Nanoscale insights into hematology: super-resolved imaging on blood cell structure, function, and pathology.

Fluorescence nanoscopy, also known as super-resolution microscopy, has transcended the conventional resolution barriers and enabled visualization of biological samples at nanometric resolutions. A series of super-resolution techniques have been developed and applied to investigate the molecular distribution, organization, and interactions in blood cells, as well as the underlying mechanisms of blood-cell-associated diseases. In this review, we provide an overview of various fluorescence nanoscopy technologies, outlining their current development stage and the challenges they are facing in terms of functionality and practicality. We specifically explore how these innovations have propelled forward the analysis of thrombocytes (platelets), erythrocytes (red blood cells) and leukocytes (white blood cells), shedding light on the nanoscale arrangement of subcellular components and molecular interactions. We spotlight novel biomarkers uncovered by fluorescence nanoscopy for disease diagnosis, such as thrombocytopathies, malignancies, and infectious diseases. Furthermore, we discuss the technological hurdles and chart out prospective avenues for future research directions. This review aims to underscore the significant contributions of fluorescence nanoscopy to the field of blood cell analysis and disease diagnosis, poised to revolutionize our approach to exploring, understanding, and managing disease at the molecular level.

Development and Evaluation of a Quantitative Systems Pharmacology Model for Mechanism Interpretation and Efficacy Prediction of Atezolizumab in Combination with Carboplatin and Nab-Paclitaxel in Patients with Non-Small-Cell Lung Cancer.

Immunotherapy has shown clinical benefit in patients with non-small-cell lung cancer (NSCLC). Due to the limited response of monotherapy, combining immune checkpoint inhibitors (ICIs) and chemotherapy is considered a treatment option for advanced NSCLC. However, the mechanism of combined therapy and the potential patient population that could benefit from combined therapy remain undetermined. Here, we developed an NSCLC model based on the published quantitative systems pharmacology (QSP)-immuno-oncology platform by making necessary adjustments. After calibration and validation, the established QSP model could adequately characterise the biological mechanisms of action of the triple combination of atezolizumab, nab-paclitaxel, and carboplatin in patients with NSCLC, and identify predictive biomarkers for precision dosing. The established model could efficiently characterise the objective response rate and duration of response of the IMpower131 trial, reproducing the efficacy of alternative dosing. Furthermore, CD8+ and CD4+ T cell densities in tumours were found to be significantly related to the response status. This significant extension of the QSP model not only broadens its applicability but also more accurately reflects real-world clinical settings. Importantly, it positions the model as a critical foundation for model-informed drug development and the customisation of treatment plans, especially in the context of combining single-agent ICIs with platinum-doublet chemotherapy.

Outcomes of Metaplastic Breast Cancer Versus Triple-Negative Breast Cancer: A Propensity Score Matching Analysis.

PURPOSE: This study aims to compare the survival outcomes of metaplastic breast cancer (MPBC) with triple-negative breast cancer (TNBC) and identify prognostic factors that influence the survival outcomes of MPBC patients and TNBC patients. METHODS: Patients with nonmetastatic MPBC or TNBC were reviewed from our database. Patients' clinicopathologic and molecular features were analyzed with respect to outcomes, including disease-free survival (DFS) and overall survival (OS). Propensity score matching (PSM) with a one-to-three matching between MPBC patients and TNBC patients was performed. RESULTS: A total of 857 female patients (76 MPBC patients and 781 TNBC patients) were included in this study. A subgroup of triple-negative MPBC (n = 60) was matched with TNBC (n = 180) cases based on patient characteristics and treatments. Kaplan-Meier analysis indicated that the MPBC group was associated with worse OS and DFS before (P = 0.0046 both) and after (P = 0.011 and P = 0.0046, respectively) PSM. Multivariable analysis revealed that a higher T stage (T > 2) (P = 0.032) and higher lymph node stage (N3 vs. N0-2, P = 0.012) were associated with worse OS after PSM. For DFS, the MPBC group (P = 0.012), higher T stage (T > 2) (P = 0.032), and higher lymph node stage (N3 vs. N0-2, P = 0.045) were associated with worse DFS. Among the 76 MPBC patients, a higher T stage and mesenchymal differentiation were associated with worse OS (pT1/2 vs. pT3/4 and mesenchymal differentiation vs. other subtypes, P = 0.007 and P = 0.011, respectively). CONCLUSIONS: Compared with TNBC, MPBC was associated with worse OS and DFS. Mesenchymal differentiation has a worse DFS than other subtypes of MPBC.

