Chumsri, Saranya

Abstract Background: APOBEC mutagenesis underlies somatic evolution and accounts for tumor heterogeneity in several cancers, including BC. In our clinical experience, HR+HER2- BC with an APOBEC signature do poorly on standard of care (SOC) first line endocrine therapy (ET) + CDK4/6 inhibitor (CDK4/6i) and need additional treatment options. Here we evaluated the characteristics of a real-world cohort for time-to-treatment discontinuation (TTD) and overall survival (OS) on SOC and immune checkpoint inhibitors (ICI).Methods: Hybrid capture-based comprehensive genomic profiling (CGP) results from 29,833 formalin-fixed paraffin-embedded tissue biopsies of all BC subtypes were analyzed. For outcomes (TTD and OS), this study used a de-identified nationwide (US-based) BC clinico-genomic database (CGDB, ~800 sites of care, 1/2011 - 12/2020). HR+HER2- metastatic BC pts who received first line ET + CDK4/6i were included (CGDB cohort).TTD was defined as the difference between the first and last drug episode within a given line of treatment (LOT). LOT were derived based on FH algorithms. OS was defined as the time from LOT start to the date of death or data cutoff. Log-rank test and Cox model were used to evaluate the difference in outcomes. To reduce the impact of confounding variables (Age at Dx, Stage at Dx, Tumor Type, Metastases sites, TMB group (≥10 vs <10), SOC treatment group, PIK3CA), inverse probability of treatment weighting (IPTW) was used. Eligible pts from Mayo Clinic and Duke University were HR+HER2- mBC with sequencing data from FMI between 9/2013-7/2020. Clinical data were manually extracted from Mayo and Duke EHR. Results: Of all 29,833 BC samples in the CGP cohort, 7.9% were APOBEC+ with high rate in invasive lobular carcinoma (ILC) 16.7% vs. 4.9% in invasive ductal carcinoma (IDC) and metastatic lesions 9.7% vs. 4.3% from breast. APOBEC+ samples had a higher median TMB 12.5 vs. 2.5 mut/Mb. In CGDB, 857 HR+HER2- BC met inclusion criteria; 69 (8%) pts were APOBEC+ and 788 (9.2%) were APOBEC-. APOBEC+ pts had significantly shorter TTD on SOC ET+CDK4/6i than APOBEC- pts, 7.8 (95% CI 4.3-14.6) vs. 12.4 (95% CI 11.2-14.1) months (p=0.0036). APOBEC+ pts also had noticeably shorter OS compared to APOBEC- pts, 32.4 (95%CI 19.8-47.4) vs. 40.5 (95%CI 36.9-45.7) months (p=0.06).Cox regression results indicate that the relative risk of shorter TTD for the APOBEC+ vs the APOBEC- was 1.6 (95%CI 1.03-2.39). Also, APOBEC+ pts had almost twice the risk that APOBEC- pts had of death (HR=1.96, 95%CI 1.2-3.3). In CGDB, there were 10 APOBEC+ pts who received ICI, 9/10 had evaluable TTD data, 4/9 were still on treatment as of Dec 2020. 5/9 received ICI monotherapy, 4 pts received ICI + chemotherapy. TTD ranged from 0.3 to 11.3 mo, 1 pt’s TTD was > 6 mo. In Mayo and Duke cohort, there were 6 pts, 5/6 received ICI + chemotherapy. The TTD was 0.9-40.5 months with longest 2 pts receiving 5-FU plus ICI (11 and 40.5 months). To better understand the ICI treatment landscape, TTD in HR+HER2- hTMB MSS APOBEC- CGDB cohort (N=6) was analyzed: 5/6 had evaluable treatment data, 4/5 finished ICI treatment, 1 pt’s TTD was > 3 mo, 0/5 had TTD > 6 mo. Conclusions: APOBEC+ occurs in ~7% of BC and is more common in ILC and metastatic lesions. APOBEC+ HR+HER2- pts had shorter TTD and OS on SOC ET+CDK4/6i relative to APOBEC- pts. However, TTD on ICI tended to be longer in APOBEC+ pts, but our data is limited, and more research is needed. CGDB APOBEC+ vs. APOBEC- with SOC 1st lineAPOBEC+ (N=69)APOBEC- (N=788)p adjusted (FDR)*Age at Dx, Median (IQR)59.0 (53.0, 65.0)56.0 (47.0, 65.0)0.102Stage at Dx0.067- 0-III52 (75.4%)505 (64.1%)- IV11 (15.9%)245 (31.1%)- Not documented6 (8.7%)38 (4.8%)Metastasis free interval, yrs, Median (IQR)5.2 (3.0, 10.5)5.1 (2.8, 9.3)0.734Tumor Grade0.104- Grade 12 (2.9%)49 (6.2%)- Grade 234 (49.3%)259 (32.9%)- Grade 311 (15.9%)163 (20.7%)- Not documented22 (31.9%)317 (40.2%)Tumor Type0*- IDC7 (10.1%)251 (31.9%)- ILC30 (43.5%)138 (17.5%)- Other /Not documented32 (46.4%)399 (50.6%)Metastases sites0.734- Bone-only17 (24.6%)169 (21.5%)- CNS11 (15.9%)104 (13.2%)- Visceral41 (59.4%)514 (65.3%)TMB, Median (IQR)11.3 (8.8, 18.8)2.5 (1.3, 3.8)0*MSI0.946- MSI-H0 (0.0%)4 (0.5%)- MSI-I0 (0.0%)1 (0.1%)- MSS68 (98.6%)728 (97.2%)- Not documented1 (1.4%)16 (2.1%)BRCA5 (7.2%)45 (5.7%)0.734PIK3CA46 (66.7%)341 (43.3%)0.001*Treatments0.005*- AI + CDK4/6i31 (44.9%)510 (64.7%)- Fulvestrant + CDK4/6i38 (55.1%)278 (35.3%) Citation Format: Saranya Chumsri, Kira Raskina, Sarah Sammons, Laura Alder, Natalie Danziger, Alexa B Schrock, Kim McGregor, Ethan Sokol. APOBEC signature, clinical characteristics, and outcome in hormone receptor-positive (HR+) HER2-negative (HER2-) breast cancer (BC) patients (pts) in real-world data (RWD) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD14-09.

