Trastuzumab-induced upregulation of a protein set in extracellular vesicles emitted by ErbB2-positive breast cancer cells correlates with their trastuzumab sensitivity.

BACKGROUND: ErbB2/HER2 oncoprotein often drives breast cancers (BCs) which are treated with the anti-ErbB2 antibody trastuzumab. The efficacy of trastuzumab-based metastatic BC therapies is routinely assessed by imaging studies. Trastuzumab typically becomes ineffective in the case of this disease and is then replaced by other drugs. Biomarkers of BC trastuzumab response could allow imaging studies and the switch to other drugs to occur earlier than is now possible. Moreover, bone-only BC metastases can be hard to measure, and biomarkers of their trastuzumab response could facilitate further treatment decisions. Such biomarkers are presently unavailable. In this study, we searched for proteins whose levels in BC cell-emitted extracellular vesicles (EVs) potentially correlate with BC trastuzumab sensitivity. METHODS: We isolated EVs from cultured trastuzumab-sensitive and trastuzumab-resistant human BC cells before and after trastuzumab treatment and characterized these EVs by nanoparticle tracking analysis and electron microscopy. We found previously that ErbB2 drives BC by downregulating a pro-apoptotic protein PERP. We now tested whether trastuzumab-induced PERP upregulation in EVs emitted by cultured human BC cells correlates with their trastuzumab sensitivity. We also used mass spectrometry to search for additional proteins whose levels in such EVs reflect BC cell trastuzumab sensitivity. Once we identified proteins whose EV levels correlate with this sensitivity in culture, we explored the feasibility of testing whether their levels in the blood EVs of trastuzumab-treated metastatic BC patients correlate with patients' response to trastuzumab-based treatments. RESULTS: We found that neither trastuzumab nor acquisition of trastuzumab resistance by BC cells affects the size or morphology of EVs emitted by cultured BC cells. We established that EV levels of proteins PERP, GNAS2, GNA13, ITB1, and RAB10 correlate with BC cell trastuzumab response. Moreover, these proteins were upregulated during trastuzumab-based therapies in the blood EVs of a pilot cohort of metastatic BC patients that benefited from these therapies but not in those derived from patients that failed such treatments. CONCLUSIONS: Upregulation of a protein set in EVs derived from cultured breast tumor cells correlates with tumor cell trastuzumab sensitivity. It is feasible to further evaluate these proteins as biomarkers of metastatic BC trastuzumab response.

Correction to: Trastuzumab-induced upregulation of a protein set in extracellular vesicles emitted by ErbB2-positive breast cancer cells correlates with their trastuzumab sensitivity.

An amendment to this paper has been published and can be accessed via the original article.

Trastuzumab beyond progression for HER2 positive metastatic breast cancer: progression-free survival on first-line therapy predicts overall survival impact.

Trastuzumab beyond first progression in the metastatic setting has been adopted based on limited data suggesting improved outcomes compared to second-line chemotherapy alone although predictive factors for preferential benefit remain elusive. We conducted a retrospective review of all patients receiving trastuzumab for HER2 + metastatic disease between Jan 1, 1999-June 15, 2011. Univariate and time to event analyses described treatment and survival patterns. Median duration of each line of therapy and overall survival times for covariates, including treatment era (pre versus post Jan 1, 2005), lines of trastuzumab-based therapy (1 versus 2 versus 3 + ), first-line chemotherapy partner (docetaxel/paclitaxel versus other) and median exposure to first-line trastuzumab-based therapy (=/> versus < cohort median) were estimated. A total of 119 patients received a median of two lines of trastuzumab-based therapy (range 1-8). Median overall survival was 21.8 months (95% CI = 14.5-27.1 m), by era was 15.6 m (95% CI = 9.7-24.8 m) versus 26.1 m (95% CI = 20.0-39.3 m; p = 0.11) and by lines of trastuzumab-based therapy received was 10.6 m (95% CI = 5.3-17.4 m) versus 13.9 m (95% CI = 9.5-27.6 m) versus 32.5 m (95% CI = 25-49.4 m) (p = 0.0014). Median overall survival was significantly longer for those receiving taxanes with trastuzumab compared to other first line partners (26.1 m, 95% CI = 17.8-31.4 m versus 14.5 m, 95% CI = 9.4-21.9 m, p = 0.02). Median overall survival with duration of first-line trastuzumab-based therapy =/> cohort median was 31.9 m (95% CI = 26.2-52.2 m) versus 10.3 m for shorter durations (95% CI = 6.9-15.6 m; p < 0.0001). Our observations support progression-free survival on first-line trastuzumab-based therapy as a clinically relevant predictive factor for overall survival benefit with the adoption of a trastuzumab beyond progression treatment strategy.

A large-scale, targeted metabolomics method for the analysis and quantification of metabolites in human plasma via liquid chromatography-mass spectrometry.

