A TMPRSS2 inhibitor acts as a pan-SARS-CoV-2 prophylactic and therapeutic.

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging variants of concern1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle5,6. Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 106 in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids7. In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern.

Following your gut: the emerging role of the gut microbiota in predicting and treating immune-related adverse events.

PURPOSE OF REVIEW: Although immune checkpoint inhibition has reshaped the therapeutic landscape leading to improved outcomes across an array of both solid and hematologic malignancies, a significant source of morbidity is caused by immune-related adverse events (irAEs) caused by these agents. RECENT FINDINGS: The gut microbiota has emerged as a biomarker of response to these agents, and more recently, also as a key determinant of development of irAEs. Emerging data have revealed that enrichment of certain bacterial genera is associated with an increased risk of irAEs, with the most robust evidence pointing to an intimate connection with the development of immune-related diarrhea and colitis. These bacteria include Bacteroides , Enterobacteriaceae, and Proteobacteria (such as Klebsiella and Proteus ) . Lachnospiraceae spp. and Streptococcus spp. have been implicated irAE-wide in the context of ipilimumab. SUMMARY: We review recent lines of evidence pointing to the role of baseline gut microbiota on the development of irAE, and the potentials for therapeutic manipulation of the gut microbiota in order to reduce irAE severity. The connections between gut microbiome signatures of response and toxicity will need to be untangled in further studies.

Physiologic colonic uptake of 18F-FDG on PET/CT is associated with clinical response and gut microbiome composition in patients with advanced non-small cell lung cancer treated with immune checkpoint inhibitors.

BACKGROUND: Immune checkpoint inhibitors (ICI) represent the backbone treatment for advanced non-small cell lung cancer (NSCLC). Emerging data suggest that increased gut microbiome diversity is associated with favorable response to ICI and that antibiotic-induced dysbiosis is associated with deleterious outcomes. 18F-FDG physiologic colonic uptake on PET/CT increases following treatment with antibiotics (ATB) and could act as a surrogate marker for microbiome composition and predict prognosis. The aim of this study was to determine if 18F-FDG physiologic colonic uptake prior to ICI initiation correlates with gut microbiome profiling and clinical outcomes in patients with advanced NSCLC. METHODS: Seventy-one patients with advanced NSCLC who underwent a PET/CT prior to ICI were identified. Blinded colonic contouring was performed for each colon segment and patients were stratified according to the median of the average colon SUVmax as well as for each segment in low vs. high SUVmax groups. Response rate, progression-free survival (PFS), and overall survival (OS) were compared in the low vs. high SUVmax groups. Gut microbiome composition was analyzed for 23 patients using metagenomics sequencing. RESULTS: The high colon SUVmax group had a higher proportion of non-responders (p = 0.033) and significantly shorter PFS (4.1 vs. 11.3 months, HR 1.94, 95% CI 1.11-3.41, p = 0.005). High caecum SUVmax correlated with numerically shorter OS (10.8 vs. 27.6 months, HR 1.85, 95% CI 0.97-3.53, p = 0.058). Metagenomics sequencing revealed distinctive microbiome populations in each group. Patients with low caecum SUVmax had higher microbiome diversity (p = 0.046) and were enriched with Bifidobacteriaceae, Lachnospiraceae, and Bacteroidaceae. CONCLUSIONS: Lower colon physiologic 18F-FDG uptake on PET/CT prior to ICI initiation was associated with better clinical outcomes and higher gut microbiome diversity in patients with advanced NSCLC. Here, we propose that 18F-FDG physiologic colonic uptake on PET/CT could serve as a potential novel marker of gut microbiome composition and may predict clinical outcomes in this population.

Transcriptome analysis reveals TMPRSS6 isoforms with distinct functionalities.

