Safety, Tolerability and Pharmacokinetics of Half-Life Extended Severe Acute Respiratory Syndrome Coronavirus 2 Neutralizing Monoclonal Antibodies AZD7442 (Tixagevimab-Cilgavimab) in Healthy Adults.

BACKGROUND: AZD7442 is a combination of extended half-life, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific neutralizing monoclonal antibodies (tixagevimab and cilgavimab). METHODS: This phase 1, first-in-human, randomized, double-blind, placebo-controlled, dose-escalation study evaluated AZD7442 administered intramuscularly (300 mg) or intravenously (300, 1000, or 3000 mg) in healthy adults (aged 18-55 years). The primary end point was safety and tolerability. Secondary end points included pharmacokinetics and antidrug antibodies. RESULTS: Between 18 August and 16 October 2020, a total of 60 participants were enrolled; 50 received AZD7442, and 10 received placebo. Adverse events (all of mild or moderate intensity) occurred in 26 participants (52.0%) in the AZD7442 groups and 8 (80.0%) in the placebo group. No infusion or injection site or hypersensitivity reactions occurred. Tixagevimab and cilgavimab had mean half-lives of approximately 90 days (range, 87.0-95.3 days for tixagevimab and 79.8--91.1 days for cilgavimab) and similar pharmacokinetic profiles over the 361-day study period. SARS-CoV-2-specific neutralizing antibody titers provided by AZD7442 were maintained above those in plasma from convalescent patients with coronavirus disease 2019 (COVID-19). CONCLUSIONS: AZD7442 was well tolerated in healthy adults, showing a favorable safety profile across all doses. Depending on the SARS-CoV-2 variant, pharmacokinetic analyses suggest the AZD7442 could offer protection for ≥6 months against symptomatic COVID-19 after a single 300-mg intramuscular administration. CLINICAL TRIALS REGISTRATION: NCT04507256.

Antibodies are proteins produced by the body in response to infections caused by microbes, including viruses. AZD7442 is a combination of 2 human antibodies, with an extended duration of effect, sourced from people who had recovered from coronavirus disease 2019 (COVID-19). These antibodies recognize a specific part (spike protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, and prevent the virus from infecting cells in the body. The current study evaluated the safety of AZD7442 in healthy volunteers. Sixty adults were given AZD7442 or placebo (salt solution) as injections into the muscle (300-mg dose) or infusions into a vein (300­3000-mg doses). The study did not find any safety issues with AZD7442, including at the highest dose. AZD7442 was measured in the blood 12 months after dosing, suggesting a long duration of protection. Following this study, AZD7442 was tested in larger clinical trials to investigate its potential in preventing and treating COVID-19. AZD7442 is currently authorized as treatment for outpatients with COVID-19 and as a preventive drug in people who may not respond well to COVID-19 vaccines and need additional protection (eg, those taking medications that dampen the immune system).

Multiplex Hybrid Antigen-Capture LC-MRM Quantification in Sera and Nasal Lining Fluid of AZD7442, a SARS-CoV-2-Targeting Antibody Combination.

AZD7442 (tixagevimab [AZD8895]/cilgavimab [AZD1061]) is a monoclonal antibody (mAb) combination in development for the prevention and treatment of coronavirus disease 2019. Traditionally, bioanalysis of mAbs is performed using ligand binding assays (LBAs), which offer sensitivity, robustness, and ease of implementation. However, LBAs frequently require generation of critical reagents that typically take several months. Instead, we developed a highly sensitive (5 ng/mL limit of quantification) method using a hybrid LBA-liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) approach for quantification of the two codosed antibodies in serum and nasal lining fluid (NLF), a rare matrix. The method was optimized by careful selection of multiple reaction monitoring, capture reagents, magnetic beads, chromatographic conditions, evaluations of selectivity, and matrix effect. The final assay used viral spike protein receptor-binding domain as capture reagent and signature proteotypic peptides from the complementarity-determining region of each mAb for detection. In contrast to other methods of similar/superior sensitivity, our approach did not require multidimensional separations and can be operated in an analytical flow regime, ensuring high throughput and robustness required for clinical analysis at scale. The sensitivity of this method significantly exceeds typical sensitivity of ∼100 ng/mL for analytical flow 1D LBA-LC-MS/MS methods for large macromolecules, such as antibodies. Furthermore, infection and vaccination status did not impact method performance, ensuring method robustness and applicability to a broad patient population. This report demonstrated the general applicability of the hybrid LBA-LC-MS/MS approach to platform quantification of antibodies with high sensitivity and reproducibility, with specialized extension to matrices of increasing interest, such as NLF.

