Moyer, Ann M.

There is large variation in response to menopausal hormone treatments among women and it remains unclear as to what genetic factors might increase risk of breast cancer in women using such treatments to relieve symptoms of menopause. SULT1A1 is a ubiquitous enzyme involved in the metabolism of steroid hormones including estrogen. A number of polymorphisms in the gene for this enzyme are associated with various cancer risks and the activity level of the enzyme is correlated closely with variation in copy number of the gene. This pilot study evaluated SNP variation and copy number of SULT1A1 in a subset of women (n=156) being screened for the Kronos Early Estrogen Prevention Study (KEEPS). All participants gave written informed consent. DNA prepared from blood was genotyped for SULT1A1 copy number, rs3760091 and rs750155 from the promoter region of SULT1A1, and rs9282861 from the coding region (SULT1A1*2 variant). In this cohort, 5.2% had 1 copy of SULT1A1, 65.2% had 2 copies, 23.9% had 3 copies, and 5.8% had 4 copies. Previously reports of the allele frequency for the three SNPs did not take into account gene copy number. When copy number was taken into account, allele frequency was significantly different, a fact that will have to be taken into account in future genotype‐phenotype correlations. (Supported by grants from the Kronos Longevity Research Institute, NIH HL90639, RR24150 and GM61388.)

Abstract Purpose: Patient-derived xenografts (PDX) are a research tool for studying cancer biology and drug response phenotypes. While engraftment rates are higher for tumors with more aggressive characteristics, it is uncertain whether engraftment is prognostic for cancer recurrence. Patients and Methods: In a prospective study of patients with breast cancer treated with neoadjuvant chemotherapy (NAC) with taxane ± trastuzumab followed by anthracycline-based chemotherapy, we report the association between breast cancer events and PDX engraftment using tumors derived from treatment naïve (pre-NAC biopsies from 113 patients) and treatment resistant (post-NAC at surgery from 34 patients). Gray test was used to assess whether the cumulative incidence of a breast cancer event differs with respect to either pre-NAC PDX engraftment or post-NAC PDX engraftment. Results: With a median follow-up of 5.7 years, the cumulative incidence of breast cancer relapse did not differ significantly according to pre-NAC PDX engraftment (5-year rate: 13.6% vs. 13.4%; P = 0.89). However, the incidence of a breast event was greater for patients with post-NAC PDX engraftment (5-year rate: 50.0% vs. 19.6%), but this did not achieve significance (P = 0.11). Conclusions: In treatment-naïve breast cancer receiving standard NAC, PDX engraftment was not prognostic for breast cancer recurrence. Further study is needed to establish whether PDX engraftment in the treatment-resistant setting is prognostic for cancer recurrence.

AbstractBackgroundCurrently, there is no biologically based rationale for drug selection in migraine prophylactic treatment.MethodsTo investigate the genetic variation underlying treatment response to verapamil prophylaxis, we selected 225 patients from a longitudinally established, deeply phenotyped migraine database (N = 5983), and collected uninterrupted quantitated verapamil treatment response data and DNA for these 225 cases. We recorded the number of headache days in the four weeks preceding treatment with verapamil and for four weeks, following completion of a treatment period with verapamil lasting at least five weeks. Whole‐exome sequencing (WES) was applied to a discovery cohort consisting of 21 definitive responders and 14 definitive non‐responders, and the identified single nucleotide polymorphisms (SNPs) showing significant association were genotyped in a separate confirmation cohort (185 verapamil treated patients). Statistical analysis of the WES data from the discovery cohort identified 524 SNPs associated with verapamil responsiveness (p < 0.01); among them, 39 SNPs were validated in the confirmatory cohort (n = 185) which included the full range of response to verapamil from highly responsive to not responsive.ResultsFourteen SNPs were confirmed by both percentage and arithmetic statistical approaches. Pathway and protein network analysis implicated myo‐inositol biosynthetic and phospholipase‐C second messenger pathways in verapamil responsiveness, emphasizing the earlier pathogenic understanding of migraine. No association was found between genetic variation in verapamil metabolic enzymes and treatment response.ConclusionOur findings demonstrate that genetic analysis in well‐characterized subpopulations can yield important pharmacogenetic information pertaining to the mechanism of anti‐migraine prophylactic medications.

