Citations

Abstract Background: Biopsy diagnoses of benign breast disease (BBD) confer a 1.5- to 4-fold increased risk of developing breast cancer (BC) compared with women without BBD. Previously, we reported that decreased numbers of specific immune cell types in lobules of BBD biopsies predicted increased BC risk, suggesting the promise of future tissue biomarker studies to define BC risk markers among BBD patients. Thus, we applied protein-based GeoMx® Digital Spatial Profiling (DSP) to BBD biopsies preceding BC (cases) and to BBD biopsies from cancer-free patients (controls) to identify possible BC risk markers, which we then evaluated in subsequent BC tissues and in surrounding normal lobules of cases. Methods: Archived pathology slides of BBD biopsies were reviewed and used to guide preparation of TMAs containing 1.0-mm diameter FFPE cores of lobules from an age- and cohort-period-matched set of 91 cases and 88 controls from the Mayo Clinic BBD Cohort. For patients who later developed BC, we prepared TMAs of BC tissue and surrounding mapped normal lobules. We applied GeoMx® DSP (immune and canonical signaling proteins) to both sets of TMAs. Following QC and data normalization, associations of case status with log-transformed biomarker expression in lobules of BBD biopsies were carried out using linear mixed modeling approaches, accounting for multiple ROIs per individual. Biomarkers significantly associated with case status (p<0.05) were further examined in BCs and adjacent normal lobules, using similar approaches. Results: The mean age at BBD biopsy was 52 years, and at BC diagnosis of cases, 61.4 years (mean time from BBD to BC was 10.2 years). A family history of BC was more frequent among cases (70% versus 43%; chi-square p=0.002). Of 72 biomarkers tested, 46 (64.4%) were evaluable after QC and normalization and 5 were associated with BC risk after adjustment for family history of BC: BCL2 (p=0.005), STING (p=0.006), CD44 (p=0.02), S100 protein (p=0.03) and pan-AKT (p=0.05); each showed higher levels in lobules of BBD biopsies of controls than cases. Three unique patterns appeared when examining these biomarkers across tissue type within cases: for BCL2 (p=5 x 10-9) and STING (p=2 x 10-19), levels were high in both BC and lobules surrounding BC but low in preceding BBD; for GAPDH (p=4 x 10-53) and pan-AKT (p=2 x 10-33), levels were high in BC, low in preceding BBD, and moderate in lobules surrounding BC; and for CD44 (p=2 x 10-6) and S100B (p=2 x 10-49), levels were low in BC, high in lobules surrounding BC and moderate in preceding BBD. Conclusions: Using a novel TMA of lobules in combination with DSP, we preliminarily identified immune-based and PI3 kinase-related protein biomarkers in BBD biopsies associated with BC risk. In case-only analyses, these markers demonstrated complex differences between lobules in BBD biopsies, subsequent BCs and adjacent normal lobules. Citation Format: Robert Alan Vierkant, Jodi M. Carter, Stacey J. Winham, Chen Wang, Jennifer M. Kachergus, Ji Shi, Raymond M. Moore, Bryan M. McCauley, Laura M. Pacheco-Spann, E A. Thompson, Derek C. Radisky, Amy C. Degnim, Mark E. Sherman. Towards prediction of breast cancer risk in benign biopsies with high-plex GeoMx spatial protein profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2203.

