Nanostring Gene Expression Profiling Research Articles

Associations of prostate tumor immune landscape with vigorous physical activity and prostate cancer progression.

Vigorous physical activity has been associated with lower risk of fatal prostate cancer. However, mechanisms contributing to this relationship are not understood. We studied 117 men with prostate cancer in the University of North Carolina Cancer Survivorship Cohort (UNC CSC) who underwent radical prostatectomy, and 101 radiation-treated prostate cancer patients in FASTMAN. Structured questionnaires administered in UNC CSC assessed physical activity. In both studies, digital image analysis of H&E-stained tissues was applied to quantify Tumor Infiltrating Lymphocytes (TILs) in segmented regions. Nanostring gene expression profiling in UNC CSC and microarray in FASTMAN were performed on tumor tissue and a 50-gene signature utilized to predict immune cell types. Vigorous recreational activity, reported by 34 (29.1%) UNC men, was inversely associated with TILs abundance. Tumors of men reporting any vigorous activity versus none showed lower gene expression-predicted abundance of Th, exhausted CD4 T cells and macrophages. T cell subsets, including Treg, Th, Tfh, exhausted CD4 T cells, and macrophages were associated with increased risk of biochemical recurrence, only among men with ERG-positive tumors. Vigorous activity was associated with lower prostate tumor inflammation and immune microenvironment differences. Macrophages and T cell subsets, including those with immunosuppressive roles and those with lower abundance in men reporting vigorous exercise, were associated with worse outcomes in ERG-positive prostate cancer. Our novel findings contribute to our understanding of the role of the tumor immune microenvironment in prostate cancer progression, and may provide insight into how vigorous exercise could affect prostate tumor biology.

High-Throughput Transcriptomics Identifies Chemoresistance-Associated Gene Expression Signatures in Human Angiosarcoma

Angiosarcomas, clinically aggressive cancers of endothelial origin, are a rare subtype of soft-tissue sarcomas characterized by resistance to chemotherapy and dismal prognosis. In this study, we aim to identify the transcriptomic biomarkers of chemoresistance in angiosarcoma. We examined 72 cases of Asian angiosarcomas, including 35 cases treated with palliative chemotherapy, integrating information from NanoString gene expression profiling, whole transcriptome profiling (RNA-seq), immunohistochemistry, cell line assays, and clinicopathological data. In the chemoresistant cohort (defined as stable disease or progression), we observed the significant overexpression of genes, including SPP1 (log2foldchange 3.49, adj. p = 0.0112), CXCL13, CD48, and CLEC5A, accompanied by the significant enrichment of myeloid compartment and cytokine and chemokine signaling pathways, as well as neutrophils and macrophages. RNA-seq data revealed higher SPP1 expression (p = 0.0008) in tumor tissues over adjacent normal compartments. Immunohistochemistry showed a significant moderate positive correlation between SPP1 protein and gene expression (r = 0.7016; p < 0.00110), while higher SPP1 protein expression correlated with lower chemotherapeutic sensitivity in patient-derived angiosarcoma cell lines MOLAS and ISOHAS. In addition, SPP1 mRNA overexpression positively correlated with epithelioid histology (p = 0.007), higher tumor grade (p = 0.0023), non-head and neck location (p = 0.0576), and poorer overall survival outcomes (HR 1.84, 95% CI 1.07–3.18, p = 0.0288). There was no association with tumor mutational burden, tumor inflammation signature, the presence of human herpesvirus-7, ultraviolet exposure signature, and metastatic state at diagnosis. In conclusion, SPP1 overexpression may be a biomarker of chemoresistance and poor prognosis in angiosarcoma. Further investigation is needed to uncover the precise roles and underlying mechanisms of SPP1.

A subpopulation of human bone marrow erythroid cells displays a myeloid gene expression signature similar to that of classic monocytes.

Erythroid cells, serving as progenitors and precursors to erythrocytes responsible for oxygen transport, were shown to exhibit an immunosuppressive and immunoregulatory phenotype. Previous investigations from our research group have revealed an antimicrobial gene expression profile within murine bone marrow erythroid cells which suggested a role for erythroid cells in innate immunity. In the present study, we focused on elucidating the characteristics of human bone marrow erythroid cells through comprehensive analyses, including NanoString gene expression profiling utilizing the Immune Response V2 panel, a BioPlex examination of chemokine and TGF-beta family proteins secretion, and analysis of publicly available single-cell RNA-seq data. Our findings demonstrate that an erythroid cell subpopulation manifests a myeloid-like gene expression signature comprised of antibacterial immunity and neutrophil chemotaxis genes which suggests an involvement of human erythroid cells in the innate immunity. Furthermore, we found that human erythroid cells secreted CCL22, CCL24, CXCL5, CXCL8, and MIF chemokines. The ability of human erythroid cells to express these chemokines might facilitate the restriction of immune cells in the bone marrow under normal conditions or contribute to the ability of erythroid cells to induce local immunosuppression by recruiting immune cells in their immediate vicinity in case of extramedullary hematopoiesis.

Strain variation in the Candida albicans iron limitation response.

Iron acquisition is critical for pathogens to proliferate during invasive infection, and the human fungal pathogen Candida albicans is no exception. The iron regulatory network, established in reference strain SC5314 and derivatives, includes the central player Sef1, a transcription factor that activates iron acquisition genes in response to iron limitation. Here, we explored potential variation in this network among five diverse C. albicans strains through mutant analysis, Nanostring gene expression profiling, and, for two strains, RNA-Seq. Our findings highlight four features that may inform future studies of natural variation and iron acquisition in this species. (i) Conformity: In all strains, major iron acquisition genes are upregulated during iron limitation, and a sef1Δ/Δ mutation impairs that response and growth during iron limitation. (ii) Response variation: Some aspects of the iron limitation response vary among strains, notably the activation of hypha-associated genes. As this gene set is tied to tissue damage and virulence, variation may impact the progression of infection. (iii) Genotype-phenotype variation: The impact of a sef1Δ/Δ mutation on cell wall integrity varies, and for the two strains examined the phenotype correlated with sef1Δ/Δ impact on several cell wall integrity genes. (iv) Phenotype discovery: DNA repair genes were induced modestly by iron limitation in sef1Δ/Δ mutants, with fold changes we would usually ignore. However, the response occurred in both strains tested and was reminiscent of a much stronger response described in Cryptococcus neoformans, a suggestion that it may have biological meaning. In fact, we observed that the iron limitation of a sef1Δ/Δ mutant caused recessive phenotypes to emerge at two heterozygous loci. Overall, our results show that a network that is critical for pathogen proliferation presents variation outside of its core functions.IMPORTANCEA key virulence factor of Candida albicans is the ability to maintain iron homeostasis in the host where iron is scarce. We focused on a central iron regulator, SEF1. We found that iron regulator Sef1 is required for growth, cell wall integrity, and genome integrity during iron limitation. The novel aspect of this work is the characterization of strain variation in a circuit that is required for survival in the host and the connection of iron acquisition to genome integrity in C. albicans.