Asymmetric Cyclopropanation with 4-Aryloxy-1-sulfonyl-1,2,3-triazoles: Expanding the Range of Rhodium-Stabilized Donor/Acceptor Carbenes to Systems with an Oxygen Donor Group.

Rhodium-catalyzed enantioselective synthesis of 1-phenoxycyclopropane-1-carbaldehydes by intermolecular cyclopropanation of terminal alkenes followed by imine hydrolysis is described. This methodology utilizes 4-aryloxy-1-sulfonyl-1,2,3-triazoles as the carbene precursors and the chiral dirhodium(II) tetracarboxylates Rh2(S-NTTL)4 or Rh2(S-DPCP)4 as the catalysts. These reactions are considered to proceed via rhodium-stabilized donor/acceptor carbene intermediates, and these studies demonstrate that a heteroatom donor group is compatible with an enantioselective transformation.

Serous borderline tumor with distant mediastinal metastasis? An Exceptional presentation of ovarian tumor.

Borderline ovarian tumor is a non-invasive lesion with an excellent prognosis. Here we report a case of 48-year-old woman with distinctive clinical presentation of metastasis of ovarian adenocarcinoma, which was an microinvasive component of a serous borderline tumor. On initial diagnosis patient did not present any clinical manifestation of ovarian tumor. Histological examination of resected ovary showed typical features of the serous borderline tumor with one very diminutive focus of invasive serous adenocarcinoma 4mm in diameter. This exceptional case shows that borderline tumors of ovary with any features of invasion could present an aggressive course with distant metastases.

Lack of CD151/integrin α3ß1 complex is predictive of poor outcome in node-negative lobular breast carcinoma: opposing roles of CD151 in invasive lobular and ductal breast cancers.

BACKGROUND: The proposed involvement of CD151 in breast cancer (BCa) progression is based on findings from studies in invasive ductal carcinoma (IDC). The IDC and invasive lobular carcinoma (ILC) represent distinct disease entities. Here we evaluated clinical significance of CD151 alone and in association with integrin α3ß1 in patients with ILC in context of the data of our recent IDC study. METHODS: Expression of CD151 and/or integrin α3ß1 was evaluated in ILC samples (N=117) using immunohistochemistry. The findings were analysed in relation to our results from an IDC cohort (N=182) demonstrating a prognostic value of an expression of CD151/integrin α3ß1 complex in patients with HER2-negative tumours. RESULTS: Unlike in the IDCs, neither CD151 nor CD151/α3ß1 complex showed any correlation with any of the ILC characteristics. Lack of both CD151 and α3ß1 was significantly correlated with poor survival (P=0.034) in lymph node-negative ILC N(-) cases. The CD151(-)/α3ß1(-) patients had 3.12-fold higher risk of death from BCa in comparison with the rest of the ILC N(-) patients. CONCLUSIONS: Biological role of CD151/α3ß1 varies between ILC and IDC. Assessment of CD151/α3ß1 might help to identify ILC N(-) patients with increased risk of distant metastases.

Association of microRNAs and pathologic response to preoperative chemotherapy in triple negative breast cancer: preliminary report.

