Analysis of Protein Glycosylation after Rapid Digestion Using Protease-Containing Membranes in Spin Columns.

Glycosylation is an important protein post-translational modification that plays a pivotal role in the bioactivity of therapeutic proteins and in the infectivity of viral proteins. Liquid chromatography with tandem mass spectrometry readily identifies protein glycans with site specificity. However, the overnight incubation used in conventional in-solution proteolysis leads to high turnaround times for glycosylation analysis, particularly when sequential in-solution digestions are needed for site-specific glycan identification. Using bovine fetuin as a model glycoprotein, this work first shows that in-membrane digestion in ∼3 min yields similar glycan identification and quantitation when compared to overnight in-solution digestion. Protease-containing membranes in a spin column enable digestion of therapeutic proteins (trastuzumab and erythropoietin) and a viral protein (SARS-CoV-2 receptor binding domain) in ∼30 s. Glycan identification is similar after in-solution and in-membrane digestion, and limited in-membrane digestion enhances the identification of high-mannose glycans in trastuzumab. Finally, stacked membranes containing trypsin and chymotrypsin allow fast sequential proteolytic digestion to site-specifically identify the glycans of SARS-CoV-2 receptor binding domain. One can easily assemble the protease-containing membranes in commercial spin columns, and spinning multiple columns simultaneously will facilitate parallel analyses.

Quality of care in the course of subcutaneous versus intravenous trastuzumab administration in patients with breast cancer: an integrated time-motion study with mixed-methods research.

OBJECTIVES: This study aimed to compare the time required and concerns raised by various perspectives of participants regarding administering subcutaneous and intravenous trastuzumab for patients with breast cancer (BC). DESIGN: This observational time-motion study design with mixed-methods research (cross-sectional surveys and semistructured interviews) was conducted. The time spent on preparing or administering trastuzumab by different healthcare professionals (HCPs) was recorded. The data were analysed by descriptive/inferential statistical analyses, followed by thematic analyses. SETTING: Outpatient and inpatient administration units of a single medical centre in Taiwan. PARTICIPANTS: The study included patients with early-stage BC who received subcutaneous or intravenous trastuzumab (n=93), and HCPs including two attending physicians, a nurse practitioner, two pharmacists and two nurses. RESULT: Based on the perspectives of patients and HCPs, the subcutaneous form of trastuzumab was more efficient, less expensive and produced less discomfort in outpatient units than inpatient units. More participants preferred the subcutaneous form over the intravenous form in both outpatient and inpatient units. Pharmacists and nurse practitioners spent threefold more time on patients when preparing and administering the intravenous form in both outpatient and inpatient units. The concerns raised by patients and HCPs varied in certain aspects, including the injection skills, speed, mental distress (eg, needle phobia) and pain associated with the subcutaneous form. Almost all patients preferred receiving the subcutaneous form in outpatient units after the initial COVID-19 outbreak. CONCLUSION: Patients with early-stage BC preferred receiving subcutaneous trastuzumab in outpatient units rather than inpatient units or the intravenous form before and after the COVID-19 outbreak. Such findings may serve as real-world evidence to facilitate better quality of care regarding administration of subcutaneous or intravenous trastuzumab in medical settings, and its feasible resolutions to balance the quality, concerns and efficiency of anticancer administration during the COVID-19 pandemic.

Atezolizumab With Neoadjuvant Anti-Human Epidermal Growth Factor Receptor 2 Therapy and Chemotherapy in Human Epidermal Growth Factor Receptor 2-Positive Early Breast Cancer: Primary Results of the Randomized Phase III IMpassion050 Trial.