Context.— Small case series have evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in formalin-fixed, paraffin-embedded tissue using reverse transcription–polymerase chain reaction, immunohistochemistry (IHC), and/or RNA in situ hybridization (RNAish). Objective.— To compare droplet digital polymerase chain reaction, IHC, and RNAish to detect SARS-CoV-2 in formalin-fixed, paraffin-embedded tissue in a large series of lung specimens from coronavirus disease 2019 (COVID-19) patients. Design.— Droplet digital polymerase chain reaction and RNAish used commercially available probes; IHC used clone 1A9. Twenty-six autopsies of COVID-19 patients with formalin-fixed, paraffin-embedded tissue blocks of 62 lung specimens, 22 heart specimens, 2 brain specimens, and 1 liver, and 1 umbilical cord were included. Control cases included 9 autopsy lungs from patients with other infections/inflammation and virus-infected tissue or cell lines. Results.— Droplet digital polymerase chain reaction had the highest sensitivity for SARS-CoV-2 (96%) when compared with IHC (31%) and RNAish (36%). All 3 tests had a specificity of 100%. Agreement between droplet digital polymerase chain reaction and IHC or RNAish was fair (κ = 0.23 and κ = 0.35, respectively). Agreement between IHC and in situ hybridization was substantial (κ = 0.75). Interobserver reliability was almost perfect for IHC (κ = 0.91) and fair to moderate for RNAish (κ = 0.38–0.59). Lung tissues from patients who died earlier after onset of symptoms revealed higher copy numbers by droplet digital polymerase chain reaction (P = .03, Pearson correlation = −0.65) and were more likely to be positive by RNAish (P = .02) than lungs from patients who died later. We identified SARS-CoV-2 in hyaline membranes, in pneumocytes, and rarely in respiratory epithelium. Droplet digital polymerase chain reaction showed low copy numbers in 7 autopsy hearts from ProteoGenex Inc. All other extrapulmonary tissues were negative. Conclusions.— Droplet digital polymerase chain reaction was the most sensitive and highly specific test to identify SARS-CoV-2 in lung specimens from COVID-19 patients.

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 (HR)-positive metastatic breast cancer (MBC), but progression of disease is inevitable. Serum thymidine kinase-1 (TK1) is a secreted marker of proliferation that is prognostic in patients (pts) with HR-positive, HER2-negative MBC and may be predictive of ET and CDK 4/6i response. PROMISE (NCT0281902) is a prospective study enrolling women with HR-positive MBC starting palbociclib (Pb) + letrozole (L) (1st line) or Pb + fulvestrant (2nd line). We undertook a comprehensive “omic” assessment of blood, tumor, urine and the fecal microbiome in order to identify novel genomic variants and pathways associated with an early decline in TK1 (measured after 2 months) and PFS. Additionally, patient derived xenografts/organoids were generated at baseline and progression to test new therapeutic approaches to overcome resistance to CDK4/6i and ET. We report the initial association between the baseline genomic landscape and baseline TK1 levels. Methods: FFPE tumor biopsies were obtained for DNA/RNA sequencing (TempusTM) and blood samples for TK1 (Divitum® assay, Biovica) were collected pretreatment (pre-Pb) and after 2 cycles of Pb + ET (post-Pb2). Both whole-exome (exome capture) sequencing (WES) and RNA-Seq used the Integrated DNA Technologies xGen Exome Research Panel v1.0 capture kit. TK1+ disease was defined as &gt; 200 Du/L and TK1- disease as below