Classification of large circulating tumor cells isolated with ultra-high throughput microfluidic Vortex technology.

Circulating tumor cells (CTCs) are emerging as rare but clinically significant non-invasive cellular biomarkers for cancer patient prognosis, treatment selection, and treatment monitoring. Current CTC isolation approaches, such as immunoaffinity, filtration, or size-based techniques, are often limited by throughput, purity, large output volumes, or inability to obtain viable cells for downstream analysis. For all technologies, traditional immunofluorescent staining alone has been employed to distinguish and confirm the presence of isolated CTCs among contaminating blood cells, although cells isolated by size may express vastly different phenotypes. Consequently, CTC definitions have been non-trivial, researcher-dependent, and evolving. Here we describe a complete set of objective criteria, leveraging well-established cytomorphological features of malignancy, by which we identify large CTCs. We apply the criteria to CTCs enriched from stage IV lung and breast cancer patient blood samples using the High Throughput Vortex Chip (Vortex HT), an improved microfluidic technology for the label-free, size-based enrichment and concentration of rare cells. We achieve improved capture efficiency (up to 83%), high speed of processing (8 mL/min of 10x diluted blood, or 800 μL/min of whole blood), and high purity (avg. background of 28.8±23.6 white blood cells per mL of whole blood). We show markedly improved performance of CTC capture (84% positive test rate) in comparison to previous Vortex designs and the current FDA-approved gold standard CellSearch assay. The results demonstrate the ability to quickly collect viable and pure populations of abnormal large circulating cells unbiased by molecular characteristics, which helps uncover further heterogeneity in these cells

PF-05280014 (a trastuzumab biosimilar) plus paclitaxel compared with reference trastuzumab plus paclitaxel for HER2-positive metastatic breast cancer: a randomised, double-blind study

BACKGROUND: This randomised, double-blind study compared PF-05280014 (a trastuzumab biosimilar) with reference trastuzumab (Herceptin®) sourced from the European Union (trastuzumab-EU), when each was given with paclitaxel as first-line treatment for HER2-positive metastatic breast cancer. METHODS: Between 4 April 2014 and 22 January 2016, 707 participants were randomised 1:1 to receive intravenous PF-05280014 plus paclitaxel (PF-05280014 group; n = 352) or trastuzumab-EU plus paclitaxel (trastuzumab-EU group; n = 355). PF-05280014 or trastuzumab-EU was administered weekly (first dose 4 mg/kg, subsequent doses 2 mg/kg), with the option to change to a 3-weekly regimen (6 mg/kg) from Week 33. Treatment with PF-05280014 or trastuzumab-EU could continue until disease progression. Paclitaxel (starting dose 80 mg/m2 ) was administered on Days 1, 8 and 15 of 28-day cycles for at least six cycles or until maximal benefit of response. The primary endpoint was objective response rate (ORR), evaluating responses achieved by Week 25 and confirmed by Week 33, based on blinded central radiology review. RESULTS: The risk ratio for ORR was 0.940 (95% CI: 0.842–1.049). The 95% CI fell within the pre-specified equivalence margin of 0.80–1.25. ORR was 62.5% (95% CI: 57.2–67.6%) in the PF-05280014 group and 66.5% (95% CI: 61.3–71.4%) in the trastuzumab-EU group. As of data cut-off on 11 January 2017 (using data up to 378 days post-randomisation), there were no notable differences between groups in progression-free survival (median: 12.16 months in the PF-05280014 group vs. 12.06 months in the trastuzumab-EU group; 1-year rate: 54% vs. 51%) or overall survival (median: not reached in either group; 1-year rate: 89.31% vs. 87.36%). Safety outcomes and immunogenicity were similar between the treatment groups. CONCLUSION: When given as first-line treatment for HER2-positive metastatic breast cancer, PF-05280014 plus paclitaxel demonstrated equivalence to trastuzumab-EU plus paclitaxel in terms of ORR. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT0198967

Estrogen/HER2 receptor crosstalk in breast cancer: combination therapies to improve outcomes for patients with hormone receptor-positive/HER2-positive breast cancer

Abstract The human epidermal growth factor receptor 2 (HER2) is overexpressed in 13–22% of breast cancers (BC). Approximately 60–70% of HER2+ BC co-express hormone receptors (HRs). HR/HER2 co-expression modulates response to both anti-HER2–directed and endocrine therapy due to “crosstalk” between the estrogen receptor (ER) and HER2 pathways. Combined HER2/ER blockade may be an effective treatment strategy for patients with HR+/HER2+ BC in the appropriate clinical setting(s). In this review, we provide an overview of crosstalk between the ER and HER2 pathways, summarize data from recently published and ongoing clinical trials, and discuss clinical implications for targeted treatment of HR+/HER2+ BC