Abstract Introduction: The immune microenvironment of the breast is critical in the development and progression of breast cancer. By analyzing the immune components of breast tissues from patients at varying degrees of breast cancer risk, we may gain enhanced understanding of breast cancer risk and mechanisms of carcinogenesis. Methods: We compared the immune cell composition in breast lobules of age-matched patients with normal and benign breast disease (BBD) at progressively lower breast cancer risk: 1) 18 women with germline mutations in (BRCA1/2); 2) women at intermediate risk (BBD), and 3) women at normal risk (Komen Tissue Bank- (KTB)). We performed immunohistochemical stains for T lymphocytes (CD4, CD8); dendritic cells (CD11c); B lymphocytes (CD20) and macrophages (CD68) and quantitated immune cell densities in up to 10 representative lobules per sample. Results: The median age of all groups was 43.8 years (range: 34-67). BRCA carriers and BBD patients had a higher percentage of fibrocystic lobules (50% fibrocystic) compared to KTB samples (15% fibrocystic), p<0.001. BRCA carriers had significantly higher densities of CD4, CD8, CD11c, and CD68 positive immune cells compared to KTB donor breast tissues, see Table. Compared with BBD patients, mutation carriers had higher densities of CD8+ cells and suggestively higher CD4+ cells, with similar results for other markers assessed. Conclusions: Preliminarily, these data showed that benign prophylactic breast tissues from BRCA mutation carriers contain increased numbers of several immune cell types versus tissues from patients at lower breast cancer risk, including BBD patients and research tissue donors. Further investigation is needed to confirm and expand these findings in relation to breast cancer risk. Table: Pairwise Wilcoxon rank-sum p-values comparing immune cell densities in breast lobules from women with BRCA germline mutations, vs BBD and KTB groups. All Lobules BRCA BBD KTB BRCA vs BBD BRCA vs KTB Median Median Median p-value p-value (IQR) (IQR) (IQR) CD4+, cells/mm2 116.3 63.8 17.7 0.08 <0.001 (46.4-183.6) (25.0-121.1) (13.3-40.6) CD8+, cells/mm2 354.4 200 150.9 0.01 <0.001 (285.5-473.2) (118.9-385) (110.1-225.0) CD11c+, % pp 3.5 3.4 0.4 0.66 <0.001 (1.9-6.8) (1.6-4.7) (0-0.6) CD20+, cells/mm2 6.2 16.7 0 0.16 0.65 (0-25.4) (0-58.3) (0-20.8) CD68, cells/mm2 237.5 219 57.8 0.99 <0.001 (122.9-281.8) (75.0-459.4) (29.2-100.0) IQR - Interquartile Range pp - pixel positive Citation Format: Joshua W. Ogony, Tanya L. Hoskin, Melody L. Stallings Mann, Stacey J. Winham, Rushin Brahmbhatt, Muhammad Asad Arshad, Alvaro Pena Jimenez, Teresa M. Allers, Mark E. Sherrman, Daniel W. Visscher, Derek C. Radisky, Amy C. Degnim. Immune biomarkers in breast tissues of patients with BRCA1/2 mutations, benign breast disease, and normal tissue donors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5947.

Abstract Arachidonic acid (AA) metabolizing enzymes, including cyclooxygenases (COX), lipoxygenases, and cytochrome P450s (CYP450), are expressed in breast cancer (BC). Elevated levels of metabolites of COX-derived, pro-inflammatory prostaglandin (PGs) metabolites in urine have been linked to increased risk of developing breast cancer (BC) in two large prospective cohorts. Further, CYP450-derived EETs in BC tumors are associated with metastasis. We hypothesize that measuring levels of AA-derived oxylipins in breast tissues could enable improved BC risk prediction, provide insights into pro-carcinogenic inflammatory mechanisms, and serve as potential surrogate endpoints in chemoprevention trials. Thus, we performed a pilot study to assess the feasibility of measuring these analytes in small breast tissue samples. We analyzed two sample sets, collected under Institutional Review Board (IRB) approved protocols: 50 normal breast tissue cores donated to the Komen Tissue Bank (KTB) via vacuum-assisted biopsy and 10 tissues removed surgically at Mayo Clinic beyond resection margins of lumpectomy samples performed for atypical ductal hyperplasia (ADH). Tissues were frozen and maintained at -80°C. Normal KTB tissue cores were split in half. Tissues for analysis were weighed, extracted in cold methanol containing a cyclooxygenase inhibitor, and assayed for a panel of 42 oxylipins using validated mass spectrometric methods with a limit of detection per analyte of 0.010 ng/mL in supernatant and CVs of approximately <11%. Analyte measurements in paired cores (ng/g tissue) were compared with Spearman correlations and intra-class correlation coefficients. Measurements in surgical samples were assessed descriptively and levels were compared with normal KTB breast tissues.Median weight of the 50 split KTB cores was 0.055 grams. 17 oxylipins were detected in the tissue samples. PGD2 demonstrated the weakest correlation between split cores (r=0.12; p=0.40), whereas 15-HETE demonstrated the strongest correlation (r=0.68; p<0.0001). ICCs ranged from non-significant to nearly perfect, with highest values for 12-HETE (ICC=0.98, 95%CI: 0.97-0.99). Exploratory analyses of the KTB cores demonstrated significant associations between two BC risk factors, alcohol use and family history of breast/ovarian cancer, and several analytes. Levels of most eicosanoids tested were substantially and highly significantly elevated in tissues surrounding ADH versus KTB normal breast tissues. We conclude that eicosanoids can be readily measured in small frozen breast tissue samples obtained via percutaneous biopsy or at surgery. Future studies to assess the associations of eicosanoid levels with BC risk factors and development of BC after a benign biopsy may inform risk prediction and potentially inhibitable inflammatory mechanisms of breast carcinogenesis. Citation Format: Ginger L. Milne, Robert A. Vierkant, Amy C. Degnim, Anna Maria Storniolo, Jill E. Henry, Laura M. Pacheco-Spann, Derek C. Radisky, Mark E. Sherman. Quantification of oxylipins as biomarkers of inflammation and cancer risk in breast tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6333.