limit of detection up to 200 Du/L. We tested the association between genomic and transcriptomic characteristics with baseline TK1 data in pretreatment samples where both WES and RNA-seq and TK1 was available. The data were analyzed using bioinformatics pipelines for somatic and germline mutations and copy number alterations. The current analysis focuses on baseline 1st-line pre-Pb omics data in conjunction with baseline TK1 levels. The database was locked for analysis on 5/29/2020. Results: Thirty-three pts (median age: 59 yrs.) were evaluable, with paired samples for TK1 in 32. Six pts had TK1+ disease pre-Pb and post-Pb2. Twenty-two pts had TK1- disease pre-Pb and post-Pb2. Four pts had a decrease in TK1 after 2 cycles of treatment that altered the classification from TK1+ to TK1-. Both baseline RNA seq and serum TK1 (n=16) were available for 4 TK1+ and 12 TK1- pts. In this group, 476 genes were differentially regulated (398 upregulated; 78 downregulated). Pathway analysis demonstrated enrichment in complement and coagulation cascade pathway, PPAR signaling pathway, and metabolism-related pathways related to up-regulation of CYP and UGT gene families. Further testing for the association of WES data with baseline TK1+ (n=8) and TK1- (n=16) disease demonstrated somatic copy number variations on chromosomes 6, 11, 12 and 15. CDK4 copy number gains were observed in 3/8 TK1+ pts and 0/16 TK1- pts. We also observed that somatic mutations (LOH, copy number and/or SNV/INDELs) were more prevalent in the TK1+ compared to the TK1- pre-Pb group in several cancer-associated genes (FAS [p=0.06] PTEN, PIK3CB, NAB2, SOX9 and FAT1 [p=0.08], TP53, and MAP2K4 [p=0.22]). Conversely, we also noted that 6/7 pts with GATA3 mutations had TK1- disease (p=0.23). Conclusions: Using a comprehensive “omics” approach, our data suggest that a secreted biomarker of proliferation (TK1) obtained prior to initiating CDK4/6i and ET for the first line treatment of HR+ MBC is associated with established and novel genes and pathways associated with prognosis of pts receiving ET and CDK 4/6i. Analysis of on-treatment (after 2 cycles) tumor RNA seq and its association with change in TK1 as well as data from the 2nd-line cohort will be presented at the meeting. Citation Format: Ciara C O Sullivan, Krishna R Kalari, Vera J Suman, Peter T Vedell, Ann Moyer, Abraham D Eyman Casey, Jason Sinnwell, Xiaojia Tang, Kevin Thompson, 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, Adrian Nordmark, Mattias Bergqvist, Brendan P McMenomy, Richard M Weinshilboum, Liewei Wang, Matthew P Goetz. Novel genomic variants and pathways associated with baseline serum thymidine kinase 1 levels in HR-positive HER2-negative MBC patients commencing palbociclib and letrozole [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 PS5-24.

Abstract Introduction: The immune microenvironment of high-risk HR+ breast cancer (BC) is poorly understood. BEAUTY is a prospective neoadjuvant chemotherapy (NAC) study of stage I-III BC patients treated with neoadjuvant weekly taxane followed by anthracycline-based chemotherapy. Among clinical luminal B BC (St. Gallen criteria) from BEAUTY, we used high-plex digital spatial profiling (DSP) to characterize the intra-epithelial tumor and stromal immune landscapes to 1) assess the impact of NAC on these landscapes and 2) identify immune biomarkers predictive of response to NAC. Methods: The tissue set included FFPE sections of resected tumors from 35 patients (median 51y; range: 21-71y) with clinically-defined luminal B BC (ER &gt; 