts had bone findings, but negative bone scan. 4 pts had PSMA+ serosal nodules. 4 pts had radiation to solitary LNs. 1 pt elected androgen deprivation and 1 pt had salvage radiation. 4 pts enrolled on a clinical study at the NCI without androgen deprivation. At 6 mos PSMA scan only 1 pt had metastatic disease (bone scan findings). No pts had LNs beyond eligibility size criteria. No pts had new visceral findings. Conclusions: These preliminary data from an ongoing study suggest PSMA+ BCR is an indolent disease process and pts are at limited risk for clinically relevant progression within 6 mos. This study continues to accrue at the NCI and will seek to better define high risk PSMA+ BCR. These preliminary data may better inform the risk/benefits of aggressive treatment of PSMA+ BCR and clinical studies in PSMA+ BCR. Clinical trial information: NCT05588128 .

Immune cells play diverse roles in cancer development. Myeloid cells are key drivers of tumor-escape mechanisms as they suppress immune responses, facilitate metastasis, and contribute to therapy resistance. In particular, macrophages can be polarized into an inflammatory M1 (anti-tumor) or anti-inflammatory M2 (pro-tumor) phenotype. M2 macrophages are associated with tumor progression, as they secrete factors that promote tumor angiogenesis, suppress T-cell activity, and correlate with poor clinical outcomes in squamous cell carcinoma (SCC). Given this context, this study aims to demonstrate the biological effects of monocytes and both M1 and M2 macrophages in squamous cell carcinoma. Our data indicate higher CD163 immunoreactivity in biopsies from SCC patients. Furthermore, we found that a conditioned medium (CM) containing bioactive compound generated by M2 macrophages enhances the proliferation and invasion of the SCC-25 cell line in vitro. Surprisingly, CM derived from blood CD14+ monocytes increased SCC-25 proliferation at the same rate of M2 macrophages-CM. M1 macrophages conditioned medium significantly enhanced the motility and decreased proliferation in Detroit 562 cells. The analysis of tumor-associated transcripts showed that both M1 and M2 conditioned medium induced high levels of EPCAM mRNA and significantly decreased the expression of MYC, an epithelial-to-mesenchymal transition marker, in SCC cell lines. Detroit cells exposed to conditioned medium from monocytes and macrophage also showed elevated SOX2 mRNA levels. The findings suggest that monocytes and macrophage mediators exert distinct biological effects on SCC cell lines.

e17038 Background: Circulating tumor cells (CTCs) with a very-small-nuclear (vsn) phenotype (i.e., vsnCTCs) in prostate cancer (PCa) represent a distinct subset of CTCs characterized by nuclei smaller than 8.5 μm. Our previous studies established a link between vsnCTCs and the presence of visceral metastasis. Emerging evidence suggests that the reduction of emerin (EMD), a nuclear envelope protein, contributes to PCa metastasis and is associated with nuclear shape instability. This study aims to validate vsnCTCs as a biomarker in metastatic castration-resistant prostate cancer (mCRPC) and investigate the correlation between EMD expression and the vsnCTC phenotype. Methods: CTCs were isolated from 93 mCRPC patients using the NanoVelcro CTC assay and categorized as either vsnCTC-positive (vsnCTC+) or vsnCTC-negative (vsnCTC-). We compared overall survival (OS) and progression-free survival (PFS) between these two groups. In vitro experiments were conducted using PCa cell lines (C4-2B, 22Rv1, and DU145) with EMD knockdown via siRNA or shRNA to study its impact on cellular phenotypes. Additionally, we measured EMD expression and nuclear size in abiraterone- and enzalutamide-resistant (Abi-R/Enza-R) C4-2B cells. Results: vsnCTC+ patients had significantly worse OS and PFS compared to vsnCTC- patients. Multivariate analysis revealed that vsnCTC+ status was independently associated with poorer OS and PFS. EMD expression was markedly reduced in CTCs from vsnCTC+ patients compared to vsnCTC- patients, with a significant positive correlation between EMD expression and CTC nuclear size. EMD knockdown in PCa cells resulted in smaller nuclei, enhanced invasion, and the upregulation of genes associated with neuronal de-differentiation (e.g., SOX2, CHGA, MYCN, and AURKA). Additionally, C4-2B Abi-R/Enza-R cells had significantly smaller nuclei and lower EMD expression, consistent with our clinical observation of the vsnCTCs association with ARSI resistance. These resistant cells also demonstrated elevated expression of neuronal de-differentiation genes. Conclusions: The presence of vsnCTCs represents a novel hallmark of an aggressive subtype of mCRPC, closely linked to EMD dysregulation and neuroendocrine differentiation. These findings highlight the potential of vsnCTCs and associated biological changes as a predictive biomarker. Our findings underscore the importance of EMD in the progression and therapeutic resistance of advanced PCa.

