Breast Cancer Tumor Microenvironment and Molecular Aberrations Hijack Tumoricidal Immunity

Breast cancer is the most common malignancy among females in western countries, where women have an overall lifetime risk of >10% for developing invasive breast carcinomas. It is not a single disease but is composed of distinct subtypes associated with different clinical outcomes and is highly heterogeneous in both the molecular and clinical aspects. Although tumor initiation is largely driven by acquired genetic alterations, recent data suggest microenvironment-mediated immune evasion may play an important role in neoplastic progression. Beyond surgical resection, radiation, and chemotherapy, additional therapeutic options include hormonal deactivation, targeted-signaling pathway treatment, DNA repair inhibition, and aberrant epigenetic reversion. Yet, the fatality rate of metastatic breast cancer remains unacceptably high, largely due to treatment resistance and metastases to brain, lung, or bone marrow where tumor bed penetration of therapeutic agents is limited. Recent studies indicate the development of immune-oncological therapy could potentially eradicate this devastating malignancy. Evidence suggests tumors express immunogenic neoantigens but the immunity towards these antigens is frequently muted. Established tumors exhibit immunological tolerance. This tolerance reflects a process of immune suppression elicited by the tumor, and it represents a critical obstacle towards successful antitumor immunotherapy. In general, immune evasive mechanisms adapted by breast cancer encompasses down-regulation of antigen presentations or recognition, lack of immune effector cells, obstruction of anti-tumor immune cell maturation, accumulation of immunosuppressive cells, production of inhibitory cytokines, chemokines or ligands/receptors, and up-regulation of immune checkpoint modulators. Together with altered metabolism and hypoxic conditions, they constitute a permissive tumor microenvironment. This article intends to discern representative incidents and to provide potential innovative therapeutic regimens to reinstate tumoricidal immunity.

Kinesin Family Member C1 (KIFC1/HSET): A Potential Actionable Biomarker of Early Stage Breast Tumorigenesis and Progression of High-Risk Lesions

The enigma of why some premalignant or pre-invasive breast lesions transform and progress while others do not remains poorly understood. Currently, no radiologic or molecular biomarkers exist in the clinic that can successfully risk-stratify high-risk lesions for malignant transformation or tumor progression as well as serve as a minimally cytotoxic actionable target for at-risk subpopulations. Breast carcinogenesis involves a series of key molecular deregulatory events that prompt normal cells to bypass tumor-suppressive senescence barriers. Kinesin family member C1 (KIFC1/HSET), which confers survival of cancer cells burdened with extra centrosomes, has been observed in premalignant and pre-invasive lesions, and its expression has been shown to correlate with increasing neoplastic progression. Additionally, KIFC1 has been associated with aggressive breast tumor molecular subtypes, such as basal-like and triple-negative breast cancers. However, the role of KIFC1 in malignant transformation and its potential as a predictive biomarker of neoplastic progression remain elusive. Herein, we review compelling evidence suggesting the involvement of KIFC1 in enabling pre-neoplastic cells to bypass senescence barriers necessary to become immortalized and malignant. We also discuss evidence inferring that KIFC1 levels may be higher in premalignant lesions with a greater inclination to transform and acquire aggressive tumor intrinsic subtypes. Collectively, this evidence provides a strong impetus for further investigation into KIFC1 as a potential risk-stratifying biomarker and minimally cytotoxic actionable target for high-risk patient subpopulations.

Genome-wide host methylation profiling of anal and cervical carcinoma

HPV infection results in changes in host gene methylation which, in turn, are thought to contribute to the neoplastic progression of HPV-associated cancers. The objective of this study was to identify joint and disease-specific genome-wide methylation changes in anal and cervical cancer as well as changes in high-grade pre-neoplastic lesions. Formalin-fixed paraffin-embedded (FFPE) anal tissues (n = 143; 99% HPV+) and fresh frozen cervical tissues (n = 28; 100% HPV+) underwent microdissection, DNA extraction, HPV genotyping, bisulfite modification, DNA restoration (FFPE) and analysis by the Illumina HumanMethylation450 Array. Differentially methylated regions (DMR; t test q<0.01, 3 consecutive significant CpG probes and mean Δβ methylation value>0.3) were compared between normal and cancer specimens in partial least squares (PLS) models and then used to classify anal or cervical intraepithelial neoplasia-3 (AIN3/CIN3). In AC, an 84-gene PLS signature (355 significant probes) differentiated normal anal mucosa (NM; n = 9) from AC (n = 121) while a 36-gene PLS signature (173 significant probes) differentiated normal cervical epithelium (n = 10) from CC (n = 9). The CC progression signature was validated using three independent publicly available datasets (n = 424 cases). The AC and CC progression PLS signatures were interchangeable in segregating normal, AIN3/CIN3 and AC and CC and were found to include 17 common overlapping hypermethylated genes. Moreover, these signatures segregated AIN3/CIN3 lesions similarly into cancer-like and normal-like categories. Distinct methylation changes occur across the genome during the progression of AC and CC with overall similar profiles and add to the evidence suggesting that HPV-driven oncogenesis may result in similar non-random methylomic events. Our findings may lead to identification of potential epigenetic drivers of HPV-associated cancers and also, of potential markers to identify higher risk pre-cancerous lesions.