10%/grade 2/Ki67 ≥ 15% or ER &gt; 10%/grade 3), and 16 paired pre-NAC biopsies. Nanostring GeoMX DSP was used to quantitate 58 immune and BC biomarker proteins in intra-epithelial, cytokeratin-positive tumor segments and adjacent stromal (cytokeratin-negative/SYTO13 (nuclear stain)-positive) segments. Data were normalized using the geometric mean of two negative controls (RbIgG and MsIgG1). A general linear model with negative binomial identified differentially-expressed (DE) proteins in pre/post-NAC tumors. Based on DE protein data and biologic function, a targeted protein subset (N=19) was evaluated in pre-NAC biopsies for associations between protein abundance and residual cancer burden (RCB) class (0-II vs. III) (Wilcoxon-rank sum test: p-value &lt; 0.025 considered significant) or RCB ‘breast only’ index (Spearman correlation). Results: Comparing tumor segments in pre-NAC specimens, intra-epithelial segments were predictably enriched in cytokeratin, ER, PR and Ki-67, but also CD127 and NY-ESO-1; whereas stromal segments were enriched in proteins associated with T cell subsets (e.g. CD3, CD4, CD8), macrophages (CD68 and CD163), antigen presentation/dendritic cells (CD11c, HLA-DR) and immune checkpoint proteins (PD-L1, PD-L2, B7-H3, TIM-3, VISTA) among others. While NAC did not alter the spatial distribution of proteins (intra-epithelial vs. stromal segments), it markedly attenuated the immune landscape, with decreased abundance of functionally-diverse immune proteins (e.g. CD45, CD3, CD4, CD127, granzyme B, CTLA4; STING, B7-H3, CD11c, and CD68, log2FC: 0.27-1.52 p &lt; 0.05), including low abundance proteins PD-1, PD-L1, PD-L2, CD20, and OX40L (log 2FC: 0.15-1.16, p &lt; 0.05). Both PR and Ki-67 decreased in post-NAC tumors whereas ER was not significantly altered (p &lt; 0.05). CD8, CD14, CD163, HLA-DR, IDO-1 and TIM-3 were not significantly altered by NAC. In the pre-NAC biopsies (which had subsequent residual tumor burdens of RCB class 0 or I (n=1 each), II (n=6), and III (n=8), and median RCB ‘breast only’ index of 3.39 (range: 0.00-38.02), 19 proteins were used for RCB analysis (CD3, CD4, CD8, CD14, CD34, CD44, CD45, CD68, CD127, CD163, CTLA4, granzyme B, STING, B7-H3, fibronectin, Ki-67, HLA-DR, SMA and TIM3). Among them, stromal CD127 was significantly higher in RCB class III than RCB class 0-II (p=0.021) and RCB ‘breast only’ index was positively correlated with intra-epithelial granzyme B (rho = 0.61; p=0.012), and negatively correlated with intra-epithelial Ki-67 (rho= -0.71; p=0.0022). Conclusion: In this series of clinical luminal B BC, NAC markedly attenuated the tumoral immune landscapes with a small set of “NAC-resistant” immune proteins. Among a targeted set, stromal CD127 was significantly higher in RCB III, and RCB breast-only index was positively correlated with intra-epithelial granzyme B. These data provide insight into the impact of NAC on HR+ BC, and identify potential immune biomarkers to predict response to neoadjuvant chemotherapy. Citation Format: Jodi M Carter, Judy C Boughey, Jun He, Vera J Suman, Xue Wang, Jennifer M Kachergus, Krishna R Kalari, Liewei Wang, Richard Weinshilboum, Ann M Moyer, Sarah A McLaughlin, Alvaro Moreno-Aspitia, Donald W Northfelt, Richard J Gray, James N Ingle, E. Aubrey Thompson, Matthew P Goetz. Neoadjuvant chemotherapy selectively alters spatially-defined immune landscapes in clinical luminal B HR+/HER2- breast cancers: Analysis of the breast cancer genome guided therapy study (BEAUTY) [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 PD7-05.