Abstract Background: Cyclin dependent 4/6 kinase inhibitors (CDK4/6i) and endocrine therapy (ET) have improved progression-free survival (PFS) and overall survival in HR+ MBC, but progression of disease ultimately occurs. Apart from HR+ status, there are no clinically available biomarkers that enable oncologists to determine prognosis and predict response to CDK4/6i. An emerging biomarker is serum thymidine kinase 1 (TK1), a secreted marker of proliferation that is prognostic in pts with HR+ HER2- MBC. High levels of TKa are associated with inferior PFS, whereas pts with low TKa levels pretreatment, or TKa levels that decrease on ET and a CDK4/6i, have superior PFS. Notably, TKa levels rebound ≥ 5 days off Pb, with resumption of cell cycling. PROMISE (NCT0281902) is a prospective study that enrolled women with HR+ MBC starting Pb + letrozole (L) in the 1st line [FL] or Pb + fulvestrant in the 2nd line [SL] setting. The trial includes a comprehensive “omic” assessment of blood, tumor, urine and the fecal microbiome to identify novel genomic variants and pathways associated with an early decline in TKa (measured after 2 months or end of cycle [C]2) and PFS. Here, we report the association between i) pre-treatment TKa (pre-TKa) levels and PFS (i.e. from registration to the 1st disease event) and ii) TKa levels at the end of C2 (C2-TKa) and PFS-2 (i.e. from the start of C3 to the 1st disease event).Methods: TKa testing was performed using the DiviTum assay (Biovica). TKa+ disease was defined as ≥ 200 Du/L and TKa- disease as below limit of detection to 200 Du/L. Log-rank test and univariate Cox modeling were used to assess the association between pre-TKa levels and PFS and between end of C2-TKa levels and PFS-2. The database was locked on June 28, 2021. Results: Of 68 pts enrolled, 4 were ineligible and pre-TKa data was unavailable for 4. Of the remaining 60 pts (45 FL, 15 SL), the percentage of pts with pre-TKa+ disease was 33.3% in FL (15/45, 95% CI: 20.0-49.0%), and 46.7% (7/15, 95% CI: 21.4-71.9%) in the SL. The median follow-up time for pts on study was 24 months (range: 2-42 months). There were 22 disease events (13 in FL, 9 in SL). In the FL setting, PFS was significantly shorter for preTKa+ pts compared to preTKa- pts (HR: 4.15, 95% CI:1.35-12.74; p=0.007), but not for SL pts (HR: 1.11, 95% CI: 0.30-4.18, p=0.875). End of C2 TKa data was obtained for pts while on Pb (n=5), or after stopping Pb as follows: 1-4 days (n=9), 5-8 days (n=28) and 9-36 days (n=11). PFS-2 was not associated with C2-TKa in the FL (p=0.834) or SL (p=0.454) settings. An analysis of TKa levels by metastatic site will be presented at the meeting.Conclusions: A secreted biomarker of proliferation (TK1) obtained prior to initiating CDK4/6i and ET for the treatment of HR+ MBC is associated with PFS in pts receiving 1st line Pb + L, but not in those receiving 2nd line Pb + fulvestrant. While the end of C2 TKa levels were not associated with PFS, the interpretability of these data are limited, given treatment delays (0-36 days) prior to starting C3 that may result in TKa rebound. Future studies evaluating the predictive nature of TKa and Pb response should focus on earlier timepoints while on drug. Citation Format: Ciara C O'Sullivan, Jun He, Jason Sinnwell, Vera J Suman, Krishna R Kalari, Peter T Vedell, Ann M Moyer, Xiaojia Tang, Kevin J Thompson, Abe Eyman Casey, Alvaro Moreno-Aspitia, Donald W Northfelt, Minetta C Liu, Tufia C Haddad, Saranya Chumsri, Brendan McMenomy, Prema Peethambaram, Kathryn J Ruddy, Karthik V Giridhar, Roberto A Leon-Ferre, Mattias Bergqvist, Adrian Nordmark, Richard M Weinshilboum, Liewei Wang, Matthew P Goetz. Serum thymidine kinase 1 activity (TKa) levels and progression-free survival (PFS) in patients (pts) with hormone receptor positive (HR+) HER2-negative metastatic breast cancer (MBC) on palbociclib (Pb) and endocrine therapy (ET) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-22.

