Citations

Abstract Introduction: Stromal tumor-infiltrating lymphocytes (sTILs) have established prognostic and predictive significance in triple-negative breast cancer (TNBC). However, the roles of other immune cells in TNBC are less well-established. We performed high-plex quantitative spatial profiling in a cohort of early-stage TNBC to 1) apply spatial context to tumoral immune landscapes and 2) identify immune proteins associated with clinical outcomes, independently of TILs and other established prognostic clinicopathologic variables, in patients (pts) treated with or without adjuvant chemotherapy (CTX). Methods: The Mayo TNBC cohort comprises pts with centrally-verified, CTX-naive tumors resected from 1985-2012. Using a cohort-based TMA, with Nanostring GeoMX DSP, we quantitated 58 proteins within spatially-distinct intra-epithelial, cytokeratin-positive tumor segments and adjacent cytokeratin-negative/nuclei-positive stromal segments. Differentially-expressed (DE) proteins were identified using a negative binomial generalized linear model (SNR>2, p< 0.05) and a target DE protein set was dichotomized (80th percentile). After adjusting for prognostic clinicopathologic variables, proteins associated with recurrence-free survival (RFS, defined as time from surgery to either local, regional, and distant recurrence, or death by any cause) were identified by performing variable selection using the Akaike Information Criterion (AIC) obtained from fitting all possible Cox proportional hazards regression models (performed separately for intra-epithelial/stromal segments, and in groups +/- adjuvant CTX. Results: From the TNBC TMA, DSP data (N=250 tumors) included 169 pts who received adjuvant CTX+ and 81 who did not (CTX-). Overall, 85/250 developed recurrent disease. In the CTX+ group, intra-epithelial tumor segments from pts without recurrent disease were enriched in 10 immune proteins, including CD8, markers involved in antigen presentation/dendritic cells (CD11c, CD40, HLA-DR) or NK cells (CD56) (FC: 1.4-2.1, p<0.05); CD14 was increased in stroma (FC: 1.5, p<0.05). In contrast, in the CTX- group, both the intra-epithelial tumor and stromal segments from pts without recurrences were enriched in immune proteins (N= 12 and 15 respectively; FC 1.6-5.5, p< 0.05) most markedly CD40, IDO1 and HLA-DR (FC: 3.2-5.5, p< 0.05). Overall, CD3, CD4, CD27, CD44, and ICOS among others were enriched only in the CTX- group; CD14 and CD56 were enriched only in the CTX+ group. Based on these spatial data, biologic function and DSP data from another set of TNBC (FinXX trial), CD11c, CD14, CD27, CD40, CD56, and IDO1 were selected for RFS analysis. After applying our model selection criterion and adjusting for pt age at surgery, tumor size, lymph node status, and sTILs, intra-epithelial CD56 was independently associated with improved RFS in the CTX+ group (HR: 0.31[0.12, 0.81]). In the CTX- group, intra-epithelial CD11c was independently associated with improved RFS (0.10 [0.01, 0.81]). Conclusion: In this early-stage TNBC cohort, spatially-distinct tumor immune landscapes were associated with RFS but differed according to receipt of CTX after surgical resection. In the patients who received CTX, the intra-epithelial compartment, rather than stromal compartment, was immune-enriched in pts without recurrences. Among a targeted protein set, intra-epithelial CD56 remained associated with improved outcomes, independent of sTILs and other clinicopathologic features. In the CTX- group, spatial landscapes were more balanced, and intra-epithelial CD11c was independently associated with improved outcomes. These data provide insight into the spatial context of intrinsic immune landscapes in TNBC, and identify candidate prognostic immune biomarkers which may inform therapeutic strategies. Citation Format: Jodi M Carter, Saranya Chumsri, David W Hillman, David M Zahrieh, Yaohua Ma, Xue Wang, Jennifer M Kachergus, Judy C Boughey, Minetta C Liu, Krishna R Kalari, JC Villasboas, Roberto A Leon Ferre, Fergus J Couch, Matthew P Goetz, E. Aubrey Thompson. Intra-epithelial tumor immune landscapes are associated with clinical outcomes in early-stage triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-01.