Background Eculizumab, a monoclonal antibody targeting C5, is an effective treatment for complement-mediated thrombotic microangiopathy (CM-TMA), also known as atypical hemolytic uremic syndrome. Although lifelong therapy has been suggested, the optimal treatment schedule is unknown, while eculizumab discontinuation does not universally lead to relapse. Data evaluating risk factors associated with an increased risk of CM-TMA recurrence following eculizumab withdrawal are scarce. Prior to the eculizumab era, complement genetic studies have been used to assess morbidity and mortality in patients with CM-TMA. Our aim was to assess the impact of complement gene variants on CM-TMA relapse rate (RR) after eculizumab discontinuation. Methods Search protocols were developed for the Ovid and PubMed databases to identify existing reports on CM-TMA and eculizumab withdrawal published before February 1st 2020. Cases were cross-referenced to eliminate duplicates. Inclusion criteria included patients diagnosed with CM-TMA who had undergone eculizumab discontinuation. Reasons for exclusion were patients with no follow-up after eculizumab withdrawal and patients lacking complement gene testing. Patients undergoing eculizumab dose extension, but not discontinuation, were not included. Results Fifteen studies including 557 individuals were retrieved. Of these, 363 (65.2%) were excluded as outlined in Figure 1 and 194 cases were included in the final analysis with a global RR of 28.9% (n=56). Distribution based on gene impacted and variant type are presented in Table 1. RR was highest among patients with CFH variants (52.5% to 60%) -particularly if involving exon 22 or nonsense mutations-, MCP/CD46 variants (38.9% to 45%) -particularly if affecting splice regions-, and cases with multiple concomitant variants (57.1%) -particularly those including CFH or a CFH-CFHR1 Hybrid (Table 2). Patients with relapse were more likely to have the presence of a complement variant (p&lt;0.001), history of previously resolved CM-TMA events (p=0.022), younger age (mean age 22.4 years for patients with relapse vs. 32.33 years for patients without relapse; p=0.002) and, within those with detected variants, multiple vs. single variants (p=0.002) and CFH-inclusive variants (p=0.003). On univariate analysis, complement gene variants (OR 4.217 95%CI 1.97-9.026; p&lt;0.001), age &lt;30 years (OR 2.346 95%CI 1.159-4.749; p=0.018), and presence of multiple variants (OR 2.06 95%CI 1.172-3.621; p=0.012) or CFH-inclusive variants (OR 3.111 95%CI 1.443-6.71; p=0.004) were associated with increased odds of CM-TMA relapse, while no prior history of additional CM-TMA events was the only factor associated with lower RR (OR 0.327 95%CI 0.123-0.87; p=0.025). Presence of complement variants (OR 4.48 95%CI 1.132-17.724; p=0.033) and CFH-inclusive variants (OR 3.045 95%CI 1.136-8.159; p=0.027) were independently associated with increased RR on multivariate analysis (Table 3). Conclusion Relapse after eculizumab discontinuation is rare in cases with no genetic variants identified but can increase to more than 80% in high-risk subgroups. Complement genetic testing is not required for CM-TMA diagnosis or for initiation of complement inhibiting therapy; however, our results demonstrate the value of complement genetic testing when stratifying risk of patients for consideration of eculizumab discontinuation. Disclosures Sridharan: Alexion: Honoraria.

Purpose: molecular testing is often indicated for recently transfused patients. However, there are no guidelines regarding the potential interference from donor DNA or whether it is necessary to wait for a period of time post-transfusion prior to genetic testing. While the majority of patients are transfused in the non-trauma setting using leukoreduced (LR) red blood cell products, the degree of leukoreduction varies among centers and is not universally practiced. Methods: whole blood units collected from anonymous donors were used in an in vitro transfusion model. One unit was split: half being leukoreduced simulating a leukopenic recipient and half left untreated. Donors were simulated by leukoreduced, partially leukoreduced (PLR), or non-leukoreduced units, transfused in 2, 5, or 16 unit equivalents. DNA from the combinations were subjected to short tandem repeat (STR) analysis for chimerism detection. Results: donor DNA was not detectable in any of the LR combinations, but detected in the PLR combinations, ranging from 0.1 to 1.5% donor DNA in the immunocompetent recipient and 6.3–27.8% in the leukopenic recipient. Non-LR donor DNA was also detected (13–95%). Conclusion: donor-derived DNA from leukoreduced blood products is unlikely to interfere with the interpretation of germline genetic testing in immunocompetent recipients but may interfere in immunocompromised recipients.