Abstract Background: Cyclin-dependent 4/6 kinase inhibitors (CDK4/6i) and endocrine therapy (ET) have improved progression-free survival (PFS) and overall survival in hormone-receptor-positive (HR+) metastatic breast cancer (MBC), but endocrine resistance is a major challenge. PROMISE [NCT0281902; n=63] is a multicenter study that enrolled women with HR+ HER2- MBC commencing palbociclib (Pb) with letrozole (1st line [1L]) or fulvestrant (2nd line [2L]), and was designed to perform a comprehensive “omic” assessment of prospectively collected biospecimens (pre-treatment (M1), at 2 months (M2), and at disease progression). The goal is to identify novel genomic variants and pathways associated with resistance to CDK4/6i and ET and PFS outcomes. Here we report the association between the proteomic, metabolomics, and lipidomics data generated from pre-Pb and 2-month serum samples and PFS. Methods: Untargeted mass spectrometry data was generated from Metabolon, assaying 1308 metabolites and 831 lipids. Additionally, 1436 proteins were assayed on the Olink platform. Cox proportional hazard models were used to evaluate the univariate hazard ratio (HR) for all features with respect to PFS. The analyses were performed on samples from 45 patients (N=33 1Lwith 9 progression events and 12 2L with 8 progression events), obtained from M1 and M2 timepoints on Pb + ET. Enrichment analysis p-values are calculated using Fisher’s exact test. Results: Proteomics: In the M1 timepoint, 93 and 43 proteins were associated with PFS in the 1L and 2L settings, respectively; inflammation genes were enriched among the 1L setting (p= 0.034); 33 proteins presented HRs ranging between 0.026 and 0.56. The FABP9 protein (HR of 1.98, 95% CI 1.02-3.83) was associated with worse PFS. Conversely, inflammation genes were not observed to be enriched in 2L. In the M2 timepoint, we observed 60 and 21 proteins significantly associated with PFS, but no biological function was enriched in 1L and 2L. Metabolites: In the M1 timepoint, metabolism of the sulfur-containing amino acids (methionine, cysteine, SAM and taurine) were enriched in the 1L setting (p= 0.035, HR range 0.15-0.33); and the branched-chain amino acids (leucine, isoleucine, and valine) were significantly associated with PFS in the 2L setting (p= 0.028, HR range 0.013-0.33). At the M2 timepoint, the amino acids were no longer enriched, but fatty acid metabolism was significantly enriched for both 1L and 2L (p= 0.048 and 0.067, respectively). Pathways involving lipids, amino acids, and xenobiotics were enriched in metabolites related to PFS (p <0.05) for both treatment lines at M1 and M2. Lipidomics: In the M1 timepoint, 10 and 19 lipids were associated with PFS for 1L and 2L, respectively. The most notable lipid associated with worse PFS in the 1L was an 18 carbon phosphatidylinositol, PI(18:1/18:2), (HR 7.34 (CI 1.27-42.50); 8 triglycerides were associated with improved PFS (HR range 0.39 and 0.55). In 2L, the 19 lipids associated with PFS included 12 phosphatidylcholines (enrichment p = 5.6X10-8). In the M2 timepoint, 15 and 8 lipids were significantly associated with PFS for 1L and 2L. An enrichment of phosphatidylinositols was observed in 1L (p= 1.2X10-5); none were observed in the 2L.Future Directions: Networks are being constructed using the proximity scores of the proteins, lipids, and metabolites associated with PFS in M1 and M2 for 1L and 2L. Network similarities and analyses will be conducted.Conclusion: Distinct multi-omic changes identified in serum samples obtained from PROMISE participants M1 and M2 on Pb correlate with disease progression in both 1L and 2L settings. Additionally, validation studies will determine the significance of these findings. Citation Format: Krishna R. Kalari, Kevin J. Thompson, Jason Sinnwell, Xiaojia Tang, Vera J. Suman, Jun He, Seul Kee Byeon, Akhilesh Pandey, Abe Eyman Casey, Peter T. Vedell, Ann M. Moyer, Alvaro Moreno-Aspitia, Donald W. Northfelt, Minetta C. Liu, Tufia C. Haddad, Saranya Chumsri, Prema Peethambaram, Kathryn J. Ruddy, Karthik V. Giridhar, Roberto A. Leon-Ferre, Richard M. Weinshilboum, Liewei Wang, Ciara C. O’ Sullivan, Matthew P. Goetz. Multiomics data reveal novel biomarkers for CDK4/6 resistance [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-03.