Abstract BACKGROUND: Brain metastatic disease occurs in 10-30% of metastatic breast cancer cases. The incidence of brain metastases is increasing, yet overall survival remains < 2 years. Treatment of brain metastases is limited with current clinical practice centered on radiation, chemotherapy and surgery. Although these treatments may prolong survival in the short term, targeting oncogenic alterations in brain metastases may deliver a more sustained clinical benefit. In this multicenter study, we comprehensively characterized DNA and RNA alterations that aberrantly drive specific oncogenic pathway activity pertinent to breast cancer brain metastases (BCBM). METHODS: RNA sequencing was performed on a cohort of patient-matched primary and resected brain metastatic tumours (45 patients; N=90 samples). Whole exome DNA sequencing (WXS) was performed for 18/45 patients (54 trios consisting of primary tumor, brain metastasis and matched normal tissue). An independent brain metastatic WXS cohort (N=21 patients) (PMID: 26410082) was also analysed resulting in a total of 39 patient samples. Recurrent somatic copy number alterations (SCNA), somatic single nucleotide variants (SNVs) and mutational signatures were identified from WXS data. Expressed gene fusions were detected computationally from RNA-Seq (N=45 cases) RESULTS: Of the 45 BCBM patients, median age at diagnosis was 51 years [25, 67], median overall survival 57 months (range 18-255) with median brain metastases free survival 34 months (range 5-216).Clinical molecular subtype of the primary tumour included 13 ER+/HER2- (29%), 16 HER2+ (35.5%) and 16 TNBC (35.5%). Regions of significant recurrent amplifications and deletions in BCBM (N=39 patients) were identified in 4q12, 10q11.21, 8p11.23, 8q23.3 and 17q12 (FDR < 0.10). Recurrent expressed gene fusions identified in known cancer driver genes were associated with chromatin modification, MAPK and HER signaling pathways. Mutational signature analysis of SNVs identified signatures associated with ageing, mismatch repair and homologous recombination deficiency (HRD) mutational processes. The relative contribution of the HRD signature was significantly increased in brain metastases compared to matched primary tumour (p < 0.05). Concordantly increased HRD in the brain metastatic transcriptome was confirmed in these patients by gene set variation analysis (GSVA) of homologous recombination pathway genes from KEGG database in RNA-Seq data. Moreover, in the extended BCBM RNA-Seq (N=45 patients) GSVA pathway scores were elevated in brain metastases relative to primary tumours, validating the functional significance of altered DNA repair defects in brain metastases CONCLUSIONS: Here, we report recurrent genetic drivers, supported by altered functional transcriptome, unique to brain metastasis that may have clinical implications for prognosis and treatment choice. Specifically, targeting defects in the homologous recombination repair mechanism may represent new therapeutic strategies and management opportunities for breast cancer brain metastases patients. Citation Format: Nicola S Cosgrove, Damir Varešlija, Stephen Keelan, Simon Furney, Jodi M Carter, Steven N Hart, Siddhartha Yadav, Arnold DK Hill, Steffi Oesterreich, Adrian V Lee, Fergus J Couch, Leonie Young. Homologous recombination deficiency represents a new therapeutic strategy for breast cancer brain metastases [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD13-01.