Abstract C012: Integration of social variables resolves subclasses of tumor microenvironment

Abstract Intro: Recent work has identified subclasses of the tumor microenvironment, with most work emphasizing a robust adaptive immune response associated with ER-negative and/or basal-like tumors. Since adaptive immune responses may influence response to chemotherapy and may be targetable by immunotherapy, understanding population-level heterogeneity in immune response is important. Methods: We leveraged the Carolina Breast Cancer Study (CBCS), a large population-based study that oversampled young (≤50 years) and Black women with invasive breast cancer (BC). We performed NanoString gene expression profiling on tissue from 1,329 BC patients, including 704 (53%) Black and 625 (47%) non-Black women, and assayed genes representative of at least 10 individual immune cell types from both adaptive and innate arms of the immune system, homologous recombination deficiency signature and TP53 mutation status. Using a latent class approach that included variables for self-reported race, triple negative status, individual measures of socioeconomic status and community-level measures of advantage and disadvantage, we identified latent classes that integrated tumor microenvironment with tumor biology, individual and community level exposures under a Cells-to-Society conceptual model of breast cancer outcomes. Results: Latent Class Analysis identified 6 integrated classes of the tumor microenvironment, further subdividing a previously identified and validated adaptive immune class into two subgroups. The two adaptive classes were distinguished by different expression levels of B-cells, CD8+ T cells, cytotoxic cells and PD-L1, and differing frequency of basal-like subtype (Adaptive Class 1 RFD [95% CI]: 19.0 [11.9, 26.2]; Adaptive Class 2 RFD [95% CI]: 32.8 [23.3,42.3]) and presence of genomic instability gene expression subtypes (Adaptive Class-1 RFD [95% CI]: 22.1 [14.0, 30.1]; Adaptive Class-2 RFD [95% CI]: 37.5 [28.1,46.2]) relative to the immune-quiet tumors. Discussion: Social variables have complex effects on tumor biology and microenvironment, but latent class models and other model-based approaches may help to integrate biological data with other social factors to identify susceptible subgroups and to optimize precision medicine in diverse populations. Citation Format: Alina M. Hamilton, Kara McCormack, Marc A. Emerson, Melissa A. Troester, Terry Hyslop. Integration of social variables resolves subclasses of tumor microenvironment [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C012.

Evaluation of a Prognostic 23-Gene Expression Panel in a Set of 390 Multiethnic Hodgkin Lymphoma Patients

Classic Hodgkin lymphoma (CHL) is a curable lymphoma with unique epidemiological features, including rare neoplastic cells, and an adolescent/young adult age (AYA) incidence peak higher in females, correlated with socioeconomic status. We previously reported that RNA expression measured in formalin-fixed paraffin-embedded (FFPE) tissues using a panel of 23 immune response genes was associated with 5-year overall survival (OS) in predominantly White/European advanced staged CHL patients treated with ABVD chemotherapy (Scott et al., 2013). Here, we conducted a study to evaluate if the 23-gene expression panel predicted 5-year OS in a real-world set of FFPE tumor tissue from racially/ethnically diverse populations in the U.S. and Mexico from the Multi- Ethnic Study of Hodgkin Lymphoma (MESH). Methods: We collected tumor blocks from 891 patients from 9 hospitals and 2 Residual Tissue Repositories in the U.S. and Mexico. Demographic and clinical data, including survival by month, age at diagnosis, birth year, race/ethnicity, sex, histology were abstracted. Patients were diagnosed from 1978 to 2018. CHL diagnoses and histological subtypes were validated by histopathology review. RNA was extracted and NanoString gene expression profiling was performed using 250-gene codeset including the published 23-gene expression (GE) panel (Scott et. al., 2013). 390 cases passed QC after applying a modified normalization protocol (Chan et.al., 2017). The multiethnic risk index was derived from a linear equation of the log 2 GE in MESH multiplied by the established regression coefficients (Scott et al., 2013). Next, we examined a dichotomized score using a cut-off value at the 75 th percentile. Both were tested for association with 5-year OS using a Cox proportional hazards model using R software 4.3.0 version adjusting for age as a continuous and categorical variable (15-39 and 40+ years), type of hospital that provided the samples, sex, EBV tumor status (assigned using EBER2), race/ethnicity, and histology. The 5-year OS was computed by right-censoring any patient that survived longer than 5 years. Proportionality assumptions were tested (using an alpha level of 0.05). Stratified models by categorical age and race/ethnicity were adjusted for patient demographical variables and effect heterogeneity was examined using a t-test. Result: The analytic set comprised 47% females and 44% Hispanic, 24% White, 20% Black, 9% Asian, and 3% Pacific Islander/Native Hawaiians. 62% were 15-39 years and 32% 40+ years at diagnosis. The multivariate analysis, excluding age, indicated an association between the risk index and 5-year OS (HR =1.41 (1.08, 1.93), p=0.01). However, including age attenuated the association (HR=1.18 (0.89, 1.57), p=0.25). When stratified by age categories, the continuous risk index was associated with 5-year OS only in the older age group: 15-39 years (HR = 0.89 (0.54, 1.47), p=0.65), and >40 years (HR = 1.47 (1.08, 2.01), p=0.02), indicating effect heterogeneity by age groups (p=0.09). We did not observe significant effect heterogeneity by EBV status (p=0.6), race/ethnicity (p=0.72), nor sex (p=0.33). When the dichotomized score was examined, the model was attenuated after adjusting for age, but stratification analysis showed an association in the older adults (HR= 2.8 (1.42, 5.51), p<0.01) with significant effect heterogeneity across age (p=0.02) (Figure 1). Evaluating the association between individual genes and OS, we found effect heterogeneity by age; two genes from the 23-gene panel indicated protective effects in the AYA but hazardous in older adults: B2M (p effect heterogeneity' =0.048) and APOL6 (p'=0.059). Individual genes with heterogeneity of association across race/ethnicity included RNF144B (p' =0.0021) with increased hazards in Whites compared to Blacks, whereas WDR83 (p'=0.018), PRF1 (p'=0.0018), LYZ (p'=0.0028) had higher hazards in Blacks compared to Whites, and RAPGEF2 (p'=0.036) had higher hazards in Hispanics compared to Whites. Conclusion: The risk index and dichotomized score derived from the previously developed 23-gene panel was significantly associated with OS among >40 year age group, with evidence of age effect heterogeneity. We detected 2 genes with an association that varied by age, and five across race/ethnicity, suggesting the importance of including diverse racial/ethnic and age groups in CHL patient populations when developing predictive models.

