Leon‐Ferre, Roberto A.

11145 Background: T-DXd is increasingly used in mBC. A T-DXd toxicity of special interest is interstitial lung disease/pneumonitis (ILD), occurring in 10-15% of pts in the DESTINY-Breast trials, and with potential lower incidence/severity in earlier line settings [Krop et al, ASCO 2023]. However, in routine practice, T-DXd may be used in pts with more heavily pre-treated mBC and previously exposed to other antibody-drug conjugates (ADC) [Premji et al SABCS 2023]. We evaluated the incidence/severity of T-DXd related ILD in mBC at Mayo Clinic. Methods: We retrospectively identified pts with mBC who received ≥1 dose of T-DXd at Mayo Clinic, Rochester, MN, between July 2022-December 2023. Demographic, mBC, and pulmonary clinical variables were abstracted from the clinical records. Data was summarized using descriptive statistics. Diagnosis of ILD was determined by treating clinicians, and severity to CTCAE V5 based on clinical documentation. Results: 77 pts with mBC received T-DXd during the study period. All were female, the majority (70, 91%) were Caucasian, and median age was 58. 53 (69%) had HER2-low mBC (37 HR+, 16 HR-) and 24 (31%) had HER2+ mBC (17 HR+, 7 HR-). 31 (40%) were active or former smokers. 11 (14%) had pulmonary comorbidities, including 6 (7.8%) with asthma, 2 (2.6%) with prior pneumonitis, and 3 (3.9%) with other pulmonary comorbidities. They had previously received a median of 1 line of endocrine therapy, and 4 lines of chemotherapy, with 20 (26%) having received another ADC. 17 (22%) developed any grade ILD [G1: 3 (4%), G2: 7 (9%), G3: 2 (3%), G4: 1 (1%), G5: 4 (5%)]. Among pts with ILD of any grade, 13 (76.5%) presented with at least 1 symptom (cough/SOB). The median number of T-DXd cycles prior to onset of any grade ILD was 6 (range 2-13), or 18 (range 6-39) weeks. 5 (29%) pts with any grade ILD received a prior ADC. Of pts with ≥G3 ILD (n=7), all were Caucasian, 2 (30%) were prior smokers, none had prior lung disease, and had received a median of 5 prior lines of chemo, with 4 (57%) having received a prior ADC. All grade ≥2 ILDs were treated with systemic steroids, and 1 also received IVIG. CT chest was used for diagnosis of all ILD. 7 pts (41%) required hospitalization, all of whom had bronchoscopy, with 5 (29%) requiring intubation. All pts with G1 ILD (3, 18%) restarted T-DXd without ILD recurrence. Conclusions: In this case series,22% of ps treated with T-DXd experienced ILD (5% G5), higher than the rate observed in the pivotal DESTINY-Breast trials, possibly due to real-world use of T-DXd in more heavily pre-treated pts and in pts with pulmonary comorbidities.