Abstract Background: About 10-20% of breast cancers (BC) are hereditary. Patients (pts) with BC and pathogenic variants (PVs) in the BRCA1 or BRCA2 gene are at increased risk of contralateral breast cancer (CBC). However, risks of CBC and recurrent BC associated with PVs in other established BC predisposition genes (e.g. PALB2, CHEK2, ATM) are less well established. We performed research genetic testing of over 4000 pts with BC treated at Mayo Clinic. Because most pts were unaware of their mutation status prior to selection of therapy, this cohort provides a unique opportunity to evaluate the natural history of BC recurrence after treatment of the index BC in pts with a deleterious mutation in predisposition genes. Methods: Women diagnosed and treated for BC at Mayo Clinic between 2001-2016 were enrolled in an IRB-approved research cohort. Pts were not selected for age of diagnosis or family history. Coding regions and consensus splice sites of established BC predisposition genes were subjected to amplicon-based deep sequencing using the QIAseq method in germline DNA extracted from peripheral blood or buccal samples. PVs in the genes were identified using GATK haplotype caller and Vardict. Patient, tumor, oncologic management and follow up data were reviewed. Herein we report on the differences in the incidence of local recurrence and CBC, among carriers and non- carriers and by predisposition gene using the cumulative incidence estimator and Fine and Gray regression to account for competing risks. Results: Of 4272 women with BC (median age 57, range 21-94), a PV in a BC predisposition gene was identified in 287 (6.7%). The most common PVs were seen in CHEK2 (67, 1.6%), BRCA1 (62, 1.5%), BRCA2 (60, 1.4%), ATM (47, 1.1%); 8 (0.2%) patients had more than one PV. Pts with any PVs presented at younger age (median 51 vs 58 years, p<0.001), were more likely to have stage IV disease (4.5% vs 2.3%, p=0.02), estrogen receptor (ER) negative BC (19.5% vs 13.5%, p=0.005), and bilateral cancer (8.8% vs 4.3%, p<0.001). ER- BC proportion varied by gene - 38.9% in pts with PVs associated with ER- BC (BRCA1, BARD1, BRIP1, RAD51C, RAD51D) and 11.9% in those with PVs associated with ER+ BC (BRCA2, ATM, CDH1, CHEK2), p<0.001. NCCN criteria for genetic testing were assessed for 3763, with 69.9% of those with PVs qualifying for testing compared to 46.3% of those without PVs (p<0.001). Among 3973 pts (249 with PVs, 3724 without) with unilateral stage 0-III cancer, 168 pts developed local recurrence, 131 pts developed CBC and 248 pts developed distant disease with median 5 years of follow-up. Patients with any PV had higher rates of local recurrence after breast conserving surgery (HR 2.3, 95% CI: 1.2-4.2, p=0.01) but overall did not have significantly higher risk of CBC if contralateral prophylactic mastectomy was not performed (HR 1.7, 95% CI: 0.9-3.2, p=0.11). Specifically, pts with PVs in genes other than BRCA1 or BRCA2 did not have a higher risk of CBC compared to pts with no PV (HR 1.0, 95% CI: 0.4-2.7, p=0.96); the comparison of CBC risk in BRCA1 or BRCA2 versus those with no PV was HR 2.9 (95% CI: 1.3-6.5, p=0.01), while BRCA1 or BRCA2 compared to those with other PVs was HR 2.7 (95% CI: 0.8-9.3, p=0.13). In pts with CHEK2 PVs, the probability of CBC at 10-years follow-up was 5.4%, which was similar to pts without a mutation (5.3%), p=0.47. Conclusion: While PVs in known BC predisposition genes increase risk of BC development, risk of CBC in carriers of PVs in non-BRCA predisposition genes with BC are not as high as seen with BRCA1 or 2 PV carriers. This information regarding future BC will be helpful in personalizing decisions on the extent of surgery in carriers of PVs in BC predisposition genes. Citation Format: Judy C. Boughey, Tanya L. Hoskin, Siddhartha Yadav, Fergus J. Couch. The influence of pathogenic variants in breast cancer predisposition genes on secondary breast cancer events in a prospectively collected cohort of breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD10-02.

Health-related quality of life (HRQOL) is an important prognostic patient-reported outcome in oncology. Because prior studies suggest that HRQOL is, in part, heritable, we performed a GWAS to elucidate genetic factors associated with HRQOL in breast cancer survivors. Physical and mental HRQOL were measured via paper surveys that included the PROMIS-10 physical and mental health domain scales in 1442 breast cancer survivors participating in the Mayo Clinic Breast Disease Registry (MCBDR). In multivariable regression analyses, age and financial concerns were significantly associated with global physical health (age: p = 1.6 × 10−23; financial concerns: p = 4.8 × 10−40) and mental health (age: p = 3.5 × 10−7; financial concerns: p = 2.0 × 10−69). Chemotherapy was associated with worse global mental health (p = 0.01). In the GWAS, none of the SNPs reached the genome-wide association significance threshold of 5 × 10−8 for associations with either global physical or global mental health, however, a cluster of SNPs in SCN10A, particularly rs112718371, appeared to be linked to worse global physical health (p = 5.21 × 10−8). Additionally, SNPs in LMX1B, SGCD, PARP12 and SEMA5A were also moderately associated with worse physical and mental health (p < 10−6). These biologically plausible candidate SNPs warrant further study as possible predictors of HRQOL.