Abstract Background: SARS-CoV-2 mRNA vaccines have been demonstrated to have robust and durable humoral immune response in healthy individuals. However, their effectiveness in immunocompromised patients, particularly cancer patients, remains less known. Newer data suggests that cancer patients may not mount adequate protective immune response after vaccination. Methods: A retrospective study of patients ≥ 18 years old who had SARS-CoV-2 spike antibody (anti-S Ab) testing after 2 doses of SARS-CoV-2 mRNA vaccines between 12-90 days at Mayo Clinic between January 1, 2021 and May 10, 2021 was performed. The Elecsys Anti-SARS-CoV-2 S electrochemiluminescence immunoassay (Roche Diagnostics, Switzerland) was used to measure the antibody response. Patients with prior COVID-19 infection and patients on immunosuppressive therapy for an indication other than cancer were excluded. Categorical variables were summarized as frequencies (percentages) and continuous variables were reported as median with range. Wilcoxon signed rank test was used to compare continuous variables between groups and Chi-squared or Fisher’s exact test was used to compare categorical variables. All tests were two-sided with p value < 0.05 considered statistically significant. The analysis was done using R program version 3.6.2. Results: Among 201 patients, 79 had breast cancer, 91 had a hematologic malignancy, 6 had other solid malignancies, and 25 had no history of cancer. All breast cancer patients on endocrine therapy or trastuzumab ± pertuzumab without chemotherapy (n=35) had anti-S Ab titer ≥ 500 U/mL. Patients on cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) appeared to have low level of anti-S Ab with 28.6% (n=4/14) had anti-S Ab titer ≤ 500 U/mL. Patients on chemotherapy also had low levels of anti-S Ab with 47% (n=14/30) having anti-S Ab titers ≤ 500 U/mL. When combining breast cancer patients on endocrine therapy alone or anti-HER2 therapy with patients without history of cancer as an immunocompetent group, there was significantly greater proportion of immunocompetent patients (97%) who had anti-S Ab titer ≥ 500 U/mL compared with only 8% of patients with hematologic malignancy and 55% of patients with solid malignancy on chemotherapy or CDK4/6i (p < 0.001). Using multivariate logistic regression analysis adjusted for age, gender, and vaccine type, patients with solid malignancies and treatment-related cytopenia, including chemotherapy and CDK4/6i, (OR 35.51 [95%CI 8.38-255.25, p < 0.001]) were more likely than immunocompetent patients to have a suboptimal anti-S Ab results ≤ 500 U/mL.Conclusion: A significant number of breast cancer patients on chemotherapies and CDK4/6i had poor humoral responses after SARS-CoV-2 mRNA vaccination. While CDK4/6i is not commonly considered as immunosuppressive therapy, breast cancer patients on CDK4/6i appeared to have suboptimal response to SARS-CoV-2 mRNA vaccine. Our study also highlights the significance of assessing antibody response after COVID-19 vaccines in these vulnerable patients. Citation Format: Saranya Chumsri, Pooja P. Advani, Tanmayi S Pai, Zhou Li, Marites Acampora, Gina A Reynolds, Alvaro Moreno-Aspitia, Christopher P Marquez, Keith L. Knutson. Antibody responses after SARS-CoV-2 mRNA vaccination in breast cancer patients [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-23-01.

Abstract Background: There is an urgent need to improve clinical trial (CT) enrollment of breast cancer (BC) patients (pts), especially from unrepresented minorities. In this regard, our study aims to better understand the characteristics and factors associated with CT enrollment to inform strategies to enhance CT accrual rates. Method: We included 1,308,767 female pts from the National Cancer Data Base diagnosed from 2010 to 2017 with known ER, PR, and HER2 status. Demographic, socioeconomic, Charlson-Deyo Score (CDS) and BC specific data were compared between CT versus non-CT enrolled pts for initial therapy. Comparisons were assessed by Chi-square test. Logistic regression was utilized to determine associations between characteristics and CT enrollment. Odds ratios (OR) and p-values are reported for each group. Results: A total of 1797 pts (0.14%) were enrolled in CT for initial BC therapy. Focusing on the CT group, majority of pts were non-Hispanic White (W;(78.2%), followed by non-Hispanic black (AA;12.2%); Hispanic (H; 5.6%); and non-Hispanic Asian (A; 4%). Most pts (29.3%) were in 50-59 age quartile. TNBC pts formed 22% of CT pts and non-TNBC were 78%. Most pts were from academic centers (54%), followed by comprehensive community cancer (CCC; 23.5%), community cancer (3.9%) and integrated network cancer (INC; 5.6%) programs. Most had private insurance (70%) versus government insurance (27%) and others (3%). Stage IV pts were only 10% of the CT group while most pts (41%) had Stage II BC. CDS of 0 was noted in 90% pts while 1.3% pts had CDS of 2 or more. Most CT pts traveled < 50 miles from treatment center (88.5%), and only 1.2% CT pts traveled 200 or more miles. Most CT pts resided in areas with highest literacy rate (30%) while 15% were from areas with lowest literacy rate. In multivariable model, non-white pts were less likely than Whites to be enrolled in CT (OR 0.81, p<0.05). The odds of CT