Targeted Therapy in Metastatic Breast Cancer: The HER2/neu Oncogene

SUMMARY: Besides surgery, radiation, chemotherapy, and endocrine treatment, immunotherapy has become an established part of systemic therapy in treating metastatic breast cancer. One of the most interesting targets for the design of anticancer therapeutics is the HER2/ErbB2 receptor which is overexpressed in about 20–25% of breast cancers. Given the poor prognosis of women whose tumors express ErbB2 (HER2) at high levels, accurate determination of the ErbB2 status should be routinely performed in women with newly diagnosed invasive breast cancer. Efficacy and safety data of numerous trials led to the approval of the monoclonal antibody trastuzumab as the first ErbB2-targeting therapy in ErbB2-positive breast cancer. However, the majority of patients who achieve an initial response to trastuzumab-based regimens for metastatic disease develop resistance within 1 year. This underlines the need for alternative or additional anti-ErbB2-targeting strategies

Abstract PS17-17: cGAS-STING pathway protein expression in human primary breast cancer

Abstract Background: The DNA-sensing nucleotidyl transferase enzyme cyclic GMP-AMP synthase (cGAS), its second-messenger product cyclic GMP-AMP (cGAMP), and the cGAMP sensor Stimulator of interferon Genes (STING) form a major cytoplasmic DNA-sensing mechanism in human cells [Barber GN. Nat Rev Immunol, 2015]. cGAMP-activated STING oligomers bind TANK-binding kinase 1 (TBK1), translocate from the endoplasmic reticulum to perinuclear vesicles, and phosphorylate transcription factor interferon (IFN) regulatory factor 3 (IRF3), resulting in IRF3 nuclear translocation to induce expression of type I IFNs such as IFNβ. This pathway is critical for controlling innate antiviral immune responses, and is implicated in anti-tumor adaptive immunity, yet little is known regarding its function in human primary breast cancer (BC). Methods: Immunohistochemistry (IHC) staining for cGAS (ab224144), STING (CST13647), TBK1 (HPA045797), IRF3 (ab25950) and IFNβ (ab238675) was performed on tissue microarrays constructed from 18 normal human breast tissue samples and 197 primary BC samples to characterize in-situ protein expression. Positive controls were normal colon, MCF10A cells, testis tissue, MCF-7 cells and normal colon for cGAS, STING, TBK1, IRF3 and IFNβ, respectively. Substitution of primary antibodies with 0.5% normal goat serum served as negative controls. Scoring of extent of the IHC-stained area was 0 for no IHC signal, 1 for 50% of tumor cells. IHC intensity was scored as 0 for no IHC signal, 1 for weak, 2 for moderate, and 3 for strong. A final IHC score was the product of extent score and intensity score, as described [Xia T, et al. Cell reports, 2016]. Categorical classification of high versus low final IHC scores was determined using the median as an unbiased cutoff. Associations between cGAS-STING pathway protein constituents was interrogated by Pearson’s test, and associations between STING pathway IHC expression and breast tumor clinicopathological features were by Chi-square. Results: Among 197 primary BCs, 51.5% were estrogen receptor (ER) positive/ human epidermal growth factor receptor 2 (HER2) negative, 28.4% were HER2 positive and 19.8% were triple negative BC (TNBC). Distinct cGAS-STING pathway expression patterns were observed among three BC subtypes (Table 1). Frequent high expression of cGAS-STING pathway constituents was observed in ER+/HER2- BC. By contrast, high cGAS expression was not associated with high STING expression or effector protein IFNβ in either HER2+ BC, or in TNBC. IRF3 and IFNβ expression was significantly positively associated with hormone receptor status (both P< .001); IFNβ expression was significantly inversely associated with higher grade (P=0.008) and higher mean Ki67 index (18 vs 10, P=0.005). High expression of IFNβ was positively correlated with higher levels of TBK1 and IRF3 (R=0.262 P<.001; R=0.347, P=<.001, respectively). Conclusion: To our knowledge, this is the first study to report cGAS-STING pathway in situ protein expression in primary BC, and demonstrates distinct expression patterns amongst different primary BC clinical subtypes. A limitation of this study is that IHC methods were insufficient for detecting post-translational modifications or translocation of STING pathway proteins. These results provide foundation for understanding the mechanisms of innate immune response in BC, as well as for developing new therapeutic strategies aimed at facilitating adaptive immune anti-tumor responses. Citation Format: Yu Zong, Xiaofei Liu, Glen N Barber, Mark D Pegram. cGAS-STING pathway protein expression in human primary breast cancer [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 PS17-17