Corrigendum: Preclinical profiles of SKB264, a novel anti-TROP2 antibody conjugated to topoisomerase inhibitor, demonstrated promising antitumor efficacy compared to IMMU-132.

[This corrects the article DOI: 10.3389/fonc.2022.951589.].

Identification and characterization of two Isatis indigotica O-methyltransferases methylating C-glycosylflavonoids.

Isatis indigotica accumulates several active substances, including C-glycosylflavonoids, which have important pharmacological activities and health benefits. However, enzymes catalyzing the methylation step of C-glycosylflavonoids in I. indigotica remain unknown. In this study, three O-methyltransferases (OMTs) were identified from I. indigotica that have the capacity for O-methylation of the C-glycosylflavonoid isoorientin. The Type II OMTs IiOMT1 and IiOMT2 efficiently catalyze isoorientin to form isoscoparin, and decorate one of the aromatic vicinal hydroxyl groups on flavones and methylate the C6, C8, and 3'-hydroxyl positions to form oroxylin A, wogonin, and chrysoeriol, respectively. However, the Type I OMT IiOMT3 exhibited broader substrate promiscuity and methylated the C7 and 3'-hydroxyl positions of flavonoids. Further site-directed mutagenesis studies demonstrated that five amino acids of IiOMT1/IiOMT2 (D121/D100, D173/D149, A174/A150R, N200/N176, and D248/D233) were critical residues for their catalytic activity. Additionally, only transient overexpression of Type II OMTs IiOMT1 and IiOMT2 in Nicotiana benthamiana significantly increased isoscoparin accumulation, indicating that the Type II OMTs IiOMT1 and IiOMT2 could catalyze the methylation step of C-glycosylflavonoid, isoorientin at the 3'-hydroxyl position. This study provides insights into the biosynthesis of methylated C-glycosylflavonoids, and IiOMTs could be promising catalysts in the synthesis of bioactive compounds.

Preclinical profiles of SKB264, a novel anti-TROP2 antibody conjugated to topoisomerase inhibitor, demonstrated promising antitumor efficacy compared to IMMU-132.

Purpose: The aim of this study was to improve the intratumoral accumulation of an antibody-drug conjugate (ADC) and minimize its off-target toxicity, SKB264, a novel anti-trophoblast antigen 2 (TROP2) ADC that was developed using 2-methylsulfonyl pyrimidine as the linker to conjugate its payload (KL610023), a belotecan-derivative topoisomerase I inhibitor. The preclinical pharmacologic profiles of SKB264 were assessed in this study. Methods: The in vitro and in vivo pharmacologic profiles of SKB264, including efficacy, pharmacokinetics-pharmacodynamics (PK-PD), safety, and tissue distribution, were investigated using TROP2-positive cell lines, cell-derived xenograft (CDX), patient-derived xenograft (PDX) models, and cynomolgus monkeys. Moreover, some profiles were compared with IMMU-132. Results: In vitro, SKB264 and SKB264 monoclonal antibody (mAb) had similar internalization abilities and binding affinities to TROP2. After cellular internalization, KL610023 was released and inhibited tumor cell survival. In vivo, SKB264 significantly inhibited tumor growth in a dose-dependent manner in both CDX and PDX models. After SKB264 administration, the serum or plasma concentration/exposure of SKB264 (conjugated ADC, number of payload units ≥1), total antibody (Tab, unconjugated and conjugated mAb regardless of the number of the payload units), and KL610023 in cynomolgus monkeys increased proportionally with increasing dosage from 1 to 10 mg/kg. The linker stability of SKB264 was significantly enhanced as shown by prolonged payload half-life in vivo (SKB264 vs. IMMU-132, 56.3 h vs. 15.5 h). At the same dose, SKB264's exposure in tumor tissue was 4.6-fold higher than that of IMMU-132. Conclusions: Compared with IMMU-132, the longer half-life of SKB264 had a stronger targeting effect and better antitumor activity, suggesting the better therapeutic potential of SKB264 for treating TROP2-positive tumors.