Enhanced Genomic Stability in Monomorphic Post-Transplant Lymphoproliferative Disorders Is Driven By Distinct Mechanisms Stratified By EBV Status

Introduction: We previously showed that HIV associated EBV negative (-) diffuse large B-cell lymphomas (DLBCL) of germinal center origins (GCB) have enhanced genomic stability and increased expression of DNA repair genes compared to their HIV negative counterparts (Maguire et al 2019). Given that immune deficiency can be induced virally or therapeutically, including the administration of immunosuppressive drugs post solid organ or hematopoietic stem cell transplant, we questioned whether these observations are specific to HIV or if they represent a more global immune impaired signature. Here we assessed the genomic stability of monomorphic DLBCL post-transplant lymphoproliferative disorders (PTLD) and the potential role of DNA repair. Methods: A total of 49 monomorphic DLBCL PTLDs (26 EBV(-), 23 EBV positive (+)) and 170 immune competent (IC) DLBCL (EBV status unknown) were studied. All were pathologically reviewed and samples with <60% tumor content were macro-dissected before RNA/DNA extraction. Cell of origin (COO) profiles were assessed using the DLBCL90 molecular assay which can distinguish primary mediastinal B-cell lymphoma (PMBL) from GCB, Activated B-cell (ABC) and unclassified (UNC) DLBCL (Ennishi et al 2019). NanoString gene expression profiling (GEP) was performed using the PanCancer Pathways panel and analyzed using NanoString nSolver Advanced Analysis. Copy number (CN) analysis was performed on a subset of samples (45 PTLD, 41 DLBCL) using the OncoScan CN assay and analyzed using Chromosome Analysis Suite (ChAS) v4.1. All p-values listed were Benjamini-Hochberg adjusted and Mann-Whitney tested for the GEP and CN data respectively. Results: In contrast to IC-DLBCL which are GCB enriched (50%), COO profiling revealed PTLD to be ABC enriched (43%). CN analyses revealed that the median CN/sample for PTLD (0.57, p<0.001), EBV(+)PTLD (0.25, p<0.001) and EBV(-)PTLD (0.80, p = 0.050) tumors were significantly less aberrant than IC-DLBCL (1.01). The data also showed that EBV(+)PTLD are less aberrant than EBV(-)PTLD tumors (0.25 vs 0.80, p<0.001). GEP pathway analysis identified DNA repair as downregulated in PTLD and EBV(+)PTLD but upregulated in EBV(-)PTLD compared IC-DLBCL. Nine DNA repair genes ( RAD51, CCNA2a, CHEK1b, POLR2Da, PCNAa, WHSC1a, GTF2H3a, ATM c, EP300 c) were differentially expressed (DE) in EBV(-)PTLD compared to IC-DLBCL (7 increased, 2 decreased, p ≤ 0.0425): where the most overexpressed gene RAD51, a critical mediator of Homologous Recombination (HR) and the Fanconi Anemia (FA) DNA repair pathways, was not DE in PTLD or EBV(+)PTLD compared to IC-DLBCL. Five a genes were increased in PTLD (p ≤ 0.0204) but not EBV(+)PTLD, 1 b was decreased in EBV(+)PTLD (p = 0.01) but not PTLD and 2 c were reduced in PTLD regardless of EBV status compared to IC-DLBCL (p ≤ 0.0024). Twenty-8 genes were DE in EBV(+)PTLD compared to IC-DLBCL. Of these, 23 were downregulated (p ≤ 0.0418) and included HR, FA and mismatch repair genes ( CHEK1, CHEK2, BRAC1/FANCS, FANCA, FANCB, FANCE, MSH2, MSH6). All but 2/23 were similarly expressed in PTLD compared to IC-DLBCL of which 11 were significant (p ≤ 0.0356). The five upregulated genes ( ZBTB32, DDB2, NBN, POLB, RBX1, p ≤ 0.0092) were also upregulated in PTLD (p ≤ 0.0042) but not EBV(-)PTLD compared to IC-DLBCL. Two additional genes ( SKP1, FBXW7) were also identified as increased in EBV(+)PTLD and PTLD (p ≤ 0.0204) compared to IC-DLBCL. In contrast, SKP1 was increased (p = 2.7E-4) and FBXW7 was decreased (p = 0.3610) in EBV(-)PTLD compared to IC-DLBCL. Together the protein products of RBX1, SKP1 and FBXW7 comprise the functional cores of a complete UPS complex, the SCF FBXW7 E3 ubiquitin ligase which have been reported to regulate the DNA damage response and promote genomic stability. Conclusions: Like HIV associated DLBCL, these results show that PTLD is more genomically stable than IC-DLBCL, and that genomic stability is further enhanced in EBV(+)PTLD which have increased expression of UPS related genes. In contrast, EBV(-)PTLD have increased expression of DNA damage repair genes, including RAD51. In summary, monomorphic DLBCL PTLD tumors have enhanced genomic stability that appears to be mediated by two distinct mechanisms stratified by EBV status. Further work is required to assess if this stratification of mechanisms is unique to PTLD or reflects a more global immune impaired EBV related signature.

A surrogate molecular approach for the detection of Philadelphia chromosome-like B-acute lymphoblastic leukemia.

Philadelphia chromosome (Ph)-like B-acute lymphoblastic leukemia (B-ALL) is a clinically significant, high-risk genetic subtype of B-ALL cases. There are few data on the incidence, characterization, and treatment outcomes of Ph-like ALL cases from low- and middle-income countries. There is a pressing need to establish a well-organized/cost-effective approach for identifying Ph-like ALL instances. Multiplex reverse transcriptase polymerase chain reaction, nCounter NanoString, and fluorescence in situ hybridization were used to detect and characterize Ph-like ALL cases among recurrent genetic abnormalities (RGA)neg B-ALL cases. At the end of induction therapy, flow cytometry-minimal residual disease (MRD) assay was used to quantify MRD positivity in Ph-like ALL cases. Of 130 newly diagnosed B-ALL cases, 25% (BCR::ABL1), 4% (ETV6::RUNX1), 5% (TCF3::PBX1), 2% (KM2TA::AFF1), and 65% RGAneg B-ALL cases were revealed by multiplex reverse transcriptase polymerase chain reaction. Among RGAneg B-ALL cases, 24% Ph-like ALL cases using nCounter NanoString were identified, with 48% CRLF2high cases with 45% CRLF2::P2RY8 and 18% CRLF2::IGH rearrangements(∼r) revealed by fluorescence in situ hybridization. In 52% of CRLF2low cases, 17% ABL1 and JAK2∼r 8% EPOR::IGH & PDGRFB∼r were identified. Ph-like ALL cases had higher total leukocyte count (p<.05), male preponderance (p<.05), and high MRD-positivity/induction failure compared with RGAneg B-ALL cases. Furthermore, in Ph-like ALL cases, 11 significant genes using quantitative polymerase chain reaction were identified and validated. CRLF2, IGJ, CEACAM6, MUC4, SPATS2L and NRXN3 genes were overexpressed and show statistical significance (p<.05) in Ph-like ALL cases. This study showed the high incidence of Ph-like ALL cases with kinase activating alterations and treatment outcomes from low- and middle-income region. Furthermore, a surrogate cost-effective multiplex panel of 11 overexpressed genes for the prompt detection of Ph-like ALL cases is proposed. Identification of recurrent gene abnormalities (RGA)neg B-acute lymphoblastic leukemia (B-ALL) cases using multiplex-reverse transcriptase polymerase chain reaction. Identification and characterization of Philadelphia (Ph)-like ALL cases using nCounter NanoString gene expression profiling and fluorescence in situ hybridization. Furthermore, Ph-like ALL cases were characterized according to CRLF2 expression and kinase-activating genomic alterations. Minimal residual disease of Ph-like ALL cases were quantified using flow cytometry-minimal residual disease assay. A surrogate molecular approach was established to detect Ph-like ALL cases from low- and middle-income countries.