207 Background: Circulating tumor cells (CTCs) with a very-small-nuclear (vsn) phenotype (i.e., vsnCTCs) in prostate cancer (PCa) represent a distinct subset of CTCs characterized by nuclei smaller than 8.5 μm. Our previous studies established a link between vsnCTCs and the presence of visceral metastasis. Emerging evidence suggests that the reduction of emerin (EMD), a nuclear envelope protein, contributes to PCa metastasis and is associated with nuclear shape instability. This study aims to validate vsnCTCs as a biomarker in metastatic castration-resistant prostate cancer (mCRPC) and investigate the correlation between EMD expression and the vsnCTC phenotype. Methods: CTCs were isolated from 93 mCRPC patients using the NanoVelcro CTC assay and categorized as either vsnCTC-positive (vsnCTC+) or vsnCTC-negative (vsnCTC-). We compared overall survival (OS) and progression-free survival (PFS) between these two groups. In vitro experiments were conducted using PCa cell lines (C4-2B, 22Rv1, and DU145) with EMD knockdown via siRNA or shRNA to study its impact on cellular phenotypes. Additionally, we measured EMD expression and nuclear size in abiraterone- and enzalutamide-resistant (Abi-R/Enza-R) C4-2B cells. Results: vsnCTC+ patients had significantly worse OS and PFS compared to vsnCTC- patients. Multivariate analysis revealed that vsnCTC+ status was independently associated with poorer OS and PFS. EMD expression was markedly reduced in CTCs from vsnCTC+ patients compared to vsnCTC- patients, with a significant positive correlation between EMD expression and CTC nuclear size. EMD knockdown in PCa cells resulted in smaller nuclei, enhanced invasion, and the upregulation of genes associated with neuronal de-differentiation (e.g., SOX2, CHGA, MYCN, and AURKA). Additionally, C4-2B Abi-R/Enza-R cells had significantly smaller nuclei and lower EMD expression, consistent with our clinical observation of the vsnCTCs association with ARSI resistance. These resistant cells also demonstrated elevated expression of neuronal de-differentiation genes. Conclusions: The presence of vsnCTCs represents a novel hallmark of an aggressive subtype of mCRPC, closely linked to EMD dysregulation and neuroendocrine differentiation. These findings highlight the potential of vsnCTCs and associated biological changes as a predictive biomarker. Our findings underscore the importance of EMD in the progression and therapeutic resistance of advanced PCa.