Biophysical and biochemical properties of PHGDH revealed by studies on PHGDH inhibitors.

The rate-limiting serine biogenesis enzyme PHGDH is overexpressed in cancers. Both serine withdrawal and genetic/pharmacological inhibition of PHGDH have demonstrated promising tumor-suppressing activities. However, the enzyme properties of PHGDH are not well understood and the discovery of PHGDH inhibitors is still in its infancy. Here, oridonin was identified from a natural product library as a new PHGDH inhibitor. The crystal structure of PHGDH in complex with oridonin revealed a new allosteric site. The binding of oridonin to this site reduced the activity of the enzyme by relocating R54, a residue involved in substrate binding. Mutagenesis studies showed that PHGDH activity was very sensitive to cysteine mutations, especially those in the substrate binding domain. Conjugation of oridonin and other reported covalent PHGDH inhibitors to these sites will therefore inhibit PHGDH. In addition to being inhibited enzymatically, PHGDH can also be inhibited by protein aggregation and proteasome-mediated degradation. Several tested PHGDH cancer mutants showed altered enzymatic activity, which can be explained by protein structure and stability. Overall, the above studies present new biophysical and biochemical insights into PHGDH and may facilitate the future design of PHGDH inhibitors.

Discovery of novel inhibitors of human phosphoglycerate dehydrogenase by activity-directed combinatorial chemical synthesis strategy.

Serine, the source of the one-carbon units essential for de novo purine and deoxythymidine synthesis plays a crucial role in the growth of cancer cells. Phosphoglycerate dehydrogenase (PHGDH) which catalyzes the first, rate-limiting step in de novo serine biosynthesis has become a promising target for the cancer treatment. Here we identified H-G6 as a potential PHGDH inhibitor from the screening of an in-house small molecule library based on the enzymatic assay. We adopted activity-directed combinatorial chemical synthesis strategy to optimize this hit compound. Compound b36 was found to be the noncompetitive and the most promising one with IC50 values of 5.96 ± 0.61 µM against PHGDH. Compound b36 inhibited the proliferation of human breast cancer and ovarian cancer cells, reduced intracellular serine synthesis, damaged DNA synthesis, and induced cell cycle arrest. Collectively, our results suggest that b36 is a novel PHGDH inhibitor, which could be a promising modulator to reprogram the serine synthesis pathway and might be a potential anticancer lead worth further exploration.

Novel Mechanistic Observations and NES-Binding Groove Features Revealed by the CRM1 Inhibitors Plumbagin and Oridonin.

The protein chromosome region maintenance 1 (CRM1) is an important nuclear export factor and drug target in diseases such as cancer and viral infections. Several plant-derived CRM1 inhibitors including plumbagin and oridonin possess potent antitumor activities. However, their modes of CRM1 inhibition remain unclear. Here, a multimutant CRM1 was engineered to enable crystallization of these two small molecules in its NES groove. Plumbagin and oridonin share the same three conjugation sites in CRM1. In solution, these two inhibitors targeted more CRM1 sites and inhibited its activity through promoting its aggregation, in addition to directly targeting the NES groove. While the plumbagin-bound NES groove resembles the NES-bound groove state, the oridonin complex reveals for the first time a more open NES groove. The observed greater NES groove dynamics may improve cargo loading through a "capture-and-tighten" mechanism. This work thus provides new insights on the mechanism of CRM1 inhibition by two natural products and a structural basis for further development of these or other CRM1 inhibitors.

