Citations

TPS3159 Background: ELU001 is a novel first-in-class nanoparticle drug conjugate that consists of ~13 folic acid targeting moieties and a payload of ~22 molecules of the topoisomerase-1 inhibitor, exatecan. Folic acid and exatecan are covalently bound by non-cleavable and cathepsin-B cleavable linkers, respectively, to short polyethylene glycol chains which surround the C’Dot’s silica core. CDCs are very small (~6 nm), allowing for more efficient penetration into solid tumors compared to ADCs and a rapid elimination by the kidneys, which is expected to lead to less toxicity than ADCs that have a longer half-life in circulation. ELU001’s high avidity is designed to promote binding to FRα on the surface of cancer cells that express any level of antigen (low to high). ELU001 then internalizes into the tumor cell and traffics to the lysosome where enzymatic cleavage releases the exatecan payload. ELU001 has demonstrated efficacy in preclinical models with low, moderate and high levels of FRα. The first-in-human trial, ELU-FRα-1, is currently recruiting patients with ovarian, endometrial, colorectal, gastric, gastroesophageal junction, triple negative breast, or non-small cell lung cancers, or cholangiocarcinoma cancers, and, in the opinion of the Investigator, have no meaningful life-prolonging therapy option available. Methods: This is a Phase 1 / 2 multicenter, open label clinical trial with two parts: Part 1 Dose Escalation and Part 2 Tumor Group Expansion Cohort(s). In Part 1, patients with cancer types with a high likelihood of having FRα overexpressing tumors, (ovarian, endometrial, colorectal, gastric, gastroesophageal junction, triple negative breast, or non-small cell lung cancers, or cholangiocarcinoma) will be enrolled. FRα expression is determined retrospectively. ELU001 is administered either weekly (QW) (once a week for 3 weeks, 1 week rest), every other week (Q2W, with no rest between cycles), or every three weeks (Q3W). Initial Part 2 expansion cohorts are planned in ovarian cancer with high FRα expression, ovarian cancer with moderate or low FRα expression, and endometrial cancer with any positive FRα expression. Here, FRα will be prospectively determined. Other cancer types may be assessed in the future. The primary objective for Part 1 is to identify the MTD/RP2D. The primary objective for Part 2 is to determine the ORR. Secondary objectives include DOR, PFS, TFST, PFS2, OS, frequency, severity and tolerability of adverse events, PK, ADA, and FRα expression assessments. Part 1 Dose Escalation will recruit about 2-5 patients at each dose for each regimen (QW; Q2W; Q3W). Part 2 Stage 1 will recruit about 15 patients per tumor group expansion cohort and is expected to begin recruitment in the second half of 2023. The study is enrolling in Part 1 Q2W Cohort 301 and Q3W Cohort A in the US. Clinical trial information: NCT05001282 .