Abstract B028: Vigorous physical activity associated changes in immune cell infiltrate are linked with reduced prostate tumour aggressiveness

Abstract Vigorous physical activity has been associated with lower risk of fatal prostate cancer and improved cancer-specific outcomes. However, the mechanisms contributing to this relationship are not understood. The current study characterized inflammation in prostate cancer tissue and evaluated features of the immune microenvironment related to physical activity and prostate cancer aggressiveness. We studied 119 men with prostate cancer participating in the University of North Carolina Health Registry/Cancer Survivorship Cohort who underwent radical prostatectomy. Structured questionnaires were administered at patients' study entry to assess physical activity. Digital image analysis of H&amp;E-stained tissue sections using QuPath was applied to quantify Tumour Infiltrating Lymphocytes (TILs) in glands and stroma of tumor and adjacent normal regions. Nanostring gene expression profiling was performed on tumour tissue and a 50-gene signature utilized to predict immune cell types. ERG gene expression was used as a surrogate of TMPRSS2:ERG fusion status. Logistic regression was used to test associations of inflammation features with tumour aggressiveness and recreational physical activity measures. Cox proportional hazards analysis was applied to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association between immune cell types and risk of prostate cancer progression, with validation in a separate study of 101 radiation-treated prostate cancer patients. Our study comprised 77% white and 23% black men, and 25% of men had Gleason grade ≥4+3, 29% men reported recreational vigorous activity prior to diagnosis of prostate cancer. Acute tumour inflammation affected 30% of men, while 53% showed acute inflammation of adjacent tissue. TILs were more abundant in glands than stroma; two-fold more in tumour and five-fold more in adjacent tissue. Frequency of inflammation was similar between ERG-positive and ERG-negative tumours. An inverse relationship was observed between recreational vigorous activity and TILs abundance. Expression of the immune gene signature was also significantly lower in men reporting any recreational vigorous activity compared to those reporting none (P=0.044), driven by reduced gene expression-predicted abundance of Th cells, exhausted CD8 T cells, macrophages and neutrophils. Moreover, T cells, Tfh, Treg, exhausted CD8 T cells and macrophages were significantly increased in men with Gleason grade ≥4+3 or clinical stage ≥III. Treg abundance was positively associated with prostate cancer progression (HR, 1.35; 95%CI 1.03-1.78) after adjusting for age and race, which was further strengthened in ERG-positive patients (HR, 1.60; 95%CI 1.03-2.47). We show that the immune landscape in prostate cancer is altered in men reporting vigorous physical activity. Immune cell types associated with an immunosuppressive microenvironment, including exhausted CD8 T cells, Tregs, and macrophages, may be potential candidate mechanisms linking recreational vigorous activity with reduced prostate tumour aggressiveness and improved outcomes. Citation Format: Lanshan Huang, Sophia Halliday, Linnea Olsson, Alina Hamilton, Erin Kirk, Laura Farnan, Adrian Gerstel, Stephanie Craig, Stephen Finn, Melissa Wilson, Melissa Troester, Eboneé Butler, Jeannette Bensen, Sara Wobker, Emma Allott. Vigorous physical activity associated changes in immune cell infiltrate are linked with reduced prostate tumour aggressiveness [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B028.

Immune Microenvironment in Sporadic Early-Onset versus Average-Onset Colorectal Cancer

The incidence of left-sided colon and rectal cancer in young people are increasing worldwide, but its causes are poorly understood. It is not clear if the tumor microenvironment is dependent on age of onset, and little is known about the composition of tumor-infiltrating T cells in early-onset colorectal cancer (EOCRC). To address this, we investigated T-cell subsets and performed gene expression immune profiling in sporadic EOCRC tumors and matched average-onset colorectal cancer (AOCRC) tumors. Left-sided colon and rectal tumors from 40 cases were analyzed; 20 EOCRC (<45 years) patients were matched 1:1 to AOCRC (70-75 years) patients by gender, tumor location, and stage. Cases with germline pathogenic variants, inflammatory bowel disease or neoadjuvant-treated tumors were excluded. For T cells in tumors and stroma, a multiplex immunofluorescence assay combined with digital image analysis and machine learning algorithms was used. Immunological mediators in the tumor microenvironment were assessed by NanoString gene expression profiling of mRNA. Immunofluorescence revealed no significant difference between EOCRC and AOCRC with regard to infiltration of total T cells, conventional CD4+ and CD8+ T cells, regulatory T cells, or γδ T cells. Most T cells were located in the stroma in both EOCRC and AOCRC. Immune profiling by gene expression revealed higher expression in AOCRC of the immunoregulatory cytokine IL-10, the inhibitory NK cell receptors KIR3DL3 and KLRB1 (CD161), and IFN-a7 (IFNA7). In contrast, the interferon-induced gene IFIT2 was more highly expressed in EOCRC. However, in a global analysis of 770 tumor immunity genes, no significant differences could be detected. T-cell infiltration and expression of inflammatory mediators are similar in EOCRC and AOCRC. This may indicate that the immune response to cancer in left colon and rectum is not related to age of onset and that EOCRC is likely not driven by immune response deficiency.

Genomic and proteomic characterization of Philadelphia-like B-lineage acute lymphoblastic leukemia: A report of Indian patients.