594 Background: Recent analyses in HER2+ and triple negative (TN) breast cancer (BC) treated with neoadjuvant systemic therapy (NAST) suggest that extent of disease at presentation (tumor [T] size and nodal [N] involvement) influences outcomes independently of response to NAST, even in the context of pathologic complete response (pCR). Given this, we evaluated the association of clinical T and N status prior to NAST with long-term outcomes in I-SPY2. Methods: We identified patients (pts) with TN and HER2+ BC who received NAST on the I-SPY2 trial with available clinical T size and N status prior to NAST, pathologic response after NAST, and follow up for recurrence and survival. We conducted univariable and multivariable (MV) Cox proportional models for Event Free (EFS), Distant Recurrence Free (DRFS), and Overall Survival (OS) in TN and HER2+ BC according to response after NAST, adjusting for residual cancer burden (RCB) category. Results: Of 1,170 pts with TN or HER2+ BC, 1,013 were eligible (TN: 623 and HER2+: 390), after 157 pts were excluded due to missing critical variables. Median follow up was 4.4 years (range 0.3-10.2). Median age was 48 for TN and 49 for HER2+ BC. Following NAST, pCR was achieved in 277 (45%) pts with TN and 197 (51%) pts with HER2+ BC. Among pts with pCR, separate MV analyses for TN and HER2+ BC showed no independent association between clinical T and N category with EFS, DRFS or OS (p values all > 0.05). However, among pts with residual disease, clinical T category (but not clinical N status) prior to NAST retained independent prognostic information in both TN and HER2+ BC in a MV model adjusting for age, RCB class, and hormone receptor status (for HER2+ BC). Compared to cT1-2, cT3-4 stage at diagnosis was associated with worse EFS, DRFS, and OS in TNBC, and with worse EFS, DRFS (but not OS) in HER2+ BC (Table). Additionally, hormone receptor status was independently associated with outcomes in HER2+ BC with residual disease (data not shown). The 5y EFS for cT1-2 vs cT3-4 according to RCB class in TNBC were: RCB0: 94% vs 95%, RCB1: 90% vs 65%, RCB2: 74% vs 44% and RCB3: 45% vs 20%. The 5y EFS for cT1-2 vs cT3-4 according to RCB class in HER2+ were: RCB0: 95% vs 88%, RCB1: 89% vs 84%, RCB2: 85% vs 64% and RCB3: 56% vs 50%. Conclusions: Tumor size (but not N involvement) prior to NAST was independently associated with outcomes in TN and HER2+ BC with residual disease, independently of RCB class. TN and HER2+ BC achieving pCR exhibited favorable outcomes regardless of extent of disease prior to NAST. These data are consistent with prior findings of the Neobioscore contributing only in the context of residual disease. Clinical trial information: NCT01042379 . [Table: see text]

Abstract Purpose: We previously reported that postmenopausal women with estrogen receptor-α–positive breast cancer receiving adjuvant anastrozole 1 mg/day (ANA1) with estrone (E1) ≥1.3 pg/mL and estradiol (E2) ≥0.5 pg/mL [inadequate estrogen suppression (IES)] had a threefold increased risk of a breast cancer event. The objective of this study was to determine if increasing anastrozole to 10 mg/day (ANA10) could result in adequate estrogen suppression (AES: E1 <1.3 pg/mL and/or E2 <0.5 pg/mL) among those with IES on ANA1. Patients and Methods: Postmenopausal women with estrogen receptor-α–positive breast cancer planning to receive adjuvant ANA1 were eligible. E1 and E2 were assessed pre- and post-8 to 10 weeks of ANA1. Those with IES were switched to 8- to 10-week cycles of ANA10 followed by letrozole 2.5 mg/day. E1 and E2 were assessed after each cycle. Anastrozole concentrations were measured post-ANA1 and post-ANA10. Primary analyses included patients who documented taking at least 80% of the planned treatment (adherent cohort). Results: In total, 132 (84.6%) of 156 eligible patients were ANA1 adherent. IES occurred in 40 (30.3%) adherent patients. Twenty-five (78.1%) of 32 patients who began ANA10 were adherent, and AES was achieved in 19 (76.0%; 90% confidence interval, 58.1%–89.0%) patients. Anastrozole concentrations post-ANA1 and post-ANA10 did not differ by estrogen suppression status among adherent patients. AES was maintained/attained in 21 (91.3%) of 23 letrozole-adherent patients. Conclusions: Approximately 30% of ANA1-adherent patients had IES. Among those who switched to ANA10 and were adherent, 76% had AES. Further studies are required to validate emerging data that ANA1 results in IES for some patients and to determine the clinical benefit of switching to ANA10 or an alternative aromatase inhibitor.