Triple negative breast cancer (TNBC) has caught the attention of oncologists worldwide because of poor prognosis and paucity of targeted therapies. Gene pathways have been widely studied, but less is known about epigenetic factors such as microRNAs (miRNAs) and their role in tailoring an individual systemic and surgical approach for breast cancer patients. The aim of the study was to examine selected miRNAs in TNBC core biopsies sampled before preoperative chemotherapy and the subsequent pathologic response in mastectomy or breast conservation specimens. Prior to treatment, core needle biopsies were collected from 11 female patients with inoperable locally advanced TNBC or large resectable tumors suitable for down-staging. In all 11 TNBC core biopsies we analyzed 19 miRNAs per sample: 512, 190, 200, 346, 148, 449, 203, 577, 93, 126, 423, 129, 193, 182, 136, 135, 191, 122 and 222 (miRCURY LNA™ Universal RT microRNA polymerase chain reaction Custom Pick & Mixpanels). The Wilcoxon signed-rank test was used to compare related samples. Ingenuity pathway analysis was used to evaluate potential functional significance of differentially expressed miRNAs. Statistical analysis showed that 3 of 19 miRNAs differed in relation to pathologic response i.e. good versus poor. These differences failed to reach statistical significance, although a trend was observed (p=0.06). Among these miRNAs, we identified-miR-200b-3p, miR-190a and miR-512-5p. In summary, our results indicate that higher miR-200b-3p, higher miR-190a and lower miR-512-5p expression levels in core biopsies sampled from TNBC patients may be associated with better pathologic response to chemotherapy and the increased feasibility of breast conserving surgery in these patients. Although these results were from a small cohort, they provide an important basis for larger, prospective, multicenter studies to investigate the potential role of miRNAs in neoadjuvant setting.

Comprehensive Summary of Safety Data on Nirsevimab in Infants and Children from All Pivotal Randomized Clinical Trials.

BACKGROUND: Nirsevimab is approved in the US for the prevention of respiratory syncytial virus (RSV) lower respiratory tract disease in neonates and infants during their first RSV season and in children aged ≤24 months who remain vulnerable to severe RSV disease through their second RSV season. We summarize a pre-specified analysis of nirsevimab safety data from three randomized controlled trials: Phase 2b (NCT02878330; healthy infants born ≥29 to <35 weeks' gestational age [wGA]); Phase 3 MELODY (NCT03979313; healthy infants born ≥35 wGA); and Phase 2/3 MEDLEY (NCT03959488; infants with congenital heart disease [CHD] and/or chronic lung disease of prematurity [CLD] or born ≤35 wGA). METHODS: Participants (randomized 2:1) received a single intramuscular dose of nirsevimab or comparator (placebo, Phase 2b/MELODY; 5× once-monthly palivizumab, MEDLEY) before their first RSV season (recipients < 5 kg, nirsevimab 50 mg; ≥5 kg, nirsevimab 100 mg). In MEDLEY, children with CHD/CLD continued to a second RSV season: first-season nirsevimab recipients received nirsevimab 200 mg; first-season palivizumab recipients were re-randomized 1:1 to receive nirsevimab 200 mg or 5× once-monthly palivizumab. RESULTS: The incidence, severity, and nature of AEs were similar across treatments (nirsevimab, n = 3184; placebo, n = 1284; palivizumab, n = 304). Most AEs were mild to moderate in severity, with ≥98% unrelated to treatment. AEs of special interest occurred infrequently (<1%): no anaphylaxis or thrombocytopenia were treatment-related, and no immune complex disease was reported. Deaths (incidence < 1.0%) were all unrelated to treatment. CONCLUSIONS: A single dose per season of nirsevimab for the prevention of RSV disease had a favorable safety profile, irrespective of wGA or comorbidities.

2023 White Paper on Recent Issues in Bioanalysis: ISR for ADA Assays, the Rise of dPCR vs qPCR, International Reference Standards for Vaccine Assays, Anti-AAV TAb Post-Dose Assessment, NanoString Validation, ELISpot as Gold Standard (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Biotherapeutics Immunogenicity & Risk Assessment; ADA/NAb Assay/Reporting Harmonization).

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19-23, 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) are published in volume 16 of Bioanalysis, issues 8 and 9 (2024), respectively.

Membrane expression of TRAIL receptors DR4, DR5, DcR1 and DcR2 in the normal endometrium, atypical endometrial hyperplasia and endometrioid adenocarcinoma: a tissue microarray study.

