Challenges and Barriers Faced by People with TB and Healthcare Workers Providing TB Care and Management - A Qualitative Study.

BACKGROUND: In tuberculosis (TB) care and management, there are practical challenges existing at the patient-provider level leading to implementation barriers at the primary care level. OBJECTIVES: The objective of the study is to explore the challenges and barriers faced by people with TB and health-care workers in TB care and management. MATERIALS AND METHODS: This study was done as a part of a community intervention study between November 2021 and December 2022. Twenty interviews were taken with treatment for TB (n = 7) and health-care personnel (n = 13). Health-care personnel include nursing staff, medical officers, laboratory technicians, community health workers, and medical personnel from tertiary care hospital. Participants were recruited across all levels of health-care systems. Interviews were carried out in the Hindi language, audio recorded, and translated to English. Participants were asked about their experiences of challenges and barriers faced during TB care and management. Qualitative data were coded, and thematic analysis was done manually. RESULTS: The challenges and barriers at the level of people with TB were issues with communication between providers and people with TB, out-of-pocket expenditure, poor adherence to medicines, lack of proper diet, gender issues, and stigma. The challenges and barriers at the level of health-care providers were a lack of infrastructure and logistics, lack of awareness, COVID-19-related issues, lack of workforce, and technical issues. CONCLUSION: Communication between providers and people with TB must be improved to improve the drug adherence and satisfaction of the end user. Proper funding must be provided for the TB programs. People with TB must be counseled properly regarding the free health care services available near their homes to prevent out-of-pocket expenditure. These will help in fast-tracking the elimination of TB.

Inflammation resolution in environmental pulmonary health and morbidity.

Inflammation and resolution are dynamic processes comprised of inflammatory activation and neutrophil influx, followed by mediator catabolism and efferocytosis. These critical pathways ensure a return to homeostasis and promote repair. Over the past decade research has shown that diverse mediators play a role in the active process of resolution. Specialized pro-resolving mediators (SPMs), biosynthesized from fatty acids, are released during inflammation to facilitate resolution and are deficient in a variety of lung disorders. Failed resolution results in remodeling and cellular deposition through pro-fibrotic myofibroblast expansion that irreversibly narrows the airways and worsens lung function. Recent studies indicate environmental exposures may perturb and deregulate critical resolution pathways. Environmental xenobiotics induce lung inflammation and generate reactive metabolites that promote oxidative stress, injuring the respiratory mucosa and impairing gas-exchange. This warrants recognition of xenobiotic associated molecular patterns (XAMPs) as new signals in the field of inflammation biology, as many environmental chemicals generate free radicals capable of initiating the inflammatory response. Recent studies suggest that unresolved, persistent inflammation impacts both resolution pathways and endogenous regulatory mediators, compromising lung function, which over time can progress to chronic lung disease. Chronic ozone (O3) exposure overwhelms successful resolution, and in susceptible individuals promotes asthma onset. The industrial contaminant cadmium (Cd) bioaccumulates in the lung to impair resolution, and recurrent inflammation can result in chronic obstructive pulmonary disease (COPD). Persistent particulate matter (PM) exposure increases systemic cardiopulmonary inflammation, which reduces lung function and can exacerbate asthma, COPD, and idiopathic pulmonary fibrosis (IPF). While recurrent inflammation underlies environmentally induced pulmonary morbidity and may drive the disease process, our understanding of inflammation resolution in this context is limited. This review aims to explore inflammation resolution biology and its role in chronic environmental lung disease(s).

Recalibrating the Non-Communicable Diseases risk prediction tools for the rural population of Western India.

