Setting Goals to Reduce Cardiovascular Risk: A Retrospective Chart Review of a Pharmacist-Led Initiative in the Workplace.

Background: Cardiovascular diseases (CVD) are the second leading cause of death in Canada with many modifiable risk factors. Pharmacists at a Canadian university delivered a novel CVD risk management program, which included goal-setting and medication management. Aim: This study aimed to describe what CVD prevention goals are composed of in a workplace CVD risk reduction program, and how might these goals change over time. Methods: A longitudinal, descriptive qualitative study using a retrospective chart review of clinical care plans for 15 patients enrolled in a CVD prevention program. Data across 6 visits were extracted from charts (n = 5413 words) recorded from May 2019-November 2020 and analyzed using quantitative content analysis and descriptive statistics. Results: Behavioural goals were most popular among patients and were more likely to change over the 12-month follow-up period, compared to health measure goals. Behavioural goals included goals around diet, physical activity (PA), smoking, medication, sleep and alcohol; health measure goals centered on weight measures, blood pressure (BP) and blood lipid levels. The most common behavioural goals set by patients were for diet (n = 11) and PA (n = 9). Over time, goals around PA, medication, alcohol and weight were adapted while others were added (e.g. diet) and some only continued. Patients experienced a number of barriers to their goal(s) which informed how they adapted their goal(s). These included environmental limitations (including COVID-19) and work-related time constraints. Conclusions: This study found CVD goal-setting in the pharmacist-led workplace wellness program was complex and evolved over time, with goals added and/or adapted. More detailed qualitative research could provide further insights into the patient-provider goal-setting experience in workplace CVD prevention.

Impact of recreational cannabis legalization on patient self-reporting of cannabis use at a pharmacist-led primary care clinic.

BACKGROUND: The Cannabis Act, introduced in Canada in 2018, legalized the use of recreational cannabis. The impact of the announcement and implementation of this act on patient self-reporting of cannabis use has not been explored. OBJECTIVE: The study objective was to determine if patient self-reported cannabis use increased after the announcement and implementation of legislative changes to legalize recreational cannabis. METHODS: A repeated cross-sectional design was used for a retrospective chart review of patients seen at a pharmacist-led primary care clinic. A convenience sample of patient records was divided into 3 panels, corresponding with the preannouncement (November 1, 2013-October 4, 2015), postannouncement (October 5, 2015-October 16, 2018), and postimplementation (October 17, 2018-October 17, 2019) stages of the legalization of recreational cannabis. Search terms used included cannabis, marijuana, marihuana, recreational drugs, natur∗, medicinal, pot, joint, oil, butter, brownies, edibles, cannabin∗, THC, tetrahydro∗, sativa, and indica (∗ = string wild card). The frequency of reporting use and the number of queries related to cannabis were assessed. The analysis of variance test and Pearson correlation (chi-square) were used to compare the 3 panels. RESULTS: A total of 298 patient charts were included in the analysis. One hundred, 99, and 99 patient charts corresponded with panels 1, 2, and 3, respectively. At each time point, 6%, 8%, and 14% of the patients reported cannabis use (P = 0.03). A statistically significant increase in topical oil use and a decrease in prescription tablet or capsule use between panels 1 and 3 (P = 0.036) were identified. CONCLUSION: This study found an increase in self-reporting of cannabis use across the 3 consecutive panels. The change in the product formulations used may reflect the various products available. Frontline pharmacists are encouraged to initiate conversations regarding cannabis use as part of routine practice.

Third-party Reimbursement of Pharmacist-Led Cardiovascular and Diabetes Preventive Health Services for Workplace Health Initiatives: A Narrative Systematic Review.

