Multi-omic characterization of ILC and ILC-like cell lines as part of ILC cell line encyclopedia (ICLE) defines new models to study potential biomarkers and explore therapeutic opportunities.

Invasive lobular carcinoma (ILC), the most common histological "special type", accounts for ∼10-15% of all BC diagnoses, is characterized by unique features such as E-cadherin loss/deficiency, lower grade, hormone receptor positivity, larger diffuse tumors, and specific metastatic patterns. Despite ILC being acknowledged as a disease with distinct biology that necessitates specialized and precision medicine treatments, the further exploration of its molecular alterations with the goal of discovering new treatments has been hindered due to the scarcity of well-characterized cell line models for studying this disease. To address this, we generated the ILC Cell Line Encyclopedia (ICLE), providing a comprehensive multi-omic characterization of ILC and ILC-like cell lines. Using consensus multi-omic subtyping, we confirmed luminal status of previously established ILC cell lines and uncovered additional ILC/ILC-like cell lines with luminal features for modeling ILC disease. Furthermore, most of these luminal ILC/ILC-like cell lines also showed RNA and copy number similarity to ILC patient tumors. Similarly, ILC/ILC-like cell lines also retained molecular alterations in key ILC genes at similar frequency to both primary and metastatic ILC tumors. Importantly, ILC/ILC-like cell lines recapitulated the CDH1 alteration landscape of ILC patient tumors including enrichment of truncating mutations in and biallelic inactivation of CDH1 gene. Using whole-genome optical mapping, we uncovered novel genomic-rearrangements including novel structural variations in CDH1 and functional gene fusions and characterized breast cancer specific patterns of chromothripsis in chromosomes 8, 11 and 17. In addition, we systematically analyzed aberrant DNAm events and integrative analysis with RNA expression revealed epigenetic activation of TFAP2B - an emerging biomarker of lobular disease that is preferentially expressed in lobular disease. Finally, towards the goal of identifying novel druggable vulnerabilities in ILC, we analyzed publicly available RNAi loss of function breast cancer cell line datasets and revealed numerous putative vulnerabilities cytoskeletal components, focal adhesion and PI3K/AKT pathway in ILC/ILC-like vs NST cell lines. In summary, we addressed the lack of suitable models to study E-cadherin deficient breast cancers by first collecting both established and putative ILC models, then characterizing them comprehensively to show their molecular similarity to patient tumors along with uncovering their novel multi-omic features as well as highlighting putative novel druggable vulnerabilities. Not only we expand the array of suitable E-cadherin deficient cell lines available for modelling human-ILC disease but also employ them for studying epigenetic activation of a putative lobular biomarker as well as identifying potential druggable vulnerabilities for this disease towards enabling precision medicine research for human-ILC.

Identifying Genomic Alterations in Patients With Stage IV Breast Cancer Using MammaSeq: An International Collaborative Study.

BACKGROUND: Identification of genomic alterations present in cancer patients may aid in cancer diagnosis, prognosis and therapeutic target discovery. In this study, we aimed to identify clinically actionable variants present in stage IV breast cancer (BC) samples. MATERIALS AND METHODS: DNA was extracted from formalin-fixed paraffin-embedded samples of BC (n = 41). DNA was sequenced using MammaSeq, a BC-specific next-generation sequencing panel targeting 79 genes and 1369 mutations. Ion Torrent Suite 4.0 was used to make variant calls on the raw data, and the resulting single nucleotide variants were annotated using the CRAVAT toolkit. Single nucleotide variations (SNVs) were filtered to remove common polymorphisms and germline variants. CNVkit was employed to identify copy number variations (CNVs). The Precision Medicine Knowledgebase (PMKB) and OncoKB Precision Oncology Database were used to associate clinical significance with the identified variants. RESULTS: A total of 41 samples from Turkish patients with BC were sequenced (read depth of 94-13,340; median of 1529). These patients were diagnosed with various BC subtypes including invasive ductal carcinoma, invasive lobular carcinoma, apocrine BC, and micropapillary BC. In total, 59 different alterations (49 SNVs and 10 CNVs) were identified. From these, 8 alterations (3 CNVs - ERBB2, FGFR1, and AR copy number gains and 5 SNVs - IDH1.R132H, TP53.E204∗, PI3KCA.E545K, PI3KCA.H1047R, and PI3KCA.R88Q) were identified to have some clinical significance by PMKB and OncoKB. Moreover, the top 5 genes with the most SNVs included PIK3CA, TP53, MAP3K1, ATM, and NCOR1. Additionally, copy number gains and losses were found in ERBB2, GRB7, IGFR1, AR, FGFR1, MYC, and IKBKB, and BRCA2, RUNX1, and RB1, respectively. CONCLUSION: We identified 59 unique alterations in 38 genes in 41 stage IV BC tissue samples using MammaSeqTM. Eight of these alterations were found to have some clinical significance by OncoKB and PKMB. This study highlights the potential use of cancer specific next-generation sequencing panels in clinic to get better insight into the patient-specific genomic alterations.