Cytoplasmic SHMT2 drives the progression and metastasis of colorectal cancer by inhibiting ß-catenin degradation.

Introduction: Serine hydroxymethyltransferase 2 (SHMT2) plays a critical role in serine-glycine metabolism to drive cancer cell proliferation. However, the nonmetabolic function of SHMT2 in tumorigenesis, especially in human colorectal cancer (CRC) progression, remains largely unclear. Methods: SHMT2 expression in human CRC cells was identified by western blot and immunofluorescence assay. The CRC cell proliferation, migration, and invasion after SHMT2 knockdown or overexpression were explored through in vitro and in vivo assays. Immunofluorescence, mRNA-seq, co-immunoprecipitation, chromatin immunoprecipitation-qPCR and immunohistochemistry assays were used to investigate the underlying mechanisms behind the SHMT2 nonmetabolic function. Results: We demonstrated that SHMT2 was distributed in the cytoplasm and nucleus of human CRC cells. SHMT2 knockdown resulted in the significant inhibition of CRC cell proliferation, which was not restored by serine, glycine, or formate supplementation. The invasion and migration of CRC cells were suppressed after SHMT2 knockdown. Mechanistically, SHMT2 interacted with ß-catenin in the cytoplasm. This interaction inhibited the ubiquitylation-mediated degradation of ß-catenin and subsequently modulated the expression of its target genes, leading to the promotion of CRC cell proliferation and metastasis. Notably, the lysine 64 residue on SHMT2 (SHMT2K64) mediated its interaction with ß-catenin. Moreover, transcription factor TCF4 interacted with ß-catenin, which in turn increased SHMT2 expression, forming an SHMT2/ß-catenin positive feedback loop. In vivo xenograft experiments confirmed that SHMT2 promoted the growth and metastasis of CRC cells. Finally, the level of SHMT2 was found to be significantly increased in human CRC tissues. The SHMT2 level was correlated with an increased level of ß-catenin, associated with CRC progression and predicted poor patient survival. Conclusion: Taken together, our findings reveal a novel nonmetabolic function of SHMT2 in which it stabilizes ß-catenin to prevent its ubiquitylation-mediated degradation and provide a potential therapeutic strategy for CRC therapy.

Ginkgo biloba Extract EGb761 Attenuates Bleomycin-Induced Experimental Pulmonary Fibrosis in Mice by Regulating the Balance of M1/M2 Macrophages and Nuclear Factor Kappa B (NF-κB)-Mediated Cellular Apoptosis.

BACKGROUND The aim of this study was to show whether the standardized Ginkgo biloba extract EGb761, a traditional Chinese medicine, has a therapeutic effect on pulmonary fibrosis (PF). MATERIAL AND METHODS Bleomycin (BLM) was used for establishing the PF mouse model. The mice were treated with a gradient of EGb761 for 28 days to determine an appropriate drug dose. On day 28, the effect of EGb761 on lung injury and inflammation was confirmed by hematoxylin and eosin and Masson staining and evaluated by pulmonary alveolitis and Ashcroft score. The balance of M1/M2 macrophages was evaluated with the respective markers inducible nitric oxide synthase and and interleukin-10 by real-time polymerase chain reaction. Furthermore, the expressions of fibrosis-associated protein alpha-smooth muscle actin (SMA), related inflammatory protein transforming growth factor (TGF)-ß1, the apoptosis-related proteins B-cell lymphoma-associated X protein (Bax), B-cell lymphoma (Bcl)-2, caspase-3, caspase-9, and phosphorylated nuclear factor (NF)-kappaB (p65) were assessed by western blot. RESULTS On day 28, PF was induced by treating with BLM, whereas EGb761 suppressed the PF of lung tissue. The BLM-induced imbalance of M1/M2 macrophages was reduced by EGb761. Furthermore, the increasing amounts of alpha-SMA and TGF-ß1 induced by BLM were suppressed by EGb761. In addition, the protein or messenger ribonucleic acid expression levels of phosphorylated NF-kappaB (p65), caspase-3, and caspase-9 were upregulated, whereas Bax and Bcl-2 were downregulated. Treatment with EGb761 restored the levels of these proteins except for caspase-9. CONCLUSIONS This study illustrated the protective effect of EGb761 on BLM-induced PF by regulating the balance of M1/M2 macrophages and NF-kappaB (p65)-mediated apoptosis. The results demonstrated the potential clinical therapeutic effect of EGb761, providing a novel possibility for curing PF.