The gold standard for the identification of Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) patients is gene expression profiling. Because of its diverse nature, its identification is extremely difficult and expensive. On the genomic and proteomic landscape of Ph-like ALL patients, there is a paucity of published literature from developing countries. The authors used digital barcoded nCounter NanoString gene expression profiling for its detection, followed by molecular and proteomic characterization using fluorescence in situ hybridization and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The authors found 32.05% Ph-like ALL patients and their median age at presentation was considerably higher than Ph-negative ALL cases (p=.0306). Furthermore, we identified 20% CRLF2 overexpressed cases having 8.33% CRLF2-IGH translocation with concomitant R683S mutation and 8.33% CRLF2-P2RY8 translocation. In 80% of CRLF2 downregulated cases, we identified 10% as having JAK2 rearrangement. Minimal residual disease-positivity was more common in Ph-like ALL cases (55.55% vs. 25% in Ph-negative ALL cases). Immunoglobulin J chain (Jchain), small nuclear ribonucleoprotein SmD1 (SNRPD1), immunoglobulin κ constant (IGKC), NADH dehydrogenase (ubiquinone) 1 α subcomplex subunit 2 (NDUFA2), histone H2AX (H2AFX), charged multivesicular body protein 4b (CHMP4B), and carbonyl reductase (NADPH) (CBR1) proteins were identified to be substantially expressed in Ph-like ALL patients, using LC-MS/MS. Gene enrichment analysis indicated that involvement of spliceosomal mediated messenger RNA splicing pathway and four microRNAs was statistically significant in Ph-like ALL patients. For the first time, we have described incidence, molecular, and proteomic characterization of Ph-like ALL, in developing nations. In developing countries, detecting Philadelphia (Ph)-like B-lineage acute lymphoblastic leukemia is complicated and challenging due to its diverse genetic landscape. There is no well-defined and cost-effective methodology for its detection. The incidence of this high-risk subtype is very high in adult cases, and there is an urgent need for its accurate detection. We have developed an online PHi-RACE classifier for its rapid detection, followed by delineating the genomic and proteomic landscape of Ph-like acute lymphoblastic leukemias for the first time in Indian patients.

BIOM-54. ANALYTICAL VALIDATION OF A TARGETED GENE EXPRESSION BIOMARKER PREDICTING MENINGIOMA OUTCOMES AND RADIOTHERAPY RESPONSES

Abstract BACKGROUND Improvements in meningioma risk stratification are needed to guide postoperative management. We previously developed and externally validated a targeted gene expression biomarker predicting meningioma outcomes and radiotherapy response using fresh frozen meningiomas. Here, we present the analytical validity, test-retest and cross-platform reproducibility, intra-tumor heterogeneity, and formalin fixed paraffin embedded (FFPE) concordance for this biomarker. METHODS Matched FFPE and fresh frozen samples from 50 meningiomas underwent RNA extraction for concordance testing using a custom Nanostring gene expression panel comprised of 34 meningioma genes and 7 housekeeping genes. Matched Nanostring gene expression profiling and RNA-sequencing was available from 173 fresh frozen meningiomas. Nanostring batch, machine, and technician variability was tested on 10 FFPE meningiomas using 2 independent batches of biomarker reagents that were processed at 2 independent laboratories. To study intra-tumor heterogeneity, 68 spatially distinct meningioma biopsies obtained under stereotactic guidance from 13 tumors were analyzed using the Nanostring gene expression biomarker. RESULTS Matched FFPE and fresh frozen meningioma samples (N=50) demonstrated high gene expression concordance (Pearson R=0.89, F-test P&amp;lt; 2.2e-16) and risk score concordance (R=0.78, F-test P&amp;lt; 1.8e-11, residual standard error 0.12). The biomarker achieved high performance on FFPE samples (local freedom from recurrence [LFFR] c-index 0.75±SEM 0.05, 5y LFFR AUC 0.84, 95% interval 0.67-0.96). Test-retest concordance on FFPE samples (N=10) across independent reagent batches, machines, and technicians was high (gene expression concordance: R=0.97; risk score concordance R=0.94). Transcript counts were concordant across Nanostring and RNA sequencing approaches (N=173 frozen samples, R=0.90). Stereotactic intra-operative sampling (N=68 samples from 13 patients) revealed low levels of intra-tumor heterogeneity (median inter-quartile risk score within tumor: 0.10±0.01). CONCLUSIONS We demonstrate analytical reproducibility and robustness of a gene expression biomarker predicting meningioma outcomes and radiotherapy responses in FFPE and frozen samples across multiple conditions and platforms.

Gas plasma-oxidized sodium chloride acts via hydrogen peroxide in a model of peritoneal carcinomatosis.

Gas plasma technology generates reactive oxygen and nitrogen species (ROS/RNS), inducing lethal oxidative damage in tumor cells. The transfer of gas plasma-derived ROS/RNS into liquids has been proposed as an innovative anti-cancer strategy targeting peritoneal carcinomatosis (PC). However, the mechanism of action is under debate. To this end, we compared gas plasma-oxidized medical-grade sodium chloride (oxNaCl) with a concentration-matched control (cmc) of NaCl enriched with equivalent concentrations of H2O2 and NO3- in several cell lines and models of PC. Strikingly, oxNaCl and cmc performed equally well in oxidation and cytotoxic activity in tumor cells in two-dimensional cultures, three-dimensional (3D) tumor spheroids, vascularized 3D tumors grown on chicken-embryo chorioallantoic membranes, and a syngeneic PC mouse model invivo. Given the importance of immunotherapies in oncology today, we focused on immunological consequences of the treatment. Again, to a similar extent, oxNaCl and cmc increased tumor cell immunogenicity and enhanced uptake by and maturation of peripheral blood monocyte-derived dendritic cells together with an inflammatory secretion profile. Furthermore, NanoString gene expression profiling revealed immune system processes and unfolded protein response-related pathways as being linked to the observed anti-tumor effects for both oxNaCl and cmc. In conclusion, gas plasma-generated oxNaCl and cmc showed equal therapeutic efficacy in our PC-related models. In light of the many promising anti-cancer studies of gas plasma-oxidized liquids and the convenient production of corresponding cmcs in large quantities as needed in clinics, our findings may spur research lines based on low-dose oxidants in peritoneal cancer therapy.

Abstract P2-13-44: HDAC6 is an unfavorable prognostic factor in HER2-positive breast cancer patients treated with adjuvant trastuzumab