The CUT/Hox transcription factor ONECUT2 (OC2) promotes lineage plasticity and is a confirmed therapeutic target in prostate cancer and several other malignancies where cell phenotype plays a substantial role in treatment resistance. OC2 governs a broad growth and lineage identity process in prostate cancer that promotes neuroendocrine (NE) differentiation, androgen receptor (AR) suppression, and the emergence of a wide range of treatment-resistant pathways. The mode of action of OC2 includes incorporation of the protein into transcription complexes at gene promoters as an activator and repressor, alteration of chromatin accessibility and epigenetic marks, and extensive alteration of large-scale chromatin modifications, such as super-enhancers and chromatin loops. Notably, OC2 may be unique among NE drivers in that it can promote AR indifference in adenocarcinoma as a direct upstream activator of the glucocorticoid receptor, thus assuming indirect control of a portion of the AR cistrome. OC2 expression and activity increase substantially following hormone therapy in association with aggressive disease in prostate and breast cancer. Experiments in model systems have shown that OC2 has a survival function in both human castration-sensitive and castration-resistant prostate cancer cells. OC2 can be targeted directly with a family of novel small-molecule inhibitors that show therapeutic efficacy in vivo in prostate, breast, and gastric cancer models, including regression of established distant metastases in mice. These findings suggest that inhibition of OC2 clinically may confer substantial therapeutic benefit in some aggressive malignancies, including in localized hormone-sensitive disease.

Extracellular vesicles (EVs) are heterogeneous in size, biogenesis, content, and function. Aggressive cancer cells release a distinct, poorly characterized, and particularly large EV subtype, namely large oncosomes (LOs). This study employs an optimized method to improve LO yields and integrates mass spectrometry and RNA sequencing (RNA-seq) to profile their molecular cargo. A consistent set of proteins enriched in LOs is identified across glioma, prostate, and breast cancer cell lines. These proteins are also present as mRNA in LOs from the prostate cancer model and are abundant in plasma LOs from 20 patients with metastasis. Single-LO RNA-seq confirms bulk LO cargo, demonstrating the utility of single-cell technologies for large vesicle analysis. Our patient study provides proof-of-principle evidence that we can use multiomics to delve into EV heterogeneity, biogenesis, and composition. It also suggests that plasma LOs help stratify patients, supporting their potential prognostic value for developing a multi-analyte approach for liquid biopsy.

Supplemental Table 2. Univariate and multivariable Cox regression analyses.

206 Background: It has been recognized that as a prostate cancer (PCa) metastasizes to bone, it begins to express bone-specific proteins such as osteopontin, bone sialoprotein, and osteocalcin. This process is known as osteomimicry. Liver metastases (LM) are associated with the poorest clinical outcomes and affect 25% of PCa patients at autopsy. We hypothesized that PCa that metastasize to the liver express liver-specific genes prior to liver metastasis – a process we have named “hepatomimicry”. Methods: We have curated a list of 23 putative genes that are both primarily expressed in the liver and found in RNA-seq data from over 3000 PCa samples. Initial sources included the PCa transcription atlas (n = 2115), West Coast/SU2C Dream Team (n = 210), neuroendocrine PCa from cBioPortal (n = 49), and TEMPUS RNA-seq data generated specifically for Cedars-Sinai (n = 88). In each dataset, for the 23 hepatomimicry genes, we used recursive feature elimination and a t-test to discriminate between PCa LM and non-liver visceral metastases. In datasets with count-level data, we performed differential gene expression analysis with DeSeq2 and gene set enrichment analysis (GSEA) on identified differentially expressed genes. Results: In three different datasets, MAT1A, ELF3, and WEE1 were found to be either optimal genes for discriminating between these two groups or significantly different by t-test and were more highly expressed in LM. 270 genes were downregulated and 437 genes were upregulated in liver metastases (FDR q-value < 0.05, log2 fold change > 1 for upregulated and < -1 for downregulated). Subsequent GSEA found that liver-specific gene sets are highly enriched in the genes upregulated in LM, and endothelial cell genes are strongly downregulated. Conclusions: The initial results indicate that a three-gene subpanel of our initial set have predictive power for LM against other visceral metastases in PCa.While hepatocyte infiltration may impact the characterization of these samples by yielding a strong signal for liver-specific gene expression signature, confirmation of this finding remains important. Ongoing work is directed at confirming this signature in single-cell RNA-seq from PCa LM. Our work may be adaptable to developing diagnostic technologies such as liquid biopsies that may enhance the identification of patients who may have occult LM or that may develop LM as part of their natural history so that interventions may be enacted in a more timely manner.