Abstract Background: Multiple studies have confirmed the central role of preexisting immune response measured by stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC). Emerging studies showed that not only the number of TILs but also the location of TILs is important. There are 3 distinct immune architectures described based on the amount and locations of TILs, namely immune enriched (IN), immune excluded (IE), and immune desert (ID). Here we evaluated outcomes and characteristics associated with each immune landscape. Methods: NanoString Digital Spatial Profiling (DSP) and CosMx, a spatial multi-omics single-cell imaging platform, were performed in 75 samples from the Mayo Clinic (MC) TNBC cohort (Leon-Ferre BCRT 2018). NanoString IO360 was performed in 114 samples from the FinXX trial ( NCT00114816) . Firstly, tumors with sTIL quantified by H&E ≤ 30% were classified as ID. The rest of the tumors with high sTIL > 30% were categorized according to the intratumoral CD8 protein expression by DSP, with IE having intratumoral CD8 in the lower median and IN having intratumoral CD8 in the upper median. Chi-square test, gene set enrichment, Cox regression, and Kaplan-Meier analysis were used. Differential expression listed as log 2-fold change (FC) was estimated from the linear mixed model with significance defined as two-sided p< 0.05. Results: ID is associated with low Ki67 < 5% (23.3% vs. 9.6% ID, p 0.02) as well as apocrine (11/13, 84.6%) and metaplastic histology (10/12, 83%). In both univariate and multivariate analysis, patients with IN had significantly improved recurrence-free survival (RFS) compared to those with ID (HR 0.37, 95%CI 0.18-0.74, p 0.005). Despite having high sTILs, IE had poor outcomes similar to ID (HR 0.84, 95%CI 0.36-1.98). Strikingly, we identified that IE patients had significantly lower plasmacytoid dendritic cells (pDCs) compared to IN (mean 0 vs. 0.26/100 tumor cells, 95%CI 0.08-0.43 , p 0.01). Using Gene Set Enrichment Analysis to evaluate differential hallmarks between IN and IE, we identified IF Nɑ and IFNγ (FDR < 0.001) responses as significantly enriched in IN group, consistent wi th the function of pDCs, which are a subset of dendritic cells specialized in secreting high levels of type I interferon. To validate this finding, we further evaluated the 11 leading edge gene IFNɑ signature in the FinXX trial. A high IFNɑ signature score was associated with significantly improved outcomes in the FinXX trial (HR 0.21, 95%CI 0.09-0.51 , p < 0.001). Similar findings were observed using Kaplan-Meier analysis in the FinXX trial with significantly improved RFS (p 0.0006) and overall survival (p 0.0001) in patients with high IFNɑ signature scores. Furthermore, we evaluated the differential gene expression unique to IN tumors in the Mayo cohort. Expressions of MHC class I and class II in tumor cells, including HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DPA1, and HLA-E, were associated with IN and significantly improved outcomes (p < 0.05) compared to ID and IE. Conclusions: Highlighting the importance of spatial context, we identified that patients with IE tumors had poor outcomes despite having high TILs. Moreover, u sing an in-depth analysis with spatially defined context, we identified the central role of pDC and the significance of IF Nɑ in TNBC. Support: Breast Cancer Research Foundation, Mayo Clinic Breast Cancer SPORE (P50CA116201-17) W81XWH-15-1-0292, P50CA015083, R35CA253187 Citation Format: Saranya Chumsri, Yi Liu, Yaohua Ma, Jodi Carter, Mark Gregory, Sarah Church, Jason Reeves, Heather Ann Brauer, Sarah Warren, Heikki Joensuu, Edith Perez, Roberto Leon-Ferre, David Hillman, Judy Boughey, James Ingle, Krishna Kalari, Fergus Couch, Matthew Goetz, Keith Knutson, E. Thompson. The spatially resolved single-cell atlas of the tumor immune architecture revealed the central role of IFN-alpha and plasmacytoid dendritic cells in triple-negative breast cancer in the Mayo Clinic cohort and FinXX trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS03-03.

