2023 White Paper on Recent Issues in Bioanalysis: EU IVDR 2017/746 Implementation/Impact, IVD/CDx/CLIA Approved Assays, High Dimensional Cytometry, Multiplexing Technologies, LBA Tissue Analysis, Vaccine Study Endpoints, Cell-Based Assays for Biomarkers, Cell Therapy and Vaccines ( - Recommendations on Development & Validation of Biomarkers, IVD, CDx, Cell-Based, Flow Cytometry, Ligand-Binding and Enzyme Assays; Advanced Critical Reagents Strategies).

The 17 Workshop on Recent Issues in Bioanalysis (17 WRIB) took place in Orlando, FL, USA on 19-23 June 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 17 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.

2022 White Paper on Recent Issues in Bioanalysis: FDA Draft Guidance on Immunogenicity Information in Prescription Drug Labeling, LNP & Viral Vectors Therapeutics/Vaccines Immunogenicity, Prolongation Effect, ADA Affinity, Risk-based Approaches, NGS, qPCR, ddPCR Assays ( - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Immunogenicity & Risk Assessment of Biotherapeutics and Novel Modalities; NAb Assays Integrated Approach).

The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. 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 16th 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.

Colchicine, Aspirin, and Montelukast - A Case of Successful Combined Pharmacotherapy for Adult Multisystem Inflammatory Syndrome in COVID-19.

Since the beginning of the COVID-19 pandemic, many therapeutic strategies have been tried, with mixed results, to prevent and treat adult multisystem inflammatory syndrome in COVID-19 (AMIS-COVID-19). The reason behind this may the complex web of highly intertwined pathophysiologic mechanisms involved in the SARS-CoV-2 infection and the corresponding human systemic response, leading to end-organ damage, disability, and death. Colchicine, high-dose aspirin, and montelukast are being investigated currently as potential modulators of AMIS-COVID-19 in patients who fail to improve with traditional therapeutic approaches.

The InSituPlex Staining Method for Multiplexed Immunofluorescence Cell Phenotyping and Spatial Profiling of Tumor FFPE Samples.

Multiplexed immunohistochemistry (mIHC) enables the detection, quantification, and localization of many markers within cell or tissue samples, leading to a better understanding of the architecture of a disease at the cellular level. Current mIHC techniques involve long staining and assay times, require dedicated and/or captive instrumentation, and entail tedious assay optimization, hindering their establishment as routine methods. Here, we demonstrate the use of the InSituPlex method for spatial profiling of immuno-oncology targets in FFPE tumor tissue with the UltiMapper™ I/O PD-L1 multiplex assay.

Comprehensive genomic profiling of relapsed and metastatic adenoid cystic carcinomas by next-generation sequencing reveals potential new routes to targeted therapies.

We hypothesized that next-generation sequencing could reveal actionable genomic alterations (GAs) and potentially expand treatment options for patients with advanced adenoid cystic carcinoma (ACC). Genomic profiling using next-generation sequencing was performed on hybridization-captured, adapter ligation libraries derived from 28 relapsed and metastatic formalin-fixed paraffin-embedded ACC. The 3230 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer were fully sequenced using the Illumina HiSeq 2000.

A high frequency of activating extracellular domain ERBB2 (HER2) mutation in micropapillary urothelial carcinoma.

Purpose: Micropapillary urothelial carcinoma (MPUC) is a rare and aggressive form of bladder cancer. We conducted genomic analyses [next-generation sequencing (NGS)] of MPUC and non-micropapillary urothelial bladder carcinomas (non-MPUC) to characterize the genomic landscape and identify targeted treatment options.

Experimental Design: DNA was extracted from 40 μm of formalin-fixed paraffin-embedded sections from 15 MPUC and 64 non-MPUC tumors.

Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing.

As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change.

Advanced urothelial carcinoma: next-generation sequencing reveals diverse genomic alterations and targets of therapy.

Although urothelial carcinoma (UC) of the urinary bladder generally portends a favorable prognosis, metastatic tumors often follow an aggressive clinical course. DNA was extracted from 40 μm of formalin-fixed, paraffin-embedded (FFPE) sections from 35 stage IV UCs that had relapsed and progressed after primary surgery and conventional chemotherapy. Next-generation sequencing (NGS) was performed on hybridization-captured, adaptor ligation-based libraries for 3320 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer to at an average sequencing depth of 1164 × and evaluated for all classes of genomic alterations (GAs).

Relapsed classic E-cadherin (CDH1)-mutated invasive lobular breast cancer shows a high frequency of HER2 (ERBB2) gene mutations.

Purpose: We queried whether comprehensive genomic profiling using a next-generation sequencing-based assay could identify novel and unanticipated targets of therapy for patients with relapsed invasive lobular carcinoma (ILC).

Experimental Design: DNA sequencing (Illumina HiSeq 2000) was conducted for 3,320 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer on indexed, adaptor-ligated, hybridization-captured libraries using DNA isolated from formalin-fixed paraffin-embedded sections from 22 histologically verified ILC.