TMPRSS6 (matriptase-2) is a type II transmembrane serine protease involved in iron homoeostasis. At the cell surface of hepatocytes, TMPRSS6 cleaves haemojuvelin (HJV) and regulates the BMP/SMAD signalling pathway leading to production of hepcidin, a key regulator of iron absorption. Although four TMPRSS6 human isoforms and three mice Tmprss6 isoforms are annotated in databases (Ensembl and RefSeq), their relative expression or activity has not been studied. Analyses of RNA-seq data and RT-PCR from human tissues reveal that TMPRSS6 isoform 1 (TMPRSS6-1) and 3 are mostly expressed in human testis while TMPRSS6-2 and TMPRSS6-4 are the main transcripts expressed in human liver, testis and pituitary. Furthermore, we confirm the existence and analyse the relative expression of three annotated mice Tmprss6 isoforms. Using heterologous expression in HEK293 and Hep3B cells, we show that all human TMPRSS6 isoforms reach the cell surface but only TMPRSS6-1 undergoes internalization. Moreover, truncated TMPRSS6-3 or catalytically altered TMPRSS6-4 interact with HJV and prevent its cleavage by TMPRSS6-2, suggesting their potential role as dominant negative isoforms. Taken together, our results highlight the importance of understanding the precise function of each TMPRSS6 isoforms both in human and in mouse.

The type II transmembrane serine protease matriptase cleaves the amyloid precursor protein and reduces its processing to ß-amyloid peptide.

Recent studies have reported that many proteases, besides the canonical α-, ß-, and γ-secretases, cleave the amyloid precursor protein (APP) and modulate ß-amyloid (Aß) peptide production. Moreover, specific APP isoforms contain Kunitz protease-inhibitory domains, which regulate the proteolytic activity of serine proteases. This prompted us to investigate the role of matriptase, a member of the type II transmembrane serine protease family, in APP processing. Using quantitative RT-PCR, we detected matriptase mRNA in several regions of the human brain with an enrichment in neurons. RNA sequencing data of human dorsolateral prefrontal cortex revealed relatively high levels of matriptase RNA in young individuals, whereas lower levels were detected in older individuals. We further demonstrate that matriptase and APP directly interact with each other and that matriptase cleaves APP at a specific arginine residue (Arg-102) both in vitro and in cells. Site-directed (Arg-to-Ala) mutagenesis of this cleavage site abolished matriptase-mediated APP processing. Moreover, we observed that a soluble, shed matriptase form cleaves endogenous APP in SH-SY5Y cells and that this cleavage significantly reduces APP processing to Aß40. In summary, this study identifies matriptase as an APP-cleaving enzyme, an activity that could have important consequences for the abundance of Aß and in Alzheimer's disease pathology.

The serine protease-mediated increase in intestinal epithelial barrier function is dependent on occludin and requires an intact tight junction.

Barrier dysfunction is a characteristic of the inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis. Understanding how the tight junction is modified to maintain barrier function may provide avenues for treatment of IBD. We have previously shown that the apical addition of serine proteases to intestinal epithelial cell lines causes a rapid and sustained increase in transepithelial electrical resistance (TER), but the mechanisms are unknown. We hypothesized that serine proteases increase barrier function through trafficking and insertion of tight junction proteins into the membrane, and this could enhance recovery of a disrupted monolayer after calcium switch or cytokine treatment. In the canine epithelial cell line, SCBN, we showed that matriptase, an endogenous serine protease, could potently increase TER. Using detergent solubility-based cell fractionation, we found that neither trypsin nor matriptase treatment changed levels of tight junction proteins at the membrane. In a fast calcium switch assay, serine proteases did not enhance the rate of recovery of the junction. In addition, serine proteases could not reverse barrier disruption induced by IFNγ and TNFα. We knocked down occludin in our cells using siRNA and found this prevented the serine protease-induced increase in TER. Using fluorescence recovery after photobleaching (FRAP), we found serine proteases induce a greater mobile fraction of occludin in the membrane. These data suggest that a functional tight junction is needed for serine proteases to have an effect on TER, and that occludin is a crucial tight junction protein in this mechanism.

Prostasin is required for matriptase activation in intestinal epithelial cells to regulate closure of the paracellular pathway.