Abstract Background: Ovarian function suppression (OFS) combined with tamoxifen (TAM) or an aromatase inhibitor (AI) is standard for premenopausal (PM) ER+, HER2- breast cancer (BC). However, > 40% PM patients (pts) are intolerant of OFS. For these pts, TAM is the only FDA approved option. In the neoadjuvant endocrine setting, pts with endocrine sensitive disease (ESD) (Ki-67 ≤ 10% at 4 weeks) have 5yr dDFS > 97%; however, only 45% of TAM pts vs >75% with AI+OFS achieve 4 wk ESD (Nitz JCO 2022). (Z)-endoxifen (ENDX) is a potent anti-estrogen superior to TAM and AI in xenograft models and with antitumor activity in endocrine-resistant postmenopausal pts. ENDX additionally targets protein kinase C beta 1 (PKCβ1) at concentrations >500 ng/mL, resulting in AKT inhibition and apoptosis (Jayaraman npj Breast Cancer 2023). We hypothesize that ENDX dual targeting of ERα and PKCβ1 will obviate the need for OFS and be non-inferior to AI plus OFS for PM pts with ER+/HER2- BC. Methods: EVANGELINE (NCT05607004) is an ongoing phase 2 multicenter neoadjuvant study with pharmacokinetic (PK) run-in assessing ENDX in PM women with ER+/HER2- BC. The primary objective for the PK run-in is to identify a dose (40 or 80 mg/day) resulting in ENDX steady state concentrations (Css) of 500-1000 ng/mL (to target both ERα and PKCβ1) without significant toxicity. Following PK run-in, the randomized phase II goal is to assess whether the ESD rate with ENDX is non-inferior to exemestane plus goserelin. Women with ESD at wk4 continue treatment for 24 weeks followed by surgery. Here we report the results from the 40 mg/day PK run-in. Results: Seven PM women (6 White, 1 Asian) aged 28-51 (median 46) received ENDX 40 mg/day. Pt characteristics on study were: ER > 90% (all pts), median Ki-67 = 13 (range 4-33%), cTstage (cT2: 6 pts, cT3: 1 pt), and tumor grade (G1: 1 pt, G2: 6 pts). The median ENDX 28 day Css (ng/mL) was 263.6 (range 180.3-376.6). One pt discontinued due to wk4 Ki-67 remaining > 10%. The remaining 6 had ESD, with either wk4 Ki-67 remaining ≤ 10% (3 pts) or decreasing to ≤ 10% (3 pts) and after 24 wks underwent surgery with surgical Ki-67 ≤ 3% (range 0-3%). MRI central review (wk12 and wk24) demonstrated target lesion decreases in all pts with 1 CR, 1 PR and 4 SD (RECIST). Treatment related toxicities included grade 3 headache (n=1), grade 2 amenorrhea (n=1), and grade 2 hot flashes (n=1). The median (range) baseline estrone (n=5) was 54 pg/mL (19-114) with median (range) fold increase from baseline of 9.0 (1.3-23.2) at wk4 and 4.7 (0.4 - 25.9) at wk24. The median baseline estradiol level (n=5) was 29 pg/mL (19-209) with median fold increases from baseline of 17.9 (0.4-57.0) at wk4 and 8.1 (0.04 - 56.6) at wk24. Additional surgical and blood biomarker data will be presented at the meeting. Conclusions: ENDX (40 mg/day) exhibits promising antitumor activity for PM ER+/HER2- BC but with ENDX Css below target. Enrollment is ongoing to the 80 mg/day dose level. Citation Format: Matthew P. Goetz, Vera J. Suman, Heather Fraser, Lida Mina, Pooja Advani, Roberto Leon-Ferre, Karthik Giridhar, Felipe Batalini, Katie N. Hunt, Swaathi Jayaraman, James Jakub, Patricia Cronin, Mara Piltin, Amy Degnim, James N. Ingle, Judy C. Boughey, Sarah Buhrow, Joel Reid, Matthew Schellenberg, John Hawse, Steven Quay. Neoadjuvant (Z)-endoxifen in premenopausal ER+, HER2- breast cancer: Evaluation of the first pharmacokinetic cohort of the EVANGELINE trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT205.