PURPOSE: To evaluate the membrane expression of DR4, DR5, DcR1 and DcR2 in the normal endometrium (NE), atypical endometrial hyperplasia (AEH) and endometrioid adenocarcinoma (EAC). METHODS: The study comprised 197 patients: 20 NE, 18 AEH and 159 EAC. Tissue microarrays were constructed. Membrane expression of DR4, DR5, DcR1 and DcR2 was examined and presented as total score (TS). RESULTS: In EAC, the membrane expression of DR4, DR5 and DcR2 was less common compared to NE (p < 0.001; p < 0.001; p = 0.018) and AEH (p < 0.001; p < 0.001; p = 0.004). In EAC the membrane expression of DcR1 did not differ when compared to NE (p = 0.055) and AEH (p = 0.173). A strong correlation was found between the type of endometrial tissue (NE/AEH/EAC) and the TS of DR4 (p < 0.001), DR5 (p < 0.001), DcR1 (p = 0.033) and DcR2 (p < 0.001). In EAC, the TS of DR4, DR5, DcR1 and DcR2 was not related to grading and staging. In EAC, the membrane expression of DR5, but not DR4, DcR1 and DcR2, was related to better disease-free survival (DFS). The overall survival (OS) was not related to membrane TRAIL receptors expression. CONCLUSIONS: The membrane expression of the receptors for TRAIL DR4, DR5, DcR1 and DcR2 is greater in NE than EAC. The level of membrane staining of the receptors in EAC is not dependent on grading and staging. In EAC patients, membrane expression of DR4, DR5, DcR1 and DcR2 are not independent predictors of survival.

[The value of Ki-67 antigen expression in tissue microarray method in prediction prognosis of patients with endometrioid endometrial cancer]. / Znaczenie ekspresji antygenu Ki-67 ocenianej metoda mikromacierzy tkankowych dla rokowania u chorych z gruczolakorakiem endometrialnym endometrium.

OBJECTIVES: To assess the prognostic significance of Ki-67 expression in the tissue microarray method (TMA) for disease free survival (DFS) and overall survival (OS) in endometrioid endometrial cancer (EEC). MATERIAL AND METHODS: The study examined 159 consecutive patients aged 37-86 (62.82 +/- 9.95) with EEC stages I-III according to FIGO, treated surgically at the Pirogow Memorial Hospital of Lodz between 2000 and 2007. Afterwards they were subsequently treated and examined at the Regional Cancer Center Copernicus Memorial Hospital of Lodz. Tissue cores 2 mm in size, in duplicate, were taken from the formalin-fixed and paraffin-embedded tissue donor blocks from surgery and constructed into the TMA recipient blocks. Using TMA method, the relationship between Ki-67 expression, DFS and OS was examined. DFS was defined as a period from primary surgery until relapse. OS was defined as a period from primary surgery until the end of the follow-up (60 months) or until the death of the patient. The study was approved by the Ethics Committee of the Medical University of Lodz (RNN/82/11/KE; KE/1673/12). RESULTS: The follow-up time varied between 3-60 months (51.42 +/- 15.87). In 31 patients (19.50%) the relapse of was diagnosed 1-59 months (24.97 +/- 16.08) after commencement of the treatment. During follow-up 32 patients (20.12%) died. DFS and OS were 80.50% and 79.88%, respectively The lack of Ki-67 expression was found in 37 cases (23.27%) while in 122 patients (76.73%) the expression was present (p < 0.001). The expression of Ki-67 in 1-10%, 11-20% and > 20% was present in 76 cases, 26 cases and 20 cases, respectively Positive correlation between the expression of Ki-67 and staging was present (r = 0.353; p < 0.001). In EEC patients with no relapse diagnosed during follow-up the expression of Ki-67 was present in 7.63 +/- 7.57% of EEC cells, when compared to 23.06 +/- 22.93% in EEC patients in relapsed disease (p < 0.001). The relationship between increased Ki-67 expression and increased grading was not statistically significant (r = 0.149; p = 0.061). The expression of Ki-67 did not depend on patient age (r = 0.040; p = 0.617). In univariate analysis negative correlation was found between the expression of Ki-67 and DFS (p < 0.001) and OS (p = 0.01). In multivariate analysis worse DFS was related to higher staging of EEC (p < 0.0 01) and increased expression of Ki-67 (p < 0.001). Worse OS was related to higher staging in multivariate analysis (p < 0.001). Ki-67 expression was not related to OS in multivariate analysis. Age of patients and grading of the EEC were not related to DFS and OS. CONCLUSIONS: The expression of the Ki-67 can significantly affect therapeutic decisions in selected EEC patients. The high Ki-67 expression in EEC patients is related to increased risk of relapse. The TMA technique is a good method for the assessment of the Ki-67 in studies conducted in EEC patients and makes it easier to carry out immunohistochemistry in large populations of patients.