3109 Background: WSD0922-Fu is an oral, central nervous system (CNS)-penetrant, small molecule, ATP non-competitive, reversible EGFR inhibitor which potently inhibits EGFR aberrations specific to non-small cell lung cancer (NSCLC) and high-grade astrocytoma (HGA). We report initial results of a first-in-human phase 1 dose-escalation trial (NCT04197934) in patients with EGFR aberrant recurrent HGA and NSCLC. Methods: Adult patients with either recurrent EGFR mutant NSCLC with CNS metastases or recurrent EGFR mutant and/or EGFR amplified HGA were enrolled. WSD0922-Fu was orally administered, and dose escalated in cohorts based on a standard 3+3 design to determine the maximum tolerated dose (MTD). Toxicities were graded per CTCAE v5.0 and preliminary efficacy was based on modified RANO criteria or RECIST 1.1 as appropriate. Results: 25 patients (17 HGA, 8 NSCLC) were enrolled and treated in six cohorts (40 mg once daily to 320 mg twice daily, 28-day cycles). The median age was 57 years (range 39-75 years). 24% were female and 36% were ECOG 0. For patients with HGA, all patients were previously treated with radiation and temozolomide; the median number of prior lines of systemic therapy was 1 (range 1-4). For patients with NSCLC, all patients were previously treated with an EGFR inhibitor (prior osimertinib 88%); the median number of prior lines of systemic therapy was 1.5 (range 1-6). The median duration of exposure to WSD0922-Fu among all patients was 54 days (range 2-258 days). The most common (≥25%) treatment-related adverse events included fatigue (68%), acneiform rash (68%), diarrhea (52%), nausea (48%) and pruritis (28%). The frequency of severe AEs was dose dependent. The most common Grade 3+ AEs (≥10%) included lymphopenia (16%), acneiform rash (12%), headache (12%) and diarrhea (12%). The MTD was defined as 160 mg twice daily. At Cycle 1 Day 15 for patients treated at this dose level, the mean terminal elimination half-life (%CV) in the plasma was 31.6 h (102%) (median 14.5 h; range 6.2-78.7 h). The mean Cmax, Tmax, oral clearance at steady state, and AUC (0-12 h) (%CV) were 4513 ng/mL (50%), 2.0 hr (69%), 6.9 L/hr/m2 (42%), and 28084 h*ng/mL (52%), respectively. Oral clearance at steady state was inversely related with dose, suggesting either inhibition or saturation of clearance processes. Among patients treated at MTD or higher dose levels and with available response data, 100% (5/5) of patients with NSCLC demonstrated clinical benefit (SD + PR) while 60% of patients had partial responses (intracranial and extracranial). Furthermore, prolonged stable disease (9 cycles) was observed in a patient with EGFRvIII mutant GBM. Conclusions: The MTD for WSD0922-Fu monotherapy is 160 mg BID. Preliminary efficacy was observed in patients with osimertinib-resistant NSCLC and EGFRvIII mutant HGA. Enrollment to expansion cohorts is ongoing. Clinical trial information: NCT04197934 .

3082 Background: CLDN6, a member of the claudin family of tight junction proteins, is expressed at high levels in multiple human malignancies and has little to no expression in normal tissues. This expression profile makes CLDN6 an ideal target for development of new therapeutics. TORL-1-23 is first-in-class ADC targeting the tumor-specific antigen CLDN6. Methods: TORL123-001 (NCT05103683) is an ongoing, 2-part, first in human study to characterize the safety, tolerability, dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), and recommended phase 2 dose (RP2D) of TORL-1-23 monotherapy in participants with advanced solid tumors. Serum pharmacokinetics (PK), immunogenicity and clinical efficacy are also assessed. TORL-1-23 is administered as a 30-minute IV infusion once every 3 weeks in 21-day cycles. During Part 1 (Dose Escalation), cohorts of 1 to 6 participants are evaluated at each dose level according to an accelerated titration design. In Part 2 (Dose Expansion), several cohorts of patients with CLDN6-expressing cancers will be evaluated to confirm the RP2D in ovarian cancer, NSCLC, and other CLDN6-expressing cancers using a CLDN6 IHC companion diagnostic. Results: As of 01FEB2023, 22 patients with ovarian (n=18), testicular (n=3), and endometrial (n=1) cancers were enrolled and treated across 8 dose levels ranging from 0.2 to 2.4 mg/kg IV every 3 weeks. 95% of pts had received ≥ 3 prior lines of treatment in the metastatic setting. The most common treatment-related adverse events were Gr1/2 fatigue (n=5), Gr1 peripheral neuropathy (n=4), and Gr1 nausea (n=3). No DLTs have been reported and no dose reductions have been required. Preliminary PK data demonstrate sustained exposure of TORL-1-23 ADC over the 21 day dosing interval and low levels of serum MMAE indicating low off-target MMAE exposure outside of tumor. Partial responses (PR) were observed in 4/17 efficacy evaluable participants with CLDN6+ disease (3 ovarian, 1 testicular). Dose escalation is ongoing and updated results will be presented. Conclusions: TORL-1-23 has a favorable safety/tolerability profile and PK characteristics with preliminary antitumor activity in pts with heavily-pretreated CLDN6-expressing ovarian and testicular cancers. Doses above the historic MTD for MMAE containing ADCs may be explored given the safety/tolerability and PK data observed up to 2.4 mg/kg. Dose finding is ongoing to identify the MTD and optimal doses for subsequent development. Clinical trial information: NCT05103683 .