Detection of ESR1 mutations in circulating cell-free DNA from patients with metastatic breast cancer treated with palbociclib and letrozole.

ESR1 mutations are frequently acquired in hormone-resistant metastatic breast cancer (MBC). CDK4/6 inhibition along with endocrine therapy is a promising strategy in hormone receptor-positive MBC. However, the incidence and impact of ESR1 mutations on clinical outcome in patients treated with CDK4/6 inhibitors have not been defined. In this study, we evaluated the frequency of ESR1 mutations in cfDNA from 16 patients with MBC undergoing palbociclib and letrozole therapy. Four common ESR1 mutations (D538G, Y537C, Y537N, and Y537S) were analyzed in serial blood draws using ddPCR. Mutation rate was 31.3% (5/16) (n=3; de novo, n=2; acquired). D538G was the most frequent mutation (n=3), followed by Y537N and Y537S (n=2). One patient showed multiple ESR1 mutations. Mutations were enriched during therapy. Progression-free survival (PFS) and overall survival (OS) were similar in patients with and without mutation detected at any given time during treatment. However, PFS was significantly shorter in patients with ESR1 mutation at initial blood draw (3.3 versus 9.0 months, P-value=0.038). In conclusion, ESR1 mutation prevalence is consistent with recent studies in hormone-refractory breast cancer. Further, treatment with palbociclib and letrozole does not prevent selection of ESR1 mutations in later lines of therapy. Larger studies are warranted to validate these findings.

Interleukin-8 as a modulator of response to bevacizumab in preclinical models of head and neck squamous cell carcinoma.

OBJECTIVES: Bevacizumab, a monoclonal antibody to VEGF-A, is under active clinical evaluation in head and neck squamous cell carcinoma (HNSCC) and appears to be a promising therapy in at least a subset of patients. However, there are no reliable predictive biomarkers to identify those patients most likely to benefit. In this study, we assessed the efficacy of bevacizumab in HNSCC xenograft models to characterize escape mechanisms underlying intrinsic resistance and identify potential biomarkers of drug response. MATERIALS AND METHODS: We evaluated the angiogenic profile of HNSCC cells from sensitive and resistant cell lines using antibody array. We further examined the role of interleukin-8 (IL-8) in contributing to resistance both in vitro and in vivo, using a loss- and gain-of-function approach. RESULTS: Angiogenic profiling indicated that resistant cells expressed higher levels of proangiogenic factors including IL-8, interleukin-1α (IL-1α), vascular endothelial growth factor (VEGF), fibroblast growth factor-a (FGF-a), and tumor necrosis factor-α (TNF-α). IL-8 was the most differentially expressed protein. IL-8 signaling compensated for VEGF inhibition in endothelial cells. Downregulation of IL-8 resulted in sensitization of resistant tumors to bevacizumab by disrupting angiogenesis and enhancing endothelial cell apoptosis. Overexpression of IL-8 in sensitive tumors conferred resistance to bevacizumab. Serum analysis of HNSCC patients treated with a bevacizumab-containing regime revealed high baseline IL-8 levels in a subset of patients refractory to treatment but not in responders. CONCLUSIONS: These results implicate IL-8 in mediating intrinsic resistance to bevacizumab in HNSCC. Hence, co-targeting of VEGF and IL-8 may help overcome resistance and enhance therapeutic efficacy.