PURPOSE: Combining standard of care (pertuzumab-trastuzumab [PH], chemotherapy) with cancer immunotherapy may potentiate antitumor immunity, cytotoxic activity, and patient outcomes in high-risk, human epidermal growth factor receptor 2 (HER2)-positive early breast cancer. We report the phase III IMpassion050 primary analysis of neoadjuvant atezolizumab, PH, and chemotherapy in these patients. METHODS: Patients with a primary tumor of > 2 cm and histologically confirmed, positive lymph node status (T2-4, N1-3, M0) were randomly assigned 1:1 to atezolizumab/placebo with dose-dense doxorubicin/cyclophosphamide, followed by paclitaxel, and PH. After surgery, patients were to continue atezolizumab/placebo and PH (total: 1 year of HER2-targeted therapy); those with residual disease could switch to ado-trastuzumab emtansine with atezolizumab/placebo. Coprimary efficacy end points were pathologic complete response (pCR; ypT0/is ypN0) rates in intention-to-treat (ITT) and programmed cell death-ligand 1 (PD-L1)-positive populations. RESULTS: At clinical cutoff (February 5, 2021), pCR rates in the placebo and atezolizumab groups in the ITT populations were 62.7% (n = 143/228) and 62.4% (n = 141/226), respectively (difference -0.33%; 95% CI, -9.2 to 8.6; P = .9551). The pCR rates in the placebo and atezolizumab groups in patients with PD-L1-positive tumors were 72.5% (n = 79/109) and 64.2% (n = 70/109), respectively (difference -8.26%; 95% CI, -20.6 to 4.0; P = .1846). Grade 3-4 and serious adverse events were more frequent in the atezolizumab versus placebo group. Five grade 5 adverse events occurred (four neoadjuvant, one adjuvant; two assigned to study treatment), all with atezolizumab. Overall, the safety profile was consistent with that of atezolizumab in other combination studies. CONCLUSION: Atezolizumab with neoadjuvant dose-dense doxorubicin/cyclophosphamide-paclitaxel and PH for high-risk, HER2-positive early breast cancer did not increase pCR rates versus placebo in the ITT or PD-L1-positive populations. PH and chemotherapy remains standard of care; longer follow-up may help to inform the long-term impact of atezolizumab.

Multidisciplinary clinical guidance on trastuzumab deruxtecan (T-DXd)-related interstitial lung disease/pneumonitis-Focus on proactive monitoring, diagnosis, and management.

Trastuzumab deruxtecan (T-DXd; DS-8201) is an antibody-drug conjugate targeting human epidermal growth factor receptor 2. Interstitial lung disease (ILD)/pneumonitis is an adverse event associated with T-DXd; in most cases, it is low grade (grade ≤ 2) and can be treated effectively but may develop to be fatal in some instances. It is important to increase patient and provider understanding of T-DXd-related ILD/pneumonitis to improve patient outcomes. Drug-related ILD/pneumonitis is a diagnosis of exclusion; other possible causes of lung injury/imaging findings must be ruled out for an accurate diagnosis. Symptoms can be nonspecific, and identifying early symptoms is challenging; therefore, diagnosis is often delayed. We reviewed characteristics of patients who developed T-DXd-related ILD/pneumonitis and its patterns, produced multidisciplinary guidelines on diagnosis and management, and described areas for future investigation. Ongoing studies are collecting data on T-DXd-related ILD/pneumonitis to further our understanding of its clinical patterns and mechanisms. SEARCH STRATEGY AND SELECTION CRITERIA: References were identified based on the guidelines used by the authors in treating interstitial lung disease and pneumonitis. Searches of the authors' own files were also completed. A search of PubMed with the search terms (trastuzumab deruxtecan) AND (interstitial lung disease) AND (guidelines) was conducted on November 1, 2021, with no restrictions based on publication date, and the two articles yielded by the search were included.

A Cohort Study on the Immunogenicity and Safety of the Inactivated SARS-CoV-2 Vaccine (BBIBP-CorV) in Patients With Breast Cancer; Does Trastuzumab Interfere With the Outcome?

