Dependoparvovirus, Aveparvovirus and Chaphamaparvovirus, and 11 different avian picornaviruses tentatively belonging to four different genera Sicinivirus, Anativirus, Megrivirus and Aalivirus. Most of these viruses were genetically different from other currently known viruses from the NCBI dataset. The study showed that the abundance and number of avian picornaviruses and parvoviruses varied considerably throughout the year, with the high number of virus reads in some of the duck samples highly suggestive of an active infection at the time of sampling. The detection and characterisation of several parvoviruses and picornaviruses from the individual duck samples also suggests co-infection, which may lead to the emergence of novel viruses through possible recombination. Therefore, as new and emerging diseases evolve, it is relevant to explore and monitor potential animal reservoirs in their natural habitat.Ducks can shed and disseminate viruses and thus play a role in cross-species transmission. In the current study, we detected and characterised various avian parvoviruses and picornaviruses from wild Pacific black ducks, Chestnut teals, Grey teals and Wood ducks sampled at multiple time points from a single location using metagenomics. We characterised 46 different avian parvoviruses belonging to three different genera  Among birds, notably wild ducks constitute a significant reservoir for viruses including, but not limited to, influenza viruses4 and coronaviruses6. Such viruses can become potential threats by spreading to other animals and humans. Thus, metagenomic characterisation of viruses from wild ducks may provide insight into viruses for which these birds may act as a natural reservoir.Birds and other animals can be reservoirs for zoonotic viruses that may have serious implications for human health and agriculture, for example, avian influenza A virus9 and may result in the emergence of new viruses through possible reassortment or recombination13. These novel viruses could have the potential to cause new virus disease with little prophylaxis or therapy to counteract, thus becoming a One-Health concern. However, very little is known about viruses circulating in wild ducks in Australia. Few studies have explored whether seasonal changes affect the presence and abundance of such viruses albeit efforts have been made to understand the avian virus community of Australian wild birds at a given time point6, and the spatiotemporal dynamics of highly pathogenic viruses such as influenza viruses from Australian waterfowl and shorebirds15.Viruses are shown to co-circulate within a community leading to co-infections16. Parvoviruses are thought to have evolved to be rather host-specific, however, variants of some parvoviruses have a broader range of hosts17. Avian parvoviruses are associated with gastrointestinal disease, especially in juvenile birds, although they have also been isolated from healthy birds16. Picornaviruses are RNA viruses and one of the most diverse family of viruses, with virions of about 30 nm in diameter18. Like parvoviruses, most picornaviruses are thought to possess a relatively narrow host species spectrum19. However, some picornaviruses like the foot-and-mouth disease virus can infect a broad range of hosts such as wild and domestic ruminants and swine20. Some avian picornaviruses like avihepatovirus and avian encephalomyelitis virus also infect a broader host range from ducks to pigeons and other birds21. Avian picornaviruses can cause infections ranging from subclinical infection to clinical signs of disease such as a drop-in egg production and decreased growth24. Little is known about the diversity, host spectrum, pathogenicity, seasonal variation and factors that affect co-circulation and co-infection of avian parvo- and picornaviruses circulating in wild ducks.Parvoviruses are amongst the smallest known DNA viruses, with virions of about 24 nm in diameter26, we will here focus only on the results for these viruses.The current study was designed to detect and characterise avian viruses present in Australian wild ducks by analysing faecal samples collected at various time points from a single location. Specifically, we wanted to determine the virus diversity, its abundance and seasonal variation in wild Australian ducks. As parvoviruses and picornaviruses were the most abundant and most often detected virusesFrom the initial data analysis, it became apparent that reads mapped to parvoviruses and picornaviruses were particularly abundant compared to other avian viruses in many of the samples included in the current study. Therefore, we have here only focused on the results for the parvoviruses and picornaviruses.Avian parvoviruses and picornaviruses were present in samples from each of the species of ducks captured, and all but one pooled sample contained more than one parvovirus and picornavirus. A total of 102 complete or partial parvovirus sequences  compared to the autumn samples . However, the abundance of parvoviruses in winter and spring/summer samples  were low, except for the juvenile Pacific black duck December 2016 sample (4.77% of reads), which may be due to the age of the birds rather than any seasonal factor, as parvoviruses prefer multiplying host cells for their replicationThe Pacific black duck sample from December 2016 (PBD12.16) contained the highest number of total parvovirus reads with 47,700 parvovirus reads/million reads (4.77%) and these belonged to at least 8 different parvovirus genomes (Table The Wood duck sample from August 2018 (WD08.18) contained the highest number of picornavirus reads with 158,400 picornavirus reads/million reads (15.84%) mapped to at least 3 different picornaviruses , Pacific black duck chaphamaparvovirus 1 (PBDCPaV1/PBD12.16) and three Chestnut teal chaphamaparvoviruses . One of the Chestnut teal chaphamaparvovirus sequences was assembled from the pooled sample (CT08.18) and two more were assembled from the individual Chestnut teal sample (CT08.18/12952).The parvovirus sequences detected in the Australian ducks were diverse and belonged to three different genera of parvoviruses: Dependoparvovirus was found in the Pacific black duck pool from December 2016 [Pacific black duck adeno-associated virus (PBDAAV/PBD12.16)]. Three partial Aveparvovirus sequences were found in the August 2018 Pacific black duck pool. These three sequences likely came from a single virus as they each represented a different section of the aveparvovirus genome [Pacific black duck aveparvovirus ]. The remaining assembled parvovirus sequences from all the duck species belonged to at least 44 viruses within the genus Chaphamaparvovirus (CPaV) [Supplementary material Parvovirinae  that likely encoded an assembly activation protein required for capsid packaging32. Although we cannot definitively say whether this dependoparvovirus was able to replicate/infect with or without a helper virus, it phylogenetically grouped with other helper-dependent adeno-associated viruses and this sample contained an avian adenovirus6.The phylogenetic analysis of the capsid protein encoded by the duck parvoviruses and picornaviruses was carried out and the genetic distance calculated to find the closest relative sequence  encoded a partial non-structural protein 1 (NS1), a partial hypothetical protein (HP) and a partial capsid protein, all most likely from a single virus. The phylogenetic analysis of the partial non-structural amino acid sequences showed that it was distantly related to KC876004.1, a pigeon Parvovirus A from China in 2011The four near full-length chaphamaparvovirus sequences and one partial sequence which contained the full NS1 were more closely related to each other (57.1–82.2% similarity) and more distantly related to currently known viruses in the NCBI dataset (56.3–69.6% similarity). They, however, still belonged to an avian lineage within the chaphamaparvovirus genus  and 1 short sequence from a Chestnut teal sicinivirus-like virus from the August pool (CTSV-like virus/505nt/CT08.18). One sequence from a Pacific black duck anativirus-like virus from August pool (PBDAnV-like virus/5691nt/PBD08.18) was identified and was the only anativirus-like virus found in the duck samples. The avian megriviruses from the duck samples included five sequences likely belonging to a single Pacific black duck megrivirus found in the August pool , seven sequences likely from three Wood duck megriviruses found in the August pool  and two sequences likely from 1 Chestnut teal megrivirus from August pool (CTMeV/CT08.18 of 583nt and 363nt long). The avian aaliviruses from the duck samples included 2 Pacific black duck aaliviruses (PBDAaV/PBD05.18 of 4768nt and 4182nt long and PBDAaV/7615nt/PBD08.18) and 1 Chestnut teal aalivirus .Phylogenetic analysis and maximum likelihood trees of the amino acid sequences of the partial polyprotein (PP) from the 26 picornavirus consensus sequences identified viruses tentatively belonging to the genera rus Fig. . The tenThe generated partial picornavirus sequences were analysed using ORFfinder, BLAST and MEGA to determine the general position and homology of the sequences and is shown in Figure S5 in the Supplementary Material 22 and even less similar (51.2–51.6%) to other anativiruses  was carried out on the amino acid sequence of the polyprotein encoded by the two partial genome consensus sequences. The 1,495 amino acids of the PBDSV-like virus/4792nt/PBD05.18 consensus sequence that encodes the RNA helicase, Peptidase C3 and RdRp of the sicinivirus was only 61.4% similar to KF979331.1, a chicken picornavirus 1 from Hong Kong in 2008The Chestnut teal sicinivirus-like virus (CTSV-like virus/505nt/CT08.18) was found to be different from the PBDSV-like virus/PBD05.18 with 88.3% similarity at the nucleotide level (BLASTN) and 97.6% similarity at the amino acid level (BLASTP).Aalivirus, Aalivirus A (GenBank Accession: KJ000696.1)35, from a domestic Pekin duck from China in 2012, with 82.8% similarity. It was even less similar (51.1–62.4%) to other picornaviruses, including MH453803, an Avihepatovirus-like virus from a dabbling duck sample from Victoria in 201336  was distantly related to the only member of the genus 37 with 99.2% similarity over 799 amino acids in RdRp region and with 98.7% similarity over 1,051 amino acids in capsid region. The Wood duck megriviruses were 96.8–99.5% similar to each other in the RdRp region than to other megriviruses from the NCBI dataset. As stated above, two RdRp partial sequences that overlap one long capsid encoding partial WDMeV genome sequence were characterised. The phylogenetic analysis of the partial capsid protein coding region of the Wood duck megriviruses may indicate an evolutionary recombination event as discussed below  has also recently been shown in silico to be present in some chaphamaparvoviruses39. Consequently, we looked for the presence of an uORF in our duck picornavirus and chaphamaparvovirus sequences using the NCBI ORFfinder  has been described and the expressed protein purified for some viruses belonging to the virus family RF as ORF has alsoRF as ORF has alsoAll the generated consensus sequences were also bioinformatically analysed further for the detection of possible motifs and nuclear translocation signals. Various motifs such as helicase , bipartite nuclear localisation signal (such as RRNYGADRAGSARTRGR in the NS1 of PBDCPaV1/PBD12.16), Arginine rich region, Glycine-rich region, Serine-rich region, Ribosomal protein S14 signature, Neutral zinc metallopeptidases, Zinc finger C2H2 type domain, C-type lectin domain, AAA+ superfamily and Malic enzyme NAD binding domain were detected in some of the parvovirus and picornavirus amino acid sequences  suggesting not only simultaneous co-circulation but indeed also co-infection. Like for the parvoviruses, the results for picornaviruses detected in the current study also suggested co-circulation and most likely co-infection. For example, in May 2018, aaliviruses were found in both the Pacific black duck and Chestnut teal samples while in August 2018, megriviruses were found in the Pacific black duck, Wood duck and Chestnut teal sample. This could suggest a seasonal co-circulation variation in the avian virome.40. Ducks are described as “trojan horses” for viruses such as influenza viruses3. They can disseminate pathogenic viruses, showing few or no signs of disease. Ducks are found in nearly all aquatic habitats and can shed and spread viruses through both the respiratory and intestinal tracts3. These facts accentuate the importance of the current study in conducting surveillance for potential virus threats to wildlife and humans from wild ducks.The ecological and biological characteristics of birds enable them to play a major role in the emergence of new viruses and cross-species transmissionThe current study shows that common wild Australian ducks  carry a diverse number of avian parvoviruses and picornaviruses. We detected and partially sequenced around 46 parvoviruses and 11 picornaviruses from the sampled ducks, with