Background TAILOR‐PCI (Tailored Antiplatelet Initiation to Lessen Outcomes due to decreased Clopidogrel Response After Percutaneous Coronary Intervention) studied genotype‐guided selection of antiplatelet therapy after percutaneous coronary intervention versus conventional therapy with clopidogrel. The presence of CYP2C19 loss‐of‐function alleles in patients treated with clopidogrel may be associated with increased risk for ischemic events. We report a prespecified sex‐specific analysis of genotyping and associated cardiovascular outcomes from this study. Methods and Results Associations between sex and major adverse cardiac events (MACE: cardiovascular death, myocardial infarction, stroke, stent thrombosis, and severe recurrent ischemia) and Bleeding Academic Research Consortium (BARC) bleeding at 12 months were analyzed using Cox proportional‐hazards models. Among 5276 randomized patients, loss‐of‐function carriers were observed in ≈36% of both sexes, and >80% of carriers were heterozygotes. At 12 months, after adjustment for baseline differences, risks of MACE (HR , 1.28 [0.97 to 1.68]; P =0.088) and BARC bleeding (hazard ratio [HR], 1.36 [0.91 to 2.05]; P =0.14) were comparable among women and men. There were no significant interactions between sex and treatment strategy for MACE interaction P value ( P int =0.59) or BARC bleeding ( P int =0.47) nor for sex and genotype (MACE P int =0.15, and BARC bleeding P int =0.60). Conclusions CYP2C19 loss‐of‐function alleles were present in ≈1 in 3 women and men. Women had similar adjusted risks of MACE and bleeding as men following percutaneous coronary intervention. Genotype‐guided therapy did not significantly reduce the risk of MACE or bleeding relative to conventional therapy for both sexes. Registration URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01742117.

In this case series, we describe a novel observation in which 4 patients with acute ischemic stroke secondary to large vessel occlusion and no history of seizure present with focal seizure activity localizable to a chronic, contralateral infarct. The explanation for this phenomenon is unknown but may be due to a combination of effects involving disrupted interhemispheric inhibitory connections and epileptogenic changes involving chronically infarcted tissue.

e19553 Background: The combination of tafa + LEN has accelerated approval in the United States (2020) and conditional approval in Canada and Europe (2021) for R/R DLBCL in ASCT-ineligible adult pts. A tolerable safety profile for R-CHOP + tafa ± LEN in pts with newly diagnosed DLBCL in the Phase Ib First-MIND study (NCT04134936) was previously reported, with numerically superior efficacy for R-CHOP + tafa + LEN (ASH 2021; #3556). We report PK, PD, and immunogenicity of R-CHOP + tafa ± LEN in this first line setting. Methods: Eligible pts ≥18 years with treatment-naïve DLBCL, IPI 2–5, and ECOG PS 0–2 were randomized 1:1 to either six 21-day (D) cycles (C) of R-CHOP (R-CHO, D1; P, D1–5) + tafa (12 mg/kg IV, D1, 8, 15) (Arm A) or R-CHOP + tafa + LEN (25 mg orally, D1–10) (Arm B). Secondary endpoints included tafa serum conc and number and percentage of pts developing anti-tafa Abs. Exploratory endpoints included natural killer (NK) cell, T-cell, and B-cell count in peripheral blood. Results: Data cut-off: 13 March 2021 for safety analysis, incl. ≥1 month follow-up after EoT visit for all pts; 15 September 2021 for efficacy. Tafa serum conc reached steady state by C3 (geometric mean trough conc: Arm A, 186.40–216.55 µg/mL; Arm B, 171.77–201.54 µg/mL) and steadily declined after treatment completion. Anti-tafa Abs were detected in 1/65 (1.5%) pts. This pt showed pre-existing tafa Abs at baseline, which decreased during treatment. Median NK cell count decreased from baseline at C1D8 but was at baseline or higher levels by EoT visit (Arm A) and C1D15 (Arm B; Table). T-cell count decreased from baseline at C1D8 in both arms but was at baseline level or higher by C1D15 (Arm A) and EoT visit (Arm B). Median B-cell count decreased from baseline to 0 cells/μL (Arm A, C1D15; Arm B, C1D8); at 6-month follow-up after EoT visit, B-cell count had recovered to measurable levels in ̃50% of pts. Conclusions: Tafa serum conc reached and maintained a therapeutic dose level in this first-line regimen and declined in line with known tafa half-life (̃16 days) after treatment completion; tafa levels were comparable between the two treatment arms. No pt developed treatment-induced or treatment-boosted anti-tafa Abs. Median NK-cell, T-cell, and B-cell counts were comparable between treatment arms in all cycles. Clinical trial information: NCT04134936. [Table: see text]