Aim: To determine the efficacy and safety of inactivated SARS-CoV-2 vaccine (BBIBP-CorV) in patients with breast cancer. Methods: In this multi- institutional cohort study, a total of 160 breast cancer patients (mean age of 50.01 ± 11.5 years old) were assessed for the SARS-CoV-2 Anti-Spike IgG and SARS-CoV2 Anti RBD IgG by ELISA after two doses of 0.5 mL inactivated, COVID-19 vaccine (BBIBP-CorV). All patients were followed up for three months for clinical COVID-19 infection based on either PCR results or imaging findings. Common Terminology Criteria for Adverse Events were used to assess the side effects. Results: The presence of SARS-CoV-2 anti-spike IgG, SARS-CoV2 anti-RBD IgG, or either of these antibodies was 85.7%, 87.4%, and 93.3%. The prevalence of COVID-19 infection after vaccination was 0.7%, 0% and 0% for the first, second and third months of the follow-up period. The most common local and systemic side-effects were injection site pain and fever which were presented in 22.3% and 24.3% of patients, respectively. Discussion: The inactivated SARS-CoV-2 vaccine (BBIBP-CorV) is a tolerable and effective method to prevent COVID-19.

Hydrophilic rhodamine B-loaded / boronic acid-modified graphene oxide nanocomposite as a substitute of enzyme-labeled second antibody for ultrasensitive detection of antibodies.

Enzyme-labeled secondary antibody is often used to amplify the output signal in the process of antibody detection. However, its preparation process is complex and time-consuming. Herein, we fabricated an innovative hydrophilic rhodamine B-loaded / boronic acid-modified graphene oxide (HRBGO) nanocomposite, used as a substitute of enzyme-labeled second antibody. The synthetic HRBGO was loaded with generous rhodamine B and modified with boronic acid. Therefore, the HRBGO could selectively label the carbohydrate chains of Fc fragment of primary antibody through specific boronate affinity recognition, and then perform signal output and amplification by releasing rhodamine B. To verify the practicability of HRBGO, trastuzumab as a humanized monoclonal antibody targeting human epidermal growth factor receptor-2 (HER2) was selected as model antibody. A glycosylation site-blocked / HER2-immobilized magnetic nanoparticles (GHMN) was also prepared for selectively capturing trastuzumab from complex samples via specific immunoaffinity. Because the glycosylation sites of HER2 can also be labeled with the HRBGO by boronate affinity recognition, these sites were blocked by a masking agent to minimize the background signal. For specific and ultrasensitive detection of trastuzumab, the integration of GHMN and HRBGO was proposed and optimized in detail. Trastuzumab detection based on HRBGO consisted of three steps: specific capture, selective labeling, and output signal. The proposed strategy provided ultrahigh sensitivity with limit of detection of 0.35 fg mL-1 and was successfully applied in the detection of trastuzumab in spiked serum sample with recovery and relative standard deviation in the range of 98.7-103.8% and 3.8-6.0%, respectively. To assess universal applicability, the HRBGO was also successfully used for the determination of anti-SARS-COV2 RBD antibody in human serum sample.

Quantitation of Trastuzumab and an Antibody to SARS-CoV-2 in Minutes Using Affinity Membranes in 96-Well Plates.

Quantitation of therapeutic monoclonal antibodies (mAbs) in human serum could ensure that patients have adequate levels of mAbs for effective treatment. This research describes the use of affinity, glass-fiber membranes in a 96-well-plate format for rapid (<5 min) quantitation of the therapeutic mAb trastuzumab and a mAb against the SARS-CoV-2 spike protein. Adsorption of a poly(acrylic acid)-containing film in membrane pores and activation of the -COOH groups in the film enable covalent-linking of affinity peptides or proteins to the membrane. Passage of mAb-containing serum through the affinity membrane results in mAb capture within 1 min. Subsequent rinsing, binding of a secondary antibody conjugated to a fluorophore, and a second rinse yield mAb-concentration-dependent fluorescence intensities in the wells. Calibration curves established from analyses on different days have low variability and allow determination of mAb levels in separately prepared samples with an average error <10%, although errors in single-replicate measurements may reach 40%. The assays can occur in diluted serum with physiologically relevant mAb concentrations, as well as in undiluted serum. Thus, the combination of 96-well plates containing affinity membranes, a microplate reader, and a simple vacuum manifold affords convenient mAb quantitation in <5 min.