the majority of the viruses being genetically different  from the other currently known viruses in the NCBI dataset. Some of the viruses described here were found in high abundance in the sample suggesting active infection at the time of sampling.41. Nevertheless, 10–100 times more parvovirus reads were found during winter and spring/summer samples compared to the autumn samples. Human parvoviruses like Parvovirus B19 that causes fifth disease or “slapped cheek” was shown to be more prevalent during these seasons also42. However, in the case of animal parvoviruses, very few studies have been conducted to determine their seasonal prevalence, especially from Australia44. The abundance of parvovirus was high in the juvenile Pacific black duck samples compared to their adult counterparts, which suggests that these duck chaphamaparvoviruses prefer juvenile hosts like any other parvovirus27. Duck picornaviruses were mainly found during late autumn to late winter months. To the best of our knowledge, the current study is the first to analyse the seasonal abundance of both duck parvoviruses and picornaviruses, especially from Australia.The duck parvoviruses and picornaviruses found varied not only in virus composition across species and time but also in their abundances, despite the ducks sharing the same habitat at the times of sampling. Duck parvoviruses were detected throughout the year, which could be because of the high virus stability exhibited by parvovirusesParvovirinae generally, the species demarcation criterium is to have the identity of NS1 protein amino acid sequences < 85%16. The picornaviruses genus demarcation criteria are to exhibit significant divergence on orthologous proteins < 66% of the capsid and < 64% of the helicase, peptidase C3 and RdRp18. The duck parvoviruses, the Pacific black duck sicinivirus-like virus and the Pacific black duck anativirus-like virus described here, fulfil these criteria and can be considered as new or novel virus species based on the genome sequence analysis. The detection of more than 30 new viruses, that are not previously described, belonging to two virus families from four duck species collected at three-time points indicates inadequate surveillance studies on wild ducks.The duck parvoviruses and the picornaviruses identified and characterised in this study showed relative similarity to other avian viruses from their respective genera. The dependoparvovirus, anativirus-like virus, aalivirus and the megrivirus characterised from the Australian duck samples had the closest similarity to other duck viruses. The aveparvovirus, chaphamaparvovirus and sicinivirus-like virus from the Australian duck samples had the closest similarity to other avian viruses from chickens or pigeons. The duck chaphamaparvoviruses from the current study consistently formed duck clusters/lineages separate to chaphamaparvoviruses from other avian hosts. Each of the sub-clusters included viruses from more than one duck species indicating that these parvovirus lineages may have evolved into individual strains or species that may have the ability to infect closely related duck species. It is also to be noted that the duck aveparvovirus and the duck sicinivirus-like virus from the current study are the first duck viruses identified in their respective genus. As per the International Committee on Taxonomy of Viruses (ICTV) for all the genera under the subfamily Evidence of virus co-circulation has been found from the faecal samples collected from different duck species at the same time point and location. For example, the presence of megriviruses and chaphamaparvoviruses in the faecal samples of Pacific black ducks, Wood ducks and Chestnut teals collected in August 2018 from the same location indicates virus co-circulation. In addition, the presence of aalivirus in both Pacific black ducks and Chestnut teals in May 2018 also indicates its co-circulation. However, these viruses were different in nucleotide and amino acid level even within the species level.46 occurring in avian host species during co-infections or due to the occurrence of gradual mutation during replication. Another example is that of the Pacific black duck aalivirus (PBDAaV/PBD05.18) showing 98.0% similarity to the KJ000696.1 Aalivirus A from China in its RdRp region while having only 92.3% similarity in its structural coding region. This suggests that this virus may have adapted to infect wild Pacific black ducks/mallards through possible recombination or gradual mutation. However, further studies are required to understand the evolution of these viruses and how that may enable cross-species infection.The simultaneous detection of related viruses in the individual sample indicates co-infection, which may lead to recombination. For example, the presence of two different non-structural Wood duck megrivirus sequences with only identical structural sequence (WDMeV/6497nt/WD08.18) leads to the speculation of two Wood duck megriviruses having the same structural protein but with different non-structural protein coding regions. This could be because of possible virus recombination47, the identification of cachavirus from dogs with diarrahea48 and the isolation of various chaphamaparvoviruses from different host faecal samples49 suggesting possible infection in the gastrointestinal area, does imply that these viruses could be pathogenic and may cause diseases in their host species. The high abundance of chaphamaparvoviruses in the juvenile Pacific black duck sample (PBD12.16) compared to their adult counterpart (PBD05.18 and PBD08.18) could also suggest that these duck chaphamaparvoviruses prefer juvenile hosts for their active infection and multiplication27.The NGS data with a high number of virus reads for some of the duck samples imply the presence of an active infection and suggest a potential health impact on the birds. It should be noted that at the collection time point no close physical examination, such as the presence of any histopathological lesions, was conducted to detect any clinical signs of disease. The high viral load detected in some of the bird samples, with the caveat that all faecal samples were subjected to virus enrichment and nucleic acid amplification, suggests substantial use of infected host cell machinery by the virus for its multiplication and expression, and consequently the presence of an active infection and potential adverse effects on cells and host. For example, with respect to chaphamaparvoviruses, the identification of mouse kidney parvovirus from mice with inclusion body nephropathy21. These symptoms in ducks, if caused by the virus, cannot be determined without close pathological examination. It has been previously shown that during autumn–winter seasons, due to limited pasture availability and low temperature, there is a possibility of body fat loss in birds50, which in turn may lead to reduced immunity51. This could be a factor that may account for susceptibility to virus infections. The abundance of the virus particles in these samples without the indication of any visible clinical signs of disease, as noted during collection time point, could also indicate the evolution of a host–pathogen relationship in these birds enabling co-infections. However, how these viruses impact the health of these birds can only be speculated at this point, and further pathological studies are required for the complete elucidation of these viruses’ pathogenicity.Another example is the 15.8% viral abundance of megriviruses in the Wood duck WD08.18 sample. Megrivirus can cause hepatitis and enteritis in turkeys6. Nevertheless, all the virus sequences described here are distantly related and do cluster together along with other avian viruses. Detection of these viruses from different duck samples or culturing and inoculation studies with the viruses could potentially better establish the identity of their hosts.Despite the detection and characterisation of 46 parvoviruses and 11 picornaviruses from these samples, there is a high possibility of the presence of many more parvoviruses and picornaviruses due to the detection of partial genome sequences. However, as shown in Figures 38 and human cytomegalovirus52 and shown in silico to be present in some chaphamaparvoviruses39. It is previously shown that in enteroviruses, the uORF encode a virus protein that facilitates virus growth in gut epithelial cells, which is the site of initial viral invasion into susceptible hosts38. However, further isolation and purification of the uORF protein only could lead to the concrete conclusion on the expression and function of this short peptide in the duck viruses. The presence of other sequence features like the helicase, RdRp, Peptidase C3 and capsid protein domain detected in silico indicate that the virus sequences described here are likely from functional avian viruses. Other virus motifs such as bipartite nuclear localisation signal54 were also detected in some of the parvoviruses and picornaviruses, although, the detailed analysis on the functions of these motifs and domains exceeds the scope of this paper. It is to be noted that the phospholipase A2 domain was not found in any of the parvoviruses detected from the duck sample, which is consistent with other avian parvoviruses16 and avian chaphamaparvovirus49.We found an upstream ORF in some of the picornaviruses and chaphamaparvoviruses in silico; however, at this point, the expression of this ORF is uncertain. The uORF could regulate the translation of the primary ORF and was detected in several viruses such as enterovirusesFinally, as stated above, the avian parvo- and picornaviruses detected and characterised from the bird samples form only a fraction of the total avian virome of these duck species. The identification of 46 different avian parvoviruses and 11 different avian picornaviruses that are distantly related to other currently known viruses from the NCBI dataset indicate the poor understanding of the avian virome and emphasise the need for further elucidation. Further analysis of the NGS reads generated from the wild duck samples is underway to determine the avian virus community of these birds and to determine the factors that influence their ecology.6) in this study to have samples from three seasonal time points for this duck species. Chestnut teal samples were collected in May, August and November 2018 . Grey teals were able to be captured and sampled in November 2018 (GT11.18) but at no other time points. Wood ducks were only captured and sampled in August 2018 (WD08.18). All samples were stored at − 80 °C within 1–3 h of collection until processing.Fresh wild duck faecal samples were collected from Wallington, south-eastern Victoria, Australia. The samples were collected in late autumn (May 2018), late winter (August 2018) and late spring/early summer (November 2018 and December 2016). Individual Pacific black ducks (PBD), Chestnut teals (CT), Grey teals (GT) and Wood ducks (WD) were able to be captured and sampled as part of this study. Pacific black ducks were captured in May and August 2018 . No wild Pacific black ducks were captured during November 2018 and consequently, we included pooled juvenile Pacific black duck samples that had been collected in December 2016 6.Samples of 3–6 individual ducks were pooled by species and collection date6. Sequencing library preparation was performed using the Ion Fragment Library kit (Life Technologies)6. Libraries were quantified and then pooled prior to loading onto Ion 530 or 540 Chips using the Ion Chef Instrument. Following template preparation, the chips were run on an Ion Torrent S5XL System (Thermo Fisher Scientific) as per company protocols. Two individual Chestnut teal samples (CT08.18/11356 and CT08.18/12952) from August 2018 and which formed part of pool CT08.18 were also processed and sequenced as described for the pooled samples, to obtain more virus sequences of a low pathogenicity H9N2 avian influenza virus detected in that sample4.The extracted nucleic acids from both aliquot A and B of the samples were subjected to cDNA synthesis and amplification with the SeqPlex RNA Amplification Kit (Sigma) as per the manufacturer’s instructions56. Briefly, all available virus sequences and all RefSeq virus sequences were downloaded from the NCBI GenBank genetic sequence database (Dec 2018), and a local BLAST database created for the two sets of sequences separately. BLASTN query against the two virus reference sequence databases was performed with an e-value cut-off of 1 × 10−10 and 1 × 10–30. A TBLASTX query against the two virus reference sequence databases was also performed with an e-value cut-off of 1 × 10−10. BLAST query results files were converted into spreadsheet files, sorted by virus matches, and a list of potential virus targets created for each sample. The list was then manually inspected to identify viruses of interest. It became apparent that parvoviruses and picornaviruses reads were particularly abundant in many of the samples, and therefore we focused further