Abstract Background Trastuzumab is the cornerstone of adjuvant therapy for HER2-positive breast cancer (BC), but due to de novo or primary resistance, &amp;gt;20% of early-stage patients become resistant. Thus, predicting the mechanisms of trastuzumab resistance would facilitate the planning of specific therapeutic strategies. We performed a case-control study of 26 HER2-positive BC patients who relapsed within 5 years of starting adjuvant treatment and 26 controls, no relapsed, analyzing an extensive gene expression profile. Methods Total RNA was isolated from formalin-fixed paraffin-embedded primary tumors with AllPrep DNA/RNA FFPE Kit (Qiagen) and quantified by Nanodrop, before performing Nanostring gene expression profiling. nCounter Breast Cancer 360 Panel (Nanostring Technologies) was used according to the manufacturer’s instructions to analyze the expression of 770 genes and important signatures for BC (e.g. PAM50). For the statistical analyses, genes were normalized using a ratio of the expression value to the geometric mean of all housekeeping genes on the panel. Housekeeper-normalized data were then log(2) transformed. These data were evaluated by unsupervised selection of genes using consensus clustering with the Partitioning Around Medoids algorithm followed by the association of cluster “representative” genes (i.e. the medoids) with respect to the presence of relapse. The optimal number of clusters was determined by inspecting the consensus matrix cumulative distribution. Given the small sample size, the association between the "representative" genes, demographic and clinical covariates and the risk of relapse was evaluated using a logistic regression model with ELASTIC-NET regularization with α and λ tuned by 5-fold cross-validation. Penalized regression coefficients β are reported. The analyses were performed using R version 4.0.4 and package “ConsensusClusterPlus” and “glmnet”. Results Patient clinical characteristics are reported in Table 1. Relapsed patients showed a higher tumor stage, larger tumor size and greater lymph node involvement than controls (Table 1). Six samples failed the quality control check and so the analyses on gene expression data were performed on 46 subjects who had a distribution of clinical covariates similar to that reported in Table 1. The PAM50 intrinsic subtype, Risk Of Recurrence (ROR) score and ROR category were not associated with relapse status (P = 0.5, 0.5 and 0.1, respectively). The consensus clustering analysis revealed that the optimal number of clusters was 5, and the corresponding medoids were: HDAC6 for cluster 1 (n1=192 genes), WNT4 for cluster 2 (n2=102 genes), BMPR2 for cluster 3 (n3=162 genes), PALB2 for cluster 4 (n4=191 genes), and PARP1 for cluster 5 (n5=84 genes). HDAC6 and BMPR2 showed the highest correlation (r = 0.53, P&amp;lt;0.001) among the 5 genes. HDAC6 showed prognostic potential in the penalized logistic regression analysis, independently of tumor stage and Body Mass Index (β: 0.57, 1.49, and 0.11, respectively). Higher HDAC6 expression was associated with a higher risk of relapse, all other conditions being equal. Conclusions We are now planning to validate these results in a larger case series and in in vitro models to verify whether HDAC6, in addition to being an unfavorable prognostic factor, could also be a new therapeutic target for trastuzumab resistance. Table 1.Patient characteristics in relation to Trastuzumab outcome.VariableAllCasesControlsP(n=52)(n=26)(n=26)n(%)n(%)n(%)Age at start adiuvant therapyMean ± sd53.4 ± 11.953.1 ± 12.653.7 ± 11.40.854Menopausal StatusPre-menopause21(40.4)10(38.5)11(42.3)0.777Post-menopause31(59.6)16(61.5)15(57.7)Stage of DiseaseI9(18.8)2(8.0)7(30.4)0.001II20(41.7)7(28.0)13(56.5)III19(39.6)16(64.0)3(13.0)Unknown413Lymph Node StatusNegative18(36.0)4(16.7)14(53.9)0.008Positive32(64.0)20(83.3)12(46.2)Unknown220Tumor SizeT128(53.9)7(26.9)21(80.8)&amp;lt;0.001T213(25.0)8(30.8)5(19.2)T36(11.5)6(23.1)0(0.0)T45(9.6)5(19.2)0(0.0)Histologic Grade12(4.1)1(4.2)1(4.0)0.889218(36.7)8(33.3)10(40.0)329(59.2)15(62.5)14(56.0)Unknown321Ki-67&amp;lt;20%17(32.7)8(30.8)9(34.6)0.768≥20%35(67.3)18(69.2)17(65.4)ERNegative14(26.9)7(26.9)7(26.9)1.000Positive38(73.1)19(73.1)19(73.1)PRNegative22(43.1)11(44.0)11(42.3)0.903Positive29(56.9)14(56.0)15(57.7)Unknown110Vascular invasionNo13(56.5)3(37.5)10(66.7)0.221Yes10(43.5)5(62.5)5(33.3)Unknown291811Adjuvant ChemotherapyAnthracyclines and taxanes24(46.2)7(26.9)17(65.4)&amp;lt;0.001Taxanes only6(11.5)4(15.4)2(7.7)Anthracyclines only10(19.2)3(11.5)7(26.9)Other12(23.1)12(46.2)0(0.0)BMI at start of Adjuvant ChemotherapyMean ± sd24.8 ± 4.025.9 ± 3.223.6 ± 4.40.039sd: standard deviation BMI: Body Mass Index Citation Format: Sara Ravaioli, Roberta Maltoni, Elisabetta Petracci, Michela Palleschi, William Balzi, Francesca Pirini, Maria Maddalena Tumedei, Michele Zanoni, Michela Cortesi, Giovanni Martinelli, Andrea Rocca, Sara Bravaccini. HDAC6 is an unfavorable prognostic factor in HER2-positive breast cancer patients treated with adjuvant trastuzumab [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 P2-13-44.

Risk stratification of gastrointestinal stromal tumors by Nanostring gene expression profiling.

The risk assessment classification schemes for gastrointestinal stromal tumors (GIST) include tumor site, size, mitotic count and variably tumor rupture. Heterogeneity in high-risk GIST poses limitations for current classification schemes. This study aims to demonstrate the clinical utility of risk stratification by gene expression profiling (GEP) using Nanostring technology. Fifty-six GIST cases were analyzed using a 231 gene expression panel. GEP results were correlated with clinical and pathological data. The prognostic performance was assessed in 34 patients with available survival data using ROC curves, Kaplan-Meier survival curves and compared with traditional risk assessment schemes. Volcano plot analysis identified seven genes with significantly higher expression (FDR < .0.05) in high-risk than in non-high-risk tumors, namely TYMS, CDC2, TOP2A, CCNA2, E2F1, PCNA, and BIRC5. Together, these transcripts exhibited significantly higher expression in high-risk tumors than in intermediate (P < 0.01), low (P < 0.001), and very low (P = 0.01) risk tumors. Receiver-operating characteristic curve analysis demonstrated area under the curve (AUC) to be 0.858 for the separation of high-risk and non-high-risk tumors. Kaplan-Meier survival analysis demonstrated improved risk stratification (log-rank test P < 0.001) compared to the current risk assessment classification (P = 0.231). In addition to current clinical and histology-based risk classification for patients with GIST, gene expression may offer complementary prognostic information.

Safety and feasibility of an in situ vaccination and immunomodulatory targeted radionuclide combination immuno-radiotherapy approach in a comparative (companion dog) setting.