The type II transmembrane serine protease matriptase is a key regulator of epithelial barriers in skin and intestine. In skin, matriptase acts upstream of the glycosylphosphatidylinositol-anchored serine protease, prostasin, to activate the prostasin zymogen and initiate a proteolytic cascade that is required for stratum corneum barrier functionality. Here, we have investigated the relationship between prostasin and matriptase in intestinal epithelial barrier function. We find that similar to skin, matriptase and prostasin are components of a common intestinal epithelial barrier-forming pathway. Depletion of prostasin by siRNA silencing in Caco-2 intestinal epithelium inhibits barrier development similar to loss of matriptase, and the addition of recombinant prostasin to the basal side of polarized Caco-2 epithelium stimulates barrier forming changes similar to the addition of recombinant matriptase. However, in contrast to the proteolytic cascade in skin, prostasin functions upstream of matriptase to activate the endogenous matriptase zymogen. Prostasin is unable to proteolytically activate the matriptase zymogen directly but induces matriptase activation indirectly. Prostasin requires expression of endogenous matriptase to stimulate barrier formation since matriptase depletion by siRNA silencing abrogates prostasin barrier-forming activity. Active recombinant matriptase, however, does not require the expression of endogenous prostasin for barrier-forming activity. Together, these data show that matriptase and not prostasin is the primary effector protease of tight junction assembly in simple columnar epithelia and further highlight a spatial and tissue-specific aspect of cell surface proteolytic cascades.

Matriptase proteolytically activates influenza virus and promotes multicycle replication in the human airway epithelium.

Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place.

Optimization of Ketobenzothiazole-Based Type II Transmembrane Serine Protease Inhibitors to Block H1N1 Influenza Virus Replication.

Human influenza viruses cause acute respiratory symptoms that can lead to death. Due to the emergence of antiviral drug-resistant strains, there is an urgent requirement for novel antiviral agents and innovative therapeutic strategies. Using the peptidomimetic ketobenzothiazole protease inhibitor RQAR-Kbt (IN-1, aka N-0100) as a starting point, we report how substituting P2 and P4 positions with natural and unnatural amino acids can modulate the inhibition potency toward matriptase, a prototypical type II transmembrane serine protease (TTSP) that acts as a priming protease for influenza viruses. We also introduced modifications of the peptidomimetics N-terminal groups, leading to significant improvements (from µM to nM, 60 times more potent than IN-1) in their ability to inhibit the replication of influenza H1N1 virus in the Calu-3 cell line derived from human lungs. The selectivity towards other proteases has been evaluated and explained using molecular modeling with a crystal structure recently obtained by our group. By targeting host cell TTSPs as a therapeutic approach, it may be possible to overcome the high mutational rate of influenza viruses and consequently prevent potential drug resistance.

Nanomolar anti-SARS-CoV-2 Omicron activity of the host-directed TMPRSS2 inhibitor N-0385 and synergistic action with direct-acting antivirals.

SARS-CoV-2 Omicron subvariants with increased transmissibility and immune evasion are spreading globally with alarming persistence. Whether the mutations and evolution of spike (S) Omicron subvariants alter the viral hijacking of human TMPRSS2 for viral entry remains to be elucidated. This is particularly important to investigate because of the large number and diversity of mutations of S Omicron subvariants reported since the emergence of BA.1. Here we report that human TMPRSS2 is a molecular determinant of viral entry for all the Omicron clinical isolates tested in human lung cells, including ancestral Omicron subvariants (BA.1, BA.2, BA.5), contemporary Omicron subvariants (BQ.1.1, XBB.1.5, EG.5.1) and currently circulating Omicron BA.2.86. First, we used a co-transfection assay to demonstrate the endoproteolytic cleavage by TMPRSS2 of spike Omicron subvariants. Second, we found that N-0385, a highly potent TMPRSS2 inhibitor, is a robust entry inhibitor of virus-like particles harbouring the S protein of Omicron subvariants. Third, we show that N-0385 exhibits nanomolar broad-spectrum antiviral activity against live Omicron subvariants in human Calu-3 lung cells and primary patient-derived bronchial epithelial cells. Interestingly, we found that N-0385 is 10-20 times more potent than the repositioned TMPRSS2 inhibitor, camostat, against BA.5, EG.5.1, and BA.2.86. We further found that N-0385 shows broad synergistic activity with clinically approved direct-acting antivirals (DAAs), i.e., remdesivir and nirmatrelvir, against Omicron subvariants, demonstrating the potential therapeutic benefits of a multi-targeted treatment based on N-0385 and DAAs.