OBJECTIVE: To summarize available literature describing third-party payer reimbursement models for pharmacist-led preventive health services as part of workplace health initiatives. METHODS: A combination of search terms related to pharmacists, preventive health, and third-party reimbursement were searched in MEDLINE, EMBASE, and PubMed. Included studies described community pharmacist-led cardiovascular and diabetes preventive health service to employees older than 18 years of age as part of a workplace health program with corresponding third-party reimbursement models. Programs that were reimbursed by government resources or studies lacking reimbursement model details were excluded. One reviewer performed level 1 screening and three reviewers analyzed included studies. RESULTS: The search criteria yielded 863 results. Sixteen articles were reviewed after level 1 screening and 13 were ineligible and excluded. Three studies with varying quality of reporting were included. Reimbursement models varied from $40 USD for a 20-minute visit to $391 to $552 USD total per patient with an average of 6 visits per patient. CONCLUSION: There is a lack of quality literature describing third-party reimbursement models for pharmacist-led preventive health services, which hinders the ability to implement a standardized model. High quality studies evaluating the cost of reimbursing pharmacist-led cardiovascular preventive health services compared to the savings to the third-party payer should be performed to inform the standardization of payment models.

Learner Perceptions of a Video Recording Activity for Patient Consultation Training in a Pharmacist-Led Primary Care Clinic Located in an Academic Institution.

Description of the problem: Development of evidence-based educational activities is needed to provide educators with the tools to aid learners in strengthening patient consultation skills in the primary care practice setting, an emerging area of practice in Canada. Objective: The objective was to develop an educational activity to bring self-awareness to fourth year pharmacy student and pharmacy resident consultation skills and to determine learner perceptions of this educational activity, including identifying the key areas of skill development that learners found were positively impacted. Description of the innovation: An innovative learning activity utilizing audio-video technology to enable recording and reviewing of learner-led patient consultations was developed and implemented within the University of British Columbia Pharmacists Clinic. Learners had the opportunity to lead 60-minute patient consultations. With patient and learner consent, patient consultations were recorded for learner viewing and self-assessment. Pharmacist preceptors supervised and assessed learner performance. Learners completed an online anonymous survey after the learning activity to evaluate its value. Critical analysis: Between September 2018 and July 2019, eight pharmacy learners, consisting of student pharmacists (5) and post-graduate pharmacy residents (3) completed the learning activity and provided their feedback. The majority of learners (87.5%) felt the learning activity was beneficial to the development of patient consultation skills. Learners gained awareness of areas requiring improvement which included appropriate questioning, clear and concise language, time management and non-verbal habits. Next steps: Adapting and modifying this learning activity to align with specific practice settings and learning objectives is feasible for other primary care practice sites offering experiential practicums.

Pharmacist-led cardiovascular risk prevention in Western Canada: a qualitative study.

OBJECTIVES: Preventing cardiovascular diseases (CVD) is a public health and policy priority, including for employers. A novel CVD risk management programme that included medication management was delivered by pharmacists to employees of a Canadian university. This qualitative study describes the experiences and perceptions of participants who received individual health consultations in this programme. METHODS: A qualitative study design using free-text responses was adopted. Data (5658 words) came from evaluation surveys completed by 119 programme participants were iteratively coded and thematically analysed. KEY FINDINGS: We identified four themes characterising participant experiences of pharmacist-led CVD prevention. Theme one was labelled self-efficacy because personalised health information and advice on CVD risk factor management empowered participants to make improvements for their health. Participants expressed a range of positive responses about the longer consultations, supportive communication and safe setting of their pharmacist-led encounters; hence, Theme two is labelled pharmacists' interpersonal skills. The wider context of the programme included a number of enabling factors (Theme three) that either supported or limited participant engagement in the programme. A number of changes to behaviour and health measures were identified and participant suggestions to expand and continue the programme further contributed to perceptions of positive programme impact (Theme four). CONCLUSIONS: This study raises questions about how external resources and broader determinants might enable, or hinder, future programme success and sustainability. It also highlights the need for greater understanding and communication of the importance of primary prevention and the role of pharmacists in CVD risk reduction and workplace health promotion.

Atrial fibrillation patients' experiences and perspectives of anticoagulation therapy changes.