Acute eosinophilic pneumonia: a fatal reaction to ado-trastuzumab.

Ado-trastuzumab emtansine (T-DM1) is a monoclonal antibody drug conjugate approved for the treatment of HER2-positive breast cancers. Presented here is a case report of a patient who developed fatal pulmonary toxicity in the form of acute eosinophilic pneumonia while undergoing treatment with T-DM1. Prior to beginning T-DM1 therapy, this patient had been treated with two HER2-targeted agents (trastuzumab, pertuzumab) per National Comprehensive Cancer Network (NCCN) guidelines. This case represents a novel presentation of toxicity associated with T-DM1 while perhaps demonstrating additive toxicity associated with multiple lines of HER2 targeted therapies.

Whole blood stability evaluation of monoclonal antibody therapeutics using volumetric absorptive microsampling.

Volumetric absorptive microsampling (VAMS) is increasingly utilized for both nonclinical and clinical pharmacokinetic studies. Currently, VAMS is employed as the sampling method for the detection of antibodies for coronavirus disease 2019. Biotherapeutics whole blood stability on VAMS presents as a critical concern for the health and pharmaceutical industries. In this follow-up to our previous publication, we evaluated daclizumab and trastuzumab whole blood sample stability on VAMS. The drug recovery data we observed at room temperature for short term and -80°C for long term was very encouraging. The knowledge could help us better understand and plan important investigation timelines, especially pandemic situations where human whole blood samples from a large population are collected and in urgent need of data analysis.

Impact of Dose Delays and Alternative Dosing Regimens on Pertuzumab Pharmacokinetics.

PERJETA (pertuzumab), administered with Herceptin (trastuzumab), is used in the treatment of human epidermal growth factor receptor 2-positive breast cancer. Pertuzumab is currently approved with an initial loading dose of 840 mg, followed by a 420-mg maintenance dose intravenously every 3 weeks. A reloading dose is required if there is a ≥6-week delay in treatment. In response to the potential treatment disruption due to COVID-19, the impact of dose delays and alternative dosing regimens on intravenous pertuzumab for human epidermal growth factor receptor 2-positive breast cancer treatment is presented. Simulations were conducted by using the validated population pharmacokinetic model for pertuzumab, and included (1) 4-, 6-, and 9-week dose delays of the 840 mg/420 mg every 3 weeks dosing regimen and (2) 840 mg/420 mg every 4 weeks and 840 mg every 6 weeks alternative dosing regimens. Simulations were compared with the currently approved pertuzumab dosing regimen. The simulations in 1000 virtual patients showed that a dose reload (840 mg) is required following a dose delay of ≥6 weeks to maintain comparable Ctrough (lowest concentration before the next dose is given) levels to clinical trials. The 840 mg/420 mg every 4 weeks and 840 mg every 6 weeks alternative dosing regimens decrease median steady-state Ctrough by ≈40% compared with the approved regimen, and <90% of patients will be above the target Ctrough . Thus, the alternative 840 mg/420 mg every 4 weeks and 840 mg every 6 weeks pertuzumab dosing regimens are not recommended. Flexibility for intravenous PERJETA-based regimens is available with an alternative route of pertuzumab administration (subcutaneous vs intravenous).

Flexible care in breast cancer.

