2021 White Paper on Recent Issues in Bioanalysis: ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry (Part 2 - Recommendations on Biomarkers/CDx Assays Development & Validation, Cytometry Validation & Innovation, Biotherapeutics PK LBA Regulated Bioanalysis, Critical Reagents & Positive Controls Generation).

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 three Main Workshops and seven 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 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. 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 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

p53 null fluorescent yellow direct repeat (FYDR) mice have normal levels of homologous recombination.

The tumor suppressor p53 is a transcription factor whose function is critical for maintaining genomic stability in mammalian cells. In response to DNA damage, p53 initiates a signaling cascade that results in cell cycle arrest, DNA repair or, if the damage is severe, programmed cell death. In addition, p53 interacts with repair proteins involved in homologous recombination. Mitotic homologous recombination (HR) plays an essential role in the repair of double-strand breaks (DSBs) and broken replication forks. Loss of function of either p53 or HR leads to an increased risk of cancer. Given the importance of both p53 and HR in maintaining genomic integrity, we analyzed the effect of p53 on HR in vivo using Fluorescent Yellow Direct Repeat (FYDR) mice as well as with the sister chromatid exchange (SCE) assay. FYDR mice carry a direct repeat substrate in which an HR event can yield a fluorescent phenotype. Here, we show that p53 status does not significantly affect spontaneous HR in adult pancreatic cells in vivo or in primary fibroblasts in vitro when assessed using the FYDR substrate and SCEs. In addition, primary fibroblasts from p53 null mice do not show increased susceptibility to DNA damage-induced HR when challenged with mitomycin C. Taken together, the FYDR assay and SCE analysis indicate that, for some tissues and cell types, p53 status does not greatly impact HR.

Tissue-specific differences in the accumulation of sequence rearrangements with age.

Mitotic homologous recombination (HR) is a critical pathway for the accurate repair of DNA double strand breaks (DSBs) and broken replication forks. While generally error-free, HR can occur between misaligned sequences, resulting in deleterious sequence rearrangements that can contribute to cancer and aging. To learn more about the extent to which HR occurs in different tissues during the aging process, we used Fluorescent Yellow Direct Repeat (FYDR) mice in which an HR event in a transgene yields a fluorescent phenotype. Here, we show tissue-specific differences in the accumulation of recombinant cells with age. Unlike pancreas, which shows a dramatic 23-fold increase in recombinant cell frequency with age, skin shows no increase in vivo. In vitro studies indicate that juvenile and aged primary fibroblasts are similarly able to undergo HR in response to endogenous and exogenous DNA damage. Therefore, the lack of recombinant cell accumulation in the skin is most likely not due to an inability to undergo de novo HR events. We propose that tissue-specific differences in the accumulation of recombinant cells with age result from differences in the ability of recombinant cells to persist and clonally expand within tissues.

Three-dimensional tissue cytometer based on high-speed multiphoton microscopy.

Image cytometry technology has been extended to 3D based on high-speed multiphoton microscopy. This technique allows in situ study of tissue specimens preserving important cell-cell and cell-extracellular matrix interactions. The imaging system was based on high-speed multiphoton microscopy (HSMPM) for 3D deep tissue imaging with minimal photodamage. Using appropriate fluorescent labels and a specimen translation stage, we could quantify cellular and biochemical states of tissues in a high throughput manner. This approach could assay tissue structures with subcellular resolution down to a few hundred micrometers deep. Its throughput could be quantified by the rate of volume imaging: 1.45 mm(3)/h with high resolution. For a tissue containing tightly packed, stratified cellular layers, this rate corresponded to sampling about 200 cells/s. We characterized the performance of 3D tissue cytometer by quantifying rare cell populations in 2D and 3D specimens in vitro. The measured population ratios, which were obtained by image analysis, agreed well with the expected ratios down to the ratio of 1/10(5). This technology was also applied to the detection of rare skin structures based on endogenous fluorophores. Sebaceous glands and a cell cluster at the base of a hair follicle were identified. Finally, the 3D tissue cytometer was applied to detect rare cells that had undergone homologous mitotic recombination in a novel transgenic mouse model, where recombination events could result in the expression of enhanced yellow fluorescent protein in the cells. 3D tissue cytometry based on HSMPM demonstrated its screening capability with high sensitivity and showed the possibility of studying cellular and biochemical states in tissues in situ. This technique will significantly expand the scope of cytometric studies to the biomedical problems where spatial and chemical relationships between cells and their tissue environments are important.