enrollment in 40-49, 50-59 and 60-69 age quartiles were 3.5, 2.7 and 2.3 times respectively that of pts 70 years or older (p<0.001). TNBC pts were 1.9 times more likely to be enrolled in CT than non-TNBC pts (p<0.001). Academic center pts were 3 times more likely (p<0.001) while INC program pts were less likely than CCC pts to be enrolled (OR 0.7, p=0.006). Pts with government insurance and other insurance were less likely to enroll in CT (OR 0.74, and 0.55, p <0.001 and p=0.003, respectively) compared to private insurance. The odds of a patient with stage II, III, IV BC enrolling in CT was about 2, 3.5 and 4 times higher than a stage I pt (p -<0.001). Pts with CDS 1 (OR 0.8, p=0.025) or 2 or more (OR 0.5, p=0.002) were less likely than CDS 0 pts to be enrolled. Pts who traveled 50-99 or 100-199 miles from the treatment center conducting CT, were 1.9 (p<0.001) and 1.5 times (p<0.05) more likely to be enrolled while those with > 200 miles were less likely (OR 0.75, p=0.442). The group of pts who belonged to a residential area with the highest literacy rate (<6.3% of no high school diploma) were 1.4 times more likely to accrue in CT (p<0.001) than those with high school diploma rate between 6.3 and 10.8%. Compared to metro areas, pts from rural areas were less likely to be enrolled (OR 0.5, p=0.04) as was a lower median income < $40,227 (OR 0.77, p= 0.016).Conclusion: CT enrollment is poor for initial therapy of BC treatment. We identify several modifiable factors including sociodemographics which are associated with BC pts not getting treated on CT. There are other factors that are non-modifiable, e.g., disease stage but may point to the need for more CT development in these areas. Addressing these disparities would lead to improved CT enrollment and overall advancement in BC management landscape. Citation Format: Pooja P Advani, Aaron Spaulding, Alexander Hochwald, Mizba Baksh, David Hodge, Saranya Chumsri, Shanada Monestime, Sikander Ailawadhi. Clinical trial enrollment for initial therapy of breast cancer: Exploring factors associated with poor accrual and significant disparities [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-11-15.

Abstract Background: This study reports incidence, timing, characteristics, and surveillance imaging of mammographic axillary adenopathy following COVID-19 vaccination. As COVID-19 immunizations continue, with possible booster vaccines upcoming, this study offers timing considerations and potential follow-up recommendations for breast imaging after vaccination. Methods: Retrospective analysis of patients (pts) who received at least one COVID-19 vaccine prior to screening (SM) or diagnostic mammography (DM) at Mayo Clinic Florida between January 15 to May 31, 2021. Vaccine-related information was queried by mammography technologists. Adenopathy was assessed by interpreting radiologists and follow-up studies were collated. Mammogram adenopathy included single enlarged node, multiple enlarged nodes, and adenopathy with soft tissue stranding. Ultrasound adenopathy included mildly prominent nodes with preserved fatty hila to rounded nodes with apparent loss of a fatty hilum. Wilcoxon rank-sum test and Fisher’s exact test were used to compare continuous and categorical variables, respectively. Multivariable logistic regression model was used to evaluate the association between days from vaccine and adenopathy. Results: Of 2349 pts, 34 (1.4%) had adenopathy (DM=6; SM=28) and 3 (0.1%) were symptomatic. Presence of axillary symptoms was associated with abnormal imaging (p<0.001) with an odds ratio of 33 in multivariable model. Median time after vaccine for pts with adenopathy was significantly shorter at 14 days compared to 33 days for pts without adenopathy (p<0.001). Incidence of adenopathy decreased as days from vaccine increased (3.4% for 0-14 days, 2.1% for 15-28 days, and 0.4% for > 28 days, p<0.001). After adjusting for being symptomatic, days from vaccine still had a significant impact on finding mammographic adenopathy (for each day after vaccine, OR=0.96, p<0.001). No significant difference was seen based on age (p=0.66), vaccine brand (p=0.66), vaccine dose (p=0.18). ROC analysis to identify a cutoff value for presence/absence of adenopathy was 0.74 (95% CI 0.67-0.81) at 22.5 days following vaccination. Additional imaging with mammogram and/or ultrasound was requested for 31 pts. These included no follow-up (n=4, 12.9%), repeat ultrasound with or without mammogram in 1-3 months (n=26, 83.9%), and biopsy (n=1, 3.2%, pt with ipsilateral breast cancer with negative results, presumably vaccine induced). To date, all pts who underwent surveillance imaging demonstrated normalization of lymph node appearance. The median time for abnormal imaging related to adenopathy to return to BI-RADS 1 or 2 was 84 (range 13-157) days. Conclusion: The incidence of COVID-19 vaccine-induced adenopathy in this study (1.4%) appeared to be lower than self-reported axillary swelling in COVID-19 vaccine trials (16%) but is still higher than the reported incidence of adenopathy on an otherwise normal SM (0.02-0.04%). The incidence of adenopathy decreased significantly over time and was not present in most pts 28 days after the vaccine. In patients with abnormal adenopathy, followup imaging showed resolution of