Treatment of patients with cancer in hospitals or clinics is resource-intensive and imposes a burden on patients. 'Flexible care' is a term that can be used to describe treatment administered outside the oncology ward, oncological outpatient clinic or office-based oncologist setting. Programmes that reduce travel burden by bringing cancer treatment to the patient's home, workplace or closer to the patient's home, in the form of satellite clinics or mobile cancer units, expand treatment capacity and are well received. Clinical trial data show that, compared with intravenous administration, subcutaneous (s.c.) administration of trastuzumab is preferred by patients with breast cancer (BC), saves healthcare professionals' (HCPs) time, reduces drug preparation and administration time and reduces direct and indirect costs. As such, s.c. trastuzumab is well suited to flexible care. The results of a Belgian study (BELIS) show that home administration of s.c. trastuzumab is feasible and preferred by patients with BC. Numerous programmes and pilot studies in Europe show that s.c. trastuzumab can be administered effectively in the patient's home, in primary care settings or local hospitals. Such programmes require planning, training, careful patient selection and technology to link patients, caregivers and specialists in oncology clinics. Once these elements are in place, flexible care offers patients with BC a choice of how treatment may be delivered and lead to improved quality of life, while reducing pressure on HCPs and hospitals. The concept of flexible care is particularly relevant amid the COVID-19 pandemic where guidelines have been developed encouraging remote care.

Clinical outcomes of radical gastrectomy following trastuzumab-based chemotherapy for stage IV HER2-positive gastric or gastroesophageal junction cancer.

PURPOSE: Patients who receive trastuzumab (T-mab) plus chemotherapy for stage IV HER2-positive gastric or gastroesophageal junction cancer sometimes respond remarkably well and can undergo radical surgery. However, the clinical outcomes of preoperative T-mab combined chemotherapy with radical gastrectomy remain unclear. We conducted this study to investigate the clinical outcomes of this multimodal treatment. METHODS: From among a total of 199 patients who received T-mab-based chemotherapy for stage IV HER2-positive gastric or gastroesophageal junction cancer between 2011 and 2018, the subjects of this retrospective analysis were 20 patients who subsequently underwent radical gastrectomy. RESULTS: Seven patients had gastroesophageal junction cancer and 13 had gastric cancer. Eleven patients had unresectable stage IV cancer and 9 had resectable metastatic disease. Chemotherapy regimens included capecitabine, cisplatin + T-mab (11 patients), and S-1, oxaliplatin + T-mab (nine patients). The median number of chemotherapy cycles before surgery was three (range, 2-62). During preoperative chemotherapy, grade 3/4 adverse events developed in six patients. None suffered grade ≥ 3b postoperative complications. The 3-year relapse-free survival (RFS) and overall survival (OS) rates were 58.9% and 89.5%, respectively. CONCLUSION: Combined preoperative T-mab-based chemotherapy and surgery appears to be safe and effective for stage IV HER2-positive gastric or gastroesophageal junction cancer, with a clinically meaningful impact on RFS and OS.

Cardio-oncology care in the era of the coronavirus disease 2019 (COVID-19) pandemic: An International Cardio-Oncology Society (ICOS) statement.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has given rise to a pandemic of unprecedented proportions in the modern era because of its highly contagious nature and impact on human health and society: coronavirus disease 2019 (COVID-19). Patients with cardiovascular (CV) risk factors and established CV disease (CVD) are among those initially identified at the highest risk for serious complications, including death. Subsequent studies have pointed out that patients with cancer are also at high risk for a critical disease course. Therefore, the most vulnerable patients are seemingly those with both cancer and CVD, and a careful, unified approach in the evaluation and management of this patient population is especially needed in times of the COVID-19 pandemic. This review provides an overview of the unique implications of the viral outbreak for the field of cardio-oncology and outlines key modifications in the approach to this ever-increasing patient population. These modifications include a shift toward greater utilization of cardiac biomarkers and a more focused CV imaging approach in the broader context of modifications to typical practice pathways. The goal of this strategic adjustment is to minimize the risk of SARS-CoV-2 infection (or other future viral outbreaks) while not becoming negligent of CVD and its important impact on the overall outcomes of patients who are being treated for cancer.