Metabolomics is the study of small molecules, primarily metabolites, that are produced during metabolic processes. Analysis of the composition of an organism's metabolome can yield useful information about an individual's health status at any given time. In recent years, the development of large-scale, targeted metabolomic methods has allowed for the analysis of biological samples using analytical techniques such as LC-MS/MS. This paper presents a large-scale metabolomics method for analysis of biological samples, with a focus on quantification of metabolites found in blood plasma. The method comprises a 10-min chromatographic separation using HILIC and RP stationary phases combined with positive and negative electrospray ionization in order to maximize metabolome coverage. Complete analysis of a single sample can be achieved in as little as 40 min using the two columns and dual modes of ionization. With 540 metabolites and the inclusion of over 200 analytical standards, this method is comprehensive and quantitatively robust when compared to current targeted metabolomics methods. This study uses a large-scale evaluation of metabolite recovery from plasma that enables absolute quantification of metabolites by correcting for analyte loss throughout processes such as extraction, handling, or storage. In addition, the method was applied to plasma collected from adjuvant breast cancer patients to confirm the suitability of the method to clinical samples.

Profiling non-small cell lung cancer reveals that PD-L1 is associated with wild type EGFR and vascular invasion, and immunohistochemistry quantification of PD-L1 correlates weakly with RT-qPCR.

Most lung cancer patients are diagnosed at an advanced stage, limiting their treatment options with very low response rate. Lung cancer is the most common cause of cancer death worldwide. Therapies that target driver gene mutations (e.g. EGFR, ALK, ROS1) and checkpoint inhibitors such anti-PD-1 and PD-L1 immunotherapies are being used to treat lung cancer patients. Identification of correlations between driver mutations and PD-L1 expression will allow for the best management of patient treatment. 851 cases of non-small cell lung cancer cases were profiled for the presence of biomarkers EGFR, KRAS, BRAF, and PIK3CA mutations by SNaPshot/sizing genotyping. Immunohistochemistry was used to identify the protein expression of ALK and PD-L1. Total PD-L1 mRNA expression (from unsorted tumor samples) was quantified by RT-qPCR in a sub-group of the cohort to assess its correlation with PD-L1 protein level in tumor cells. Statistical analysis revealed correlations between the presence of the mutations, PD-L1 expression, and the pathological data. Specifically, increased PD-L1 expression was associated with wildtype EGFR and vascular invasion, and total PD-L1 mRNA levels correlated weakly with protein expression on tumor cells. These data provide insights into driver gene mutations and immune checkpoint status in relation to lung cancer subtypes and suggest that RT-qPCR is useful for assessing PD-L1 levels.

Comparative performance of different methods for circulating tumor cell enrichment in metastatic breast cancer patients.

The isolation and analysis of circulating tumor cells (CTC) has the potential to provide minimally invasive diagnostic, prognostic and predictive information. Widespread clinical implementation of CTC analysis has been hampered by a lack of comparative investigation between different analytic methodologies in clinically relevant settings. The objective of this study was to evaluate four different CTC isolation techniques-those that rely on surface antigen expression (EpCAM or CD45 using DynaBeads® or EasySep™ systems) or the biophysical properties (RosetteSep™ or ScreenCell®) of CTCs. These were evaluated using cultured cells in order to calculate isolation efficiency at various levels including; inter-assay and inter-operator variability, protocol complexity and turn-around time. All four techniques were adequate at levels above 100 cells/mL which is commonly used for the evaluation of new isolation techniques. Only the RosetteSep™ and ScreenCell® techniques were found to provide adequate sensitivity at a level of 10 cells/mL. These techniques were then applied to the isolation and analysis of circulating tumor cells blood drawn from metastatic breast cancer patients where CTCs were detected in 54% (15/28) of MBC patients using the RosetteSep™ and 75% (6/8) with ScreenCell®. Overall, the ScreenCell® method had better sensitivity.

Molecular profiling of non-small cell lung cancer.

Lung cancer is generally treated with conventional therapies, including chemotherapy and radiation. These methods, however, are not specific to cancer cells and instead attack every cell present, including normal cells. Personalized therapies provide more efficient treatment options as they target the individual's genetic makeup. The goal of this study was to identify the frequency of causal genetic mutations across a variety of lung cancer subtypes in the earlier stages. 833 samples of non-small cell lung cancer from 799 patients who received resection of their lung cancer, were selected for molecular analysis of six known mutations, including EGFR, KRAS, BRAF, PIK3CA, HER2 and ALK. A SNaPshot assay was used for point mutations and fragment analysis searched for insertions and deletions. ALK was evaluated by IHC +/- FISH. Statistical analysis was performed to determine correlations between molecular and clinical/pathological patient data. None of the tested variants were identified in most (66.15%) of cases. The observed frequencies among the total samples vs. only the adenocarcinoma cases were notable different, with the highest frequency being the KRAS mutation (24.49% vs. 35.55%), followed by EGFR (6.96% vs. 10.23%), PIK3CA (1.20% vs. 0.9%), BRAF (1.08% vs. 1.62%), ALK (0.12% vs. 0.18%), while the lowest was the HER2 mutation (0% for both). The statistical analysis yielded correlations between presence of a mutation with gender, cancer type, vascular invasion and smoking history. The outcome of this study will provide data that helps stratify patient prognosis and supports development of more precise treatments, resulting in improved outcomes for future lung cancer patients.