Murine syngeneic tumor models have revealed efficacious systemic antitumor responses following primary tumor in situ vaccination combined with targeted radionuclide therapy to secondary or metastatic tumors. Here we present studies on the safety and feasibility of this approach in a relevant translational companion dog model (n = 17 dogs) with advanced cancer. The three component of the combination immuno-radiotherapy approach were employed either separately or in combination in companion dogs with advanced stage cancer. In situ vaccination was achieved through the administration of hypofractionated external beam radiotherapy and intratumoral hu14.18-IL2 fusion immunocytokine injections to the index tumor. In situ vaccination was subsequently combined with targeted radionuclide therapy using a theranostic pairing of IV 86Y-NM600 (for PET imaging and subject-specific dosimetry) and IV 90Y-NM600 (therapeutic radionuclide) prescribed to deliver an immunomodulatory 2 Gy dose to all metastatic sites in companion dogs with metastatic melanoma or osteosarcoma. In a subset of dogs, immunologic parameters preliminarily assessed. The components of the immuno-radiotherapy combination were well tolerated either alone or in combination, resulting in only transient low grade (1 or 2) adverse events with no dose-limiting events observed. In subject-specific dosimetry analyses, we observed 86Y-NM600 tumor:bone marrow absorbed-dose differential uptakes ≥2 in 4 of 5 dogs receiving the combination, which allowed subsequent safe delivery of at least 2 Gy 90Y-NM600 TRT to tumors. NanoString gene expression profiling and immunohistochemistry from pre- and post-treatment biopsy specimens provide evidence of tumor microenvironment immunomodulation by 90Y-NM600 TRT. The combination of external beam radiotherapy, intratumoral immunocytokine, and targeted radionuclide immuno-radiotherapy known to have activity against syngeneic melanoma in murine models is feasible and well tolerated in companion dogs with advanced stage, spontaneously arising melanoma or osteosarcoma and has immunomodulatory potential. Further studies evaluating the dose-dependent immunomodulatory effects of this immuno-radiotherapy combination are currently ongoing.

Abstract 2701: Molecular profiling to assess the immune response to neoadjuvant SABR in early breast cancer

Abstract Radiation therapy is used successfully in the treatment of breast cancer. Stereotactic radiation therapy (SBRT) is well established in the treatment of lung cancer, brain metastases and various other metastatic sites. Localized radiotherapy can promote dendritic cells maturations and activation and enhances phagocytosis of cells by antigen-presenting cells (APCs) increased presentation of tumor-associated antigenic peptides, and T cells being primed from naïve towards memory phenotypes In this study we evaluated the ability to implement a three fraction SBRT regimen for low-risk primary carcinoma of the breast prior to lumpectomy and profiled the immune microenvironment using NanoString's GeoMx Digital Spatial Profiling (DSP) platform and we performed NanoString gene expression profiling using the Human v.1.1 PanCancer immune profiling panel. NanoString's DSP platform was used to analyze 25 patient samples from the SIGNAL 2.0 clinical trial. For analysis, region of interest were selected comparing the tumor microenvironment (TME, CD45+ve) and tumor (pan cytokeratin) in pre and post treated FFPE slides. A panel of 60-antibodies were analyzed in each region. For gene expression profiling total RNA was extracted from the frozen tissue and the human V.1.1 PanCancer Immune Profiling Panel was used. We observed notable differences in the immune microenvironment gene expression patterns in samples pre- and post-treatment. We identified a total of 175 differentially expressed genes (DEGs) using a 5% adjusted p-value across the entire patient population regardless of which radiation arm. Pathway analysis identified genes associated with activation of immune response, signaling by interleukins and adaptive immune response were significantly changed in the post treatment samples. Deconvolution of the immune cell mRNA gene expression data demonstrated that there were significant changes in the cellular composition after radiotherapy. There were significant increase in expression levels of macrophages, dendritic cells, neutrophils and CD8 T cells post radiation treatment. In conclusion we have demonstrated through proteomic and transcriptomic profiling, SBRT elicits an immune response with increases in the innate response. Citation Format: Melanie Spears, Vida Talebian, Linda Liao, Megan Hopkins, Kalan Lynn, Michael Lock, Anat Kornecki, John M. Bartlett, Muriel Brackstone. Molecular profiling to assess the immune response to neoadjuvant SABR in early breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2701.

Response to the EZH2 Inhibitor Tazemetostat Is Independent of Cell of Origin Determined Via Hans Imunohistochemistry or Nanostring Lymphoma Subtyping Test in EZH2 Wild-Type DLBCL Patients

Response to the EZH2 Inhibitor Tazemetostat Is Independent of Cell of Origin Determined Via Hans Imunohistochemistry or Nanostring Lymphoma Subtyping Test in EZH2 Wild-Type DLBCL Patients

Ibrutinib Inhibits cGVHD Pathogenic Pre-Germinal Center B-Cells and Follicular Helper Cells While Preserving Immune Memory and Th1 T-Cells