analyses on these virus families.NGS data analyses were carried out as described earlier56. AssemblerSPAdes 5.6.0 plugin on the Ion Torrent Server was used to generate contigs from the sequence reads of each sample as described earlier6. The contigs were queried against the two virus reference sequence databases using BLASTN and TBLASTX to identify virus sequences as described above and using the same e-value cut-offs. Picornavirus and parvovirus contigs of length greater than 500 nucleotides were then used as references in TMAP plugin and trimmed to regions with a mapping quality of 20 or higher and a coverage depth of at least 2 unless specified. Nearly complete sequences for some viruses were also generated by assembling overlapping sequences and by using MEGA7 and magicblast57 from contigs and consensus sequences generated from different NGS runs. These sequences were again subjected to TMAP and consensus sequences generated from mapped sequences using IGV to determine the depth of coverage.The NGS reads were mapped against reference genomes of parvoviruses and picornaviruses identified to be of interest using the TMAP plugin on the Ion Torrent server. The full or partial consensus sequences of the viruses were obtained from TMAP using Integrative Genomics Viewer software (IGV)  as described earlierThe use of the non-ultracentrifuged samples acted as a control to differentiate endogenous sequences compared to sequences coming from exogenous virion. The sequences more abundant in the ultracentrifuged samples and those which did not provide any evidence of host genome sequences attached were considered likely to be from virus genomes.59 , NCBI ORFfinder60, ScanProsite61 and PSORT II62. The presence of these features in the expected location in the virus sequences was used as an additional confirmation that reliable consensus sequences had been generated from the reads.Open reading frames (ORFs), and features of the virus genomes such as promotors, motifs and nuclear locating signals were identified in silico using Basic Local Alignment Search Tool63 using the MEGA 764/MEGACC65 software. Maximum Likelihood phylogenetic analyses were conducted using the MEGA 7/MEGACC software by first identifying the best evolutionary model for generating a phylogenetic tree before creating the phylogenetic trees. The robustness of different nodes was assessed by bootstrap analysis using 1,000 replicates for amino acid alignments. The number of nucleotide/amino acid differences between sequences was calculated using the same software.Relevant sequences related to the partial and complete virus consensus sequences identified here were selected from the NCBI GenBank database using BLASTN. Nucleotide and protein sequences were aligned using Clustal-WSupplementary information


Carbon dioxide (CO2) laser surgery as a conservative tool plays a peculiar role in the management of head and neck cancer. Numerous patients who were candidates for transoral laryngeal microsurgery have forced us to eliminate frozen-section evaluation of surgical margins and use a magnified view of the larynx. The present study evaluated surgeon-judged negative margins with permanent microscopic pathologic evaluation. In this cross-sectional study, we evaluated the permanent pathologic margins of the resected laryngeal specimen which were considered negative by judgment of surgeons. Patients consisted of 61 pathologic proven T1-T2 laryngeal squamous cell carcinoma (SCC) cases. In all patients, tumor resection was performed via a transoral route with CO2 laser, and no residual laryngeal tumor was observed according to judgment of the surgeon. The patients with positive margin (s) underwent another resection. Patients were followed up for 18 months for tumor recurrence.The obtained results demonstrated that pathologic margins were reported in 6 patients, with the deep margin being the most common positive margin. During the 18-month follow-up, 8 cases of recurrence were detected.Judgment of the surgeon was in agreement with permanent pathologic evaluation in transoral laryngeal laser resection at the early stages of laryngeal SCC in most cases. Nevertheless, it is suggested that further direct studies be conducted to evaluate the frozen section on oncologic outcomes in transoral laser surgery for laryngeal cancer.  Head and neck squamous cell carcinoma (HNSCC) is the eights leading cancer by incidence worldwide . LaryngeThe timely treatment and complete eradication of this cancer with surgery plays a key role in the long-term prognosis of patients. There have been numerous studies investigating the efficacy and outcomes of various treatment modalities for early glottic malignancies -4. LaserBased on extensive studies, it is nowadays an acceptable practice to remove laryngeal lesions in the T1 and T2 stages by microscopic transoral surgery using laser as a regular method ,7. Microscopic methods have received increasing attention due to the complications and consequences of open surgeries, the need to spend more time and cost, as well as prolonged surgery duration ,5. The cTherefore, numerous efforts have been made to find ways to ensure that the margin of the lesion is clean and free from tumor cells after transoral laryngeal microsurgery resection. Some of these methods include in vivo staining, immunofluorescence staining, narrow-band imaging based on the vascular bed and blood flow, and frozen section margin evaluation during operation to ensure complete removal of tumorMoreover, a microscopic view during operation has seemingly a high correlation with the negative peripheral margin of the lesion ,3,5,7-9.The present study described the18-month follow-up of 61 patients with T1 and T2 laryngeal squamous cell carcinoma (SCC) who were treated with laser surgery, and margins were just evaluated by surgeon judgment during resection. 2 laser with high magnification. Neither did the patients have open surgery nor additional treatment, including radiotherapy. After the completion of tumor removal and aggressive rubbing of surgical site with brain cotton (for eliminating carbonated cells), cold-cup biopsy forceps was performed on five samples of margins , and they were sent for permanent pathologic evaluation. Frozen section evaluation of margin was not performed since one of the advantages of laser resection without reconstruction had been proposed as resection performed by a second-look procedure  and an informed consent form was obtained. Before the commencement of the study, the patients were provided by the purpose of the study, and the informed consent was obtained; thereafter, they entered into the study. Demographic characteristics of the patients, including age, gender, and location of the primary lesion were recorded. Patients underwent surgical removal of laryngeal lesions with CO The results of permanent pathological examinations were recorded and another resection with laser was carried out using second look procedure in case of positive margins on permanent pathologic evaluation. After discharge from hospital, the patients underwent regular follow-up laryngoscopy one month after the surgery and then every 3 months. It is worth noting that laryngeal biopsies were performed under general anesthesia at the suspicion of recurrence in the follow up examination.Out of 61 patients enrolled in the present study, 57 cases were male and 4 subjects were female. The mean age of patients was reported as 56.9 years (30- 81 years). 44 patients had T1 tumors and 17 cases had T2 tumors. The location of the tumor was glottic in 42 patients, while the involved site was supraglottic in 19 patients. In 55 cases, all the samples sent from marginal laryngeal lesions were demonstrated to be free from cancer cells, and the resected margins were positive in 6(8.9%) subjects. For these patients, the positive marginal tumor was resected again the sample was sent to the pathology department for further evaluation. Deep margins were found to be the most common margin samples involved with cancer cells, followed by the samples of the anterior and inferior margins. During the 18-month follow-up, eight recurrences were detected (13.1% of cases), five of which were glottis cancer, and the other three were supraglottic cancer. The mean relapse time was obtained as 10 months, and the relapse was not associated with patients’ gender (P= 0.421) . The patients who relapsed were candidates for radiotherapy and no recurrence was detected in further follow-up.Moreover, no significant relationship was observed between the primary site of the tumor and recurrence (P=0.6) . Data analysis indicated no significant relationship between tumor T and its recurrence probability . On the other hand, there was no significant relationship between the stage and the recurrence time. Finally, there was no correlation between the status of histopathological margins and the relapse of the lesion .The present study evaluated surgeon reliability to predict surgical margin status in transoral laser resection of T1 and T2 laryngeal cancer. The findings indicated that the difference in surgeons’ judgments about tumor margin using microscopic magnification was only 8.9%.Today, the use of laser in the treatment of early stages of the laryngeal SCC is universally approved which is due to the success of this method in organ preservation and local tumor control. Nonetheless, it is of utmost importance to ensure that the lesion is completely removed from cancerous tissue with a safe margin (about 1-4 mm) .To this end, various methods have been proposed, such as the frozen section procedure, use of in vivo staining, and an immunofluorescence staining, as well as the reliance on the surgeon's microscope vision using a high magnification of the microscope during surgery , 3, 7-9.In a study conducted by Hendriksam et al., 15 patients with T1 and T 2 glottic SCC underwent surgical resection through transoral laser resection. They showed that the evaluation of the resected specimen margin had the worst correlation with the site of the tumor resection margins. They proposed that margin evaluation of resected specimen may be biased by thermal shrinkage of Laser beam and carbonization. Neither did they use the frozen section nor they mention clearly how they managed positive margins. The current study did not find the significantly worse outcome in positive margins and margin assessment technique was similar to techniques correlated with outcome. Three is a possibility that our approach will be different with positive margins .In a study carried out by Wong et al. (2013), 29 patients  underwent surgery based on surgeon's and were compared with the histopathological exams. The histopathologically examined margins were negative in 14 patients. In 10 patients, the margins of tumor were inconclusive due to burning and carbonization of the margin of the lesion. The results of histological studies demonstrated positive marginal involvement in 5 patients. It is worthy to note that five cases of recurrence were reported during the follow up period (In a study carried out by Blanch et al. 2007, 357 patients who underwent transoral laryngeal microsurgery were assigned to three groups: negative margins, positive margins, and unknown margins; subsequently, relapsing was followed up for 24 months . There wAlong the similar lines, a study was conducted by Fang et al. to evaluate the predictive value of in-operative frozen section in relapsing and survival of patients with SCC undergoing lesion removal using CO2 laser. The relapse was investigated in patients with a negative margins during the first transoral laryngeal microsurgery process and those requiring re-transoral laryngeal microsurgery due to positive frozen-section during surgery . 75 patiIn a study performed by Piazza et al. on 147 patients with glottis in early-stage, the frozen section was not used during resection; however, the entire specimen was sent for permanent pathology evaluation. In the mentioned study, the patients had 26.5 positive margins; nonetheless, the important point was that they would follow a single positive superficial margin. It worth noting that similar to the present study, this study weakened the usage of the frozen section during surgery .As evidenced by the obtained results, laser can be widely used as a reliable method in the treatment of early stages of SCC in larynx. The achievement of proper local control and good prognosis with much less complications and much less time and cost is one of the advantages of this method over invasive surgical procedures or radiotherapy. The current study demonstrated than we can only rely on the surgeon judgment during the removal of the lesion by laser for the achievement of tumor-free margins.In summary, microscopic magnified surgeon evaluation has good concordance with permanent pathologic evaluation in transoral laryngeal laser resection at early stages. Further direct studies need to evaluate the frozen section on oncologic outcomes in transoral laryngeal cancer resection.