[The value of progesterone and estrogen receptors expression in tissue microarray method in prognosis of patients with endometrioid endometrial cancer]. / Znaczenie ekspresji receptorów progesteronowych i estrogenowych ocenianej metoda mikromacierzy tkankowych dla rokowania u chorych z gruczolakorakiem endometrioidalnym endometrium.

OBJECTIVES: To assess prognostic significance of progesterone receptors (PR) and estrogen receptors (ER) expression in the tissue microarray (TMA) technique for disease free survival (DFS) and overall survival (OS) in endometrioid endometrial cancer (EEC). MATERIAL AND METHODS: The study included 151 consecutive patients, aged 37-86 years (62.80 +/- 9.99), with the EEC in stages I-III (FIGO), treated surgically at the Pirogow Memorial Hospital of Lodz between 2000 and 2007. Afterwards, they were subsequently treated and examined at the Regional Cancer Center, Copernicus Memorial Hospital of Lodz. Tissue cores 2 mm in size, in duplicate, were taken from the formalin-fixed and paraffin-embedded tissue donor blocks from surgery and constructed into the TMA recipient blocks. Using TMAs, the expression of PR and ER was examined and presented as Total Score (TS). The TS was determined by adding the intensity and marker distribution scores in a given case. The relationship between PR and ER expression, DFS and OS was examined. DFS was defined as the period from primary surgery until relapse. OS was defined as the period from primary surgery until the end of the follow-up (60 months) or until the death of the patient. The study was approved by the Ethics Committee of the Medical University of Lodz (RNN/82/11/KE). RESULTS: Lack of the PR and ER expression was found in 46 cases (30.46%) and 67 cases (44.37%), respectively. The expression of the PR and ER was weak in 24 cases (15.89%) and 22 cases (14.57%), respectively. Strong PR and ER expression was found in 81 patients (53.65%) and 62 patients (41.06%), respectively. Follow-up after surgery varied from 3 to 60 months (50.95 +/- 16.36). In 30 patients (19.87%) relapse was diagnosed 1-54 months (22.17 +/- 15.59) after surgery. During follow-ups, 29 patients (19.21%) died. In univariate analysis better DFS was related to the presence of PR (p = 0.010), higher TS of PR (HR = 0.81; 95% CI 0.71-0.94), the presence of ER (p = 0.001) and higher TS of ER (HR = 0.88; 95% CI 0.78-0.99). DFS differed significantly between the groups: without PR and ER expression (A), with presence of the PR but not ER expression (B), with the ER but not PR expression (C) and with the PR and ER expression (D) (p = 0.004). In univariate analysis OS was not related to PR expression (p = 0.110), TS of PR (HR = 0.89; 95% CI 0.80-1.02) and ER expression (p = 0.070). TS of ER was connected to better OS (HR = 0.83; 95% CI 0.72-0.96). The OS differed between groups A, B, C and D (p = 0.006). In multivariate analysis variants of PR/ER expression influenced the DFS (p = 0.039) and OS (p = 0.016). CONCLUSIONS: The expression of the PR and ER can significantly affect therapeutic decisions in selected patients with EEC. In EEC, common assessment of PR and ER expression is of higher prognostic value, than compared to single evaluation of PR and ER receptors.