BACKGROUND: Atrial fibrillation (AF) patients' experiences with changes in their oral anticoagulant (OAC) therapy are understudied. OBJECTIVE: The objective of this study was to qualitatively describe AF patients' experiences and perspectives of changes made to their OAC therapy (switches or discontinuations). METHODS: A thematic analysis was performed on systematically-collected qualitative data from AF patients who experienced a therapy change (switching or discontinuing an OAC) as part of their participation in a large 2-year prospective observational study. RESULTS: A total of 56 participants met the inclusion criteria. Six themes emerged from the data: 1. reasons for switch or discontinuation of therapy, 2. attitudes towards changes in therapy attributes, 3. challenges with taking medications after therapy change, 4. relief from perceived burden of medication after discontinuation, 5. patients' limited involvement in decision-making, and 6. inadequate education and follow up. Patients were found to request changes in therapy based on their subjective experience with it (rather than clinically justified reasons). They were found to have limited knowledge about their medications, differing reactions to changes in their therapy attributes after a switch, an overall negative attitude towards taking medications, adherence challenges after switching from once daily to twice daily medication, feelings of being excluded from the decision-making process about their therapy changes and feelings of being unsupported after these changes. CONCLUSIONS: There are clear opportunities to improve patients' experiences with OAC therapy changes through improved shared decision-making and patient education/counselling.

Assessment of Burnout among Canadian Pharmacists Working in Team-based Primary Care Settings.

BACKGROUND: Burnout syndrome is well-documented among healthcare professionals across various practice settings. There has been recent expansion of Canadian pharmacists into team-based primary care and burnout in this setting has not been assessed. Our objective was to assess workplace burnout and to identify factors that play a role in perpetuating or diminishing it. METHOD: An online survey to assess burnout was developed using the Maslach Burnout Inventory (MBI) tool and questions regarding pharmacist background and practice. Invitations to complete the survey were sent to Canadian pharmacists working in team-based primary care settings on November 26, 2019 via a national primary healthcare listserv. RESULTS: A total of 31/433 completed responses were collected. The main analysis focused on the personal accomplishment (PA) domain as it had an adequate response rate. The PA domain had a median score of 5.0 (95% CI 4.69-5.22). We compared medians of the PA domain across different groups of each categorical variable. We found that the number of years working in primary care settings was positively associated with a higher PA domain score (p= 0.029). DISCUSSION: PA was higher in pharmacists who have been practicing in a primary care setting for longer; however, burnout rates could not be properly assessed due to the limited response rate. CONCLUSION: This is the first study to assess burnout among Canadian team-based primary care pharmacists. Personal accomplishment was higher in those who have been practicing in a primary care setting for longer. Future studies should consider alternate methods to evaluate burnout in this population.

Evaluating the Effect of a Patient Decision Aid for Atrial Fibrillation Stroke Prevention Therapy.

BACKGROUND: Stroke prevention therapy decisions for patients with atrial fibrillation (AF) are complex and require trade-offs, but few validated patient decision aids (PDAs) are available to facilitate shared decision making. OBJECTIVE: To evaluate the effects of a novel PDA on decision-making parameters for AF patients choosing stroke prevention therapy. METHODS: We developed an evidence-based individualized online AF PDA for stroke prevention therapy and evaluated it in a prospective observational pilot study. The primary outcome was decisional conflict. Secondary outcomes were knowledge, usability/acceptability, patient preferences, effects on therapy choices, and participant feedback. RESULTS: 37 participants completed the PDA. The PDA could be completed independently and was well accepted. It significantly decreased the mean decisional conflict score ( P < 0.001) and all its subscales and increased participant AF knowledge ( P = 0.02). 76% of participants indicated that their individualized therapy attribute ranking was congruent with their values. The PDA-generated best-match therapy was chosen by 70% of participants in decision 1 (no therapy, aspirin, or oral anticoagulant), and 17% for decision 2 (choice of anticoagulant). Among AF patients, 60% chose a different drug than that currently prescribed to them. Conclusion and Relevance: Our PDA was effective for reducing decisional conflict, increasing patient knowledge, eliciting patients' values, and presenting therapy options that aligned with patients' values and preferences. Using the PDA revealed that many patients have therapy preferences different from their currently prescribed treatment. The PDA is a practical and potentially valuable tool to facilitate decision making about stroke prevention therapy for AF.