Targeted biologic therapies demonstrate similar efficacies in rheumatoid arthritis despite distinct mechanisms of action. They also exhibit a ceiling effect, with 10% to 20% of patients achieving remission in clinical trials. None of these therapies target synovial fibroblasts, which drive and maintain synovitis. Seliciclib (R-roscovitine) is an orally available cyclin-dependent kinase inhibitor that suppresses fibroblast proliferation, and is efficacious in preclinical arthritis models. We aim to determine the toxicity and preliminary efficacy of seliciclib in combination with biologic therapies, to inform its potential as an adjunctive therapy in rheumatoid arthritis.TRAFIC is a non-commercial, multi-center, rolling phase Ib/IIa trial investigating the safety, tolerability, and efficacy of seliciclib in patients with moderate to severe rheumatoid arthritis receiving biologic therapies. All participants receive seliciclib with no control arm. The primary objective of part 1 (phase Ib) is to determine the maximum tolerated dose and safety of seliciclib over 4 weeks of dosing. Part 1 uses a restricted 1-stage Bayesian continual reassessment method based on a target dose-limiting toxicity probability of 35%. Part 2 (phase IIa) assesses the potential efficacy of seliciclib, and is designed as a single arm, single stage early phase trial based on a Fleming-A’Hern design using the maximum tolerated dose recommended from part 1. The primary response outcome after 12 weeks of therapy is a composite of clinical, histological and magnetic resonance imaging scores. Secondary outcomes include adverse events, pharmacodynamic and pharmacokinetic parameters, autoantibodies, and fatigue.The study has been reviewed and approved by the North East - Tyne & Wear South Research Ethics Committee (reference 14/NE/1075) and the Medicines and Healthcare Products Regulatory Agency (MHRA), United Kingdom. Results will be disseminated through publication in relevant peer-reviewed journals and presentation at national and international conferences.Trials Registration: ISRCTN, ISRCTN36667085. Registered on September 26, 2014; http://www.isrctn.com/ISRCTN36667085Current protocol version: Protocol version 11.0  Around a third of people with RA have stopped working within 2 years of onset and around a half by 10 years, with significant costs to the economy in sick leave and work-related disability. Suboptimally controlled joint inflammation in RA leads to damage, deformity, disability, and impaired quality of life. Chronic inflammation reduces life expectancy by increasing the risk of cardiovascular disease. Advances in RA management strategies and targeted biologic therapies have contributed to significantly improved prognosis and outcomes for people with RA. However, there remains significant unmet clinical need. There is no cure for RA and, even with the best available therapies, only 20% to 30% of patients achieve remission, while 5% to 10% are refractory to all current treatments.Rheumatoid arthritis (RA) is a chronic inflammatory arthritis characterized by joint pain, swelling, and damage. which secrete pro-inflammatory mediators and tissue-destructive enzymes not specifically targeted by current therapies. A therapy that addresses the abnormal behaviour of RA synovial fibroblasts, particularly with potential additional actions on monocytes/macrophages and neutrophils,,7 could have a unique niche in RA management.Despite modulating different implicated immune pathways, current targeted therapies exhibit remarkably similar response rates in clinical trials. On average, 50% to 60% of participants improve by 20% (American College of Rheumatology (ACR) 20 response), 30% to 40% improve by 50% (ACR50) and 10% to 20% improve by 70% (ACR70). As current therapies target primary immune cells and cytokines, an untested hypothesis is that RA synovial fibroblasts may themselves drive and maintain synovitis, explaining the apparent “ceiling effect” of current therapies, which do not target these cells. RA synovial fluid also contains abundant neutrophils, However altered cytokine and tissue destructive enzymes due to dysregulation of these pathways has also been reported in RA synovial fibroblasts.–11 Seliciclib (R-roscovitine) is an orally available cyclin dependent kinase inhibitor that selectively targets CDK2, CDK7, and CDK9.,13 In addition to directly suppressing fibroblast proliferation by inhibiting CDK2, seliciclib induces p21,,9 an endogenous CDK inhibitor whose activity is down-regulated in RA synovial fibroblasts.,15 Furthermore, inhibition of CDK7 and CDK9 reduces transcription of MCL1, which reduces viability of synovial fibroblasts, neutrophils, and macrophages. In vitro seliciclib causes growth inhibition and apoptosis of abnormally dividing cells, leaving normally dividing cells unaffected.–20 Seliciclib and related CDKIs have demonstrated good efficacy and potency in preclinical models of arthritis.–25 Additionally, genetic studies have implicated CDK inhibition as a plausible therapeutic strategy in RA, and support investigation of CDK inhibition in RA. Specific interactions of conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs) or biologic disease-modifying anti-rheumatic drugs (bDMARDs) with seliciclib are not predicted. Seliciclib has a reported toxicity profile similar to existing csDMARDs used to treat RA, and without the myelosuppression seen with other CDK inhibitors. Preclinical studies suggest combining CDK inhibition with cytokine blockers has additive effects and may not increase immune suppression. Determination of the toxicity profile and preliminary efficacy of seliciclib in combination with bDMARD therapies within a true-to-life pharmacological context (in which concomitant csDMARD use is permitted), will provide important insight into its potential acceptability as an adjunctive therapy in RA.Cell proliferation is dependent on orderly progression through the cell cycle, which is regulated by tightly controlled formation and activation of complexes comprising cyclin proteins and cyclin-dependent kinases (CDKs). Dysregulation of cyclin-CDK pathways has been demonstrated in many tumors, and their manipulation consequently exploited to develop anti-cancer drugs.22.1TRAFIC is an investigator-led, multi-center, rolling phase Ib/IIa trial investigating the safety, tolerability, and efficacy of seliciclib as an addition to existing bDMARD therapy in patients with moderate-severe RA. All eligible participants will receive seliciclib, the investigational medicinal product (IMP), with no randomization or control arm. based on a target dose-limiting toxicity (DLT) probability of 35% , or determination of unacceptable toxicity leading to cessation of the trial. It is planned that up to 7 cohorts of 3 participants each will be treated, with each cohort receiving 1 of 5 possible doses of the IMP. The trial design allows for early stopping if sufficient evidence of MTD has been achieved or if the lowest IMP dose is too toxic. Part 1 of the study has been completed and clinical data will be published in a separate manuscript.The primary objective of part 1 of the study (phase Ib) is to determine the maximum tolerated dose (MTD) and safety of seliciclib over 4 weeks when given in RA as an addition to existing bDMARD therapy. The MTD was established using a restricted 1-stage Bayesian Continual Reassessment Method (CRM) recruiting a total of 18 participants to receive the MTD recommended from part 1 for a maximum of 12 weekly cycles. At the conclusion of part 2, further investigation is warranted if the observed number of clinical responses at least equals a pre-specified critical number according to the statistical design. As this is the first trial investigating seliciclib in this indication as a repurposed drug and given the novel mechanism of action, the decision to continue will also be based on pharmacodynamic (PD) biomarkers and pharmacokinetic (PK) parameters, to avoid rejecting a potentially active drug which has not achieved pragmatic clinical measures of efficacy. A secondary aim, given seliciclib's potentially unique mode of action in RA, is to identify sensitive and robust outcome measures for future RA trials of seliciclib and similar agents in this indication.The primary objective of part 2 (phase IIa) is to assess the potential efficacy, using a composite response, of seliciclib as an addition to existing therapy following 12 weeks of treatment. Part 2 is designed as a single arm, single stage early phase trial based on a Fleming–A’Hern design In addition, the 28 joint disease activity score (DAS28) cut-off for inclusion in part 2 of the study was reduced from ≥4.0 to ≥3.2 as this was identified as a barrier to recruitment and 3.2 was felt to be a more clinically relevant cut-off.When we wrote the initial protocol, there was a significant unmet need for patients who had received tumor necrosis factor (TNF) blockade but who had not reached remission. Whilst the same was true for patients receiving other bDMARDs, more patients were receiving anti-TNF therapy at that stage because this treatment had been licensed for significantly longer than other biologic agents. The initial study protocol was therefore limited to patients receiving TNF inhibitors. However, recently more patients are being “cycled” from a TNF inhibitor to another bDMARD as part of their standard care and existing treatment guidelines, thereby reducing the pool of patients suitable for TRAFIC and significantly impacting on study recruitment. In other words, the “unmet need” has now been shifted downstream to include patients receiving other biologic therapies. We therefore amended the protocol (approved June 2019) to broaden the inclusion criteria to incorporate patients on 2 other bDMARDs (tocilizumab and abatacept) supported by preclinical data.2.2Adults (≥18 years old) fulfilling the 1987 ACR or 2010 ACR/European League Against Rheumatism (EULAR) criteria for RA who are currently taking a bDMARD therapy for at least 3 months as part of standard clinical care and who have ongoing moderate-severe disease activity are eligible. In part 1 of the study, the bDMARD was limited to anti-TNF therapy, which was amended in June 2019 to include abatacept or tocilizumab. The DAS28 cut-off in part 1 was ≥3.2. In part 2, the initial proposed DAS28 cut-off was ≥4.0 plus clinical synovitis in at least 3 joints, which was amended to ≥3.2 plus at least 1 joint amenable to ultrasound-guided synovial biopsy in June 2019. The current protocol allows anti-TNF or 2 other bDMARD therapies (abatacept or tocilizumab) to be used either as monotherapy or with permitted background csDMARDs .Full inclusion and exclusion criteria for current protocol v11.0  are shown in Table 2.3The study has been reviewed and given favourable ethical opinion by the North East – Tyne & Wear South Research Ethics Committee (reference number 14/NE/1075). Clinical trial authorization was given by the Medicines and Healthcare Products Regulatory Agency, United Kingdom. The study is being conducted in compliance with Good Clinical Practice, the Research Governance Framework for Health and Social Care, and national legislation implementing the EU Clinical Trials Directive (2001/20/EC) and subsequent amendments. Patients wishing to take part provide written informed consent by signing and dating a trial specific consent form, obtained prior to any study-specific procedures. The study is registered in the ISRCTN registry (identifier 36667085).2.4Data will be collected using standardized data entry and management system. An overview of the schedule of enrolment and timing and types of data collected in parts 1 and 2 of the study are shown in Tables 2.52.5.1Part 1: The MTD is defined as the dose that is closest to the dose at which 35% of participants experience a DLT over a 4-week treatment period with seliciclib. A DLT is defined as the cessation of IMP due to adverse events or reactions (AE/AR) occurring during the treatment period. AEs and ARs are attributed  to seliciclib and other therapies and categorised as mild, moderate or severe. These can be either symptomatic AEs or ARs  or abnormal laboratory parameters or investigations. DLT is based upon the patient's request to stop treatment, regardless of the severity classification, or an abnormal laboratory parameter necessitating cessation of treatment. In the event of several AEs/ARs contributing to the decision to discontinue IMP, only a single DLT will be recorded for the purposes of dose pathway decision making.,33 histological score (macrophage number in the sublining layer of the synovium)–37 and magnetic resonance imaging (MRI) score (rheumatoid arthritis MRI scoring system (RAMRIS)).