vaccine-induced adenopathy in most patients by 3 months. Adenopathy(N=34)No Adenopathy (N=2315)P valueAge0.66N342314Median (Range)63.5 (35.0, 83.0)64.0 (30.0, 94.0)Q1, Q351.5, 72.555.0, 71.0Exam type<0.001DM6 (13.3%)39 (86.7%)SM28 (1.2%)2276 (98.8%)Symptomatic<0.001Missing032Yes3 (23.1%)10 (76.9%)No31 (1.3%)2273 (98.7%)Vaccine brand0.66Missing119mRNA-127319 (1.7%)1117 (98.3%)BNT162b214 (1.2%)1138 (98.8%)Other0 (0.0%)41 (100.0%)Vaccine dose0.18Missing0181st9 (2.2%)409 (97.8%)2nd25 (1.3%)1888 (98.7%)Days from vaccine<0.001Median (Range)14.0 (1.0, 91.0)33.0 (0.0, 239.0)Q1, Q38.2, 20.816.0, 61.0Days from Vaccine<0.0010-1418 (3.4%)519 (96.6%)15-2811 (2.1%)513 (97.9%)>285 (0.4%)1283 (99.6%) Citation Format: Kristin Robinson, Santo Maimone, Pooja P Advani, Zhuo Li, Denise Gococo-Benore, Neda Qosja, Ashita Mummareddy, Ahmed M Ashai, Annamaria Wilhelm, Robert Maxwell, Martha Wasserman, Neema Patel, Andrey Morozov, Saranya Chumsri. Incidence and time to resolution of axillary adenopathy on mammography after COVID-19 vaccination [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-03-19.

Abstract Background: Data supporting high tumor mutational burden (TMB-H) as a lone biomarker for an immune responsive tumor microenvironment (TME) or of response to immune checkpoint inhibition (ICI) in metastatic breast cancer (MBC) is weak; yet the tumor agnostic approval in TMB-H advanced tumors makes ICI an option in the clinic. PDL-1 positivity (PDL-1+) is the only consistent biomarker for ICI benefit in metastatic triple-negative BC (mTNBC). We sought to further evaluate concurrent predictors of an immune responsive or non-responsive TME within TMB-H MBC. Methods: Tumor samples (N=5621) obtained from patients with MBC were analyzed by next-generation sequencing (NGS) of DNA (592-gene panel or whole exome sequencing) and RNA (whole transcriptome sequencing) at Caris Life Sciences (Phoenix, AZ). Friends of Cancer Research TMB Harmonization Project (Merino et al., 2020) recommendations, with the TMB-H threshold set to ≥ 10 muts/Mb, were used. PDL-1+ (≥1% immune cells stained) assessed by IHC (Ventana SP142). Deficient-mismatch repair (dMMR) and high microsatellite instability (MSI-H) were determined by IHC and NGS, respectively. Gene expression profiling assessed 21 immune-related signatures and TMEs using MCP-counter (Betch et al., 2016) and quanTIseq (Finotello et al., 2019). Immune cell types were classified as immune-responsive (CD8+ and CD4+ T cells, B cells, myeloid dendritic cells, M1 macrophages, NK cells, and neutrophils) or non-responsive (Tregs, M2 macrophages, and monocytes). P-values adjusted by the Benjamini-Hochberg procedure. Exploratory p-values reported for sub-analyses of TMB-H responsive vs nonresponsive TME and PDL1+ vs PDL1- subgroups. Results: TMB-H was identified in 461/5621 (8.2%) MBC samples. TMB-H tumors exhibit significant enrichment of dMMR/MSI-H (7 vs 0%, p<0.0001) and IHC-PDL1+ (36 vs 28%, p<0.05) compared to TMB-L. TMB-H was not associated with increased abundance or proportion of immune responsive cell types or immune response gene signatures (e.g. antigen presentation), yet positive trends were observed. To identify predictive biomarkers of immune responsive (hot) or non-responsive (cold) TME profiles within TMB-H MBC, we generated a composite TME score representing the overall proportion of hot vs cold immune cell types. APOBEC mutagenesis was common in TMB-H tumors (72%) and was more frequently associated with an immune cold TME (77 vs 66% in hot TME subgroup, p=0.068). Within TMB-H tumors, cold TMEs were enriched with mutations (muts) in PIK3CA (61 vs 47%), CHEK2 (5 vs 0%), CBFB (4 vs 0%), amplifications (amp) of CCND1 (13 vs 4%), FGF19 (16 vs 5%), FGF4 (11 vs 4%), and MMLT6 (6 vs 0%), and expression of IHC-AR (75 vs 58%) compared to hot TMEs (all p<0.05). Hot TMEs had increased frequency of MSI-H (12 vs 5%) and KMT2D muts (12 vs 3%) compared to cold TMEs (all p<0.05). Amongst TMB-H and TMB-L tumors, PDL1+ was significantly associated with increased proportion of immune responsive cell types (ie CD8+) compared to PDL1- tumors (0.14 vs -0.08 TME score, p<0.0001). Comparison of TMB-H tumors by PDL1 status revealed enrichment of TP53 (67 vs 53%) and B2M (8 vs 0%) muts in PDL1+ tumors, while IHC-ER positivity (68 vs 54%) and CDH1 muts (32 vs 16%) were increased in PDL1- tumors (all p<0.05). Conclusions: High TMB alone does not strongly correlate with immune infiltrate or immune response gene signatures in MBC. Within TMB-H MBC, concurrent mutations in MSI-H and KMT2D are associated with an immune responsive TME while mutations in PIK3CA, CHEK2, CBFB, amplifications of CCND1, FGF19, FGF4, and MMLT6, and expression of IHC-AR are associated with an immune non-responsive TME. Co-occurring biomarkers within TMB-H breast cancer warrant evaluation in prospective cohorts for response or resistance to ICI to help develop composite biomarkers in breast cancer. Citation Format: Sarah Sammons, Andrew Elliott, Jeremy Force, Saranya Chumsri, Carey Anders, Antoinette R Tan, Daniel Magee, Jia Zeng, W. Michael Korn, Mustafa Kahsraw, Evanthia T Roussos Torres. Concurrent predictors of an immune responsive tumor microenvironment within tumor mutational burden-high breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-08.