Cardiovascular Risk Reduction in the Workplace With CAMMPUS (Cardiovascular Assessment and Medication Management by Pharmacists at the UBC Site).

BACKGROUND: Cardiovascular (CV) disease is a leading cause of death despite being largely preventable. Employers increasingly offer preventive health programs in the workplace, and pharmacists are well suited to provide these programs. OBJECTIVE: To evaluate the impact of a pharmacist-led service on CV risk in University of British Columbia (UBC) employees. METHODS: This was a prospective observational pre-and-post design study, with participants as their own controls. Employees >18 years of age in the UBC health plan with a Framingham Risk Score (FRS) ≥10% or ≥1 medication-modifiable CV risk factor were included. Participants received a baseline assessment, individualized consultation for 12 months, and a final assessment by a pharmacist at the UBC Pharmacists Clinic. The primary end point was FRS reduction. RESULTS: Baseline assessment of 512 participants between September 2015 and October 2016 yielded 207 (40%) participants, of whom 178 (86%) completed the 12-month intervention. Participants were 54% female and 55% Caucasian, with an average age of 51 (SD = 9.1) years. FRS at baseline was <10 in 45.8%, 10 to 19.9 in 37.9%, and ≥20 in 16.4% of participants. Over 12 months, significant reductions in average FRS (from 11.7 [SD = 7.7] to 10.7 [SD = 7.3]; P = 0.0017) and other parameters were observed. Significant improvements in quality of life (EQ5D change of 0.031 [95% CI = 0.001, 0.062] P = 0.023) and medication adherence (MMAS-8 change of 0.42 [ P = 0.019]) were also noted. CONCLUSIONS AND RELEVANCE: UBC employees had improvements in health markers, self-reported quality of life, and medication adherence after receiving a 12-month pharmacist-led intervention. Pharmacists are encouraged to provide CV risk reduction services in workplaces.

Induction of autophagy is an early response to gefitinib and a potential therapeutic target in breast cancer.

Gefitinib (Iressa(®), ZD1839) is a small molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. We report on an early cellular response to gefitinib that involves induction of functional autophagic flux in phenotypically diverse breast cancer cells that were sensitive (BT474 and SKBR3) or insensitive (MCF7-GFPLC3 and JIMT-1) to gefitinib. Our data show that elevation of autophagy in gefitinib-treated breast cancer cells correlated with downregulation of AKT and ERK1/2 signaling early in the course of treatment. Inhibition of autophagosome formation by BECLIN-1 or ATG7 siRNA in combination with gefitinib reduced the abundance of autophagic organelles and sensitized SKBR3 but not MCF7-GFPLC3 cells to cell death. However, inhibition of the late stage of gefitinib-induced autophagy with hydroxychloroquine (HCQ) or bafilomycin A1 significantly increased (p<0.05) cell death in gefitinib-sensitive SKBR3 and BT474 cells, as well as in gefitinib-insensitive JIMT-1 and MCF7-GFPLC3 cells, relative to the effects observed with the respective single agents. Treatment with the combination of gefitinib and HCQ was more effective (p<0.05) in delaying tumor growth than either monotherapy (p>0.05), when compared to vehicle-treated controls. Our results also show that elevated autophagosome content following short-term treatment with gefitinib is a reversible response that ceases upon removal of the drug. In aggregate, these data demonstrate that elevated autophagic flux is an early response to gefitinib and that targeting EGFR and autophagy should be considered when developing new therapeutic strategies for EGFR expressing breast cancers.

The combination of gefitinib and RAD001 inhibits growth of HER2 overexpressing breast cancer cells and tumors irrespective of trastuzumab sensitivity.