Anticancer activity of dietary xanthone α-mangostin against hepatocellular carcinoma by inhibition of STAT3 signaling via stabilization of SHP1.

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers worldwide. The dietary xanthone α-mangostin (α-MGT) exhibits potent anti-tumor effects in vitro and in vivo. However, the anti-HCC effects of α-MGT and their underlying mechanisms are still vague. Aberrant activation of signal transducer and activator of transcription 3 (STAT3) is involved in the progression of HCC. We therefore investigated whether α-MGT inhibited the activation of STAT3 and thereby exhibits its anti-HCC effects. In this study, we found that α-MGT significantly suppressed cell proliferation, induced cell cycle arrest, and triggered apoptosis in HCC cells, including HepG2, SK-Hep-1, Huh7, and SMMC-7721 cells in vitro, as well as inhibiting tumor growth in nude mice bearing HepG2 or SK-Hep-1 xenografts. Furthermore, α-MGT potently inhibited the constitutive and inducible activation of STAT3 in HCC cells. In addition, α-MGT also suppressed IL-6-induced dimerization and nuclear translocation of STAT3, which led to inhibition of the expression of STAT3-regulated genes at both mRNA and protein levels. Mechanistically, α-MGT exhibited effective inhibition of the activation of STAT3's upstream kinases, including JAK2, Src, ERK, and Akt. Importantly, α-MGT increased the protein level of Src homology region 2 domain-containing phosphatase-1 (SHP1), which is a key negative regulator of the STAT3 signaling pathway. Furthermore, α-MGT enhanced the stabilization of SHP1 by inhibiting its degradation mediated by the ubiquitin-proteasome pathway. Knockdown of SHP1 using siRNA obviously prevented the α-MGT-mediated inhibition of the activation of STAT3 and proliferation of HCC cells. In summary, α-MGT exhibited a potent anti-HCC effect by blocking the STAT3 signaling pathway via the suppression of the degradation of SHP1 induced by the ubiquitin-proteasome pathway. These findings also suggested the potential of dietary derived α-MGT in HCC therapy.

GEF-independent Ran activation shifts a fraction of the protein to the cytoplasm and promotes cell proliferation.

Ran (Ras-related nuclear protein) plays several important roles in nucleo-cytoplasmic transport, mitotic spindle formation, nuclear envelope/nuclear pore complex assembly, and other functions in the cytoplasm, as well as in cellular transformation when switched on. Unlike other members of the GTPase superfamily, Ran binds more tightly to GDP than to GTP due to the presence of an auto-inhibitory C-terminal tail. Multiple missense mutations in the C-terminus of Ran occur in cancers, but their biological significance remains unclear. Here, the quantitative GDP/GTP binding preference of four engineered mutations with unstable C-termini was analyzed using a devised mant-GDP dissociation assay. The results showed that the impact of different C-terminal mutations depends on multiple factors. Although these mutants were more GTP-loaded in human cells, they were shown to be more cytoplasmic, and to support nuclear transport with minimally or partially reduced efficiency. Further, several Ran cancer mutants were compromised in autoinhibition, slightly more GTP-bound, more cytoplasmic, and enhanced the proliferation of A549 and HeLa cells in vitro. Thus, our work reveals a new route of Ran activation independent of guanine nucleotide exchange factor (GEF), which may account for the hyper-proliferation induced by Ran cancer mutations.