3079 Background: Elraglusib (9-ING-41), a novel inhibitor of GSK-3β, has been evaluated in >230 patients (pts) with advanced malignancies including metastatic melanoma in the 1801 phase 1/2 trial (NCT03678883). Pts with melanoma treated with single agent elraglusib demonstrated clinical benefit including a durable complete response despite multiple prior lines of therapy including checkpoint inhibitors (CPI). Methods: We collected data from numerous -omic modalities in the 1801 trial to characterize markers of elraglusib response with bioinformatics and machine learning (ML). Genomic panels were collected on 105 pts. The most frequent tumor types included colorectal cancer (n=35), pancreas (n=31), lung (n=15) and metastatic melanoma (n=12). Pts were treated with elraglusib as a single agent or in combination with chemotherapy. Pathway based features were generated by quantifying the percent of each Reactome pathway that was mutated. These pathway features were iteratively evaluated for their performance in shallow neural networks predicting patient RECIST with each model using 5-15 randomly selected features at a time. Publicly available genomic data from an anti-PD-1 trial in patients with metastatic melanoma (Hugo et al., 2017) was transformed into pathway-based features and input into the final model to predict elraglusib response in this population. Results: Our final pathway based genomic model for elraglusib response performed with a training accuracy of 93% and a test accuracy of 88% on the 1801 patients. We then tested the model on metastatic melanoma patients who were treated with anti-PD-1 therapy. We found that patients from Hugo et al. were predicted to respond to elraglusib (22/26). In addition, most patients with tumors resistant to anti-PD-1 therapy were also predicted to respond to elraglusib (10/12). Finally, we tested the model on metastatic melanoma patients treated with elraglusib in the 1801 study that had failed CPI and for whom -omic data was available. The model correctly identified 5/5 patients that demonstrated clinical benefit (1 CR, ongoing>1400 days; 4 prolonged OS of 107, 256, 357, and 556 days). Conclusions: We applied robust in-silico modeling developed using 1801 trial data to develop a rationale for the development of elraglusib in the treatment of metastatic melanoma including patients that failed or were refractory to anti-PD-1 treatment. This model correctly identified metastatic melanoma patients in 1801 with good clinical outcomes that had failed prior CPI treatment. Further, we present this in silico approach as a robust way to repurpose prior clinical study genomics and ML modeling for population enrichment during the clinical development of novel drugs. Clinical trial information: NCT03678883 .