A phase 1 study of concurrent cabozantinib and cetuximab in recurrent or metastatic head and neck squamous cell cancer.

OBJECTIVES: Epidermal growth factor receptor (EGFR) inhibition with cetuximab is a standard treatment for head and neck squamous cell carcinoma (HNSCC). Activation of the receptor tyrosine kinases AXL, MET and VEGFR can mediate resistance to cetuximab. Cabozantinib, a multikinase inhibitor (MKI) targeting AXL/MET/VEGFR, has demonstrated antitumor activity in preclinical models of HNSCC. This investigator- initiated phase I trial evaluated the safety and efficacy of cetuximab plus cabozantinib in patients with recurrent/metastatic (R/M) HNSCC. MATERIALS AND METHODS: Patients received cetuximab concurrently with cabozantinib daily on a 28-day cycle. Using a 3 + 3 dose-escalation design, the primary endpoint was to determine the maximally tolerated dose (MTD) of cabozantinib. Secondary endpoints included overall response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) RESULTS: Among the 20 patients enrolled, most had prior disease progression on immune checkpoint inhibitors (95 %), platinum-based chemotherapy (95 %), and cetuximab (80 %). No dose-limiting toxicities were recorded and the MTD for cabozantinib was established to be 60 mg. Grade ≥ 3 adverse events occurred in 65 % of patients (n = 13). ORR was 20 %, with 4 partial responses (PRs). Two PRs were observed in cetuximab-naïve patients (n = 4), with an ORR of 50 % in this subgroup. In the overall population, DCR was 75 %, median PFS was 3.4 months and median OS was 8.1 months. CONCLUSION: Cetuximab plus cabozantinib demonstrated a manageable toxicity profile and preliminary efficacy in patients with heavily treated R/M HNSCC. The combination of cetuximab with MKIs targeting the AXL/MET/VEGFR axis warrants further investigation, including in cetuximab-naïve patients.

Gut microbiome predicts gastrointestinal toxicity outcomes from chemoradiation therapy in patients with head and neck squamous cell carcinoma.

OBJECTIVES: Chemoradiation (CRT) in patients with locally advanced head and neck squamous cell cancer (HNSCC) is associated with significant toxicities, including mucositis. The gut microbiome represents an emerging hallmark of cancer and a potentially important biomarker for CRT-related adverse events. This prospective study investigated the association between the gut microbiome composition and CRT-related toxicities in patients with HNSCC, including mucositis. MATERIALS AND METHODS: Stool samples from patients diagnosed with locally advanced HNSCC were prospectively collected prior to CRT initiation and analyzed using shotgun metagenomic sequencing to evaluate gut microbiome composition at baseline. Concurrently, clinicopathologic data, survival outcomes and the incidence and grading of CRT-emergent adverse events were documented in all patients. RESULTS: A total of 52 patients were included, of whom 47 had baseline stool samples available for metagenomic analysis. Median age was 62, 83 % patients were men and 54 % had stage III-IV disease. All patients developed CRT-induced mucositis, including 42 % with severe events (i.e. CTCAE v5.0 grade ≥ 3) and 25 % who required enteral feeding. With a median follow-up of 26.5 months, patients with severe mucositis had shorter overall survival (HR = 3.3, 95 %CI 1.0-10.6; p = 0.02) and numerically shorter progression-free survival (HR = 2.8, 95 %CI, 0.8-9.6; p = 0.09). The gut microbiome beta-diversity of patients with severe mucositis differed from patients with grades 1-2 mucositis (p = 0.04), with enrichment in Mediterraneibacter (Ruminococcus gnavus) and Clostridiaceae family members, including Hungatella hathewayi. Grade 1-2 mucositis was associated with enrichment in Eubacterium rectale, Alistipes putredinis and Ruminococcaceae family members. Similar bacterial profiles were observed in patients who required enteral feeding. CONCLUSION: Patients who developed severe mucositis had decreased survival and enrichment in specific bacteria associated with mucosal inflammation. Interestingly, these same bacteria have been linked to immune checkpoint inhibitor resistance.