HER3, p95HER2, and HER2 protein expression levels define multiple subtypes of HER2-positive metastatic breast cancer.

Trastuzumab is effective in the treatment of HER2/neu over-expressing breast cancer, but not all patients benefit from it. In vitro data suggest a role for HER3 in the initiation of signaling activity involving the AKT­mTOR pathway leading to trastuzumab insensitivity. We sought to investigate the potential of HER3 alone and in the context of p95HER2 (p95), a trastuzumab resistance marker, as biomarkers of trastuzumab escape. Using the VeraTag® assay platform, we developed a dual antibody proximity-based assay for the precise quantitation of HER3 total protein (H3T) from formalin-fixed paraffin-embedded (FFPE) breast tumors. We then measured H3T in 89 patients with metastatic breast cancer treated with trastuzumab-based therapy, and correlated the results with progression-free survival and overall survival using Kaplan­Meier and decision tree analyses that also included HER2 total (H2T) and p95 expression levels. Within the sub-population of patients that over-expressed HER2, high levels of HER3 and/or p95 protein expression were significantly associated with poor clinical outcomes on trastuzumab-based therapy. Based on quantitative H3T, p95, and H2T measurements, multiple subtypes of HER2-positive breast cancer were identified that differ in their outcome following trastuzumab therapy. These data suggest that HER3 and p95 are informative biomarkers of clinical outcomes on trastuzumab therapy, and that multiple subtypes of HER2-positive breast cancer may be defined by quantitative measurements of H3T, p95, and H2T.

Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark) for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens.

We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7-10 times greater than conventional immunohistochemistry (IHC) (HercepTest). The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC) or on indirect assessments of gene amplification (FISH).

Comparison of central HER2 testing with quantitative total HER2 expression and HER2 homodimer measurements using a novel proximity-based assay.

The accuracy and reliability of immunohistochemical analysis and in situ hybridization for the assessment of HER2 status remains a subject of debate. We developed a novel assay (HERmark Breast Cancer Assay, Monogram Biosciences, South San Francisco, CA) that provides precise quantification of total HER2 protein expression (H2T) and HER2 homodimers (H2D) in formalin-fixed, paraffin-embedded tissue specimens. H2T and H2D results of 237 breast cancers were compared with those of immunohistochemical studies and fluorescence in situ hybridization (FISH) centrally performed at the Mayo Clinic, Rochester, MN. H2T described a continuum across a wide dynamic range ( approximately 2.5 log). Excluding the equivocal cases, HERmark showed 98% concordance with immunohistochemical studies for positive and negative assay values. For the 94 immunohistochemically equivocal cases, 67% and 39% concordance values were observed between HERmark and FISH for positive and negative assay values, respectively. Polysomy 17 in the absence of HER2 gene amplification did not result in HER2 overexpression as evaluated quantitatively using the HERmark assay.

Differential survival following trastuzumab treatment based on quantitative HER2 expression and HER2 homodimers in a clinic-based cohort of patients with metastatic breast cancer.

BACKGROUND: We have recently described the correlation between quantitative measures of HER2 expression or HER2 homodimers by the HERmark assay and objective response (RR), time-to progression (TTP), and overall survival (OS) in an expanded access cohort of trastuzumab-treated HER2-positive patients with metastatic breast cancer (MBC) who were stringently selected by fluorescence in situ hybridization (FISH). Multivariate analyses suggested a continuum of HER2 expression that correlated with outcome following trastuzumab. Here we investigate the relationship between HER2 expression or HER2 homodimers and OS in a clinic-based population of patients with MBC selected primarily by IHC. METHODS: HERmark, a proximity-based assay designed to detect and quantitate protein expression and dimerization in formalin-fixed paraffin-embedded (FFPE) tissues, was used to measure HER2 expression and HER2 homodimers in FFPE samples from patients with MBC. Assay results were correlated with OS using univariate Kaplan-Meier, hazard function plots, and multivariate Cox regression analyses. RESULTS: Initial analyses revealed a parabolic relationship between continuous measures of HER2 expression and risk of death, suggesting that the assumption of linearity for the HER2 expression measurements may be inappropriate in subsequent multivariate analyses. Cox regression analyses using the categorized variable of HER2 expression level demonstrated that higher HER2 levels predicted better survival outcomes following trastuzumab treatment in the high HER2-expressing group. CONCLUSIONS: These data suggest that the quantitative amount of HER2 expression measured by Hermark may be a new useful marker to identify a more relevant target population for trastuzumab treatment in patients with MBC.