3054 Background: Tumor Macrophage Fusion Cells (TMFCs) are hybrids fusions of tumor cells and macrophage/immune cells that have recently been documented in advanced solid tumors, such as metastatic Breast Cancer (mBC). Further, TMFCs are found in the blood of patients (pts) as a CD45+/CD14+ binucleated subtype of standard circulating tumor cells (CTCs), which are Cytokeratin+ (CK+) & CD45 negative, but distinct from highly hyperploidy cells known as cancer associated macrophage-like cells. However, no study has explored the clinical meaning of TMFCs in blood. To better elucidate the clinical meaning of TMFCs, we prospectively pooled 137 mBC pt samples prior to induction of new therapy, ie baseline (BL), and after therapy induction (FU). Both TMFCs or standard CTCs were identified and enumerated to analyze their prognostic value for progression-free survival (PFS) and overall survival (OS). Methods: In this prospective study, we collected 7.5 mL blood samples from n=137 mBC pts enrolled in prospective trials from multiple institutions before starting new treatment lines for newly progressive mBC. If possible, an optional FU sample was collected (n=73) after BL (median=4.9 weeks). TMFCs & CTCs were isolated using a CellSieve microfilter and differentiated by staining for CK, CD45, CD14, and DAPI. Cox proportional regression hazard ratios (HRs) with 95% Confidence Intervals (CI) for PFS & OS by univariate & multivariate analyses were based on RECIST v1.1, determined by local institutional pathologist over 24 months (M). Results: TMFCs were detected in 26.3% (n=36/137) at BL and associated with significantly worse PFS (HR=3.3, CI 1.9-5.7, p<0.001) & OS (HR=2.7, CI 1.4-5.1, p=0.005). TMFCs were also detected in 27.4% (n=20/73) at FU (n=73) and prognostic for PFS (HR=5.2, CI 2.3-11.9, p<0.001) & OS (HR=5.1, 1.8-14.2, p=0.004). Further, pts with increases in TMFCs between BL and FU had worse PFS (HR=4.3, CI 1.9-10.1, p=0.001) & OS (HR=4.5, CI 1.6-12.7, p=0.011). In contrast, pts with CTCs were found in 39.4% (n=54/137) at BL, which were prognostic for worse PFS (HR=2.0, CI 1.3-3.2, p=0.005) and not prognostic for OS (HR=1.7, CI 0.9-3.0, p=0.103). At FU, pts with CTCs were found in 38.4% (n=28/73), which were prognostic for worse PFS (HR=3.6, CI 1.8-7.3, p=0.001) & OS (HR=3.2, CI 1.3-8.0, p=0.021). Overall at BL, pts without CTCs or TMFCs had a 5.7 M mPFS & >24M mOS, CTCs alone had a 5.0 M mPFS & 20.3 M mOS, TMFCs had 2.0 M mPFS & 5.8 M mOS. At FU, pts without CTCs or TMFCs had a 5.8 M mPFS & 14.5 M mOS, CTCs alone had a 3.3 M mPFS & 14.6 M mOS, while TMFCs pts had a 1.7 M mPFS & 4.8 M mOS. Conclusions: TMFCs appear to represent a CTC subgroup with additional prognostic value to standard CTCs, that may correlate with significantly faster PFS and OS versus pts with CTCs alone, or without CTCs. Subtyping treatment regimens is ongoing to determine if the TMFC populations are responsive to specific therapy types in these mBC pts.

e14683 Background: Immune checkpoint inhibitors (ICIs) have altered the landscape of cancer treatment. ICIs are associated with immune mediated diarrhea and colitis (IMDC) which can be challenging to diagnose and differentiate from other etiologies of diarrhea. Clostridium difficile infection (CDI) is a common cause of diarrhea in patients with cancer and can have similar symptoms. In a similar study, CDI was found to be relatively common in ICI-treated cancer patients and especially those with IMDC requiring immunosuppressants. Our study evaluates the incidence of CDI and IMDC in patients with ICI exposure and compares clinical characteristics between the two to determine risk factors that may predispose patients to CDI or IMDC. Methods: This is a retrospective, single-center study of cancer patients on an ICI from 1/1/2011 to 4/7/2022 who were tested for Clostridium difficile with stool nucleic acid amplification tests during or after treatment with an ICI. Patients were determined to have CDI if clinically suspected and had to have a positive stool GDH Ag along with positive C. difficile toxins A & B or a positive stool C. difficile toxin B gene. Patients with positive GDH Ag in the stool but with negative toxins A&B were determined to be colonized. The diagnosis of IMDC was based on the clinical presentation and had to be made by the treating oncologist. Results: 375 patients met our study criteria and got CDI testing at any point during or after ICI exposure. 27 patients tested positive for CDI, whereas 24 were colonized. 105 patients were determined to have IMDC and 97/105 received treatment with steroids or immunosuppressants. Among patients determined to have IMDC, 6 were colonized for clostridium difficile. There were no patients with IMDC who were also positive for clostridium difficile. 71 patients were on a dual ICI regimen whereas 304 patients were on a single ICI. In patients on the dual ICI regimen, 67.6% were diagnosed with IMDC when compared to patients on a single ICI regimen where 18.8% of patients were diagnosed with IMDC (p<0.0001). The absolute lymphocyte count on the day of CDI testing was 1.185 ± 0.066 in the IMDC group when compared to 0.948 ± 0.044 in the non-IMDC group (p=0.003). The mean duration of diarrhea was longer in the IMDC group at 16.905 ± 1.211 days when compared to the CDI group at 9.852 ± 2.387 days (p= 0.0094). Smoking status, sex, history of autoimmunity, and history of prior immune related adverse events did not impact incidence of IMDC or CDI. Conclusions: IMDC was a common cause of diarrhea in patients with ICI exposure who were tested for CDI. We did not find IMDC to be a risk factor for CDI. IMDC has a longer duration of diarrhea and colitis symptoms when compared to CDI. The absolute lymphocyte count on symptom onset is higher in patients with IMDC when compared to patients with CDI and may be useful in identifying patients with IMDC earlier.