PURPOSE:Circulating tumor cells (CTC) with a very-small-nuclear phenotype (vsnCTC) in prostate cancer are characterized by nuclei smaller than 8.5 μm. Our previous studies established an association between vsnCTCs and visceral metastasis. Reduction of emerin (EMD), a nuclear envelope protein, contributes to prostate cancer metastasis and nuclear shape instability. In this study, we investigated the correlation between EMD expression and the vsnCTC phenotype and its clinical impact. EXPERIMENTAL DESIGN:We analyzed CTCs from 93 patients with metastatic castration-resistant prostate cancer and categorized them as either vsnCTC+ or vsnCTC- and compared overall survival and progression-free survival. C4-2B, 22Rv1, and DU145 with EMD knockdown were developed and characterized by nuclear size and gene expression by gene set enrichment analysis. Abiraterone- and enzalutamide-resistant C4-2B cells were also characterized by nuclear size and EMD expression. RESULTS:Patients who were vsnCTC+ had significantly worse overall survival and progression-free survival compared with patients who were vsnCTC-. EMD expression was markedly reduced in CTCs from patients who were vsnCTC+ compared with patients who were vsnCTC-, with a significant positive correlation between EMD expression and CTC nuclear size. EMD knockdown in prostate cancer cells resulted in smaller nuclei, enhanced invasion, and the upregulation of genes associated with lineage plasticity. Additionally, abiraterone- and enzalutamide-resistant C4-2B cells had smaller nuclei and lower EMD expression. vsnCTC+ cells also showed enhanced platinum sensitivity. CONCLUSIONS:The presence of vsnCTCs represents a novel hallmark of an aggressive subtype of metastatic castration-resistant prostate cancer closely linked to EMD loss and lineage plasticity. These findings highlight the importance of EMD dysregulation in the vsn phenotype, disease progression, and therapeutic resistance in patients with prostate cancer.

AIM:To investigate the role of CD105 in mediating drug resistance to EGFR-targeted therapy in non-small cell lung cancer (NSCLC). METHODS:Imaging mass cytometry was conducted on 66 NSCLC tumors, 44 of which had EGFR mutations. We correlated clinical variables, including overall survival, with CD105 (endoglin) expression, a co-receptor for bone morphogenetic protein (BMP) signaling. Two osimertinib-resistant EGFR-mutant cell lines were developed to study the effects of EGFR and CD105 disruption. Single cell RNA sequencing of the isogenic parental and osimertinib resistant lines was performed. Additionally, ATAC sequencing and Single Cell ENergetIc metabolism by profiling Translation inHibition analysis (SCENITH) was used to assess promoter chromatin status and glycolytic state. RESULTS:We found a negative correlation between CD105 expression and overall survival in patients. Treatment with osimertinib or EGFR knockdown significantly elevated CD105 expression in EGFR-mutant cell lines. Single-cell RNA sequencing identified a subset of cells with heightened endothelial characteristics and altered pyrimidine metabolism, associated with osimertinib resistance. These cells exhibited a slow-cycling behavior, characterized by elevated chromatin condensation and reduced glycolysis. Combining osimertinib with carotuximab, a CD105 neutralizing antibody, significantly reduced the slow-cycling transcriptomic signature, increased chromatin accessibility, and restored glycolysis compared to osimertinib treatment alone. Mass spectrometry confirmed that carotuximab re-engaged EGFR signaling by coupling it with CD105. Consequently, carotuximab re-sensitized resistant tumors to osimertinib by increasing their mitotic index and ERK signaling in mouse models. CONCLUSION:Carotuximab effectively reduced the slow-cycling cell population and restored osimertinib sensitivity, offering a promising strategy for managing refractory NSCLC.