Safety of Re-dosing Nirsevimab Prior to RSV Season 2 in Children With Heart or Lung Disease.

In children with congenital heart disease and/or chronic lung disease entering their second respiratory syncytial virus (RSV) season, 200 mg nirsevimab had a similar safety profile to that of palivizumab and resulted in nirsevimab serum exposures associated with efficacy in healthy infants, supporting efficacy in this population at risk of severe RSV disease.

Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody-Drug Conjugate.

Deamidation, a common post-translational modification, may impact multiple physiochemical properties of a therapeutic protein. MEDI7247, a pyrrolobenzodiazepine (PBD) antibody-drug conjugate (ADC), contains a unique deamidation site, N102, located within the complementarity-determining region (CDR), impacting the affinity of MEDI7247 to its target. Therefore, it was necessary to monitor MEDI7247 deamidation status in vivo. Due to the low dose, a sensitive absolute quantification method using immunocapture coupled with liquid chromatography-tandem mass spectrometry (LBA-LC-MS/MS) was developed and qualified. We characterized the isomerization via Electron-Activated Dissociation (EAD), revealing that deamidation resulted in iso-aspartic acid. The absolute quantification of deamidation requires careful assay optimization in order not to perturb the balance of the deamidated and nondeamidated forms. Moreover, the selection of capture reagents essential for the correct quantitative assessment of deamidation was evaluated. The final assay was qualified with 50 ng/mL LLOQ for ADC for total and nondeamidated antibody quantification, with qualitative monitoring of the deamidated antibody. The impact of deamidation on the pharmacokinetic characteristics of MEDI7247 from clinical trial NCT03106428 was analyzed, revealing a gradual reduction in the nondeamidated form of MEDI7247 in vivo. Careful quantitative biotransformation analyses of complex biotherapeutic conjugates help us understand changes in product PTMs after administration, thus providing a more complete view of in vivo pharmacology.

Gene expression and pathologic response to neoadjuvant chemotherapy in breast cancer.

Pathologic complete response after neoadjuvant systemic treatment appears to be a valid surrogate for better overall survival in breast cancer patients. Currently, together with standard clinicopathologic assessment, novel molecular biomarkers are being exhaustively tested in order to look into the heterogeneity of breast cancer. The aim of our study was to examine an association between 23-gene real-time-PCR expression assay including ABCB1, ABCC1, BAX, BBC3, BCL2, CASP3, CYP2D6, ERCC1, FOXC1, GAPDH, IGF1R, IRF1, MAP2, MAPK 8, MAPK9, MKI67, MMP9, NCOA3, PARP1, PIK3CA, TGFB3, TOP2A, and YWHAZ receptor status of breast cancer core biopsies sampled before neoadjuvant chemotherapy (anthracycline and taxanes) and pathologic response. Core-needle biopsies were collected from 42 female patients with inoperable locally advanced breast cancer or resectable tumors suitable for downstaging, before any treatment. Expressions of 23 genes were determined by means of TagMan low density arrays. Analysis of variance was used to select genes with discriminatory potential between receptor subtypes. We introduced a correction for false discovery rates (presented as q values) due to multiple hypothesis testing. Statistical analysis showed that seven genes out of a 23-gene real-time-PCR expression assay differed significantly in relation to pathologic response regardless of breast cancer subtypes. Among these genes, we identified: BAX (p = 0.0146), CYP2D6 (p = 0.0063), ERCC1 (p = 0.0231), FOXC1 (p = 0.0048), IRF1 (p = 0.0022), MAP2 (p = 0.0011), and MKI67 (p = 0.0332). The assessment of core biopsy gene profiles and receptor-based subtypes, before neoadjuvant therapy seems to predict response or resistance and to define new signaling pathways to provide more powerful classifiers in breast cancer, hence the need for further research.

Comparison of prognosis in patients with endometrioid endometrial cancer staged IB in FIGO 1988 and 2009 classifications.