Importance The association of tumor-infiltrating lymphocyte (TIL) abundance in breast cancer tissue with cancer recurrence and death in patients with early-stage triple-negative breast cancer (TNBC) who are not treated with adjuvant or neoadjuvant chemotherapy is unclear. Objective To study the association of TIL abundance in breast cancer tissue with survival among patients with early-stage TNBC who were treated with locoregional therapy but no chemotherapy. Design, Setting, and Participants Retrospective pooled analysis of individual patient-level data from 13 participating centers in North America (Rochester, Minnesota; Vancouver, British Columbia, Canada), Europe (Paris, Lyon, and Villejuif, France; Amsterdam and Rotterdam, the Netherlands; Milan, Padova, and Genova, Italy; Gothenburg, Sweden), and Asia (Tokyo, Japan; Seoul, Korea), including 1966 participants diagnosed with TNBC between 1979 and 2017 (with follow-up until September 27, 2021) who received treatment with surgery with or without radiotherapy but no adjuvant or neoadjuvant chemotherapy. Exposure TIL abundance in breast tissue from resected primary tumors. Main Outcomes and Measures The primary outcome was invasive disease-free survival [iDFS]. Secondary outcomes were recurrence-free survival [RFS], survival free of distant recurrence [distant RFS, DRFS], and overall survival. Associations were assessed using a multivariable Cox model stratified by participating center. Results This study included 1966 patients with TNBC (median age, 56 years [IQR, 39-71]; 55% had stage I TNBC). The median TIL level was 15% (IQR, 5%-40%). Four-hundred seventeen (21%) had a TIL level of 50% or more (median age, 41 years [IQR, 36-63]), and 1300 (66%) had a TIL level of less than 30% (median age, 59 years [IQR, 41-72]). Five-year DRFS for stage I TNBC was 94% (95% CI, 91%-96%) for patients with a TIL level of 50% or more, compared with 78% (95% CI, 75%-80%) for those with a TIL level of less than 30%; 5-year overall survival was 95% (95% CI, 92%-97%) for patients with a TIL level of 50% or more, compared with 82% (95% CI, 79%-84%) for those with a TIL level of less than 30%. At a median follow-up of 18 years, and after adjusting for age, tumor size, nodal status, histological grade, and receipt of radiotherapy, each 10% higher TIL increment was associated independently with improved iDFS (hazard ratio [HR], 0.92 [0.89-0.94]), RFS (HR, 0.90 [0.87-0.92]), DRFS (HR, 0.87 [0.84-0.90]), and overall survival (0.88 [0.85-0.91]) (likelihood ratio test, P < 10e-6). Conclusions and Relevance In patients with early-stage TNBC who did not undergo adjuvant or neoadjuvant chemotherapy, breast cancer tissue with a higher abundance of TIL levels was associated with significantly better survival. These results suggest that breast tissue TIL abundance is a prognostic factor for patients with early-stage TNBC.

Abstract Background: Previous studies demonstrated that obesity is associated with poor outcomes and significantly worse pathological complete response (pCR) in triple-negative breast cancer (TNBC) treated with conventional chemotherapy. Emerging studies described a phenomenon termed the obesity paradox, which referred to improved outcomes observed in obese patients treated with immune checkpoint inhibitors (ICIs). Our study aimed to determine distinct immune features in TNBC across body mass index (BMI). Methods: Characteristics and outcomes of TNBC patients treated with neoadjuvant pembrolizumab-based chemotherapy at Mayo Clinic were collected. Normal BMI was defined as 18-25 kg/m2, overweight 25-30, and obese BMI ≥ 30. NanoString CosMx, a spatial multi-omics single-cell imaging (SMI), was performed in 75 samples from the Mayo Clinic TNBC cohort (Leon-Ferre BCRT 2018). Differential expression as log 2-fold change (FC) was estimated from the linear mixed model with significance defined as two-sided p< 0.05. Results: Between 2021-2023, there were 108 TNBC patients treated with neoadjuvant pembrolizumab. 