Abstract Background: The erythropoietin (EPO) receptor activation can stimulate tumor proliferation and may be implicated in resistance to anti-HER2 therapies. EPO administration can be used for the management of chemotherapy-induced anemia. We aim to assess if EPO intake during the adjuvant anti-HER2 treatment impacts the outcomes of HER2-positive early breast cancer (EBC) patients. Methods: For this post hoc analysis of the ALTTO trial (NCT00490139), we categorized two cohorts according to the use of EPO during adjuvant anti-HER2 treatment: i) at least one EPO dose (EPO-cohort) vs ii) no EPO (non-EPO-cohort). We compared their baseline characteristics to identify potential confounders. The primary endpoint disease-free-survival (DFS), and the secondary endpoint overall survival (OS) were compared between these two cohorts, with subgroup analysis according to menopausal status, tumor characteristics, and anti-HER2 treatment. Kaplan Meier method, log-rank test, and Cox regression model adjusted for confounders were performed for DFS and OS (removal criterion p<0.10). The frequency of cardiovascular and thromboembolic adverse events (AEs) of EPO interest was compared between both cohorts. Results: Out of the 8,381 patients recruited in the ALTTO trial, 123 (1.5%) received EPO concomitantly with adjuvant treatment, 59 in the single HER2 blockade (Lapatinib (L)-alone or Trastuzumab (T)-alone) and 64 in dual blockade (T→L or T+L) arms. The median age of EPO-cohort patients was 54 (range: 27-74) years, and 39% were premenopausal, while most had pathologic tumor size >2 cm (56.9%), positive nodal status (58.5%), positive hormone receptor (HR) status (61.8%), and poorly differentiated or undifferentiated histologic grade (64.2%). Baseline characteristics were similar in both cohorts, except for the timing of chemotherapy, for which 93.5% of patients from the EPO-cohort were enrolled in the concurrent designs (2/2B) vs 44.2% in the non-EPO-cohort (p<0.001). Median FU was 6.9 years, with 6,409 (76.5%) patients still on follow-up. For the entire ALTTO cohort, the estimated 3- and 7-year DFS were 88.3% (95% CI: 87.6% - 89.0%) and 80.1% (95% CI: 79.1% - 81.0%), respectively. No difference in DFS by EPO administration (Yes/No) was observed (log-rank p=0.70). The effect of EPO remained non-significant when controlled for the four stratification factors and all other characteristics (p=0.91). The effect of EPO administration on DFS was significant for the subgroup of premenopausal women (p=0.041), favoring the EPO-cohort, noting that this result is based on small event numbers. The interaction was found significant in Cox analysis (p=0.024), remaining significant after adjustment for important prognostic variables (p=0.032). For the entire ALTTO cohort, 758 (9.0%) deaths occurred, with 3-year OS estimate 95.7% (95% CI: 95.2% - 96.1%) and 7-year OS estimate 89.9% (89.2% - 90.6%). The OS results are consistent with those for DFS regarding the lack of a significant association with EPO and the significance (p<0.10) of menopausal status and EPO interaction. In the EPO-cohort (n=123), eight (6.5%) AEs of EPO interest were recorded, while from the non-EPO-cohort (n=8,147), 417 (5.1%) reported similar AEs. Three fatal and 25 life-threatening AEs of EPO interest were recorded in the non-EPO-cohort, and none in the EPO-cohort. Conclusion: EPO administration to patients receiving adjuvant anti-HER2 treatment for HER2-positive EBC was safe and not associated with a harmful impact on survival outcomes, however the sample size of patients who received EPO is rather small. These findings contrast with data suggesting poorer outcomes with EPO, therefore further investigation of tumour microenvironment is needed to better understand these results, particularly in the premenopausal subgroup. Citation Format: Diogo Martins-Branco, Marie Kassapian, Véronique Debien, Rafael Caparica, Daniel Eiger, Urania Dafni, Charitini Andriakopoulou, Sarra El-Abed, Miguel Izquierdo, Malou Vicente, Saranya Chumsri, Martine Piccart-Gebhart, Alvaro Moreno-Aspitia, Ann Søegaard Knop, Janine Lombard, Evandro de Azambuja. The impact of erythropoietin administration concomitantly with adjuvant anti-HER2 treatment on the patients’ outcome: Sub-analysis of the ALTTO study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-14-05.