BACKGROUND: The aim of the present study was to recalibrate the effectiveness of Indian Diabetes Risk Score (IDRS) and Community-Based Assessment Checklist (CBAC) by opportunistic screening of Diabetes Mellitus (DM) and Hypertension (HT) among the people attending health centres, and estimating the risk of fatal and non-fatal Cardio-Vascular Diseases (CVDs) among them using WHO/ISH charts. METHODS: All the people aged ≥ 30 years attending the health centers were screened for DM and HT. Weight, height, waist circumference, and hip circumferences were measured, and BMI and Waist-Hip Ratio (WHR) were calculated. Risk categorization of all participants was done using IDRS, CBAC, and WHO/ISH risk prediction charts. Individuals diagnosed with DM or HT were started on treatment. The data was recorded using Epicollect5 and was analyzed using SPSS v.23 and MedCalc v.19.8. ROC curves were plotted for DM and HT with the IDRS, CBAC score, and anthropometric parameters. Sensitivity (SN), specificity (SP), Positive Predictive Value (PPV), Negative Predictive Value (NPV), Accuracy and Youden's index were calculated for different cut-offs of IDRS and CBAC scores. RESULTS: A total of 942 participants were included for the screening, out of them, 9.2% (95% CI: 7.45-11.31) were diagnosed with DM for the first time. Hypertension was detected among 25.7% (95% CI: 22.9-28.5) of the participants. A total of 447 (47.3%) participants were found with IDRS score ≥ 60, and 276 (29.3%) with CBAC score > 4. As much as 26.1% were at moderate to higher risk (≥ 10%) of developing CVDs. Area Under the Curve (AUC) for IDRS in predicting DM was 0.64 (0.58-0.70), with 67.1% SN and 55.2% SP (Youden's Index 0.22). While the AUC for CBAC was 0.59 (0.53-0.65). For hypertension both the AUCs were 0.66 (0.62-0.71) and 0.63 (0.59-0.67), respectively. CONCLUSIONS: IDRS was found to have the maximum AUC and sensitivity thereby demonstrating its usefulness as compared to other tools for screening of both diabetes and hypertension. It thus has the potential to expose the hidden NCD iceberg. Hence, we propose IDRS as a useful tool in screening of Diabetes and Hypertension in rural India.

Integrating transcriptomic and proteomic data for accurate assembly and annotation of genomes.

Complementing genome sequence with deep transcriptome and proteome data could enable more accurate assembly and annotation of newly sequenced genomes. Here, we provide a proof-of-concept of an integrated approach for analysis of the genome and proteome of Anopheles stephensi, which is one of the most important vectors of the malaria parasite. To achieve broad coverage of genes, we carried out transcriptome sequencing and deep proteome profiling of multiple anatomically distinct sites. Based on transcriptomic data alone, we identified and corrected 535 events of incomplete genome assembly involving 1196 scaffolds and 868 protein-coding gene models. This proteogenomic approach enabled us to add 365 genes that were missed during genome annotation and identify 917 gene correction events through discovery of 151 novel exons, 297 protein extensions, 231 exon extensions, 192 novel protein start sites, 19 novel translational frames, 28 events of joining of exons, and 76 events of joining of adjacent genes as a single gene. Incorporation of proteomic evidence allowed us to change the designation of more than 87 predicted "noncoding RNAs" to conventional mRNAs coded by protein-coding genes. Importantly, extension of the newly corrected genome assemblies and gene models to 15 other newly assembled Anopheline genomes led to the discovery of a large number of apparent discrepancies in assembly and annotation of these genomes. Our data provide a framework for how future genome sequencing efforts should incorporate transcriptomic and proteomic analysis in combination with simultaneous manual curation to achieve near complete assembly and accurate annotation of genomes.

A draft map of the human proteome.

The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.

Widespread somatic L1 retrotransposition occurs early during gastrointestinal cancer evolution.

Somatic L1 retrotransposition events have been shown to occur in epithelial cancers. Here, we attempted to determine how early somatic L1 insertions occurred during the development of gastrointestinal (GI) cancers. Using L1-targeted resequencing (L1-seq), we studied different stages of four colorectal cancers arising from colonic polyps, seven pancreatic carcinomas, as well as seven gastric cancers. Surprisingly, we found somatic L1 insertions not only in all cancer types and metastases but also in colonic adenomas, well-known cancer precursors. Some insertions were also present in low quantities in normal GI tissues, occasionally caught in the act of being clonally fixed in the adjacent tumors. Insertions in adenomas and cancers numbered in the hundreds, and many were present in multiple tumor sections, implying clonal distribution. Our results demonstrate that extensive somatic insertional mutagenesis occurs very early during the development of GI tumors, probably before dysplastic growth.

Tissue matrix arrays for high-throughput screening and systems analysis of cell function.

