Lung cancer, especially non-small cell lung cancer (NSCLC), poses a significant global health challenge due to its high prevalence and poor prognosis despite treatment advancements, including immunotherapy. While programmed death-ligand 1 (PD-L1) expression is a commonly used biomarker, its limitations justify exploration of alternative markers like the neutrophil-to-lymphocyte ratio (NLR), absolute neutrophil count (ANC) and derived NLR (dNLR). This retrospective study aims to directly compare NLR, ANC and dNLR as predictive biomarkers in first-line NSCLC immunotherapy, shedding light on their prognostic implications and potential clinical utility.

This retrospective single-center study included 70 consecutive patients diagnosed with metastatic NSCLC, treated in first-line with immune checkpoint inhibitors (ICIs) between September 2015 and March 2023 at the University Hospital of Reims, France. Baseline clinical characteristics and hematological values were collected, and survival analysis, including progression-free survival (PFS) and overall survival (OS), was performed based on RECIST (Response Evaluation Criteria in Solid Tumors) criteria. NLR and dNLR were calculated, and their predictive performances were assessed.

Baseline characteristics revealed a median age of 65.5 years, predominantly adenocarcinoma histology (82.9%), and high PD-L1 expression (≥50%) in 61.4% of cases. Neither NLR, ANC nor dNLR showed significant associations with known clinical outcome influencers like age, PD-L1 expression, or performance status, but dNLR correlated significantly with initial response (P=0.02). While NLR ≥5 was significantly associated with shorter PFS and OS (P=0.03 and P<0.001, respectively), dNLR >2.5 (P=0.008) or ANC >7.5 (P=0.02) showed significance in predicting poorer OS only. Optimal cut-off values were determined as 5.0 for NLR [area under the curve (AUC) =0.570], 9.00 for ANC (AUC =0.683) and 2.496 for dNLR (AUC =0.610) for OS prediction. Cox regressions revealed no significant association between either biomarker and clinical or histological cofactors. Subgroup analyses suggested NLR's predictive consistency across various subgroups, whereas dNLR and ANC showed limited performance. Both biomarkers demonstrated significant association with OS in patients exposed to ICI alone, but not with chemotherapy combination.

The results underscore the potential of NLR as a predictor of survival and progression in NSCLC patients treated with immunotherapy, while dNLR and ANC demonstrate more limited interest. However, larger prospective studies are needed to confirm these observations and further elucidate their clinical utility.

Cancer is a state of immunological imbalance associated with chronic inflammation and local immunosuppression. Introducing immune checkpoint inhibitors was a breakthrough in cancer treatment. However, the treatment outcomes remain unsatisfactory, and many patients still progress after the initial response. The study aimed to assess whether serum neopterin (NEO), an indicator of cellular immune activation, could be used as a predictor of the long-term benefits of drugs blocking the programmed cell death protein 1 pathway (anti-PD-1/PD-L1 drugs). We enrolled 103 patients with non-small cell lung cancer (NSCLC) treated with anti-PD-1/PD-L1s. Serum was collected at baseline and at the end of each treatment cycle for the first three months of immunotherapy. NEO concentrations were determined with a validated high-performance liquid chromatography assay and correlated with treatment outcomes. Low-NEO status (i.e., serum NEO levels ≤ 71.65 nM at the end of the 3rd treatment cycle and ≤ 66.84 nM at the end of the 4th treatment cycle) increased the odds of ≥ 12-month benefits (odds ratio, OR = 11.70, p < 0.001), and decreased the hazard of NSCLC progression (hazard ratio, HR = 0.327, p < 0.001) and treatment failure (adjusted HR = 0.450, p < 0.05). Patients with low-NEO status had three times longer progression-free survival (PFS, 17.3 vs. 5.9 months) and three times longer time to treatment failure (TTF, 16.3 vs. 5.5 months) compared to other patients. Baseline NEO levels could not discriminate between patients who had and lacked the long-term benefits of treatment. In conclusion, the on-treatment serum NEO concentrations could be a biomarker of the long-term benefits of the anti-PD-1/PD-L1 treatment in advanced NSCLC.

Primary and secondary resistance to immune checkpoint blockade (ICB) reduces its efficacy. The mechanisms underlying immunotherapy resistance are highly complex. In non-small cell lung cancer (NSCLC), these mechanisms are primarily associated with the loss of programmed cell death-ligand 1 (PD-L1) expression, genetic mutations, circular RNA axis and transcription factor regulation, antigen presentation disorders, and dysregulation of signaling pathways. Additionally, alterations in the tumor microenvironment (TME) play a pivotal role in driving immunotherapy resistance. Primary resistance is mainly attributed to TME alterations, including mutations and co-mutations, modulation of T cell infiltration, enrichment of M2 tumor-associated macrophages (M2-TAMs) and mucosal-associated invariant T (MAIT) cells, vascular endothelial growth factor (VEGF), and pulmonary fibrosis. Acquired resistance mainly stems from changes in cellular infiltration patterns leading to "cold" or "hot" tumors, altered interferon (IFN) signaling pathway expression, involvement of extracellular vesicles (EVs), and oxidative stress responses, as well as post-treatment gene mutations and circadian rhythm disruption (CRD). This review presents an overview of various mechanisms underlying resistance to ICB, elucidates the alterations in the TME during primary, adaptive, and acquired resistance, and discusses existing strategies for overcoming ICB resistance.