The utility of BRAFV600E mutation-specific antibody for colon cancers with microsatellite instability.

This study's objective was to assess the performance of immunohistochemical staining with the BRAF mutation-specific antibody (clone VE1) in tissue from colon cancers, including those with a high degree of microsatellite instability (MSI-H). VE1 was applied to tissue microarrays of 152 colon cancers with known MSI status. Results of immunohistochemical analyses were scored as negative, positive, or equivocal. The results of VE1 immunohistochemical analysis were compared with BRAF mutation analysis by PCR. Fifteen of the 152 cases (10%) were positive with VE1 immunohistochemical analysis, 8 were equivocal, and 129 were negative. There was a single false-negative case and no false positives were identified when 74 VE1-positive or VE1-negative cases were tested by the BRAF PCR testing. Of the 8 equivocal VE1 cases identified, 3 were BRAF-positive. In the 17 MSI-H colon cancers, VE1 immunohistochemical analysis resulted in 7 true-positive, 9 true-negative, and 1 false-negative case when compared with PCR results. The sensitivity and specificity of VE1 in the MSI-H colon cancer group were determined to be 88% and 100%, respectively. The BRAF positivity rate by VE1 immunohistochemical analysis in MSI-H colon cancers is consistent with that of published cohorts, which use molecular assays, and the accuracy of positive or negative VE1 staining is high. A small subset of equivocal cases (5% in our cohort) with heterogenous staining requires confirmation by the BRAF mutation analysis. We propose a testing algorithm for Lynch syndrome screening in MSI-H colorectal cancers that incorporates VE1 immunohistochemical analysis with PCR testing for equivocal cases.

Ephrin b2 receptor and microsatellite status in lymph node-positive colon cancer survival.

BACKGROUND: Ephrin B2 receptor (EphB2) is a target of the canonical wnt pathway implicated in colorectal carcinogenesis, and its down-regulation may be associated with adverse prognosis. We evaluated its prognostic value in resected colon cancer stratified by microsatellite status and other clinicopathologic characteristics. METHODS: We identified all cases of resected stage III colon cancer from 1995 to 2009 managed in the Capital Health district of Nova Scotia. Tissue microarrays were constructed and immunohistochemistry (IHC) for tumor EphB2 staining assigned into quartiles. Microsatellite status was evaluated by IHC for MutL homolog 1 (MLH1) and MutS homolog 2 (MSH2). Microsatellite stable tumors were defined as both MLH1/MSH2 (+/+); tumors staining otherwise were classified with microsatellite instability (MSI-H). Primary and secondary outcomes were disease-free survival (DFS) and overall survival (OS), respectively. RESULTS: We identified 159 cases with sufficient tissue for microarray analysis having a median follow-up of 3.47 years (range, 0.14-14). Median age was 61, 52% were male, 40% had an event, and 29% died. MSI-H was present in 18 (13%). Univariate analysis of EphB2 expression on DFS and OS showed a hazard ratio (HR) of 2.00 (P = .01) and 2.14 (P = .03), respectively. Multivariate analysis of EphB2 expression on DFS and OS showed an HR of 2.24 and 2.23, respectively, with tumor IHC ≤ 50%. CONCLUSIONS: In this cohort, decreased EphB2 expression was an independent prognostic factor for recurrence and death and may have prognostic relevance in tumors with MSI-H. However, this would require prospective validation in a larger study.

Microsatellite instability status does not predict total lymph node or negative lymph node retrieval in stage III colon cancer.

The relationship between higher total lymph node resection number in colorectal cancer resection specimens and improved overall survival is well known. Recent studies describe an association between a high rate of microsatellite instability and a high total lymph node count in colorectal cancer. Higher lymph node retrieval may potentially explain the improved survival seen in cancers with microsatellite instability. We investigate whether these associations can be validated in a cohort of American Joint Committee on Cancer stage III colon cancers. Medical records from 200 cases of stage III colon cancer resection specimens were reviewed, and sufficient tissue was available for 168. Expression of DNA mismatch repair proteins was determined by immunohistochemistry, and microsatellite status, by polymerase chain reaction. The mean total lymph node count in cases with microsatellite instability versus microsatellite stable tumors (15.9 versus 16.9; P = .664) and the mean number of negative lymph nodes in each respective category (12.2 versus 13.6; P = .522) were not significantly different. There was no difference between microsatellite stable cases and cases with microsatellite instability when total lymph node counts (P = .953) or negative lymph node counts (P = .381) were analyzed with respect to percentage of cases above and below the medians. This cohort of stage III colon cancers does not support a significant relationship between microsatellite status and a higher retrieval of total or negative lymph nodes. Although microsatellite instability is associated with improved overall survival in our cohort (P = .026), the reason for this does not appear to be related to higher numbers of retrieved lymph nodes.