Applications of fluorescence for detecting rare sequence rearrangements in vivo.

Homologous recombination (HR) is an important pathway for the accurate repair of potentially cytotoxic or mutagenic double strand breaks (DSBs), as well as double strand ends that arise due to replication fork breakdown. Thus, measuring HR events can provide information on conditions that induce DSB formation and replicative stress. To study HR events in vivo, we previously developed Fluorescent Yellow Direct Repeat (FYDR) mice in which a recombination event at an integrated transgene yields a fluorescent signal. Recently, we published an application of these mice demonstrating that fluorescent recombinant cells can be directly detected within intact pancreatic tissue. Here, we show that in situ imaging is a more sensitive method for detecting exposure-induced recombinant cells, yielding statistical significance with smaller cohorts. In addition, we show inter-mouse and gender-dependent variation in transgene expression, examine its impact on data interpretation, and discuss solutions to overcoming the effects of such variation. Finally, we also present data on enhanced yellow fluorescent protein (EYFP) expression, showing that several tissues, in addition to the pancreas, may be amenable for in situ detection of recombinant cells in the FYDR mice. The FYDR mice provide a unique tool for identifying genetic conditions and environmental exposures that induce genotoxic stress in a variety of tissues.

Age-dependent accumulation of recombinant cells in the mouse pancreas revealed by in situ fluorescence imaging.

Mitotic homologous recombination (HR) is critical for the repair of double-strand breaks, and conditions that stimulate HR are associated with an increased risk of deleterious sequence rearrangements that can promote cancer. Because of the difficulty of assessing HR in mammals, little is known about HR activity in mammalian tissues or about the effects of cancer risk factors on HR in vivo. To study HR in vivo, we have used fluorescent yellow direct repeat mice, in which an HR event at a transgene yields a fluorescent phenotype. Results show that HR is an active pathway in the pancreas throughout life, that HR is induced in vivo by exposure to a cancer chemotherapeutic agent, and that recombinant cells accumulate with age in pancreatic tissue. Furthermore, we developed an in situ imaging approach that reveals an increase in both the frequency and the sizes of isolated recombinant cell clusters with age, indicating that both de novo recombination events and clonal expansion contribute to the accumulation of recombinant cells with age. This work demonstrates that aging and exposure to a cancer chemotherapeutic agent increase the frequency of recombinant cells in the pancreas, and it also provides a rapid method for revealing additional factors that modulate HR and clonal expansion in vivo.

A single acute exposure to a chemotherapeutic agent induces hyper-recombination in distantly descendant cells and in their neighbors.

Homologous recombination can induce tumorigenic sequence rearrangements. Here, we show that persistent hyper-recombination can be induced following exposure to a bifunctional alkylating agent, mitomycin C (MMC), and that the progeny of exposed cells induce a hyper-recombination phenotype in unexposed neighboring cells. Residual damage cannot be the cause of delayed recombination events, since recombination is observed after drug and template damage are diluted over a million-fold. Furthermore, not only do progeny of MMC-exposed cells induce recombination in unexposed cells (bystanders), but these bystanders can in turn induce recombination in their unexposed neighbors. Thus, a signal to induce homologous recombination can be passed from cell to cell. Although the underlying molecular mechanism is not yet known, these studies reveal that cells suffer consequences of damage long after exposure, and that can signal unexposed neighboring cells to respond similarly. Thus, a single acute exposure to a chemotherapeutic agent can cause long-term changes in genomic stability. If the results of these studies of mouse embryonic stem (ES) cells are generally applicable to many cell types, these results suggest that a relatively small number of cells could potentially induce a tissue-wide increase in the risk of de novo homologous recombination events.

In vivo recombination after chronic damage exposure falls to below spontaneous levels in "recombomice".