Quantitation of HER2 expression or HER2:HER2 dimers and differential survival in a cohort of metastatic breast cancer patients carefully selected for trastuzumab treatment primarily by FISH.

The selection of patients with HER2-positive breast cancer for treatment with trastuzumab is based on the measurement of HER2 protein expression by immunohistochemistry, or the presence of HER2 gene amplification by fluorescence in situ hybridization (FISH). By using multivariate analyses, we investigate the relationship between quantitative measurements of HER2 expression or HER2:HER2 dimers and objective response (Response Evaluation Criteria in Solid Tumors), time to progression, and breast cancer survival after trastuzumab treatment in a cohort of patients with metastatic breast cancer who were primarily selected for treatment by FISH. The VeraTag assay, a proximity-based assay designed to quantitate protein expression and dimerization in formalin-fixed, paraffin-embedded tissue specimens, was used to measure HER2 protein expression and HER2:HER2 dimer levels. In a Cox proportional hazards analysis, higher HER2 expression or HER2:HER2 dimer levels were both correlated with longer survival (P=0.0058 and P=0.016, respectively) after treatment with trastuzumab in a population of patients that were either FISH-positive (90%) or immunohistochemistry 3+ (10%). Patients with higher levels of HER2 expression or HER2:HER2 dimers seemed to derive little benefit from the addition of concomitant chemotherapy to trastuzumab, whereas those with lower levels benefited significantly [interaction test P=0.43 (HER2 expression), P=0.27 (HER2:HER2 dimers)]. These data suggest that more quantitative or functional measurements of HER2 status may facilitate the development of more personalized treatment strategies for patients with metastatic breast cancer.

A novel proximity assay for the detection of proteins and protein complexes: quantitation of HER1 and HER2 total protein expression and homodimerization in formalin-fixed, paraffin-embedded cell lines and breast cancer tissue.

The availability of drugs targeting the EGFR/HER/erbB signaling pathway has created a need for diagnostics that accurately predict treatment responses. We have developed and characterized a novel assay to provide sensitive and quantitative measures of HER proteins and homodimers in formalin-fixed, paraffin-embedded (FFPE) cell lines and breast tumor tissues, to test these variables. In the VeraTag assay, HER proteins and homodimers are detected through the release of fluorescent tags conjugated to specific HER antibodies, requiring proximity to a second HER antibody. HER2 protein quantification was normalized to tumor area, and compared to receptor numbers in 12 human tumor cell lines determined by fluorescence-activated cell sorting (FACS), and with HER immunohistochemistry (IHC) test categories and histoscores in cell lines and 170 breast tumors. HER1 and HER2 expression levels determined by the VeraTag assay are proportional to receptor number over more than a 2 log10 range, and HER homodimer levels are consistent with crosslinking and immunoprecipitation results. VeraTag HER2 measurements of breast tumor tissue and cell lines correlate with standard IHC test categories (P<0.001). VeraTag HER2 levels also agree with IHC histoscores at lower HER2 protein levels, but are continuous and overlapping between IHC test categories, extending the dynamic range 5-fold to 10-fold at higher HER2 levels. The VeraTag assay specifically and reproducibly measures HER1 and HER2 protein and homodimers in FFPE tissues. The continuous measure of HER2 protein levels over a broad dynamic range, and the novel HER2 homodimer measure, are presently being assessed as predictive markers for responses to targeted HER2 therapy.