Tumor-agnostic genomic and clinical analysis of BRAF fusions identify actionable targets.

BACKGROUND: Even though BRAF fusions are increasingly detected in standard multigene next-generation sequencing panels, few reports have explored their structure and impact on clinical course. PATIENTS/METHODS: We collected data from patients with BRAF fusion-positive cancers identified through a genotyping protocol of 97,024 samples. Fusions were characterized and reviewed for oncogenic potential (in-frame status, non-BRAF partner gene, intact BRAF kinase domain). RESULTS: We found 241 BRAF fusion-positive tumors from 212 patients with 82 unique 5' fusion partners spanning 52 histologies. 39 fusion partners were not previously reported, and 61 were identified once. BRAF fusion incidence was enriched in pilocytic astrocytomas, gangliomas, low-grade neuroepithelial tumors, and acinar cell carcinoma of the pancreas. 24 patients spanning multiple histologies were treated with MAPK-directed therapies of which 20 were evaluable for RECIST. Best response was partial response (N=2), stable disease (N=11), and progressive disease (N=7). The median time on therapy was 1 month with MEK plus BRAF inhibitors ([N=11], range 0-18 months) and 8 months for MEK inhibitors ([N=14], range 1-26 months). 9 patients remained on treatment for longer than 6 months [pilocytic astrocytomas (N=6), Erdheim-Chester disease (N=1), extraventricular neurocytoma (N=1), melanoma (N=1)]. Fifteen patients had acquired BRAF fusions. CONCLUSIONS: BRAF fusions are found across histologies and represent an emerging actionable target. BRAF fusions have a diverse set of fusion partners. Durable responses to MAPK therapies were seen, particularly in pilocytic astrocytomas. Acquired BRAF fusions were identified after targeted therapy underscoring the importance of post-progression biopsies to optimize treatment at relapse in these patients.

RET-Altered Cancers-A Tumor-Agnostic Review of Biology, Diagnosis and Targeted Therapy Activity.

RET alterations, such as fusions or mutations, drive the growth of multiple tumor types. These alterations are found in canonical (lung and thyroid) and non-canonical (e.g., gastrointestinal, breast, gynecological, genitourinary, histiocytic) cancers. RET alterations are best identified via comprehensive next-generation sequencing, preferably with DNA and RNA interrogation for fusions. Targeted therapies for RET-dependent cancers have evolved from older multikinase inhibitors to selective inhibitors of RET such as selpercatinib and pralsetinib. Prospective basket trials and retrospective reports have demonstrated the activity of these drugs in a wide variety of RET-altered cancers, notably those with RET fusions. This paved the way for the first tumor-agnostic selective RET inhibitor US FDA approval in 2022. Acquired resistance to RET kinase inhibitors can take the form of acquired resistance mutations (e.g., RET G810X) or bypass alterations.

Essential role of endocytosis of the type II transmembrane serine protease TMPRSS6 in regulating its functionality.

The type II transmembrane serine protease TMPRSS6 (also known as matriptase-2) controls iron homeostasis through its negative regulation of expression of hepcidin, a key hormone involved in iron metabolism. Upstream of the hepcidin-regulated signaling pathway, TMPRSS6 cleaves its target substrate hemojuvelin (HJV) at the plasma membrane, but the dynamics of the cell-surface expression of the protease have not been addressed. Here, we report that TMPRSS6 undergoes constitutive internalization in transfected HEK293 cells and in two human hepatic cell lines, HepG2 and primary hepatocytes, both of which express TMPRSS6 endogenously. Cell surface-labeled TMPRSS6 was internalized and was detected in clathrin- and AP-2-positive vesicles via a dynamin-dependent pathway. The endocytosed TMPRSS6 next transited in early endosomes and then to lysosomes. Internalization of TMPRSS6 is dependent on specific residues within its N-terminal cytoplasmic domain, as site-directed mutagenesis of these residues abrogated internalization and maintained the enzyme at the cell surface. Cells coexpressing these mutants and HJV produced significantly decreased levels of hepcidin compared with wild-type TMPRSS6 due to the sustained cleavage of HJV at the cell surface by TMPRSS6 mutants. Our results underscore for the first time the importance of TMPRSS6 trafficking at the plasma membrane in the regulation of hepcidin expression, an event that is essential for iron homeostasis.