–40 A patient receiving seliciclib is defined as responding if they achieve 2 out of 3 of:Part 2: Response rate at 12 weeks as a composite measure using clinical scores ,1.EULAR moderate response or ACR20 response2.2 at the end of dosingA reduction of sublining macrophage number of ≥20% or an absolute mean reduction of >200 macrophages/mm3.A reduction of RAMRIS synovitis of ≥0.5 units or osteitis score of ≥0.2 units at the end of dosing2.6Both study parts record and report:1.all AEs,2.PD biomarkers in peripheral blood,3.PK parameters in peripheral blood,4.relationship between PD and PK markers with each other and with AEs,5.longitudinal changes in FACIT Fatigue Scale and6.longitudinal changes in circulating autoantibodies.Part 2 also records and reports:1.response rate after 1, 2, 3, 6, and 9 weeks of therapy;2.relationship between PD and PK markers and efficacy,3.change in synovial metabolism assessed by fluorodeoxyglucose uptake by inflamed synovium measured by fluorodeoxyglucose positron emission tomography at baseline and at week 12 ;4.change from baseline in urinary metabolomic profiles during treatment; and5.molecular changes in the synovium consistent with seliciclib PD activity.2.7Seliciclib is the IMP. The selected dose range and schedule is based on healthy control and oncology studies in which more than 450 participants have received between 50 and 3600 mg seliciclib per day in a variety of intermittent dosing schedules. Using the selected 4 days per week schedule, the longest treatment period was 111 weeks. The terminal half-life is 3 to 4 hours and primary route of metabolism is via cytochromes 3A4 and 2B6, the major metabolite being much less active. Tolerability in previous studies was related to daily dose and number of consecutive dosing days. DLTs were reversible below 1600 mg of seliciclib daily and included fatigue, nausea and vomiting, elevated liver enzymes, and hypokalaemia. Non-limiting toxicities included anorexia and elevated serum creatinine. Most AEs were mild to moderate in severity, dose-related, and generally occurred during the first 3 weeks of therapy. Such symptoms are familiar to RA patients, either as a manifestation of the disease itself or as side effects of commonly prescribed csDMARDs. Hence the experience to date, particularly lack of myelosuppression, suggests that seliciclib should be acceptable and well tolerated in RA.The IMP is supplied as a 200 mg strength capsule . Cyclacel Pharmaceutical provides the bulk IMP. Penn Pharmaceuticals Ltd is responsible for treatment packaging and labelling. The IMP is prescribed by a trial clinician according to the protocol and dispensed according to local pharmacy policy. Participants return all IMP supplies in their original packaging (even if empty) to the trial clinician or pharmacist at each visit. All returned or unused IMP is stored in pharmacy until the end of the trial, or until the trial manager has completed appropriate reconciliation.In part 1, participants receive 200, 400, 600, 800, or 1000 mg seliciclib daily for 4 consecutive days every week for 4 weeks. Participants are assigned to the dose levels in cohorts of 3, with 400 mg seliciclib assigned to the first cohort. Participants in part 1 receive IMP treatment for 4 weekly cycles (in the absence of DLT) and, once the third subject of a cohort has attended their final visit, the chief investigator, principal investigators from each site, trial manager and trial statistician discuss any AEs/ARs and DLTs and make the decision whether or not to open the subsequent cohort, according to the CRM algorithm, with sponsor and Data Monitoring Committee (DMC) approval. An independent DMC undertakes independent review with the purpose of monitoring safety and efficacy endpoints. Dose cannot be escalated or de-escalated by more than 200 mg per cohort, and cohorts are enrolled until the MTD was determined, the maximum sample size reached, or if the lowest dose (200 mg) is determined to be too toxic.In part 1 of the trial, a DLT triggers patient withdrawal and informs the CRM algorithm. In part 2, seliciclib is withheld in the presence of severe DLTs, until the toxicity resolves to mild, with the exception of anaemia, hypokalaemia, or other laboratory abnormality that can be managed adequately by supplementation or medications. Dosing is recommenced at the next lower dose, and all dose reductions are permanent. In the event of a non-severe DLT, dosing with seliciclib may be withheld until the toxicity resolves. At this point drug is restarted at the same dose unless the treatment delay is 2 weeks or more, in which case a dose reduction is mandated as above.Participants may not receive any other investigational drugs while participating in this trial but may receive existing standard therapies and supportive treatment. As nausea and vomiting are commonly reported by participants treated with seliciclib, prophylactic use of an anti-emetic is permissible but not mandatory.2.8All analyses are predefined and documented in the statistical analysis plan approved by the chief investigator. The statistical design will be reported in detail in a separate manuscript. Briefly, part 1 will report the recommended MTD, the Bayesian posterior probability of DLT at each dose level  and the posterior probability that the DLT rate at dose level 1 (200 mg) is greater than the target level of 35%. The number of participants experiencing DLT at each dose level, together with the proportion of participants with DLT at that dose level, will be reported. Secondary outcome measures including the relationship of PD biomarkers and PK parameters will be presented graphically.Part 2 will assess potential efficacy of treatment based on a composite outcome of response following 12 cycles of treatment. Individual components of the composite response outcome and all AEs will also be reported descriptively. Secondary outcome measures including PK parameters and PD biomarkers will be presented graphically.2.9Part 1 includes a maximum of 21 participants based on a maximum of 7 cohorts each consisting of 3 participants. The decision to recruit 3 participants per cohort was based on the proposed 35% DLT rate. The performance of the Bayesian design based on 21 participants was assessed via simulations under several clinically relevant scenarios in terms of accuracy of selecting the true MTD, optimal allocation, and average percentage of participants being treated at a dose higher than the MTD. and assumes a composite response rate to reject seliciclib (p0) <25% and a response rate to investigate seliciclib further (p1) >50%. The justification to investigate seliciclib further is based on observing a minimum critical number of responses. As an early phase trial, the error levels have been inflated but restricted to an acceptable level of 15% alpha (type 1) and 20% beta (type 2). With these stated parameters the target recruitment for part 2a is calculated as 18 participants.Part 2 is based on a Fleming–A’Hern designAs this is the first trial investigating seliciclib as a repurposed drug for RA and, given the novel mechanism of action, the decision to continue will also be based on PD biomarkers and PK parameters to minimise the risk of rejecting a potentially active drug which has not achieved pragmatic clinical measures of efficacy. In terms of synovial PD biomarkers, the decision to continue will be informed by the following observations and evidence:1.Polymerase chain reaction of synovial tissue for genes of interest relevant to fibroblast biology, inflammation, cell cycle, and apoptosis, applied to mRNA extracted from synovial tissue;2.PD effects of seliciclib on the synovial fibroblast using immunohistochemistry and appropriate dual staining with fibroblast markers, including reagents that recognise phospho-specific isoforms of RNA polymerase II and the retinoblastoma protein, which are targets of CDK7/9 and CDK2, respectively.3.Markers of cell proliferation, such as Ki-67, within the synovial fibroblast population.If any of these criteria suggest a positive PD effect of seliciclib in the synovium, particularly if the IMP is well tolerated, then a decision may be taken to progress to a further study with a more focussed dose range and using outcome measures informed by the current study.2.10The trial is monitored by an independent DMC consisting of 2 physicians not connected to the trial, 1 acting as chair, and 1 statistician. The purpose of this committee is to undertake independent review and monitor efficacy and safety endpoints. The committee convened prior to the start of recruitment to discuss the dose transition pathway as recommended by the proposed CRM. The DMC members are updated after completion of each cohort in part 1. The DMC also convenes at the end of part 1, during recruitment of part 2 and at trial completion, with additional interim meetings at the request of the DMC members in light of emerging safety data.A Trial Steering Committee (TSC) provides overall supervision of the trial. The TSC consists of an independent chairperson, an independent clinician plus principal investigators, a Cyclacel representative and lay representation. In addition, representatives from the Trial Management Group, sponsor and funder will be invited. Requests for data sharing will be reviewed and considered by the TSC in conjunction with the chief investigator and sponsor.The trial may be subject to inspection and audit by The Newcastle upon Tyne Hospitals NHS Foundation Trust under their remit as Sponsor, and other regulatory bodies to ensure adherence to GCP.2.11Two patient research partners have been involved throughout this project. They have contributed to the initial project design and protocol, reviewed and improved the information sheet for participants, and have taken part in regular project steering group meetings. The patient research partners will review the results of this study, contribute to the drafting of lay summaries for all manuscripts and will advise and participate in the dissemination of results to lay audiences.2.12The trial may be prematurely discontinued on the basis of new safety information, or for other reasons given by the DMC and/or TSC, sponsor, regulatory authority, or Ethics Committee concerned. There are 2 early stopping rules included in part 1 to allow for early termination if:1.there is a high probability that the posterior probability of DLT at the lowest dose is greater than the target DLT rate, indicating that the lowest dose is too toxic or2.sufficient patients have already been allocated at the current MTD, which would also be the recommended dose level for the next cohort if the trial continues.3The TRAFIC study aims to repurpose seliciclib to address an area of unmet need in RA. This is the first study of a CDK inhibitor or other therapeutic agent targeting the synovial fibroblast in RA, and employs efficient trial designs that additionally avoid the requirement for control arms in part 1 or part 2 of the study.,41 the MTD for RA cannot be assumed to be the same. Patients with RA will have received a number of immunomodulating DMARD therapies, anti-inflammatories and analgesics as