PURPOSE: Since 2009 the new FIGO Staging System of endometrial cancer, which changed the previous FIGO 1988 Staging System, has been in use. The aim of the study was to compare prognosis in patients with endometrioid endometrial cancer at stage IB of the 2009 FIGO Staging System and of the 1988 FIGO Staging System. METHODS: We analyzed 173 patients: 108 patients (group A) at stage IB in FIGO 1988 Staging System, and 68 patients (group B) at stage IB in FIGO 2009 Staging System from 262 consecutive endometrioid endometrial cancer patients. The disease-free survival (DFS) and overall survival (OS) were compared between these groups. RESULTS: The DFS rate was 96.3 % in group A and it was 87.7 % in group B (p = 0.029). Relapses were observed in 12 patients (6.4 %) from 6 to 57 months (mean 28.1; SD = 14.6) after initial surgery, and occurred in four patients from group A (3.7 %) and eight patients from group B (12.3 %) (p = 0.032). The OS rate was 94.4 % in group A and it was 83.1 % in group B (p = 0.018). During follow-up, 17 patients (9.8 %) died: six patients from group A (5.6 %), and 11 patients from group B (16.9 %). CONCLUSIONS: Stage IB in FIGO 2009 Staging System is associated with worse prognosis compared to stage IB according to FIGO 1988 classification. There seems to be a need to use exclusively the new FIGO 2009 classification worldwide to avoid therapeutic mistakes, which can be caused by diverse nomenclature.

[Effectiveness of tissue microarray technique for the assessment of estrogen and progesterone receptors expression in endometrioid endometrial cancer--preliminary report]. / Efektywnosc zastosowania techniki mikromacierzy tkankowych do oceny ekspresji receptorów estrogenowych i progesteronowych w gruczolakoraku endometrioidalnym endometrium--doniesienie wstepne.

OBJECTIVES: To assess the effectiveness of the donor-block biopsies with a 2 mm-size needle in endometrioid endometrial cancer (EEC) in the tissue microarray (TMA) technique and the application of the TMA for estrogen receptors (ER) and progesterone receptors (PR) expression in EEC. MATERIAL AND METHODS: The study examined EEC tissues from 60 patients. Tissue cores, 2 mm in size, in duplicate, were taken from the formalin-fixed and paraffin-embedded tissue donor blocks and constructed into the TMA recipient block. The presence of EEC tissue in the TMAs was analyzed, and the ER and PR expressions were examined. RESULTS: EEC tissue in TMAs was confirmed in 56 cases (93.33%). In 49 of them (81.67%), both cores presented with cancer tissues. In 4 cases (6.67%) EEC tissue was absent. All cases with ECC present on the TMA slides were appropriate for the ER and PR analysis. In 29 EEC cases (51.98%) both ER and PR were expressed. In 3 cases (5.36%) only ER was expressed, in 8 cases (14.29%) only PR was expressed, and in 16 cases (28.57%) ER and PR were assessed as negative. CONCLUSIONS: Two 2 mm-sized tissue cores from donor-block biopsies constructed into the TMA recipient block were sufficient to diagnose EEC and enabled the assessment of ER and PR expression in 93.3% of the cases. The use of the described TMA technique makes the immunohistochemical study of EEC easier and more time-efficient.

Critical reagents for ligand-binding assays: process development methodologies to enable high-quality reagents.

Development of biotherapeutics require pharmacokinetic/pharmacodynamic (PK/PD) and immunogenicity assays that are frequently in a ligand-binding assay (LBA) format. Conjugated critical reagents for LBAs are generated conjugation of the biotherapeutic drug or anti-drug molecule with a label. Since conjugated critical reagent quality impacts LBA performance, control of the generation process is essential. Our perspective is that process development methodologies should be integrated into critical reagent production to understand the impact of conjugation reactions, purification techniques and formulation conditions on the quality of the reagent. In this article, case studies highlight our approach to developing process conditions for different molecular classes of critical reagents including antibodies and a peptide. This development approach can be applied to the generation of future conjugated critical reagents.