Abstract Background: The importance of the antitumor immune response in TNBC is well established. TNBC with higher TILs are less likely to recur and more responsive to systemic therapy. Likewise, PD-L1+ TNBC are more likely to benefit from chemoimmunotherapy. However, TNBC is highly heterogeneous. Of the TNBC molecular subtypes, LAR TNBC is less sensitive to systemic therapy, has lower TILs and lower rates of PD-L1 positivity. The role of other immune related proteins in LAR TNBC is not well established. Here, we evaluated differentially expressed (DE) immune related proteins in the stromal and intratumoral compartments of LAR vs non-LAR TNBC tumors. Methods: We used the Nanostring GeoMX DSP platform to quantitate 58 proteins within spatially distinct intraepithelial, cytokeratin (CK)-positive tumor segments and adjacent CK-negative/nuclei-positive stromal segments in 248 TNBC tumors included in a tissue microarray generated from a cohort of pts with centrally confirmed TNBC who underwent breast surgery without prior neoadjuvant therapy. A subset (n=111) underwent bulk tumor RNA sequencing and were classified as LAR or non-LAR TNBC. DE proteins were identified using a negative binomial generalized linear model (SNR>2, p<0.05). A targeted set of DE proteins was dichotomized at the 80th percentile. Results: Of 111 TNBC tumors, 17 (15%) were LAR and 94 (85%) non-LAR. Compared to non-LAR TNBC, pts with LAR TNBC were older (age ≥50: 82% vs 52%, p<0.01), with tumors that were more often of apocrine histology (35% vs 0%, p <0.01), grade 1-2 (24% vs 1%, p<0.01), and had lower Ki67 (Ki67 ≤15: 24% vs 11%, p=0.06). Most tumors were T1-2 (94% vs 93%, p=0.82) and N0 (53% vs 62%, p=0.09), respectively. As expected, expression of most immune-related proteins was higher in the stromal vs the intratumoral compartment for both LAR and non-LAR TNBC. When focusing on the stromal compartment, expression of multiple immune related proteins was significantly lower in LAR compared to non-LAR TNBC, including the pan-leukocyte marker CD45 (log-2 fold change [log2FC]: 0.552, p=0.05), the macrophage marker CD14 (log2FC: 0.834, p=0.06), CD44 (lof2FC: 0.637, p=0.07), and the immune checkpoint proteins IDO1 (log2FC: 0.914, p=0.04), VISTA (log2FC: 0.471, p=0.07), ICOS (log2FC: 0.444, p=0.08), and STING (log2FC: 0.544, p=0.09). Proteins with expression levels too low for comparisons included PD-L1, LAG3, FOXP3 and BCL-2. When focusing on the intratumoral compartment, expression of most immune-related proteins was very low in both LAR and non-LAR TNBC. Like in the stromal compartment, CD45 expression was lower in LAR TNBC (log2FC: 0.78, p=0.02). Expression of the immune checkpoint B7-H3 was lower in LAR TNBC (log2FC: 0.737, p=0.02), while expression of the T cell marker CD127 was higher (log2FC: -0.528, p=0.34). With regards to relevant non-immune markers, expression of Ki67 was lower in LAR TNBC (log2FC: 0.5498, p=0.05), consistent with the clinical assay. Conclusion: In this ultra high-plex spatial analysis, we provide first insights into the differential expression at the protein level of several targetable immune checkpoint molecules in LAR vs non-LAR TNBC. The lower expression of several immune related proteins in LAR TNBC is consistent with the hypothesis that LAR TNBC exhibits a “cold” immune microenvironment compared to other TNBC subtypes, potentially rendering itself less susceptible to immunotherapy-based strategies. These data support the need to consider TNBC molecular subtypes in future evaluations of immune-based therapeutic approaches. Funding: This work was supported by NIH grant P50CA116201 to RLF, JMC, KRK, FJC, DZ, JNI, and MPG; BCRF grant 19-161 to EAT and NCATS grant CTSA KL2 TR002379 to RLF. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH Citation Format: Roberto A Leon-Ferre, Jodi M. Carter, David M. Zahrieh, David W. Hillman, Saranya Chumsri, Yaohua Ma, Jennifer M. Kachergus, Xue Wang, Judy C. Boughey, Minetta C. Liu, James N. Ingle, Krishna R. Kalari, Jose C. Villasboas Bisneto, Fergus J. Couch, E. Aubrey Thompson, Matthew P. Goetz. Digital spatial profiling of immune-related proteins in luminal androgen receptor (LAR) vs non-LAR triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-01.