e14674 Background: Myocarditis is an uncommon but serious adverse event (AE) of immune checkpoint inhibitor (ICI) therapy. We aimed to identify factors associated with ICI-related myocarditis in cancer patients using pooled data from multiple clinical trials. Methods: Data on 25,965 adults receiving at least one dose of an ICI (nivolumab [NIVO] or ipilimumab [IPI])-containing regimen were extracted from corporate safety and clinical trial databases. Patients missing documentation of AEs, indication, or trial ID were excluded. Myocarditis events were identified using MedDRA preferred terms. A nested case-control study design with controls who had no myocarditis selected at random from the risk sets and matched 1:5 on ICI regimen, duration of exposure to ICI regimen, and trial ID was used. Descriptive statistics were used to derive time to onset of myocarditis and to investigate patterns of myositis and rhabdomyolysis relative to myocarditis. A multivariable conditional logistic regression model was used to identify factors associated with myocarditis. Covariates included age, sex, race/ethnicity, medical history (cardiovascular disease, diabetes, sleep apnea), smoking, cancer type, medication use (within the prior 6 months), and co-occurrences of other AEs (pneumonitis, colitis, and hepatitis). Results: Myocarditis was documented in 122/24,106 (0.5%) patients meeting study eligibility criteria. A total of 732 patients (122 cases and 610 matched controls) from 46 trials were included in the analyses. Mean (SD) age was 63 (12) years; 65% of patients were male, 76% Caucasian, and 52% had a history of cardiovascular disease. NIVO + IPI was the most commonly used regimen (36%), followed by NIVO + relatlimab (25%) and NIVO + chemotherapy (14%). Median (Q1-Q3) time to onset of myocarditis was 38 (26-77) days from first dose of ICI. Myositis was reported in 18/122 (15%) cases and in 2/610 (0.3%) controls (p<0.001). Among the 18 cases, a majority (83%) developed myositis within 3 days before the onset of myocarditis. Rhabdomyolysis was reported in 4/122 (3%) cases, all within a month before myocarditis, and in none of the controls (p=0.001). In adjusted analyses, older age (odds ratio 1.38 per 10-year increase, 95% CI 1.12-1.70) and hepatitis (5.69, 2.24-14.44) were significantly associated with the development of myocarditis. Conclusions: In this large cohort of ICI-treated cancer patients, myocarditis had an early onset and was associated with older age and co-occurrences of hepatitis. Due to a low incidence among controls, rhabdomyolysis and myositis could not be included as covariates in adjusted analyses; however bivariate results showed strong associations between myocarditis and rhabdomyolysis or myositis. These findings suggest that co-occurrences of myositis, rhabdomyolysis, or hepatitis are associated with myocarditis and may originate from a more generalized ICI myopathy.