Comparison of Titer and Signal to Noise (S/N) for Determination of Anti-drug Antibody Magnitude Using Clinical Data from an Industry Consortium.

During biotherapeutic drug development, immunogenicity is evaluated by measuring anti-drug antibodies (ADAs). The presence and magnitude of ADA responses is assessed using a multi-tier workflow where samples are screened, confirmed, and titered. Recent reports suggest that the assay signal to noise ratio (S/N) obtained during the screening tier correlates well with titer. To determine whether S/N could more broadly replace titer, anonymized ADA data from a consortium of sponsors was collected and analyzed. Datasets from clinical programs with therapeutics of varying immunogenicity risk levels (low to high), common ADA assay platforms (ELISA and MSD) and formats (bridging, direct, solid-phase extraction with acid dissociation), and titration approaches (endpoint and interpolated) were included in the analysis. A statistically significant correlation between S/N and titer was observed in all datasets, with a strong correlation (Spearman's r > 0.8) in 11 out of 15 assays (73%). For assays with available data, conclusions regarding ADA impact on pharmacokinetics and pharmacodynamics were similar using S/N or titer. Subject ADA kinetic profiles were also comparable using the two measurements. Determination of antibody boosting in patients with pre-existing responses could be accomplished using similar approaches for titer and S/N. Investigation of factors that impacted the accuracy of ADA magnitude measurements revealed advantages and disadvantages to both approaches. In general, S/N had superior precision and ability to detect potentially low affinity/avidity responses compared to titer. This analysis indicates that S/N could serve as an equivalent and in some cases preferable alternative to titer for assessing ADA magnitude and evaluation of impact on clinical responses.

2021 White Paper on Recent Issues in Bioanalysis: TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccine Assays; Immunogenicity of Biotherapeutics and Novel Modalities; Integrated Summary of Immunogenicity Harmonization).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.

[Endometrioid endometrial cancer--the prognostic value of selected clinical and pathological parameters]. / Gruczolakorak endometrium typu endometrioidalnego--analiza wartosci prognostycznej wybranych parametrów klinicznych i histopatologicznych.

OBJECTIVES: To assess the relationship between selected clinical and pathological factors and disease free survival (DFS) and overall survival (OS) in endometrioid endometrial cancer patients. MATERIAL AND METHODS: A retrospective review of 262 patients aged 37-86 (6.0 +/- 9.0) was performed. Selected clinical and pathological data were correlated with DFS and OS. RESULTS: Follow-up was 8-123 months (64.9 +/- 27.1). In 4 patients (1.5%) clinical progression was diagnosed during the treatment. In 43 patients (16.4%) relapse was diagnosed 2-61 months (23.9 +/- 15.7) after commencing treatment. DFS and OS were 82.1% and 81.3% respectively. In univariate analysis worse DFS was related to older patients (p = 0.007) and non-radical surgery (p < 0.001). In multivariate analysis worse DFS was related to older patients (HR = 1.058; 95% CI = 1.024-1.093; p < 0.001), younger at menopause (HR = 0.910; 95% CI = 0.851-0.973; p = 0.006), with higher staging (HR = 2.639; 95% CI = 1.968-3.539; p < 0.001) operated non-radically (HR = 0.220; 95% CI = 0.096-0.504; p < 0.001). In univariate analysis worse OS was connected with older patients (p = 0.018), diabetes type II (p = 0.019) and non-radical surgery (p < 0.001). In multivariate analysis worse OS was related to younger age at menopause (HR = 0.932; 95% CI = 0.873-0.996; p = 0.039), diabetes type II (HR = 2.372; 95% CI = 1.260-4.466; p = 0.008), higher staging (HR = 2.053; 95% CI = 1.482-2.845; p < 0.001), and non-radical surgery (HR = 0.240; 95% CI = 0.091-0.636; p = 0.004). CONCLUSIONS: Relapsed endometrial cancer developed in 90.7% during four years after commencing treatment. In 79.1% of these patients distant metastases were present. Most significant prognostic factors were radicality of surgery age of patients and staging. The presence of diabetes type II and early menopause were connected with worse prognosis.