part of their previous treatment. Furthermore, patients with RA may exhibit a different tolerance to adverse effects than patients with cancer. The MTD dose for seliciclib in patients with RA therefore has to be established in part 1 of this study before assessment of efficacy can be undertaken.While seliciclib has been tested in patients with malignancy, and DAS28 <2.6 representing remission.The potential target RA patient population for seliciclib was extensively discussed. There are multiple csDMARD and bDMARD therapies, with established efficacy and safety, available for the treatment of early and moderate-severe active RA. In general, patients who fail to respond to initial csDMARDs are rapidly escalated to biologic therapies. Despite the distinct mechanisms of action of bDMARDs, their efficacy is surprisingly similar in RA clinical trials, including a therapeutic “ceiling” such that only 10% to 20% of patients achieve remission. The hypothesis under test in TRAFIC is that these unexpected phenomena could be explained if a proportion of RA disease activity were attributable to the synovial fibroblast. This cell is an integral element of RA “pannus”, the tissue that erodes bone and cartilage; it also secretes pro-inflammatory cytokines and chemokines, thereby potentially sustaining inflammation in the presence of potent biologic therapies. Current RA therapies do not target synovial fibroblasts, hence the rationale for adding seliciclib to existing therapeutic regimes in patients with a partial response to biologic therapies. The DAS28 cut-offs for inclusion in this study was chosen to reflect ongoing disease activity and for being feasible for recruitment. Part 1 was designed to determine the MTD of seliciclib, and a DAS28 score of ≥3.2 was chosen. In part 2, a higher DAS28 score of ≥4.0 was initially selected to maximise efficacy assessment but subsequently this was reduced to ≥3.2 to improve recruitment. DAS28 scores of ≥3.2 indicate moderate disease activity, with DAS28 <3.2 representing low disease activity In contrast to rule-based designs, CRM designs utilise all available accumulating data to inform decisions regarding dosing for successive patients. Part 2 is designed as a proof of concept trial with a go/no-go decision for a further randomized phase II/III trial. This single arm, single stage, early-phase trial uses the Fleming–A’Hern design which incorporates specification of a desired level of response and a level of response below which the treatment would be considered ineffective.Part 1 utilised a Bayesian model-based CRM dose finding design used more frequently in oncology. CRM designs have been shown to be more efficient than the often used rule-based designs, such as the 3 + 3; CRM designs require fewer patients on average and treat more patients at potentially therapeutic doses at or near to the MTD.,36 The RAMRIS criteria for MRI improvement reflect those observed in a 12 week phase II study of fostamatinib (Syk kinase inhibitor) in RA, albeit a much larger study than the current one. The RAMRIS score in the placebo group of that study increased (indicating disease progression). We believe that fluorodeoxyglucose positron emission tomography, reflecting synovial metabolism, could also provide a useful outcome measure predicted to reduce after seliciclib. However, as a non-validated outcome measure this is optional for part 2 participants and not included in the Fleming–A’Hern decision process.The part 2 outcomes were chosen to reflect both symptoms and biomarkers. The latter are particularly important for a study design that does not include a placebo or control group. ACR responses are composite scores reflecting swollen and tender joints, an acute phase biomarker, physician, and patient global assessments, an assessment of function  and an assessment of pain. ACR20 was initially developed to reflect an improvement in phase II/III clinical trials that was unlikely to represent a placebo response. Similarly, a moderate EULAR response is likely to reflect a true benefit. Nonetheless, in a single arm study where all patients receive active treatment, a strong placebo response is anticipated. Hence the inclusion of validated biomarkers based on a reduction of macrophages in the synovial sublining and MRI evidence of improvement in synovitis and/or osteitis. In previous studies, prednisolone and a CCR1 antagonist resulted in 40% to 50% reduction in sublining macrophages over 12 to 14 weeks in controlled studies, while in contrast patients receiving placebo or stable background therapy demonstrated a 10% to 25% increase in sublining macrophages.TRAFIC is the first trial to use a cell cycle inhibitor to target the synovial fibroblast in patients with RA. It is also innovative in employing CRM to identify the MTD in part 1, and a single arm Fleming–A’Hern design to seek evidence of efficacy in part 2. Both are efficient trial designs that should expedite further development if evidence of safety and efficacy emerges. Lessons learned from the recruitment challenges in this study will also inform study design of subsequent studies arising from this line of work, and similar studies in RA.3.1Part 1 of the study is completed and part 2 is ongoing using the MTD of seliciclib identified in part 1. Recruitment for part 2 has been slow, necessitating amendments to the background bDMARD  and DAS28 cut-off inclusion criteria as outlined.The authors thank all participating sites and investigators for their support with the clinical study and the patients for their participation in the study.All authors read and approved the final manuscript.SS is a principal investigator, contributed to the study design and drafted the manuscript. DDS is a Trial Statistician and drafted the manuscript. JDI is the chief investigator. He conceived the study and designed the trial. MM is Trial Manager and contributed to writing the study protocol. AC was Trial Manager and contributed to study design, protocol development and reviewed the manuscript. SF liaised with the principal investigators to provide information on seliciclib and advise on the appropriate timing for PD sampling. She also assisted with the grant application and conducted the pharmacodynamic analysis of patient samples. MC is the Trial Statistician. He contributed to the design of part 1 and has contributed to the writing of this manuscript. AGP, CDB, WFN, AF and IBM are principal investigators, contributed to the study design and have reviewed the manuscript.


The characterization of target binding interactions is critical at each stage of antibody therapeutic development. During early development, it is important to design fit-for-purpose in vitro molecular interaction characterization (MIC) assays that accurately determine the binding kinetics and the affinity of therapeutic antibodies for their targets. Such information enables PK/PD (pharmacokinetics/pharmacodynamics) modeling, estimation of dosing regimens, and assessment of potency. While binding kinetics and affinities seem to be readily obtained, there is little discussion in the literature on how the information should be generated and used in a systematic manner along with other approaches to enable key drug development decisions. The introduction of new antibody modalities poses unique challenges to the development of MIC assays and further increases the need to discuss the impact of developing context-appropriate MIC assays to enable key decision making for these programs. In this paper, we discuss for the first time the challenges encountered when developing MIC assays supporting new antibody modalities. Additionally, through the presentation of several real case studies, we provide strategies to overcome these challenges to enable investigational new drug (IND) filings. Since the first approved clinical therapeutic in 1985  and hundEarly clinical therapeutic products were dominated by chimeric ,6, humanD), can be derived; this is the concentration of the ligand required to occupy or compete 50% of the binding sites. MIC assays that are commonly used to determine the binding affinity of therapeutics to their targets can be classified into two categories: cell-based equilibrium assays and non-cell-based assays that rely on purified or recombinant target proteins. Cell-based equilibrium assays are well-established for the determination of the binding affinity of a ligand to cell-expressed target proteins . The majTechnologies that are commonly used to develop non-cell-based assays for affinity measurement include surface plasmon resonance (SPR)  using cell-based equilibrium assays as it may take extended periods of time to reach equilibrium and the cell viability can be severely compromised. SPR is capable of measuring affinity in a wide range , and is thus suitable for studying most biomolecular interactions. Additionally, it provides kinetic information that can be very useful for understanding the biological processes behind an interaction [MIC assays in each category have strengths and weaknesses. Cell-based equilibrium assays are often considered, since cell-expressed receptors are generally deemed more biologically relevant than the recombinantly expressed proteins. However, depending on the availability of cell lines, receptor expression levels and the biophysical properties of the evaluated therapeutics, it is not always feasible to develop a reliable cell-based assay for affinity measurement. Cell-based assays are often run at 4 °C or 25 °C to minimize receptor internalization. As affinity can be highly temperature dependent , KD valueraction . As SPR Compared to conventional mAbs, MIC assay strategies for new antibody modalities are more complex. The complex molecular structure and properties of these molecules create additional challenges for characterizing the binding of these molecules to their targets. For many bispecific antibodies, both of the therapeutic targets are expressed on cells, which might have low expression levels and also hard to purify as recombinant proteins. Often, a combination of multiple MIC assays is required to fill in the gaps and provide confidence in the data. For conjugated antibody modalities such as ADCs, PEGylated antibody fragments, and immuno-PET imaging agents, characterization of the target binding of both unconjugated and conjugated forms is needed to assess the extent to which conjugation impacts binding. The additional characterization not only increases the number of interaction pairs to be evaluated, but could also pose challenges to the development of MIC assays, since the conjugation process can change molecular properties such as increased hydrophobicity of ADCs ,40. Furtp-aminobenzyloxycarbonyl (MC-VC-PAB), that joins the antibody and MMAE together. The mechanism of action of anti-MSLN ADC is similar to that of other ADCs: following target-specific binding of the ADC on the cancer cell surface, the ADC can be taken into the cell and the cytotoxic drug is released, resulting in disruption of the microtubule network, inhibition of cell division and growth, and tumor cell death [The anti-mesothelin (MSLN) ADC consists of three moieties: the antibody that recognizes MSLN, a protein that is over-expressed on cell surfaces in several types of cancer , monometll death ,43,44. Tll death ,46.There are several unique aspects of this program. The clinical lead antibody only binds to human MSLN, so a surrogate antibody was produced in order to assess its safety in the species used for toxicity studies, cynomolgus monkey and rat. Additionally, it was found that the glycosylation level of the MSLN extracellular domain (ECD) affected the surrogate antibody binding to human MSLN in vitro, but had no impact on the lead antibody binding to the human target. The goal of the IND-enabling MIC study was to evaluate binding affinities of the lead and surrogate antibodies to their targets. In addition, it was important to assess the impact