Background: Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Treatment relies upon corticosteroids and immunosuppressive agents with limited efficacy and considerable side-effects. Evolving research points to the complex involvement of immune T, B, and myeloid cells in the pathogenesis of cGVHD. Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) which inhibits the activation of B-cells, specific T-cell subsets, and certain myeloid cells that are implicated in cGVHD. Substantial clinical benefit was demonstrated in an open-label phase 1b/2 study of ibrutinib in patients with cGVHD that had failed frontline therapy (Miklos et al ASH 2016). Based upon this data, ibrutinib is indicated by the US FDA for the treatment of adult patients with cGVHD after failure of one or more lines of systemic therapy.Methods: Blood samples were collected before and multiple timepoints after starting ibrutinib 420mg daily therapy from 42 subjects with cGVHD that had failed frontline therapy (Miklos et al ASH 2016). Correlative analyses included Nanostring gene expression profiling, Luminex and ELISA based plasma testing, CyTOF phospho-immunophenotyping, and ex-vivo T-cell stimulation assays.Results: Initial data from 8 patients using targeted, Nanostring gene expression analysis showed ibrutinib reduced inflammatory genes including NFkB-1 and chemokines CXCL10, CCL7 and CCL3 (2.6, 2.3, 25, and 1.8-fold decrease over first 3 months, respectively). Luminex plasma quantification supported gene expression profiling results, and unveiled several additional chemotactic, inflammatory, and fibrotic factors which were down modulated by ibrutinib therapy including CCL22, CXCL9, CXCL10, IL-8, sCD25, CCL4, and ST2 (69-80% of patients showed decreasing trend). 42-parameter CyTOF single cell analysis revealed a 10-fold reduction in absolute numbers of cGVHD implicated pre-germinal center B-cells (CD19+, CD27+, CD38+, IgD+), and notably diminished Tfh, Th17, and total B cells (2.6, 1.5, and 1.4-fold decrease over first 90 days, respectively) following ibrutinib therapy. Relative numbers of CD4 and CD8 T-cells, class-switched B-cells, and CD14+ monocytes remained unchanged, and iNKT, Th1, and Treg cells increased incrementally (1.8, 1.2, and 1.1-fold at day 90, respectively). IgG plasma levels persisted while IgM significantly decreased corroborating an ibrutinib germinal center effect that did not deplete long-lived plasma cells. Furthermore, T cell reactivity against influenza increased, and antibodies against EBV and tetanus remained unchanged. Strikingly, single cell phosphorylation analysis showed BTK and ITK signaling was attenuated following ibrutinib treatment in defined B and T cell subsets (Figure 1). Specifically, PLCγ1/2 activation was simultaneously diminished in the pre-germinal center and plasmablast B-cell subsets.Conclusions: Our examination of the biologic and cellular mechanisms which support the clinical results of ibrutinib in the setting of cGVHD demonstrate that ibrutinib selectively inhibits cGVHD pathogenic pre-germinal center B-cells and Tfh cells, while preserving immune memory and Th1 T-cells. These data are especially salient given the clinical efficacy of ibrutinib in cGVHD, and the meticulous nature of the correlative studies designed to elucidate the multifactorial mechanism of action in this life-threatening disease. [Display omitted] DisclosuresSahaf:Pharmacyclics: Research Funding; Stanford: Patents & Royalties. Tebaykin:Stanford University: Patents & Royalties. Hopper:Pharmacyclics: Employment. Cheung:Pharmacyclics: Employment; AbbVie: Equity Ownership. Bittencourt:Pharmacyclics: Employment, Research Funding; AbbVie: Equity Ownership, Research Funding. Cutler:Astellas: Consultancy; Incyte: Consultancy; Bristol-Myers Squibb: Consultancy; Kite: Consultancy; Pharmacyclics: Consultancy; Pfizer: Consultancy. Arora:Takeda Oncology: Consultancy. Waller:Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Cerus: Equity Ownership; Helocyte: Consultancy; National Institutes of Health: Research Funding; Katz Foundation: Research Funding; AMGEN: Consultancy; PRA: Consultancy; Coulter Foundation: Research Funding; Celldex: Consultancy; Chimerix: Equity Ownership; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding. Jagasia:Janssen: Consultancy, Research Funding; Mallinckrodt: Consultancy; Therakos: Consultancy, Research Funding. Flowers:Pharmacyclics: Consultancy. Logan:Novartis: Consultancy, Research Funding; Incyte: Consultancy; Kite: Research Funding; Astellas: Research Funding; Jazz: Consultancy; Amgen: Consultancy; Pharmacyclics: Research Funding; Shire: Consultancy. Jaglowski:Pharmacyclics: Consultancy, Research Funding. Lih:AbbVie: Equity Ownership; Pharmacyclics: Employment. Solman:Pharmacyclics LLC, an AbbVie Company: Employment, Other: Travel, Accommodations, Expenses; AbbVie: Equity Ownership. Lal:Gilead Sciences: Employment; AbbVie: Equity Ownership; The Permanente Medical Group (TPMG): Employment; Pharmacyclics LLC, an AbbVie Company: Employment. Styles:Pharmacyclics LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership. Hill:Amgen: Employment, Equity Ownership, Patents & Royalties; Pharmacyclics: Employment; AbbVie: Equity Ownership; Miravant Medical: Patents & Royalties. James:AbbVie: Equity Ownership; Pharmacyclics LLC, an AbbVie Company: Employment. Bendall:Fluidigm Sciences: Consultancy, Other: Travel, Accomodations, Expenses; Bluebird Bio: Other: Travel, Accomodations, Expenses, Research Funding; Ionpath, Inc.: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Boehringer-ingelheim: Consultancy, Other: Travel, Accomodations, Expenses; Pharmacyclics: Research Funding. Dubovsky:Pharmacyclics LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership.

Abstract SS1-05: Racial differences in breast cancer immune microenvironments

Abstract Background: Black women suffer 40% higher mortality from breast cancer (BC) compared to non-Hispanic white women, and the underlying causes of these disparities remain uncertain. Growing evidence supports the importance of the immune microenvironment in BC survival, but immune response differences by race are poorly understood. We sought to characterize the immune microenvironment of BC to evaluate how phenotypes of immune response vary across race and tumor intrinsic subtype to impact clinical outcomes. Methods: We leveraged the Carolina Breast Cancer Study (CBCS), a large population-based study that oversampled young (≤50) and black women with invasive breast cancer and collected tumor tissue on &amp;gt;95% of participants. We curated a 48-gene panel representative of 13 individual immune cell types, and performed NanoString gene expression profiling on tissue from 1957 BC patients, including 1033 (53%) Black and 924 (47%) Non-Black women. Consensus clustering was used to identify phenotypes of immune response, and individual immune cell scores were calculated as the median expression of cell-type markers. Immune phenotypes and cell-type scores were compared against validated protein markers and H&amp;E-based quantification of TILs from corresponding tissue microarrays (TMAs). We estimated associations of immune classes with BC intrinsic subtype, ROR-PT scores (calculated as low, medium and high), age and race using relative frequency differences (RFDs), adjusting for age and tumor stage. Cell type scores were compared by race using Welch’s t-tests and adjusted for multiple comparisons with the Benjamani-Hochberg procedure. Results: We identified three BC immune phenotypes primarily defined by features related to an Adaptive-enriched, Innate-enriched, or a Quiet immune microenvironment. These expression-based groups correlated with histological evidence of immune cells from corresponding TMAs. Similarly, expression-based cell scores correlated strongly with protein-based quantification of immune cells from corresponding TMAs (e.g. B-cell score vs. CD19 immunofluorescence, rho=0.75; ICOS RNA counts vs. protein; Rho=0.76). Both Adaptive-enriched and Innate-enriched tumors were associated with high ROR-PT scores [RFD for Adaptive-enriched vs. Quiet: 24.1% (95% CI 19.3-28.8); Innate-enriched vs. Quiet RFD: 13.1 (95% CI 9.1-17); frequencies: 37.2%, 26.2% and 10% for Adaptive, Innate and Quiet, respectively], the basal-like intrinsic subtype [RFD for Adaptive-enriched vs. Quiet: 19.4% (95% CI 14.3-24.6); Innate-enriched vs. Quiet RFD: 9.8 (95% CI 5.6-14.1); frequencies: 43.4%, 27.4% and 8.9% respectively] and Black race [RFD for Adaptive-enriched vs. Quiet: 16.1% (95% CI 10.3-21.9); Innate-enriched vs. Quiet RFD: 9.5 (95% CI 3.9-15); frequencies 60.5%, 53.5% and 42.5% respectively]. After adjusting for tumor subtype, the Adaptive-enriched class remained associated with Black race (RFD for Adaptive-Enriched vs Quiet: 7.5% (95% CI 1.4-13.6). Conversely, tumors in the immune-quiet group were primarily non-basal (90%) with low ROR-PT scores (86.7%). Within the Adaptive-enriched class, Black women displayed decreased CD8 T cell scores (p=0.05) but increased T-reg cell scores (p=0.02) relative to Non-Black women. Conclusion: Immune response appears to be intricately related to race and tumor subtype, with black women having strong associations with adaptive-enriched and innate-enriched immune microenvironments. Differences in CD8 T cell and Treg expression suggest that even within broad classes of immune response, racial differences in specific cell-type distributions exist. Immune response differences may be targetable to improve treatment response, and therefore it is important to identify race- and subtype-specific differences in immune microenvironments. Citation Format: Alina M Hamilton, Linnea T Olsson, Benjamin C Calhoun, Katherine A Hoadley, Melissa A Troester. Racial differences in breast cancer immune microenvironments [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 SS1-05.