e14576 Background: Immune checkpoint inhibitors (ICI) have revolutionized the treatment of many solid malignancies. ICI appears particularly effective when tumors exhibit high microsatellite instability (MSI-H) and/or mismatch repair deficient (dMMR) status. Although the response is typically quite good in this setting, there are scenarios where benefit is less than anticipated. In this study, we aimed to explore the efficacy of ICIs in patients with MSI-H/dMMR cancers with peritoneal metastasis (PM). Methods: We conducted a retrospective chart review to identify patients (pts) with PM from primary MSI-H/dMMR cancers treated with immunotherapy between 2010 and 2022 at Mayo Clinic. Data was collected to include pt demographics, tumor characteristics, pathology, tumor markers, imaging, mutational profile, and previous and subsequent treatments. The primary endpoint was disease control rate (DCR), defined as objective response rate (RECIST v1.1 criteria) or stable disease. Results: Forty-six pts with MSI-H/dMMR and PM treated with ICIs. Most of our cohort were female (61%) with a median age at diagnosis of 61 years. Colorectal cancer was the most common primary site (69.6%), followed by endometrial (6.5%). Most pts received treatment with PD-1 antibodies as second-line therapy following systemic chemotherapy. DCR of the PM was 71.6 % (n= 13 PR, 9 CR, and 9 SD), with a median duration of response of 12.6 months. Thirteen pts (28.4%) had progressive peritoneal disease on ICI with a median PFS of 2.2 months. Ascites was a poor prognosticator and predicted a lack of benefit from ICI (HR 6.6, 95% CI=2.24-19.9, p= 0.006). Mucinous and signet ring histology were common in pts with peritoneal progression (61.5%); however, this was not statistically significant (HR=1.6; 95%CI= 0.4383 to 3.899, p 0.5). Multivariate analysis for the presence or absence of BRAF and/or RAS mutations was not statistically significant in predicting response. Neither was the specific MMR gene mutation(i.e., MLH1 or PMS2). Interestingly, discordant responses between the peritoneum and other sites of metastasis to immunotherapy were seen in 13 pts (28.4%), primarily as primary peritoneal progression compared to other tumor sites. Conclusions: In our cohort, the DCR of metastatic peritoneal disease in MSI-H/dMMR tumors was approximately 71%. The presence of ascites was identified as a poor prognosticator in this setting and was associated with a lack of response/benefit from ICIs. Histologic subtype (i.e., mucinous/signet ring) did not show statistical significance in predicting ICI response. The subpopulation with discordant response to ICI suggests that peritoneal immunobiology may differ from other sites. Further research on immunotherapy response in the peritoneum is needed for this special subset of pts.

e14555 Background: 9-ING-41 (elraglusib) is a novel inhibitor of GSK-3β that has shown early signs of clinical benefit in a phase 1/2 clinical trial (ACT1801; NCT03678883). Early data support a multi-modal mechanism of action for elraglusib that targets both the tumor and immune system. Thus, the discovery and validation of biomarkers of drug sensitivity to elraglusib is a high priority. Here, we take inspiration from the literature on checkpoint inhibitor therapy and explore the potential of cytokines to predict elraglusib clinical response. Methods: Peripheral plasma samples from 59 patients enrolled in the 1801 phase 1/2 trial of elraglusib were assayed for 40 circulating cytokine levels. Patients advanced solid tumors refractory to standard treatments and received either elraglusib (1.0-15.0 mg/kg) as a single agent or in combination with other chemotherapies. Samples were taken from time points prior to elraglusib administration (pre-dose), after cycle 1, or after cycle 2. Each cytokine time point combination was used to stratify patients into high and low categories using an optimal cut-point determined using maximally selected rank statistics. Kaplan Meier and Cox Proportional Hazards analysis was used to determine the efficacy of each cytokine as a biomarker. The change in cytokine values between pre-dose and cycle 1 or pre-dose and cycle 2 was evaluated in a similar manner. Results: A total of 142 samples spanning eight different tumor histologies were collected and analyzed. A total of 240 cytokine-timepoint combinations were evaluated, and 53 significantly stratified patients by overall survival (p-value < 0.05). Of those, 35 showed that higher levels of the given cytokine correlated with a survival benefit and 18 a survival decrease. Notable among the cytokines demonstrating survival benefits were Granzyme B at Cycle 1 Day 4 (HR: 9.54, p-value:0.015) and TGF β at Cycle 1 Day 4 (HR: 8.48, p-value:0.0063). Strong hazard ratios were also observed for cytokines that correlated with survival decreases including TRAIL-R2 at Cycle 1 Day 2 (HR: 9.56e-02, p-value:0.0001), and IL-10 at Cycle 1 Day 4 (HR: 8.77e-02, p-value:0.007). Delta between timepoints was also able to stratifying patients by overall survival. Conclusions: Plasma cytokine profiling is a promising approach for discovery and validation of biomarkers prognostic for elraglusib clinical benefit. Numerous cytokines measured pre-dose stratified patients into significant survival groups suggesting a potential strategy for patient enrichment in clinical trials of elraglusib. These initial findings are being expanded by 1) increasing the cohort to accommodate histology specific investigations; 2) examining which cytokines are elraglusib response-specific versus drug-agnostic; and 3) combining cytokines in multivariate and machine learning models for optimal predictive capacity. Clinical trial information: NCT03678883 .