BACKGROUND: HER2-positive breast cancers exhibit high rates of innate and acquired resistance to trastuzumab (TZ), a HER2-directed antibody used as a first line treatment for this disease. TZ resistance may in part be mediated by frequent co-expression of EGFR and by sustained activation of the mammalian target of rapamycin (mTOR) pathway. Here, we assessed feasibility of combining the EGFR inhibitor gefitinib and the mTOR inhibitor everolimus (RAD001) for treating HER2 overexpressing breast cancers with different sensitivity to TZ. METHODS: The gefitinib and RAD001 combination was broadly evaluated in TZ sensitive (SKBR3 and MCF7-HER2) and TZ resistant (JIMT-1) breast cancer models. The effects on cell growth were measured in cell based assays using the fixed molar ratio design and the median effect principle. In vivo studies were performed in Rag2M mice bearing established tumors. Analysis of cell cycle, changes in targeted signaling pathways and tumor characteristics were conducted to assess gefitinib and RAD001 interactions. RESULTS: The gefitinib and RAD001 combination inhibited cell growth in vitro in a synergistic fashion as defined by the Chou and Talalay median effect principle and increased tumor xenograft growth delay. The improvement in therapeutic efficacy by the combination was associated in vitro with cell line dependent increases in cytotoxicity and cytostasis while treatment in vivo promoted cytostasis. The most striking and consistent therapeutic effect of the combination was increased inhibition of the mTOR pathway (in vitro and in vivo) and EGFR signaling in vivo relative to the single drugs. CONCLUSIONS: The gefitinib and RAD001 combination provides effective control over growth of HER2 overexpressing cells and tumors irrespective of the TZ sensitivity status.

Multivalent rituximab lipid nanoparticles as improved lymphoma therapies: indirect mechanisms of action and in vivo activity.

AIMS: The activity of therapeutic antibodies can be enhanced by creating multivalent constructs, such as antibody lipid nanoparticles (LNPs). Here, we examine differences between rituximab (Ritux) and Ritux-LNPs in terms of their indirect mechanisms of action: complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). MATERIALS & METHODS: We employed two mantle-cell lymphoma cell lines, Z138 and JVM2, which exhibit different in vivo sensitivities to Ritux along with variable expression levels of cell-surface proteins that regulate ADCC and CDC. RESULTS: In both cell lines, CDC and ADCC were found to be significantly enhanced after treatment with Ritux-LNPs compared with Ritux. In vivo efficacy studies, however, suggested that the therapeutic activities of Ritux and Ritux-LNPs were equivalent, which was subsequently explained in part by pharmacokinetic studies indicating rapid elimination of Ritux-LNP. CONCLUSION: Although indirect and direct mechanisms of multivalent Ritux are enhanced, its further development requires methods to improve its circulation lifetime.

Development of a liposomal nanoparticle formulation of 5-fluorouracil for parenteral administration: formulation design, pharmacokinetics and efficacy.

5-Fluorouracil (5-FU) is a small, very membrane permeable drug that is poorly retained within the aqueous compartment of liposomal nanoparticles (LNP). To address this problem a novel method relying on formation of a ternary complex comprising copper, low molecular weight polyethylenimine (PEI) and 5-FU has been developed. More specifically, in the presence of entrapped copper and PEI, externally added 5-FU can be efficiently encapsulated (>95%) in DSPC/Chol (1,2-Distearoyl-sn-Glycero-3-Phosphocholine/cholesterol; 55:45 mol%) liposomes (130-170 nm) to achieve drug-to-lipid ratios of 0.1 (mol:mol). Drug release studies completed using this LNP formulation of 5-FU demonstrated significant improvements in drug retention in vitro and in vivo. Plasma concentrations of 5-FU were 7- to 23-fold higher when the drug was administered intravenously to mice as the LNP 5-FU formulation compared to free 5-FU. Further, the therapeutic effects of the LNP 5-FU formulation, as determined in a HT-29 subcutaneous colorectal cancer model where treatment was given QDx5, was greater than that which could be achieved with free 5-FU when compared at equivalent doses. This is the first time an active loading method has been described for 5-FU. The use of ternary metal complexation strategy to encapsulate therapeutic agents may define a unique platform for preparation of LNP drug formulations.

Silencing Bcl-2 in models of mantle cell lymphoma is associated with decreases in cyclin D1, nuclear factor-kappaB, p53, bax, and p27 levels.