Cell and protein arrays have demonstrated remarkable utility in the high-throughput evaluation of biological responses; however, they lack the complexity of native tissue and organs. Here we spotted tissue extracellular matrix (ECM) particles as two-dimensional (2D) arrays or incorporated them with cells to generate three-dimensional (3D) cell-matrix microtissue arrays. We then investigated the responses of human stem, cancer and immune cells to tissue ECM arrays originating from 11 different tissues. We validated the 2D and 3D arrays as representative of the in vivo microenvironment by means of quantitative analysis of tissue-specific cellular responses, including matrix production, adhesion and proliferation, and morphological changes after culture. The biological outputs correlated with tissue proteomics, and network analysis identified several proteins linked to cell function. Our methodology enables broad screening of ECMs to connect tissue-specific composition with biological activity, providing a new resource for biomaterials research and further understanding of regeneration and disease mechanisms.

Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling.

Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in the ProteomeXchange with identifier PXD002411 (http://proteomecentral.proteomexchange.org/dataset/PXD002411).

Prosthetic Aortic Valve Endocarditis Without Evidence of Vegetation.

Despite significant technological advances, the diagnosis of infective endocarditis (IE) remains a major challenge, and the condition continues to be associated with significant morbidity and mortality. Valvular vegetations have long been the diagnostic and pathologic hallmarks of IE. However, IE can be diagnosed even in the absence of vegetations using the modified Duke criteria. Vegetation-negative endocarditis is rare, and to the present authors' knowledge no cases of septic emboli in the absence of valvular vegetations have been reported. Herein is reported a case of prosthetic aortic valve endocarditis associated with both clinical and radiologic evidence of septic emboli, but in the absence of vegetations on both repeated transesophageal echocardiography and pathologic evaluation. This case highlights the importance of maintaining a high clinical suspicion and a low threshold for the surgical replacement of a possibly infected valve, in patients that meet other clinical criteria for IE, even in the absence of detectable valvular vegetations.

Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer.

Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers.

Annotation of the zebrafish genome through an integrated transcriptomic and proteomic analysis.

Accurate annotation of protein-coding genes is one of the primary tasks upon the completion of whole genome sequencing of any organism. In this study, we used an integrated transcriptomic and proteomic strategy to validate and improve the existing zebrafish genome annotation. We undertook high-resolution mass-spectrometry-based proteomic profiling of 10 adult organs, whole adult fish body, and two developmental stages of zebrafish (SAT line), in addition to transcriptomic profiling of six organs. More than 7,000 proteins were identified from proteomic analyses, and ∼ 69,000 high-confidence transcripts were assembled from the RNA sequencing data. Approximately 15% of the transcripts mapped to intergenic regions, the majority of which are likely long non-coding RNAs. These high-quality transcriptomic and proteomic data were used to manually reannotate the zebrafish genome. We report the identification of 157 novel protein-coding genes. In addition, our data led to modification of existing gene structures including novel exons, changes in exon coordinates, changes in frame of translation, translation in annotated UTRs, and joining of genes. Finally, we discovered four instances of genome assembly errors that were supported by both proteomic and transcriptomic data. Our study shows how an integrative analysis of the transcriptome and the proteome can extend our understanding of even well-annotated genomes.

Erythema Necroticans - A Case Report.

Erythema Nodosum Leprosum (ENL) is characterized by evanescent, erythematous, painful raised nodules which fade within 48-72 hours. Necrotic and ulcerative forms are rare presentations of severe ENL. A 27 year old male patient presented with multiple erythematous nodules on trunk and extremities associated with high grade fever, joint pain and pedal edema. Patient developed ulceration of nodules associated with pain and burning sensation over another 3 days. Slit smear showed clumps of granular bacilli. Biopsy showed superficial dermis showing edema with dense focal perivascular infiltrate of lymphocytes, macrophages and few scattered neutrophils. Fite-Faraco stain was negative. Patient was diagnosed as a case of erythema necroticans and started on oral steroids and thalidomide. The histological findings illustrate the need to consider leprosy diagnosis in necrotizing vasculitis even when Virchow's cells are not found in the infiltrate. Thalidomide is the drug of choice in such cases. This patient showed a marked response to the drug with healing of all ulcers within 2 weeks of starting thalidomide.

Quantitative phosphoproteomic analysis of IL-33-mediated signaling.