Abstract Diffuse midline glioma (DMG) of the pons remains the leading cause of death among pediatric patients with brain tumors, despite numerous attempts to intensify treatment. While standard treatment includes 54Gy in 30 fractions of radiation over six weeks of time, nearly all patients progress within the treatment field, and many experience symptomatic radionecrosis with steroid dependence. Symptom improvement typically begins after 20Gy to the tumor. Both hypofractionation and reirradiation after recurrence have been found to be safe for patients with DMG. Our study aims to enroll patients with newly diagnosed pontine DMG aged >1 year (no maximum age), and collect molecular information about DMG via stereotactic biopsy, followed by a short 2 week course of 25Gy in 10 fractions of radiation, with volumes guided by MRI with tractography reconstruction and FDOPA PET radiotracer uptake. Patients are followed closely and can complete the 25Gy in 10 fraction radiation course up to 3 times total for meeting radiographic and clinical progression criteria. Our primary endpoint is to estimate the time to progression from diagnosis to after receiving the second 25Gy course, and to compare this to the historical 7 month standard from diagnosis to progression after one 54Gy course. We aim to improve the time interval where patients are asymptomatic at home while minimizing time receiving daily treatments. Other endpoints will include the patient quality of life, caregiver quality of life, PFS intervals after each course, overall survival, and toxicity.

Abstract Structural discovery of guanine nucleotide exchange factor (GEF) protein complexes is likely to become increasingly relevant with the development of new therapeutics targeting small GTPases and development of new classes of small molecules that inhibit protein‐protein interactions. Syx (also known as PLEKHG5 in humans) is a RhoA GEF implicated in the pathology of glioblastoma (GBM). Here we investigated protein expression and purification of ten different human Syx constructs and performed biophysical characterizations and computational studies that provide insights into why expression of this protein was previously intractable. We show that human Syx can be expressed and isolated and Syx is folded as observed by circular dichroism (CD) spectroscopy and actively binds to RhoA as determined by co‐elution during size exclusion chromatography (SEC). This characterization may provide critical insights into the expression and purification of other recalcitrant members of the large class of oncogenic—Diffuse B‐cell lymphoma (Dbl) homology GEF proteins. In addition, we performed detailed homology modeling and molecular dynamics simulations on the surface of a physiologically realistic membrane. These simulations reveal novel insights into GEF activity and allosteric modulation by the plekstrin homology (PH) domain. These newly revealed interactions between the GEF PH domain and the membrane embedded region of RhoA support previously unexplained experimental findings regarding the allosteric effects of the PH domain from numerous activity studies of Dbl homology GEF proteins. This work establishes new hypotheses for structural interactivity and allosteric signal modulation in Dbl homology RhoGEFs.

Abstract PET imaging plays an essential role in achieving earlier and more specific diagnoses of dementia syndromes, important for clinical prognostication and optimal medical management. This has become especially vital with the recent development of pathology-specific disease-modifying therapy for Alzheimer disease, which will continue to evolve and require methods to select appropriate treatment candidates. Techniques that began as research tools such as amyloid and tau PET have now entered clinical use, making nuclear medicine physicians and radiologists essential members of the care team. This review discusses recent changes in the understanding of dementia and examines the roles of nuclear medicine imaging in clinical practice. Within this framework, multiple cases will be shown to illustrate a systematic approach of FDG PET interpretation and integration of PET imaging of specific molecular pathology including dopamine transporters, amyloid, and tau. The approach presented here incorporates contemporary understanding of both common and uncommon dementia syndromes, intended as an updated practical guide to assist with the sophisticated interpretation of nuclear medicine examinations in the context of this rapidly and continually developing area of imaging.