Molecular mechanisms responsible for lymphoma resistance to apoptosis often involve the bcl-2 pathway. In this study, we investigated the cell signaling pathways activated in bcl-2-overexpressing human mantle cell lymphoma cell lines (JVM-2 and Z-138) that have been treated with oblimersen, a molecular gene silencing strategy that effectively suppresses bcl-2 in vitro and in vivo. Z-138 cells expressed higher levels of bcl-2 and were more sensitive to the effects of bcl-2 silencing, mediated by oblimersen or bcl-2 small interfering RNA, in vitro. Tumors derived following injection of Z-138 cells were sensitive to oblimersen as judged by decreases in tumor growth rate and decreases in cell proliferation (as measured by Ki-67). Immunohistochemistry and Western blot analysis of oblimersen-treated Z-138 tumors revealed a dose-dependent decrease in bcl-2 levels and an associated increase in the proapoptotic proteins caspase-3 and caspase-9. Silencing bcl-2 in Z-138 xenografts revealed an associated dose-dependent suppression of bax, a decrease in nuclear factor-kappaB and phospho-nuclear factor-kappaB, and transient loss of p53 levels. Coimmunoprecipitation studies suggest that the latter observation is mediated by an association between bcl-2 and phospho-mdm2. Bcl-2 silencing also led to p27 down-regulation and coimmunoprecipitation studies point to a role for bcl-2 in regulation of p27 localization/degradation. Bcl-2 silencing was also correlated with loss of cyclin D1a protein levels but not cyclin D1b levels. Coimmunoprecipitation studies indicate that bcl-2 may mediate its effects on cyclin D1a via interaction with p38 mitogen-activated protein kinase as well as a previously unreported interaction between bcl-2 and cyclin D1a.

Modulation of cancer cell survival pathways using multivalent liposomal therapeutic antibody constructs.

Various methods have been explored to enhance antibody-based cancer therapy. The use of multivalent antibodies or fragments against tumor antigens has generated a great deal of interest, as various cellular signals, including induction of apoptosis, inhibition of cell growth/survival, or internalization of the surface molecules, can be triggered or enhanced on extensive cross-linking of the target/antibody complex by the multivalent form of the antibody. The goal of the studies reported here was to develop multivalent antibody constructs via grafting of antibody molecules onto liposome membranes to enhance antibody activity. Using trastuzumab and rituximab as examples, up to a 25-fold increase in the antibody potency in cell viability assay was observed when the antibodies were presented in the multivalent liposome formulation. Key cell survival signaling molecules, such as phosphorylated Akt and phosphorylated p65 nuclear factor-kappaB, were down-regulated on treatment with multivalent liposomal trastuzumab and liposomal rituximab, respectively. Potent in vivo antitumor activity was shown for liposomal trastuzumab. The data presented here showed the potential of liposome technology to enhance the therapeutic effect of antibodies via a mechanism that modulates cell survival through clustering of the target/antibody complex.

Cytogenetic characterization and gene expression profiling of the trastuzumab-resistant breast cancer cell line JIMT-1.

Resistance to the HER-2 targeting drug trastuzumab can be observed clinically, but the lack of suitable experimental models hampers studies of resistance mechanisms. We characterized a HER-2-positive carcinoma cell line (JIMT-1) derived from a 62-year-old breast cancer patient which was clinically resistant to trastuzumab. Multicolor fluorescence in situ hybridization revealed a complex hyperdiploid karyotype with numerous marker chromosomes and unbalanced translocations. Comparative genomic hybridization (CGH) revealed numerous regions of copy number aberration (CNA). Further analysis by array CGH identified 27 regions of CNA (16 amplified, 11 deleted). Thirty-eight percent of the genes in the amplified regions were overexpressed, compared to only 9% in regions of normal copy number ratios (CNR). Accordingly, 26% of the genes in the deleted regions were underexpressed, compared to 10% in regions of normal CNR. Most amplified and overexpressed genes were located on chromosome 1 as well as on 8q, 12q14.1, 17q11 approximately q21, and 20q13. In 17q11 approximately q21, we identified two separate amplicons, the HER-2 amplicon and a previously unreported amplicon at 17q21.31. Several aberrant genes are implicated in cancer development (e.g., JUN, CDK4, and SLUG protooncogenes, as well as the drug/hormone-metabolizing genes GSTM1 and CYP24). We conclude that cytogenetic and expression profiling of JIMT-1 revealed several new features that need further characterization and may shed light on trastuzumab resistance.