of MMAE conjugation on target binding of the anti-MSLN antibodies. D values. The high assay background is likely caused by significant non-specific binding of the ADC antibodies to the cell surface, since ADC antibodies overall are more hydrophobic than unconjugated antibodies [As the binding of the surrogate antibody to human MSLN is affected by the glycosylation level of MSLN ECD proteins, it is expected that the binding of the surrogate antibody to the cynomolgus monkey and rat MSLN would also be affected. For this reason, cell-based radiolabeled equilibrium assays were chosen to evaluate the MSLN antibodies binding to their targets. tibodies ,40, a phE. coli were first tested in the SPR assay as these proteins are non-glycosylated. The MSLN ECD proteins expressed in E. coli were provided in acidic buffers (pH 3.0) to improve solubility, and were in denatured and reduced forms. Although the proteins were successfully used in an ELISA-based PK assay as a coat reagent to capture MSLN ADC molecules, it is necessary to evaluate whether the MSLN ECD proteins exhibit binding properties suitable for SPR analysis. To address the question, the proteins were immobilized on a sensor chip and MSLN antibodies were injected as analytes. As shown in max) (E) values for all three tested target ECD proteins were found to be much lower than the theoretically calculated Rmax (T), and the ratios of Rmax (E) to Rmax (T) for all three targets were less than 20%, much lower than that generally recommended (over 70–80%) by the manufacturer. As lysine is not involved in the key binding epitope of the MSLN antibodies, the unacceptably low binding responses were likely caused by the impaired binding properties of the purified ECD proteins instead of being introduced by the amine coupling immobilization reaction, although modification of lysine residues could lead to steric hindrance and affect the protein binding indirectly. To investigate the cause of the low binding responses in the SPR assay, recombinant human and cynomolgus monkey MSLN ECD expressed in Chinese Hamster Ovary (CHO) cells were produced, and the fractions with the lowest levels of glycosylation were collected and tested in the assay. As shown in max (E) and Rmax (T) for human and cynomolgus monkey MSLN ECD were 85% and 78%, respectively. This result suggested that it is unlikely that the amine coupling procedure modified the binding properties of the MSLN ECD proteins. Moreover, it showed that the quality of the E. coli-produced MSLN ECD proteins were not suitable for use in the MIC assay.To circumvent the impact of glycosylation on the binding, MSLN ECD proteins expressed in SDS-PAGE characterization of the ECD proteins expressed in CHO cells showed that the fraction with the lowest amount of glycosylation was still very heterogeneous, with multiple bands on gels (data not shown). To minimize the impact of heterogeneity on data analysis, the MSLN ECD proteins were immobilized on the sensor chip, and the conjugated antibodies were injected as analyte in the flow. As each MSLN ECD bears one binding site for the MSLN antibody, it is possible that one antibody could bind to two immobilized MSLN ECD molecules despite the effort of immobilizing MSLN ECD at a low density on the sensor chip. For this reason, both monovalent and bivalent analyte binding models were evaluated for describing the binding interactions. A much greater discrepancy was observed between the experimental data and the fitted curves using a 1:1 monovalent binding model than was obtained from the bivalent binding model in all cases (data not shown), suggesting that the 1:1 binding model did not adequately describe the interactions between MSLN antibodies and MSLN ECD. D1 values determined using a bivalent binding model for the parental and ADC-conjugated lead MSLN antibodies binding to recombinant human MSLN ECD were 1.4 and 2.3 nM, respectively. The values for the parental and ADC-conjugated surrogate antibodies binding to recombinant cynomolgus monkey MSLN ECD were 7.9 and 11.9 nM, respectively. These affinity data were similar to those derived from the radiolabeled equilibrium assays. Under the experimental conditions described herein, the binding affinities and the kinetics of the conjugated MSLN antibodies were not significantly different than those observed with the unconjugated antibodies. As shown in In this case study, both the cell-based radiolabeled equilibrium assay and the SPR assays were used to cross-validate and provide confidence in measured affinities. This strategy enabled a successful IND filing by confirming the appropriate dose selection in the cynomolgus monkey toxicity study. It also highlighted the importance of producing and characterizing each of the reagents employed to facilitate the development of an MIC assay. 1G7 is a humanized bispecific antibody directed against the ECDs of the fragment crystallizable receptor-like 5 (FcRH5) and the epsilon chain of cluster of differentiation 3 (CD3ε) antigens. FcRH5 is an Ig superfamily transmembrane protein; its expression is highly restricted to the B-cell lineage, including plasma cells and all multiple myeloma (MM) tumor cells . CD3 is In this study, it was necessary to characterize the binding affinities of 1G7 to both FcRH5 and CD3 from both human and cynomolgus monkey to verify receptor occupancy (RO) of a proposed first-in-human dose and to justify the species selection for toxicity studies. Preliminary binding data generated at the early discovery stage of the program suggested that 1G7 binding to both targets is temperature dependent. As the binding affinity value is important to verify RO in the first human study, it is important to derive accurate affinity data at physiologically relevant temperature (37 °C). On the other hand, like many other transmembrane proteins, recombinant FcRH5 and CD3ε ECD proteins are very difficult to express and purify. Despite all efforts, high quality recombinant cynomolgus monkey ECD proteins for both targets were unavailable. Considering these challenges, the MIC assay strategy for this program was to combine results from both cell-based assays and the SPR assay to enhance data confidence. D value of 1G7 binding to the recombinant human CD3εγ ECD was 6.9 nM. This format, however, could not be used to determine 1G7 binding to recombinant FcRH5 ECD because there was no detectable binding response of 1G7 to immobilized recombinant human FcRH5 ECD, indicating that the ECD protein lost binding activity after being immobilized on the sensor chip. For this reason, the binding kinetics and affinities of 1G7 to the recombinant human FcRH5 ECD was evaluated with an alternative format in which 1G7 was indirectly captured on the sensor chip via a monoclonal antibody against the Fc domain of human IgGs, and the ECD proteins were injected as analytes. An indirect capture format is often used in SPR analysis to evaluate protein–protein interactions [D value of 1G7 to the recombinant human FcRH5 ECD was 7.5 nM.In the SPR assay, the binding kinetics and affinity of 1G7 to recombinant human CD3 was determined using a format in which the recombinant human CD3 containing covalently linked extracellular domains of ε and γ subunits (CD3εγ ECD) was immobilized on the sensor chip and 1G7 was injected in the flow as analyte. The recombinant human CD3εγ ECD was prone to aggregation, and this format was found less sensitive to aggregation of human CD3εγ ECD. During the purification process, only the monomeric fraction of the recombinant human CD3εγ ECD was collected and stored. However, prior to the SPR study, size exclusion chromatography with multi-angle static light scattering (SEC-MALS) analysis revealed that this material still contained a high amount of aggregates (~30%). Although aggregates were removed again and the protein was reformulated in a buffer that minimizes aggregate formation, it was not clear whether aggregates would form when the recombinant human CD3εγ ECD was used in the SPR binding study. As shown in ractions . The indD values of 1G7 binding to human and cynomolgus monkey FcRH5 expressed in SV40 transformed fibroblasts (SVT2) cell lines were 3.1 nM and 8.2 nM, respectively [D value of 1G7 binding to human CD3ε expressed on the Jurkat T-cell line was 2.6 nM. Binding affinity of 1G7 to cynomolgus monkey CD3ε was not determined when the assay was needed. In order to bridge the 1G7 binding results between human and cynomolgus monkey CD3ε, a flow cytometry assay was developed to compare binding responses of 1G7 to human and cynomolgus monkey CD3ε expressed on CD8-positive T cells sorted from peripheral blood mononuclear cells (PBMCs). As shown in ectively . The aveIn this case study, SPR and cell-based assays including flow cytometric and radiolabeled equilibrium assays were combined to determine binding affinity of FcRH5/CD3 antibody binding to its bispecific targets. The strategy addressed the unique challenges of the program and mitigated the potential risk of delayed timelines caused by production of challenging proteins and assay development. This study highlighted the need to characterize reagents right before they are used in the MIC assays.Factor D (FD) is a highly specific chymotrypsin-like serine protease that is a rate-limiting enzyme of the complement alternative pathway. The substrate for FD is another alternative pathway serine protease, factor B (FB). Following cleavage by FD, FB is converted into the proteolytically active factor Bb, whereupon it initiates the alternative complement pathway . IncreasD values determined using a 1:1 binding model for the binding of CCFD9260S to human and cynomolgus monkey FD were 136 and 369 pM, respectively, and the KD values for anti-FD Fab were 168 and 394 pM, respectively. These data verified the Tox species selection, and also proved that PEG conjugation did not affect anti-FD binding to FD targets as CCFD9260S had very similar binding affinity and kinetics compared with the unconjugated anti-FD Fab. The goals of the MIC study were to support the IND filing in several ways: (1) To evaluate the binding affinities and kinetics of CCFD9260S with human and cynomolgus monkey FD proteins and (2) to evaluate whether PEG conjugation impacted anti-FD Fab binding to its targets. Since FD is secreted in soluble form, an SPR binding assay was chosen. To compare CCFD9260 and the unconjugated anti-FD Fab binding to FD, it is preferable to keep them in solution as analytes so that the molecules are not modified. In this case, the final PEGylated material was very heterogeneous. The number of conjugated Fab molecules on each PEG ranged from fewer than 6 to greater than 8, with ~60% of the material having 7 or 8 Fab molecules. This requires CCFD9260 and the unconjugated anti-FD to be captured or immobilized on the sensor chip as ligand. The multivalency and the heterogeneity of the PEGylated material would otherwise add challenges to data analysis and prevent accurate determination of kinetics and affinities when injected as the analyte. As mentioned in case study 2, the indirect capture format offers multiple advantages over the direct immobilization format. The requirement of using the indirect format, though, is that the ligand protein must be tightly captured by the capturing antibodies so that it generates a stable baseline for the binding interaction between the ligand and the analyte molecules. In this case, an indirect capture format was first attempted, where the PEGylated anti-FD and unconjugated anti-FD Fab were captured by an anti-human IgG Fab antibody  on the sensor chip, and recombinant FD proteins were injected as analytes. As shown in This case study provides a strategy for assessing the binding interaction of multivalent and heterogeneous antibody fragments to its target antigen, and demonstrated that understanding technical limitations of different assay designs is critical to the development of a successful fit-for-purpose MIC strategy. 