25.9% were obese, 32.4% were overweight, and 41.7% had normal BMI. In contrast to previous studies with conventional chemotherapy, obese patients numerically had a higher pCR rate of 71.4% with pembrolizumab-based chemotherapy compared to 55.6% in normal and 62.9% in overweight patients. However, this difference did not reach statistical significance (obese vs. normal weight, OR 2.26, 95%CI 0.89-6.06, p 0.094). We further evaluated spatially-resolved immune features associated with obesity using CosMx SMI in 75 samples from the MC TNBC cohort. Obese patients had significantly higher intratumoral B cells (log2FC 1.88, p 0.017), stromal B cells (log2FC 2.79, p 0.001), stromal plasmablasts (log2FC 1.94, p 0.047), and stromal macrophages (log2FC 1.80, p 0.017) compared to patients with normal BMI. Obese patients also had significantly lower intratumoral myeloid dendritic cells (mDC) (log2FC -1.27, p 0.037). Similar trends were observed after standardizing cell counts per 100 tumor or stromal cells, including higher intratumoral B cells (log2FC 3.00, p 0.001), intratumoral plasmablasts (log2FC 2.00, p 0.006), intratumoral natural killer (NK) cells (log2FC 1.66, p 0.044), and stromal B cells (log2FC 1.50, p 0.008). Differential gene analyses for intratumoral B cells revealed that genes (CD74, HLA-C, HLA-DRB1, HLA-DRA, HLA-DRB) associated with the positive regulation of type 2 immune or chemokine response were upregulated. Conclusions: Using in-depth analysis with spatially defined context, our study showed that obese TNBC patients had significantly higher B cell infiltration, which is associated with improved outcomes with ICIs. Future research on how obesity alters the immune microenvironment is needed to identify potential pathways to improve outcomes with ICIs in TNBC. Citation Format: Songhan Pang, Yi Liu, Yaohua Ma, Zhuo Li, Roberto A. Leon-Ferre, David Zahrieh, David W. Hillman, Judy C. Boughey, James N. Ingle, Krishna R. Kalari, Fergus J. Couch, Matthew P. Goetz, Keith L. Knutson, Saranya Chumsri, E. Aubrey Thompson. Tumor-immune microenvironment associated with obesity in triple-negative breast cancer in the Mayo Clinic cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1162.

Abstract Background: Dysregulated Notch signalling contributes to breast cancer development and progression, but validated tools to measure the level of Notch signalling in breast cancer subtypes and in response to systemic therapy are largely lacking. A transcriptomic signature of Notch signalling would thus be warranted, and in this report, we have established such a classifier. Methods: To generate the signature, we first identified Notch-regulated genes from six basal-like breast cancer cell lines subjected to elevated and reduced Notch signalling by culturing on immobilized Notch ligand Jagged1 or blockade of Notch by g-secretase inhibitors, respectively. From this cadre of Notch-regulated genes, we developed candidate transcriptomic signatures that were trained on a breast cancer patient dataset (the TCGA-BRCA cohort) and a broader breast cancer cell line cohort and sought to validate in independent data sets. Results: An optimal 20-gene transcriptomic signature was selected. We validated the signature on two independent patient datasets (METABRIC and Oslo2) and it showed an improved coherence score and tumour specificity compared with previously published signatures. Furthermore, the signature score was particularly high for basal-like breast cancer, indicating an enhanced level of Notch signalling in this subtype. The signature score was increased after neoadjuvant treatment in the PROMIX and BEAUTY patient cohorts, and a lower signature score generally correlated with better clinical outcome. Conclusions: The 20-gene transcriptional signature has the potential to better stratify patients and to evaluate the response of future Notch-based therapies for breast cancer. Citation Format: Eike-Benjamin Braune, Felix Geist, Xiaojia Tang, Krishna Kalari, Judy Boughey, Liewei Wang, Roberto A. Leon-Ferre, Antonino B. D’Assoro, James N. Ingle, Matthew P. Goetz, Kang Wang, Theodoros Foukakis, Anita Seshire, Dirk Wienke, Urban Lendahl. Identification of a Notch transcriptomic signature for breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B013.