Interleukin-33 (IL-33) is a novel member of the IL-1 family of cytokines that plays diverse roles in the regulation of immune responses. IL-33 exerts its effects through a heterodimeric receptor complex resulting in the production and release of proinflammatory cytokines. A detailed understanding of the signaling pathways activated by IL-33 is still unclear. To gain insights into the IL-33-mediated signaling mechanisms, we carried out a SILAC-based global quantitative phosphoproteomic analysis that resulted in the identification of 7191 phosphorylation sites derived from 2746 proteins. We observed alterations in the level of phosphorylation in 1050 sites corresponding to 672 proteins upon IL-33 stimulation. We report, for the first time, phosphorylation of multiple protein kinases, including mitogen-activated protein kinase activated protein kinase 2 (Mapkapk2), receptor (TNFRSF) interacting serine-threonine kinase 1 (Ripk1), and NAD kinase (Nadk) that are induced by IL-33. In addition, we observed IL-33-induced phosphorylation of several protein phosphatases including protein tyrosine phosphatase, nonreceptor-type 12 (Ptpn12), and inositol polyphosphate-5-phosphatase D (Inpp5d), which have not been reported previously. Network analysis revealed an enrichment of actin binding and cytoskeleton reorganization that could be important in macrophage activation induced by IL-33. Our study is the first quantitative analysis of IL-33-regulated phosphoproteome. Our findings significantly expand the understanding of IL-33-mediated signaling events and have the potential to provide novel therapeutic targets pertaining to immune-related diseases such as asthma where dysregulation of IL-33 is observed. All MS data have been deposited in the ProteomeXchange with identifier PXD000984 (http://proteomecentral.proteomexchange.org/dataset/PXD000984).

Phosphoproteomic analysis reveals compensatory effects in the piriform cortex of VX nerve agent exposed rats.

To gain insights into the toxicity induced by the nerve agent VX, an MS-based phosphoproteomic analysis was carried out on the piriform cortex region of brains from VX-treated rats. Using isobaric tag based TMT labeling followed by titanium dioxide enrichment strategy, we identified 9975 unique phosphosites derived from 3287 phosphoproteins. Temporal changes in the phosphorylation status of peptides were observed over a time period of 24 h in rats exposed to a 1× LD50, intravenous (i.v.) dose with the most notable changes occurring at the 1 h postexposure time point. Five major functional classes of proteins exhibited changes in their phosphorylation status: (i) ion channels/transporters, including ATPases, (ii) kinases/phosphatases, (iii) GTPases, (iv) structural proteins, and (v) transcriptional regulatory proteins. This study is the first quantitative phosphoproteomic analysis of VX toxicity in the brain. Understanding the toxicity and compensatory signaling mechanisms will improve the understanding of the complex toxicity of VX in the brain and aid in the elucidation of novel molecular targets that would be important for development of improved countermeasures. All MS data have been deposited in the ProteomeXchange with identifier PXD001184 (http://proteomecentral.proteomexchange.org/dataset/PXD001184).

Functional annotation of proteome encoded by human chromosome 22.

As part of the chromosome-centric human proteome project (C-HPP) initiative, we report our progress on the annotation of chromosome 22. Chromosome 22, spanning 51 million base pairs, was the first chromosome to be sequenced. Gene dosage alterations on this chromosome have been shown to be associated with a number of congenital anomalies. In addition, several rare but aggressive tumors have been associated with this chromosome. A number of important gene families including immunoglobulin lambda locus, Crystallin beta family, and APOBEC gene family are located on this chromosome. On the basis of proteomic profiling of 30 histologically normal tissues and cells using high-resolution mass spectrometry, we show protein evidence of 367 genes on chromosome 22. Importantly, this includes 47 proteins, which are currently annotated as "missing" proteins. We also confirmed the translation start sites of 120 chromosome 22-encoded proteins. Employing a comprehensive proteogenomics analysis pipeline, we provide evidence of novel coding regions on this chromosome which include upstream ORFs and novel exons in addition to correcting existing gene structures. We describe tissue-wise expression of the proteins and the distribution of gene families on this chromosome. These data have been deposited to ProteomeXchange with the identifier PXD000561.

Proteomic analysis of human osteoarthritis synovial fluid.