Gender differences in Barrett’s esophagus and progression of disease: a systematic review and meta-analysis

Summary It is known that Barrett’s esophagus (BE) and esophageal adenocarcinoma occur more commonly in men. What is unknown are the prevalence of BE and rates of neoplastic progression in women. Our aim was to determine the prevalence of Barrett’s and its progression to esophageal cancer in women through systematic review and meta-analysis. A comprehensive search was conducted using PubMed, Scopus, and Google Scholar. Studies were included that reported prevalence rates of BE or progression rates to neoplastic disease stratified by gender. Barrett’s was defined by updated criteria as salmon-colored mucosa ≥1 cm proximal to the gastroesophageal junction. Pooled rates and odds ratios (ORs) at 95% confidence interval (CI) of the prevalence of BE and its progression to neoplastic disease were calculated. Ten studies with 19,337 patients (50.6% women) reported on prevalence and six studies with 5137 patients (24.3% women) reported on neoplastic progression of disease between genders. The rate of BE in women was 1.29% ([95% CI: 0.76–2.19], I2 = 91%) compared to men at 4.66% ([95% CI: 3.31–6.53], I2 = 89%); OR: 0.33 ([95% CI: 0.27–0.42], I2 = 0%). The rate of annual progression of Barrett’s to high-grade dysplasia or adenocarcinoma was 0.62% ([95% CI: 0.22–1.75]) in women compared to 1.54% ([95% CI: 0.83–2.81], I2 = 96%) in men; OR: 0.44 ([95% CI: 0.30–0.65], I2 = 22%). This study demonstrates a 70% lower rate of prevalence and a 60% lower rate of neoplastic progression of Barrett’s in women. Future BE guidelines should tailor screening and surveillance practices by gender.

TAMI-26. INVESTIGATING TUMOR MICROENVIRONMENT METABOLIC DEPENDENCIES IN GLIOBLASTOMA

Metabolic reprogramming is a hallmark of cancer. Malignant cells must acquire metabolic adaptations in response to a multitude of intrinsic and extrinsic factors to fuel neoplastic progression. Mutations or changes in metabolic gene expression can impose nutrient dependencies in tumors, and even in the absence of metabolic defects, cancer cells can become auxotrophic for particular nutrients or metabolic byproducts generated by other cells in the tumor microenvironment (TME). Altered metabolism in GBM is becoming an increasingly promising area of research to identify novel therapeutic targets and biomarkers, as metabolic rewiring can occur across numerous genotypes. The unique features of the brain TME pose a difficult challenge when studying GBM and other primary brain cancers – currently, the availability of nutrients in the brain, as well as how they influence or are influenced by tumor metabolism, are not well understood. Our group has identified a subgroup of gliomas, hereafter termed TME-dependent, which can only form tumors in the brain TME. While genetically heterogeneous, these tumors share transcriptional identities linked to oligodendrocyte precursor cell (OPC) and neuronal lineages. Systematic molecular profiling of over 75 patient tumors and their matched cell culture and brain orthotopic xenograft derived models revealed that TME-dependent tumors display lipid metabolic signatures linked to signaling and interactions with surrounding neurons and glial cells. Collectively, these data emphasize the metabolic heterogeneity within GBM, and reveal a subset of gliomas that lack metabolic plasticity in fatty acid biosynthetic programs, indicating a potential brain-microenvironment specific metabolic dependency linked to transcriptional identity that can be targeted for therapy.

NIMG-57. MANAGEMENT OF TECTAL GLIOMAS IN ADULTS

BACKGROUND Tectal gliomas (TG) are rare tumors occurring primarily in children but also found in adults during workup of various neurological symptoms. Surgery is not required in asymptomatic cases, so histopathological information is sparse. No consensus on timing of imaging surveillance or management has been established. OBJECTIVE We seek to standardize neuroimaging, including MRI protocol and surveillance time intervals, and clinical management of symptoms and disease progression, including surgery, radiotherapy, and chemotherapy. METHODS At our institution, patients with TG were identified through a search of radiology reports and clinic notes between 1989 and 2020. Initial and serial MRI exams were evaluated for tumor size, enhancement, edema, hydrocephalus, and other radiographic features. When tissue was available, cellularity, mitotic activity, and morphology were described. We documented neurological symptoms and signs potentially related to the tumor. RESULTS 37 cases were identified: 22 female, 15 male; 5 children, 32 adults. Age of diagnosis ranged from 7 to 69 years. Presenting symptoms included headache (59%), visual symptoms (35%), and imbalance (14%), less commonly: seizure, weakness, nausea/vomiting, and dizziness. Surgical procedures included biopsy (9), resection (7), endoscopic third ventriculostomy (15), and shunt placement (11). Eight patients received radiotherapy, including IMRT, CyberKnife, GammaKnife, and Zap-X (all adults; 4 at diagnosis, 3 at progression, 1 at diagnosis and again at progression). Four patients received chemotherapy (all adults; 1 at diagnosis, 3 at progression), all with temozolomide. One additionally received bevacizumab for radionecrosis. Three patients died with progressive disease, two following treatment and one without. Of interest, 5 adult patients developed signs of parkinsonism during their follow-up period. CONCLUSION Management of TG encompasses both neoplastic progression and symptom control, either from local compression or infiltrative disease. We have developed an algorithm for imaging surveillance and treatment, including MRI protocol, definition of progressive disease, and indications for antineoplastic therapies.