e18807 Background: CUP represents both a diagnostic and a management challenge. Despite improvements through sophisticated imaging modalities, specific and sensitive testing using immunohistochemistry, a concrete determination of the site of origin of the primary tumor can still be a diagnostic challenge Hereby, we described the genomic landscape of patients with CUP treated at our institution. Methods: Retrospective study of patients with CUP treated at Mayo Clinic Rochester MN from 2010 to 2021. Next generation sequencing (NGS) information was abstracted, and alterations were ranked into different levels of actionability following the Onco KB framework. All data were analyzed by the SPSS version 22.0 software (SPSS, Chicago, IL, United States). Results: From a total of 214 patients, we identified 67 (32%) patients in whom NGS was performed. Among patients with available NGS the median age was 64 years (range: 35-86) and 51% were female. the most prevalent histology included adenocarcinoma (39%), carcinoma NOS (31%), and neuroendocrine tumors (12%). NGS on tumor (t-NGS) was available in 56 (84 %) patients, circulating tumor NGS (b-NGS) was available in 12 (18%) patients, and only 1 patient had both testing available. Among the 56 patients with available t-NGS, MSI-high was detected in 9 patients, whereas among the 12 patients with available b-NGS, MSI-high was detected in 8 patients; yielding an MSI-high detection rate of 16% and 67% for t-NGS and b-NGS. In this cohort, there were a total of 474 genomic alterations detected from which 103 (22 %) were able to be ranked into one therapeutic level following the OncoKB framework. Genomic alterations were most observed among patients with carcinoma (n = 50, 48.53%) adenocarcinoma (n = 33, 32.03 %), squamous cell carcinoma (n = 3, 2.91 %) and neuroendocrine tumor (n = 2, 1.94%). Following the OncoKB framework, 39-level 1 genomic alterations were detected in a total of 14 genes. The most common level 1 genomic alterations comprised the BRCA2 (20.51%), NF1 ATM (17.95%) and HER2/NEU (ERBB2) (15.38%) genes. Therapeutic level 2 included 51 genomic alterations in 13 genes of which KRAS (17.65%), BRAF (15.69%), and BRCA2 (15.69%) were the most prevalent mutated genes. Additionally, level R comprised 13 genomic alterations in the following genes: KRAS (69.23) NRAS (23.08%) and PDGFRA (7.69%). Conclusions: CUP are malignancies rich in potentially actionable alterations, and NGS of tissue and blood samples can inform systemic therapy decisions. Alteration-specific targeted therapy may be an option for a substantial proportion of patients. NGS of tumor tissue or blood should be considered for all patients with CUP.

e18803 Background: CUPs are a heterogeneous group of metastatic cancers for which a standardized diagnostic work-up fails to identify the site of origin at the time of diagnosis. CUP accounts for 3%-5% of all cancer diagnoses worldwide, and the natural history of patient with CUP differs from that of patients (pts) with cancer with known primary tumor. Here we report and summarize the clinical features of patients with CUP treated at Mayo Clinic. Methods: Retrospective study of patients treated at Mayo Clinic Rochester MN from 2010 to 2021. Patient demographics, tumor-related variables, treatment-related variables, and clinical data were extracted. Descriptive statistics were used, and all data were analyzed by the SPSS version 22.0 software (SPSS, Chicago, IL, United States). Results: From a total of 214 pts, 207 pts were included, as primary tumor site was identified in 7 patients after initial evaluation. The pts in this study were predominantly white (87%) of female (50.2%) gender with a mean age of 63 (SD: 13) without history of smoking (51%) and either private or public insurance (82.6%). The predominant histology was adenocarcinoma (30%), followed by neuroendocrine neoplasms (27%), and carcinoma NOS (not otherwise specified) (25%). The most prevalent grade in this cohort was grade III with poorly differentiated tumor cells. All pts had diagnostic imaging and the most used imaging modalities included CT (81.2%), and PET-CT (43.5%). In our cohort, the 3 most common sites of metastases included lymph nodes (57.5%), liver (37.8%), lung (34.3%), and bone (23.7%); and in 64% of pts, there was a specific presentation pattern including liver-predominant (24.6%), lymph node-predominant (24.6%), bone-predominant (7.7%), and peritoneal carcinomatosis (7.7%). The majority of pts received systemic therapy (69%) as first line of therapy and the most frequent first line regimens included platinum-doublet (26.5%), gemcitabine-based regimen (20.7%), 5FU-based regimens (18%) and somatostatin analogs (16.7%); whereas salvage radiation and ablation were offered to 11% of pts and surgery to 17%. The median follow-up time was 22 months (IQR: 45) with a median survival time of 23 months and the 3-year cumulative survival rate was 30%. Conclusions: The heterogenous nature of CUPs and lack of specific immunohistochemical signature prevents primary tissue of origin diagnoses in CUP pts. Without the identification of a primary origin site, treatment is restricted to generic chemotherapy with limited benefit as demonstrated in this cohort.