PI3K Inhibition for Squamous Cell Head and Neck Carcinoma.

ABSTRACT: The phosphoinositide 3-kinase (PI3K) pathway is aberrantly activated in most head and neck squamous cell carcinomas, making it a prized target for targeted therapy development. Multiple PI3K inhibitors have been studied in early phase trials, with unfavorable risk-benefit ratios in molecularly unselected patient populations. Buparlisib, a potent pan-class I PI3K inhibitor, shows promising efficacy in combination with paclitaxel for advanced head and neck squamous cell carcinoma. Results of the phase III BURAN trial are awaited.

Targeting HRAS in Head and Neck Cancer: Lessons From the Past and Future Promise.

ABSTRACT: HRAS mutations define a unique biologic subset of head and neck squamous cell carcinoma. Oncogenic HRAS is uniquely dependent on posttranslational farnesylation for membrane localization and activation of downstream signaling. Tipifarnib, a farnesyltransferase inhibitor, demonstrated encouraging antitumor activity for HRAS mutant head and neck squamous cell carcinoma and modest activity for HRAS mutant salivary gland cancer. New combination strategies to circumvent intrinsic and acquired resistance to TFIs are being investigated.

Functionally impaired isoforms regulate TMPRSS6 proteolytic activity.

TMPRSS6 is a type II transmembrane serine protease involved in iron homeostasis expressed as 4 isoforms in humans. TMPRSS6 isoform 2 downregulates hepcidin production by cleaving hemojuvelin and other surface proteins of hepatocytes. The functions of catalytically impaired isoforms 3 and 4 are still unknown. Here we demonstrate that TMPRSS6 isoforms 3 and 4 reduce the proteolytic activity of isoform 2 and uncover the ability of isoforms to interact. Moreover, we identified 49 potential protein partners common to TMPRSS6 isoforms, including TfR1, known to be involved in iron regulation. By co-expressing TMPRSS6 and TfR1, we show that TfR1 is cleaved and shed from the cell surface. Further, we demonstrate that TMPRSS6 isoforms 3 and 4 behave as dominant negative.

Upfront DPYD Genotyping and Toxicity Associated with Fluoropyrimidine-Based Concurrent Chemoradiotherapy for Oropharyngeal Carcinomas: A Work in Progress.

Background: 5-FU-based chemoradiotherapy (CRT) could be associated with severe treatment-related toxicities in patients harboring at-risk DPYD polymorphisms. Methods: The studied population included consecutive patients with locoregionally advanced oropharyngeal carcinoma treated with carboplatin and 5-FU-based CRT one year before and after the implementation of upfront DPYD*2A genotyping. We aimed to determine the effect of DPYD genotyping on grade ≥3 toxicities. Results: 181 patients were analyzed (87 patients before and 94 patients following DPYD*2A screening). Of the patients, 91% (n = 86) were prospectively genotyped for the DPYD*2A allele. Of those screened, 2% (n = 2/87) demonstrated a heterozygous DPYD*2A mutation. Extended genotyping of DPYD*2A-negative patients later allowed for the retrospective identification of six additional patients with alternative DPYD variants (two c.2846A>T and four c.1236G>A mutations). Grade ≥3 toxicities occurred in 71% of the patients before DPYD*2A screening versus 62% following upfront genotyping (p = 0.18). When retrospectively analyzing additional non-DPYD*2A variants, the relative risks for mucositis (RR 2.36 [1.39-2.13], p = 0.0063), dysphagia (RR 2.89 [1.20-5.11], p = 0.019), and aspiration pneumonia (RR 13 [2.42-61.5)], p = 0.00065) were all significantly increased. Conclusion: The DPYD*2A, c.2846A>T, and c.1236G>A polymorphisms are associated with an increased risk of grade ≥3 toxicity to 5-FU. Upfront DPYD genotyping can identify patients in whom 5-FU-related toxicity should be avoided.