Results: A total of 75 genomic alterations were identified with an average of 3.

Combination antiangiogenic therapy in advanced breast cancer: a phase 1 trial of vandetanib, a VEGFR inhibitor, and metronomic chemotherapy, with correlative platelet proteomics.

This phase 1 study evaluated the safety and tolerability of antiangiogenic therapy using vandetanib and metronomic cyclophosphamide and methotrexate in metastatic breast cancer. Eligible patients had metastatic breast cancer with 0-4 prior chemotherapy regimens. All received cyclophosphamide 50 mg daily, methotrexate 2.

Targeted next-generation sequencing of advanced prostate cancer identifies potential therapeutic targets and disease heterogeneity.

Background: Most personalized cancer care strategies involving DNA sequencing are highly reliant on acquiring sufficient fresh or frozen tissue. It has been challenging to comprehensively evaluate the genome of advanced prostate cancer (PCa) because of limited access to metastatic tissue.

Objective: To demonstrate the feasibility of a novel next-generation sequencing (NGS)-based platform that can be used with archival formalin-fixed paraffin-embedded (FFPE) biopsy tissue to evaluate the spectrum of DNA alterations seen in advanced PCa.

Suppression of heat shock protein 27 induces long-term dormancy in human breast cancer.

The mechanisms underlying tumor dormancy have been elusive and not well characterized. We recently published an experimental model for the study of human tumor dormancy and the role of angiogenesis, and reported that the angiogenic switch was preceded by a local increase in VEGF-A and basic fibroblast growth factor. In this breast cancer xenograft model (MDA-MB-436 cells), analysis of differentially expressed genes revealed that heat shock protein 27 (HSP27) was significantly up-regulated in angiogenic cells compared with nonangiogenic cells.

Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies.

Applying a next-generation sequencing assay targeting 145 cancer-relevant genes in 40 colorectal cancer and 24 non-small cell lung cancer formalin-fixed paraffin-embedded tissue specimens identified at least one clinically relevant genomic alteration in 59% of the samples and revealed two gene fusions, C2orf44-ALK in a colorectal cancer sample and KIF5B-RET in a lung adenocarcinoma. Further screening of 561 lung adenocarcinomas identified 11 additional tumors with KIF5B-RET gene fusions (2.0%; 95% CI 0.

Isolation and proteomic analysis of platelets by SELDI-TOF MS.

Many growth factors, leukotrines, and biological ligands are not circulating free in plasma or serum, except in the case of late or disseminated disease. During early tumor growth and angiogenesis, platelets actively and selectively sequester regulators of angiogenesis and, as such, the platelet protein content can be used as a marker of early tumor growth or angiogenesis. With the recent increase in the clinical use of biologic modifiers in cancer and chronic disease therapy, the search for markers of early disease, therapeutic response, and/or recurrence has suggested that analysis of platelet proteins may be more relevant and accurate.

Simultaneous and exact interval estimates for the contrast of two groups based on an extremely high dimensional variable: application to mass spec data.

Motivation: Analysis of high-throughput proteomic/genomic data, in particular, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) data and microarray data, has led to a multitude of techniques aimed at identifying potential biomarkers. Most of the statistical techniques for comparing two groups are based on qualitative measures such as P-value. A quantitative way such as interval estimation for the contrasts of two groups is more appealing.

Mutations in ribonuclease L gene do not occur at a greater frequency in patients with familial prostate cancer compared with patients with sporadic prostate cancer.

Several genetic loci are suspected to be involved in hereditary prostate cancer, including the hereditary prostate cancer 1 (HPC1) locus at chromosome 1q24-25. The ribonuclease L (RNase L) gene has been reported as the putative hereditary prostate cancer gene located at HPC1. If this is the case, mutations of RNase L should be found at a greater frequency in familial cancers than in sporadic prostate cancers.

Elevated levels of prostate-specific antigen (PSA) in prostate cancer cells expressing mutant p53 is associated with tumor metastasis.

The underlying basis for rising levels of prostate-specific antigen (PSA) in prostate cancer is not fully understood, but attention has turned to the possibility that loss of normal p53 function might be directly involved. We have investigated the relationship between p53 function and PSA expression using in vitro and in vivo approaches. Three prostate cancer-derived p53 mutants (F134L, M237L, R273H) were introduced into LNCaP prostate cancer cells and stable transfectants established.

Alterations of p53 are common in early stage prostate cancer.

Introduction: Mutations in the p53 tumor suppressor gene are generally believed to be a late event in the progression of prostate cancer, and are associated with androgen-independence, increased angiogenesis, metastasis, recurrence, and a worse prognosis. In this review, we examine the current literature available on p53 mutations found in prostate cancer and focus on stages A (T1) and B (T2) of the disease. The alteration of genes involved in p53 regulation are also examined, as well as animal models that support an early role for p53 in the initiation and development of prostate cancer.