BACKGROUND: Osteoarthritis is a chronic musculoskeletal disorder characterized mainly by progressive degradation of the hyaline cartilage. Patients with osteoarthritis often postpone seeking medical help, which results in the diagnosis being made at an advanced stage of cartilage destruction. Sustained efforts are needed to identify specific markers that might help in early diagnosis, monitoring disease progression and in improving therapeutic outcomes. We employed a multipronged proteomic approach, which included multiple fractionation strategies followed by high resolution mass spectrometry analysis to explore the proteome of synovial fluid obtained from osteoarthritis patients. In addition to the total proteome, we also enriched glycoproteins from synovial fluid using lectin affinity chromatography. RESULTS: We identified 677 proteins from synovial fluid of patients with osteoarthritis of which 545 proteins have not been previously reported. These novel proteins included ADAM-like decysin 1 (ADAMDEC1), alanyl (membrane) aminopeptidase (ANPEP), CD84, fibulin 1 (FBLN1), matrix remodelling associated 5 (MXRA5), secreted phosphoprotein 2 (SPP2) and spondin 2 (SPON2). We identified 300 proteins using lectin affinity chromatography, including the glycoproteins afamin (AFM), attractin (ATRN), fibrillin 1 (FBN1), transferrin (TF), tissue inhibitor of metalloproteinase 1 (TIMP1) and vasorin (VSN). Gene ontology analysis confirmed that a majority of the identified proteins were extracellular and are mostly involved in cell communication and signaling. We also confirmed the expression of ANPEP, dickkopf WNT signaling pathway inhibitor 3 (DKK3) and osteoglycin (OGN) by multiple reaction monitoring (MRM) analysis of osteoarthritis synovial fluid samples. CONCLUSIONS: We present an in-depth analysis of the synovial fluid proteome from patients with osteoarthritis. We believe that the catalog of proteins generated in this study will further enhance our knowledge regarding the pathophysiology of osteoarthritis and should assist in identifying better biomarkers for early diagnosis.

Model-based evaluation of the Canberra Hospital Acute Care Surgical Unit : acute care surgery: a case of one size fits all?

PURPOSE: Surgical services in Australia are under sustained and growing pressure. The global implementation of acute care surgery services has been shown to facilitate the timeliness of acute surgery. The question is: Do acute care surgical units fit major regional centers like ours? The current study coincides with the introduction of a Surgical Assessment and Planning Unit (SAPU) at the Canberra Hospital and compares patient outcomes before vs. after the introduction of the SAPU, using acute appendicitis as the model illness. METHODS: We reviewed patients presenting to the Canberra Hospital Emergency Department with a preliminary diagnosis of acute appendicitis before vs. after the introduction of an acute care surgical unit. RESULTS: The subjects were 150 patients, ranging in age from 16 to 97 years. The mean time from presentation at casualty to surgical review and the surgical review itself was reduced by 19 and 26 %, respectively (p < 0.05). Time to the operating table and the percentage of after-hours operations were reduced by 8 and 40 %, respectively. There was a significant reduction in the utilization of abdominal ultrasonography after the implementation of the SAPU. CONCLUSIONS: The implementation of a SAPU has benefited the management of patients with acute surgical conditions. Ultimately, patient care is enhanced, with patients being reviewed, admitted, and treated earlier.

Whole exome sequencing uncovers HRAS mutations as potential mediators of resistance to metronomic chemotherapy.

OBJECTIVES: The aim of this pilot study is to identify the genetic factors that contribute to the response of metronomic chemotherapy in head and neck squamous cell carcinoma (HNSCC) patients using whole-exome sequencing (WES). This study would facilitate the identification of predictive biomarkers, which would enable personalized treatment strategies and improve treatment outcomes for patients with HNSCC. MATERIALS AND METHODS: We have selected patients with recurrent head and neck cancer who underwent metronomic chemotherapy. Sequential tumor biopsies were collected from the patients at different stages of treatment to capture the genomic alterations and tumor evolution during metronomic chemotherapy and sequenced using WES. RESULTS: We identified several known HNSCC hallmark genes reported in COSMIC, including KMT2B, NOTCH1, FAT1, TP53, HRAS, CASP8, and CDKN2A. Copy number alteration analysis revealed amplifications and deletions in several oncogenic and tumor suppressor genes. COSMIC Mutational Signature 15 associated with defective DNA mismatch repair was enriched in 73% of HNSCC samples. Further, the comparison of genomic alterations between responders and non-responders identified HRAS gene uniquely mutated in non-responders that could potentially contribute to resistance against metronomic chemotherapy. DISCUSSION: Our findings corroborate the molecular heterogeneity of recurrent HNSCC tumors and establish an association between HRAS mutations and resistance to metronomic chemotherapy, suggesting HRAS as a potential therapeutic target. Combining HRAS inhibitors with metronomic regimens could improve treatment sensitivity in HRAS-mutated HNSCC patients. Further studies are needed to fully elucidate the genomic mechanisms underlying the response to metronomic chemotherapy.