STEM-08. ST6Gal1-MEDIATED SILALYLATION IS CRITICAL FOR GLIOBLASTOMA GROWTH

Although altered cell surface glycosylation was one of the earliest modifications observed in neoplastic progression, this facet of cancer cell biology has received meager attention, particularly in brain tumors. Among the various glycosyltransferases present in human cells, golgi sialyltransferase ST6Gal1 [beta-galactoside alpha-2,6-sialyltransferase 1] adds sialic acid residues in α2-6 linkage to membrane-bound and secreted N-glycans. ST6Gal1 is known to be pro-tumorigenic in epithelial cancers where it can promote epithelial to mesenchymal transformation, tumor-initiating cell (TIC) phenotypes, and survival of cells exposed to stressors such as chemo- and radiotherapy, hypoxia, or serum starvation. However, roles for this potent TIC regulator have not been well explored in GBM as experiments in standard cell lines suggested ST6Gal1 was epigenetically silenced. To explore our hypothesis that ST6Gal1-mediated α2,6 sialylation is elevated in Brain Tumor Initiating Cells (BTICs) and promotes GBM growth, we utilized GBM patient-derived xenografts (PDXs). ST6Gal1 is expressed in GBM PDX tissue sections and elevated in stem-like BTICs in comparison to differentiated GBM cells or astrocytes. Knockdown of ST6Gal1 in BTICs decreased growth and neurosphere formation capacity in vitro, suggesting that ST6Gal1 regulates BTIC maintenance. Similarly, cells isolated directly from PDXs that were sorted for high and low expression of α2,6 sialylation showed that α2,6 sialylationhigh GBM PDX have elevated neurosphere formation capacity and growth. Further, immunocompromised mice injected with sorted α2,6 sialylationhigh PDX cells had significantly lower survival compared to mice injected with α2,6 sialylationlow cells. Using proteomic analysis of ST6Gal1 KD vs NT PDX, we identified novel regulators of cancer stem cell biology directly modulated by ST6Gal1. As we identified a small subset of IDHwt GBMs with ST6Gal1 and SOX2 amplification, we are generating a novel gliomagenesis model with conditional ST6Gal1 overexpression. Together, our data strongly implicates ST6Gal1 as a regulator of GBM BTIC maintenance and GBM growth.

CSIG-20. CANNABIDIOL CONVERTS NFKB INTO A TUMOR SUPPRESSOR IN GLIOBLASTOMA WITH DEFINED ANTIOXIDATIVE PROPERTIES

The transcription factor NFKB drives neoplastic progression of many cancers including primary brain tumors (glioblastoma; GBM). Precise therapeutic modulation of NFKB activity can suppress central oncogenic signalling pathways in GBM, but clinically applicable compounds to achieve this goal have remained elusive. In a pharmacogenomics study with a panel of transgenic glioma cells we observed that NFKB can be converted into a tumor suppressor by the non-psychotropic cannabinoid Cannabidiol (CBD). Subsequently, we investigated the anti-tumor effects of CBD, which is used as an anticonvulsive drug (Epidiolex) in pediatric neurology, in a larger set of human primary GBM stem-like cells (hGSC). For this study we performed pharmacological assays, gene expression profiling, biochemical and cell-biological experiments. We validated our findings using orthotopic in vivo models and bioinformatics analysis of human GBM-datasets. We found that CBD promotes DNA binding of the NFKB subunit RELA and simultaneously prevents RELA-phosphorylation on serine-311, a key residue which permits genetic transactivation. Strikingly, sustained DNA binding by RELA lacking phospho-serine 311 was fully sufficient to mediate hGSC cytotoxicity. Widespread sensitivity to CBD was observed in a cohort of hGSC defined by low levels of reactive oxygen-species (ROS), while high ROS-content in other tumors blocked CBD induced hGSC death. Consequently, ROS levels served as predictive biomarker for CBD-sensitive tumors. This evidence demonstrates how a clinically approved drug can convert NFKB into a tumor suppressor and suggests a promising repurposing option for GBM-therapy