All forms of cancer are initiated by heritable changes in gene expression. Although point mutations have been studied extensively, much less is known about homologous recombination events, despite its role in causing sequence rearrangements that contribute to tumorigenesis. Although transgenic mice that permit detection of point mutations have provided a fundamental tool for studying point mutations in vivo, until recently, transgenic mice designed specifically to detect homologous recombination events in somatic tissues in vivo did not exist. We therefore created fluorescent yellow direct repeat mice, enabling automated detection of recombinant cells in vivo for the first time. Here, we show that an acute dose of ionizing radiation induces recombination in fluorescent yellow direct repeat mice, providing some of the first direct evidence that ionizing radiation induces homologous recombination in cutaneous tissues in vivo. In contrast, the same total dose of radiation given under chronic exposure conditions suppresses recombination to levels that are significantly below those of unexposed animals. In addition, global methylation is suppressed and key DNA repair proteins are induced in tissues from chronically irradiated animals (specifically AP endonuclease, polymerase beta, and Ku70). Thus, increased clearance of recombinogenic lesions may contribute to suppression of homologous recombination. Taken together, these studies show that fluorescent yellow direct repeat mice provide a rapid and powerful assay for studying the recombinogenic effects of both short-term and long-term exposure to DNA damage in vivo and reveal for the first time that exposure to ionizing radiation can have opposite effects on genomic stability depending on the duration of exposure.

"Recombomice": the past, present, and future of recombination-detection in mice.

Homology directed repair (HDR) provides an efficient strategy for repairing and tolerating many types of DNA lesions, such as strand breaks, base damage, and crosslinks. Recombinational repair and lesion avoidance pathways that involve homology searching are integral to normal DNA replication. Indeed, it is estimated that at least ten HDR events take place each time a mammalian cell divides. HDR is associated with the transfer and exchange of DNA sequences. Usually, homologous sequences are aligned perfectly and flanking sequences are not exchanged. However, those sequence misalignments and exchanges that do occur can lead to rearrangements that contribute to cancer (e.g. deletions, inversions, translocations or loss of heterozygosity (LOH)). In order to reveal genetic and environmental factors that modulate HDR in mammals, several approaches have been used to detect recombination events in vivo. Here, we briefly review three methods for detecting homologous recombination in mice, namely: sister chromatid exchange (SCE), LOH, and recombination at tandem repeats. We conclude with a more detailed description of the recently developed "Fluorescent Yellow Direct Repeat" (FYDR) mouse model, which exploits enhanced yellow fluorescent protein (EYFP) for detecting mitotic homologous recombination in vivo. Applications of the FYDR mice are described, as well as the broader potential for using fluorescent proteins to detect recombination in various tissues/cell types in vivo.

Spontaneous mitotic homologous recombination at an enhanced yellow fluorescent protein (EYFP) cDNA direct repeat in transgenic mice.

A transgenic mouse has been created that provides a powerful tool for revealing genetic and environmental factors that modulate mitotic homologous recombination. The fluorescent yellow direct-repeat (FYDR) mice described here carry two different copies of expression cassettes for truncated coding sequences of the enhanced yellow fluorescent protein (EYFP), arranged in tandem. Homologous recombination between these repeated elements can restore full-length EYFP coding sequence to yield a fluorescent phenotype, and the resulting fluorescent recombinant cells are rapidly quantifiable by flow cytometry. Analysis of genomic DNA from recombined FYDR cells shows that this mouse model detects gene conversions, and based on the arrangement of the integrated recombination substrate, unequal sister-chromatid exchanges and repair of collapsed replication forks are also expected to reconstitute EYFP coding sequence. The rate of spontaneous recombination in primary fibroblasts derived from adult ear tissue is 1.3 +/- 0.1 per 106 cell divisions. Interestingly, the rate is approximately 10-fold greater in fibroblasts derived from embryonic tissue. We observe an approximately 15-fold increase in the frequency of recombinant cells in cultures of ear fibroblasts when exposed to mitomycin C, which is consistent with the ability of interstrand crosslinks to induce homologous recombination. In addition to studies of recombination in cultured primary cells, the frequency of recombinant cells present in skin was also measured by direct analysis of disaggregated cells. Thus, the FYDR mouse model can be used for studies of mitotic homologous recombination both in vitro and in vivo.