Decreased accessibility and lack of activation of ErbB2 in JIMT-1, a herceptin-resistant, MUC4-expressing breast cancer cell line.

Overexpression of erbB2 in breast tumors is associated with poor prognosis and is a target of receptor-oriented cancer therapy. Trastuzumab (Herceptin), a monoclonal antibody against a membrane-proximal epitope in the extracellular region of erbB2, shows a therapeutic effect against a fraction of erbB2-amplified breast tumors. Unfortunately, resistance to Herceptin is common, and its cause is as yet unclear. Here we investigated the properties of erbB2 in a Herceptin-resistant cell line, JIMT-1, established from a breast cancer patient showing erbB2 gene amplification and primary resistance to Herceptin. The expression profile of erbB proteins, Herceptin-induced erbB2 internalization, and down-regulation in JIMT-1 were similar to those in Herceptin-sensitive lines. However, the mean number of Herceptin Mab binding sites in JIMT-1 was 1/5 that of the expressed erbB2 molecules, although 5% to 10% of the cells showed a approximately 10-fold higher Herceptin binding than the main population. Herceptin Fab and Mab 2C4, an antibody binding to an epitope in the ectodomain further removed from the membrane, bound more efficiently to JIMT-1 cells than Herceptin Mab, implying that erbB2 was partly masked. The expression of MUC4, a membrane-associated mucin that according to reports contributes to the masking of membrane proteins, was higher in JIMT-1 than in Herceptin-sensitive lines, and its level was inversely correlated with the Herceptin binding capacity of single cells. Knockdown of MUC4 expression by RNA interference increased the binding of Herceptin. Western blotting showed a low level of proteolytic processing, shedding, and tyrosine phosphorylation of erbB2 in JIMT-1. The latter finding may explain its Herceptin-resistant phenotype characterizing both the low and high Herceptin binding subpopulations. We conclude that masking of erbB2 in JIMT-1 leads to diminished Herceptin binding and isolation of erbB2 from its normal interaction and activation partners.

Characterization of a novel cell line established from a patient with Herceptin-resistant breast cancer.

Clinical resistance to the HER-2 oncogene-targeting drug trastuzumab (Herceptin) exists, but studies of the resistance mechanisms are hampered by the lack of suitable experimental model systems. We established a carcinoma cell line (designated JIMT-1) from a pleural metastasis of a 62-year old patient with breast cancer who was clinically resistant to trastuzumab. JIMT-1 cells grow as an adherent monolayer and form xenograft tumors in nude mice. JIMT-1 cells have an amplified HER-2 oncogene, which showed no identifiable mutations in its coding sequence. JIMT-1 cells overexpress HER-2 mRNA and protein, and the levels of HER-1, HER-3, and HER-4 mRNA and protein were similar to the trastuzumab-sensitive cell line SKBR-3. The cell line lacks expression of hormone receptors (estrogen receptors and progesterone receptors) and is phenotypically of epithelial progenitor cell origin, as evidenced by immunohistochemical positivity for both cytokeratins 5/14 and 8/18. JIMT-1 cells were insensitive to trastuzumab and another HER-2-inhibiting drug, pertuzumab (2C4), in vitro and in xenograft tumors. Small molecule tyrosine kinase inhibitors Ci1033 and ZD1839 inhibited the JIMT-1 cell growth but to a lesser degree than in trastuzumab-sensitive BT-474 cells. The lack of growth inhibition was rationalized by the unaltered Akt phosphorylation in JIMT-1 cells. Erk1/2 phosphorylation was slightly reduced but still evident in JIMT-1 cells. We conclude that the JIMT-1 cell line provides a valuable experimental model for studies of new trastuzumab-resistance mechanisms.