e14548 Background: Micronuclei (MN) are small, excised DNA fragments resulting from biological DNA repair mechanisms in response to internal chromosomal instability, such as those caused by genotoxic events (i.e. chemotherapy and radiation), and whose formation in tumor stromal cells in blood circulation is prognostic for worse clinical outcomes (Fenech 2020). Further, mutagenic events occurring during the formation of MN are associated with the development of cancer neoantigens, which are a major element in the programmed cell death ligand (PD-L1) pathway that influences response to immunotherapies. We hypothesized that MN can be detected in metastatic breast cancer (mBC) patients (pts) within circulating stromal cells (CStCs) that prognosticates for clinical outcomes and may associate with PD-L1 cell expression. Methods: We initiated a prospective pilot study using a compiled set of n=124 mBC samples, from multiple institutions, enrolled prior to starting new lines of treatment for progressive mBC. We enumerated MN formation found in CStCs from whole peripheral blood (7.5mL), filtered for CStCs, then stained for PD-L1 and DAPI. DAPI was used to identify MN, defined by small (<3 µm) DAPI+ circular formations within the cytoplasm, separate from primary nuclei. In conjunction, all cells were stained with PD-L1 with expression normalized to background and evaluated against MN by T-test analysis. MN presence was compared to all available clinical variables, including induction of PD-L1 immunotherapy. Pts’ progression-free survival (PFS) and overall survival (OS) hazard ratios (HRs) were analyzed by censored univariate and multivariate analyses based on RECIST v1.1 over 24 months. Results: MN were identified in 56% (n=70/124) of CStCs and their presence was significantly prognostic for worse PFS (HR=2.4, CI= 1.53-3.76, p<0.001) & OS (HR= 1.89 CI=1.07-3.34, p=0.039) over 24 months. Overall, mPFS of pts with MN was 2.8 months and without MN was 12.1 months. The mOS of pts with MN was 12.7 months and without MN was 20.8 months. Additionally, MN positive CStCs had a significantly higher PD-L1 expression than MN negative CStCs (p<0.001), with 67% of MN positive pts (n=46/66) having high PD-L1 expression while 30% of MN positive pts (n=20/66) having low PD-L1. Further, evaluation of PD-L1 in CStCs showed a linear relationship (R=0.9806) between MN number and PD-L1 in cells, with 0 MN average=0.016, 1 MN=0.200, 2 MN=0.318, and ≥3 MN=0.400. Conclusions: These initial findings suggest that MN are detected in pts with advanced mBC and may identify pts with faster progression and worse survival outcomes. Further, the number of MN in CStCs appears to correlate with higher PD-L1 expression within cells. Follow up clinical information and subtyping studies are ongoing to also evaluate of the effect of MN positive CStCs on PD-L1 immunotherapies in these pts.