89Zr (ZED8) [CD8 is a transmembrane glycoprotein characteristic of effector T lymphocytes, that are believed to be central to the anti-tumor activity of cancer immunotherapy (CIT) . Imagingr (ZED8)  . As saturation of receptors is required to derive reliable KD values using cell-based equilibrium assay, an alternative SPR binding assay that can accurately compare the binding affinities of the CD8 antibodies to human and cynomolgus monkey CD8 proteins was needed.In order to verify the dose selection in the IND-enabling GLP toxicity study in cynomolgus monkey, it is critical to derive accurate binding affinities of CD8 antibodies to cynomolgus monkey and human CD8 targets. In addition, it is important to evaluate the impact of the DFO conjugation on the binding of CD8 antibody to its targets. Since CD8 is a cell surface protein, and ZED8 itself already has the radioisotope max less than 5 RU) were immobilized on the sensor chip to encourage monovalent interaction. In the SPR assay, the binding kinetics and affinities of the CD8 antibodies to recombinant CD8α ECD proteins were first evaluated using an assay format (format 1) in which the recombinant human and cynomolgus monkey CD8α ECD proteins were immobilized on a sensor chip, and the CD8 antibodies were injected as analytes. This format was preferred because CD8 is expressed on the surface of cells and the available recombinant CD8α ECD Fc-fusion proteins contained two binding domains. Thus, the binding interaction would be a simple 1:1 binding. However, it was found that the data could not be described well by a 1:1 binding model (data not shown), indicating that there were multiple populations involved in the binding. This is possibly caused by heterogeneous population of CD8α ECD proteins after being immobilized on the sensor chip via amine coupling chemistry. Thus, an alternative assay format (format 2) was explored where the CD8 antibodies were immobilized on a sensor chip and CD8α ECD proteins were injected as analytes. Since antibodies are well-known to have excellent stability properties, they might be more consistent in their orientation after being immobilized on the sensor chip. A potential issue of using this format is that bivalent binding interactions could happen because of the bivalent nature of the recombinant CD8α ECD fusion proteins, which could cause the determined affinity to appear higher . To circD values of 5.06 [D values of 65.81 and 56.74 nM, respectively. No significant or systematic discrepancy was observed between the experimental data and the fitted curves, suggesting that the 1:1 binding model adequately described the interactions. The CD8 antibody to be evaluated in the cynomolgus monkey GLP study, CED8, bound to the recombinant human CD8α ECD fusion protein with an affinity 13-fold higher than that to the recombinant cynomolgus monkey CD8α ECD fusion protein, a difference attributed to a slower dissociation rate. The data supported the dose selection for the IND-enabling cynomolgus monkey GLP toxicity study, and also demonstrated that the DFO conjugation did not have an impact on the CD8 antibody binding to its targets.As shown in  of 5.06  and 5.09This study highlighted the importance of designing fit-for-purpose MIC assays so that the derived affinity data addresses specific needs in support of toxicity studies during development. Fit-for-purpose MIC assay strategies are critical during early development of therapeutic antibodies to enable key decisions. The successful design of the MIC assay strategy for a program rely on many factors, including target biology, biophysical properties of the therapeutics, mechanism of interaction, relevant binding species, as well as logistical considerations such as accessibility of cell lines, supply of assay reagents, timeline, and resources. As affinity measurement is context-dependent, it is critical to choose the assay design  that is most relevant to the question(s) to be addressed. Therefore, a sound MIC strategy that is based on the thorough understanding of the needs and the technical feasibility should be developed prior to in-depth technical work. This outcome is best achieved through discussions with team members from different functions such as pharmacology, toxicology, biology, and manufacturing. In addition, because MIC assay results are dependent on reagents used, it is highly desirable to assess the quality of critical reagents right before they are used in the MIC assays. This practice is especially important for reagents that are prone to aggregate, have gone through multiple freeze-thaw cycles, or are generated for the first time with unknown stabilities. It is worth pointing out that reagents that work in other types of assays do not necessarily possess the quality required for MIC assays. Therefore, besides characterizing MIC assay reagents by traditional analytical tools such as SDS-PAGE, SEC-MALS, and mass spectrometry, it is highly desirable to evaluate binding activity of proteins when conducting MIC assays with appropriate assay format.The introduction of new antibody modalities provides various advantages over conventional antibodies, meanwhile poses unique challenges to the development of MIC assays primarily due to their complex structural and molecular properties. Compared to conventional mAbs, traditional strategies involving single MIC assay development is unlikely sufficient to address the development need of these programs any more. This may require MIC strategies that apply a diversity of traditional and unconventional approaches to overcome challenges that are associated with these new modalities and to address their specific needs. With additional new antibody-related modalities in development covering a wide range of diversified platforms ,58,59, t


The COVID-19 pandemic has been declared a public health emergency of international concern; this has caused excessive anxiety among health care workers. In addition, publication bias and low-quality publications have become widespread, which can result in the dissemination of unreliable findings.This paper presents the protocol for a meta-analysis with the following two aims: (1) to examine the prevalence of anxiety among health care workers and determine whether it has increased due to the COVID-19 pandemic, and (2) to investigate whether there has been an increase in publication bias.2 statistic. The effect size  and a 95% CI for each paper will also be calculated. We will use a moderator analysis to test for the effect of COVID-19 on health care workers’ anxiety levels and detect publication bias in COVID-19 studies. We will also assess publication bias using the funnel plot and Egger regression. In case of publication bias, if studies have no homogeneity, the trim-and-fill procedure will be applied to adjust for missing studies.All related studies that were published/released from 2015 to 2020 will be searched in electronic databases . The risk of bias in individual studies will be assessed using the STROBE  checklist. The heterogeneity of the studies will be assessed using the IDatabase searches will commence in November 2020. The meta-analysis will be completed within 2 months of the start date.This meta-analysis aims to provide comprehensive evidence about whether COVID-19 increases the prevalence of anxiety among health care workers and whether there has been an increase in publication bias and a deterioration in the quality of publications due to the pandemic. The results of this meta-analysis can provide evidence to help health managers to make informed decisions related to anxiety prevention in health care workers.PRR1-10.2196/24136 The COVID-19 pandemic has affected more than 18.9 million individuals and resulted in over 709,000 deaths globally . To tideThe COVID-19 pandemic has affected many aspects of people’s lives, especially their mental health -7. WhileAlthough some researchers have investigated health care workers’ anxiety levels during the COVID-19 pandemic ,14, manyIn addition, since the onset of the outbreak, knowledge about COVID-19 is direly needed, and medical journals have drastically accelerated the publication process for COVID-19–related articles to accelerate knowledge acquisition ,16. In tWe have generated the following two hypotheses:COVID-19 makes health care workers more anxious and thus the studies related to COVID-19 will have a larger effect size. We will investigate this by comparing studies related to COVID-19 vs studies unrelated to it.Publication bias in COVID-19–related studies is widespread. We will investigate this by comparing unpublished preprints about COVID-19 with published journal papers about the disease.This study will follow the PRISMA  guidelines . We willANDStudies will be included only if they meet the following inclusion criteria: (1) written in English (which will be decided based on the research team’s unified considerations); (2) related to “anxiety among health care workers”; (3) quantitative research designs; (4) submitted during 2015 to 2020; (5) include standardized measures of anxiety with published psychometric data and reasonable evidence of reliability and validity; (6) include a clear description of methods used to assess and score standardized measurement instruments; and (7) include publicly available effect sizes  or values that can be calculated .The exclusion criteria are: (1) studies with insufficient data, (2) duplicate sources, (3) research with unclear methods, and (4) publications about other outbreaks.First, duplicate papers that are found in multiple databases will be removed. Subsequently, screening of the titles and abstracts will be conducted, and papers will be removed based on the inclusion and exclusion criteria. Furthermore, the full text of the papers will be checked, and article information will be extracted using a preprepared extraction table that includes the article’s title, authors’ names, scales used, year of submission, country, sample size, whether the study has been published, whether the study relates to COVID-19, and the effect size . The article review and data extraction processes will be performed independently by two of the authors. When there is a disagreement between them, the other authors will resolve the conflict.We will use the STROBE  checklist to assess the quality of observational studies . The che2 statistical index, which ranges from 0 to 100; the larger the index, the more heterogeneous are the findings. The categories encompassed by the index will be defined based on the test developed by Higgins et al [First, the heterogeneity of the studies will be determined using the Ins et al  to measuP value less than the significance level (α=.05) suggests that publication bias is present. If publication bias is present, and studies have no homogeneity, the trim-and-fill procedure will be applied to adjust these missing studies [Subsequently, we will use a moderator analysis to test for the effect of COVID-19 on health care workers’ anxiety levels (related vs unrelated to COVID-19), and publication bias in COVID-19 studies . We will also assess publication bias using the funnel plot and Egger regression . For the studies .α=.05.Finally, sensitivity analyses will be performed to assess the influence of each individual study on the pooled effect size. The statistical significance level is defined as Database searches will commence in November 2020. The meta-analysis will be completed within 2 months.This paper presents a protocol for a meta-analysis that aims to provide comprehensive evidence about whether the COVID-19 pandemic increases the prevalence of anxiety among health care workers and whether there has been an increase in publication bias and a deterioration in the quality of publications due to the pandemic. The results of this meta-analysis can provide evidence to help health managers to make informed decisions for preventing anxiety in health care workers.