Abstract Background: It is not known whether modifiable lifestyle factors that predict survival after invasive breast cancer differ by subtype. Methods: We analyzed data for 121,435 women diagnosed with breast cancer from 67 studies in the Breast Cancer Association Consortium with 16,890 deaths (8,554 breast cancer specific) over 10 years. Cox regression was used to estimate associations between risk factors and 10-year all-cause mortality and breast cancer–specific mortality overall, by estrogen receptor (ER) status, and by intrinsic-like subtype. Results: There was no evidence of heterogeneous associations between risk factors and mortality by subtype (Padj &gt; 0.30). The strongest associations were between all-cause mortality and BMI ≥30 versus 18.5–25 kg/m2 [HR (95% confidence interval (CI), 1.19 (1.06–1.34)]; current versus never smoking [1.37 (1.27–1.47)], high versus low physical activity [0.43 (0.21–0.86)], age ≥30 years versus &lt;20 years at first pregnancy [0.79 (0.72–0.86)]; &gt;0–&lt;5 years versus ≥10 years since last full-term birth [1.31 (1.11–1.55)]; ever versus never use of oral contraceptives [0.91 (0.87–0.96)]; ever versus never use of menopausal hormone therapy, including current estrogen–progestin therapy [0.61 (0.54–0.69)]. Similar associations with breast cancer mortality were weaker; for example, 1.11 (1.02–1.21) for current versus never smoking. Conclusions: We confirm associations between modifiable lifestyle factors and 10-year all-cause mortality. There was no strong evidence that associations differed by ER status or intrinsic-like subtype. Impact: Given the large dataset and lack of evidence that associations between modifiable risk factors and 10-year mortality differed by subtype, these associations could be cautiously used in prognostication models to inform patient-centered care.

Purpose Integrative therapies such as yoga are potential treatments for many psychological and physical symptoms that occur during and/or after treatment for cancer. The purpose of the current study was to evaluate the patient-perceived benefit of yoga for symptoms commonly experienced by breast cancer survivors. Methods 1,049 breast cancer survivors who had self-reported use of yoga on a follow up survey, in an ongoing prospective Mayo Clinic Breast Disease Registry (MCBDR), received an additional mailed yoga-focused survey asking about the impact of yoga on a variety of symptoms. Differences between pre- and post- scores were assessed using Wilcoxon Signed Rank Test. Results 802/1,049 (76%) of women who were approached to participate, consented and returned the survey. 507/802 (63%) reported use of yoga during and/or after their cancer diagnosis. The vast majority of respondents (89.4%) reported some symptomatic benefit from yoga. The most common symptoms that prompted the use of yoga were breast/chest wall pain, lymphedema, and anxiety. Only 9% of patients reported that they had been referred to yoga by a medical professional. While the greatest symptom improvement was reported with breast/chest wall pain and anxiety, significant improvement was also perceived in joint pain, muscle pain, fatigue, headache, quality of life, hot flashes, nausea/vomiting, depression, insomnia, lymphedema, and peripheral neuropathy, (all p-values <0.004). Conclusion Data supporting the use of yoga for symptom management after cancer are limited and typically focus on mental health. In this study, users of yoga often reported physical benefits as well as mental health benefits. Further prospective studies investigating the efficacy of yoga in survivorship are warranted.

Approximately 5% to 10% of women diagnosed with breast cancer will have a pathogenic variant (PV) in a hereditary cancer susceptibility gene, and this has significant implications for the management of these patients and their relatives. Despite the benefits of genetic testing, many eligible patients with breast cancer never undergo testing because of various barriers, including complicated testing criteria such as those from the National Comprehensive Cancer Network (NCCN). In 2019, the American Society of Breast Surgeons (ASBrS) proposed germline genetic testing for all patients with breast cancer to increase the identification of PV carriers. In 2020, a Mayo Clinic study highlighted the limitations of these 2 genetic testing guidelines (NCCN and ASBrS) and proposed a hybrid approach of testing all women diagnosed with breast cancer by the age of 65 years and using NCCN criteria for older patients. This commentary presents an updated analysis of the Mayo Clinic data and discusses the rationale for using the age of 60 years rather than 65 years as the cutoff for this hybrid approach. Using an age at diagnosis of ≤60 or ≤65 years for the universal testing of patients with breast cancer detected more PVs (11.9% [16 of 134] and 15.7% [21 of 134], respectively) in comparison with using the NCCN criteria. Lowering the age for universal testing from 65 to 60 years maintained the sensitivity of detecting a PV at >90% while sparing testing for an additional 10% of women. Compared with the testing of all patients, the hybrid approach would allow 31% of all women with breast cancer to forgo testing and result in fewer variants of uncertain significance identified and, therefore, would decrease the chance of harm from misinterpretation of these variants.

Chromosome region maintenance protein1 (CRM1) mediates protein export from the nucleus and is a new target for anti-cancer therapeutics. Broader application of KPT-330 (selinexor), a first in class CRM1 inhibitor recently approved for multiple myeloma and diffuse large B-cell lymphoma (DLBCL), has been limited by substantial adverse effects (AEs). To address this clinical problem, we focused on identifying novel strategies to boost the potency, reduce toxicity, and broaden the applicability of CRM1 inhibitors to a wider range of malignancies. We discovered that salicylates could markedly enhance the anti-tumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. KPT-330 was chosen as the prototypical CRM1 inhibitor given its current FDA approval status and characterized pharmacokinetics; and choline salicylate (CS) was chosen as the prototypical salicylate given its favorable pharmacokinetics and reduced antiplatelet, renal, neurological and gastrointestinal AEs in humans compared to other salicylates. By using cell lines belonging to different hematologic malignancies and solid tumors, we demonstrated ex vivo that the combination of KPT-330 and CS (K+CS) could induce unique and significant antitumor effect at much lower dose of KPT-330 (at 25% of the dose used in the clinic), thereby potentially mitigating prohibitive clinical AEs (Figure 1a-d, and e-g). This significant synergetic antitumor effect observed with K+CS ex vivo was also validated in vivo by using an NSG mouse model of mantle cell lymphoma (Figure 1h). Moreover, the K+CS combination did not show this potent toxic effect on non-malignant cells in vivo and was safe without inducing toxicity to normal organs in NSG mice. Mechanistically, protein profiling through mass spectroscopy revealed that K+CS uniquely affects the cellular pathways of DNA damage repair, DNA synthesis and cell cycle progression. Studies involving immunoblotting, cell cycle analysis, immunofluorescence microscopy assessing nucleocytoplasmic molecular export and DNA damage, reporter assay to assess homologous recombination repair proficiency and immunohistochemistry showed that, compared to KPT-330 treatment alone, K+CS decreased the expression of CRM1, Rad51 and thymidylate synthase proteins in vitro and in vivo, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, and importantly a unique cell cycle arrest in S-phase, thus leading to cell apoptosis. These effects on cellular proteins and pathways were unique to K+CS treatment and were not observed with KPT-330 or CS single agent treatment. Pathway analyses through RNA sequencing also paralleled the findings of proteomic studies thus further validating the unique effect of K+CS treatment on the aforementioned cellular pathways. Importantly, K+CS treatment exerted unique and significant antitumor effect ex vivo on primary malignant cells obtained from patient's with high-risk hematologic malignancies such as double hit DLBCL, BTK and BCL2 inhibitor resistant mantle cell lymphoma, TP53 deleted/mutated chronic lymphocytic leukemia and high-risk multiple myeloma thus signifying its broader applicability as a treatment option for these aggressive hematologic malignancies where there is a dire need to find new treatment strategies (Figure 1i). In summary, we describe a unique all-oral drug combination with a novel constellation of mechanisms of action on cellular pathways that are exploited by cancer cells. K+CS represents a new class of therapy for multiple cancer types and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for therapy of blood cancers. Disclosures Witzig: Karyopharm Therapeutics: Research Funding; Acerta: Research Funding; Incyte: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Research Funding; MorphSys: Consultancy; Immune Design: Research Funding; Spectrum: Consultancy.