Immune checkpoint inhibitors (ICIs) enhance the tumor-killing ability of T-cells in non-small cell lung cancer (NSCLC), improving overall survival (OS) and revolutionizing treatment for advanced stages. However, challenges remain, such as low response rates and the lack of effective markers for selecting candidates. This study evaluated the impact of hemoglobin, albumin, and platelet (HALP), neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) on the efficacy of immunotherapy and survival outcomes in advanced NSCLC. Additionally, it aimed to develop a nomogram based on these parameters. Clinical and hematological data from NSCLC patients who received immunotherapy were analyzed. Efficacy was assessed using the immune Response Evaluation Criteria in Solid Tumors (iRECIST), and progression-free survival (PFS) and OS were evaluated. Prediction models incorporated baseline and post-treatment HALP, NLR, and PLR values. The 203 patients had a median follow-up of 16 months, a median PFS (mPFS) of seven months (6.0-8.0), while the median OS (mOS) was not reached (24.0-not available). Pretreatment PLR (PLR0) was associated with a higher disease control rate (DCR) (odds ratio [OR] = 0.258), while initial immunotherapy and NLR after four treatment cycles (NLR4C) significantly improved the objective response rate (ORR). Cox regression analysis showed that pretreatment HALP (HALP0), HALP after four cycles of treatment (HALP4C), and pretreatment NLR (NLR0) significantly impacted PFS. Additionally, HALP0, NLR0, and PLR after four treatment cycles (PLR4C) were associated with OS. The C-indices for PFS and OS were 0.823 and 0.878, respectively, indicating good predictive accuracy. HALP, NLR, and PLR at various time points effectively predicted immunotherapy response in advanced NSCLC patients, with low HALP combined with high NLR and PLR indicating a poor prognosis. These findings could serve as the basis for stratified randomized controlled trials (RCTs) in the future.

Lung cancer (LC) continues to pose the highest mortality and exhibits a common prevalence among all types of cancer. The genetic interaction between human eukaryotes and microbial cells plays a vital role in orchestrating every physiological activity of the host. The dynamic crosstalk between gut and lung microbiomes and the gut-lung axis communication network has been widely accepted as promising factors influencing LC progression. The advent of the 16s rDNA sequencing technique has opened new horizons for elucidating the lung microbiome and its potential pathophysiological role in LC and other infectious lung diseases using a molecular approach. Numerous studies have reported the direct involvement of the host microbiome in lung tumorigenesis processes and their impact on current treatment strategies such as radiotherapy, chemotherapy, or immunotherapy. The genetic and metabolomic cross-interaction, microbiome-dependent host immune modulation, and the close association between microbiota composition and treatment outcomes strongly suggest that designing microbiome-based treatment strategies and investigating new molecules targeting the common holobiome could offer potential alternatives to develop effective therapeutic principles for LC treatment. This review aims to highlight the interaction between the host and microbiome in LC progression and the possibility of manipulating altered microbiome ecology as therapeutic targets.

Baseline thyroid function, as measured by the fT3 to fT4 ratio, has been shown to influence the prognosis of advanced cancer patients receiving active treatments. Although immune checkpoint blockade can alter the balance of thyroid hormones, this interaction has not been thoroughly investigated. The present research sought to determine whether changes in the fT3/fT4 ratio could affect the survival outcomes of patients with advanced non-small cell lung cancer (NSCLC) who were undergoing pembrolizumab-based therapies. This study included patients with metastatic NSCLC who received pembrolizumab as upfront treatment, either alone or in combination with platinum-based chemotherapy. Relevant data were gathered before the start (time point 1) and after 12 weeks (time point 2) of treatment. From April 2018 to May 2023, we enrolled 258 eligible patients, 156 (60.5%) and 102 (39.5%) of whom were treated with single-agent or combination therapy, respectively. We stratified patients into two groups based on baseline fT3 and fT4 values [euthyroid cohort defined by fT3 and fT4 both within the normal range vs. euthyroid sick syndrome cohort defined by low fT3 and/or fT4 levels]. We examined the differences in progression-free survival (PFS) and overall survival (OS) by univariate and multivariate analyses. After applying propensity-score matching, we considered 88 relevant cases in each cohort. Longitudinal comparison of fT3/fT4 ratios showed a significant increase in the median value after pembrolizumab-based therapy (p < 0.001). We computed ROC curves to analyze the correlation between fT3/fT4 ratios and survival outcomes. The relative AUC values were not viable in predicting a positive outcome at the first time point. Conversely, assessment at the second time point revealed a significant association with PFS [AUC 0.82 (95% CI 0.75-0.89), p < 0.001] and OS [AUC 0.81 (95% CI 0.75-0.88), p < 0.001]. After a median follow-up of 20.2 (95% CI 16.2-24.2) months, the median PFS for the low and high fT3/fT4 ratio groups was 4.1 (95% CI 3.0-5.1) and 15.3 (95% CI 10.3-20.1) months, respectively (p < 0.001). The median OS for the low and high fT3/fT4 ratio groups was 6.7 (95% CI 4.9-8.5) and 19.6 (95% CI 16.4-22.8) months, respectively (p < 0.001). The multivariate analysis revealed that a low fT3/fT4 ratio was independently associated with shorter PFS [HR 2.51 (1.66-3.78); p < 0.001] and OS [HR 2.18 (1.43-3.34); p < 0.001]. After the optimal weighting of prognostic factors according to thyroid function impairment, the fT3/fT4 ratio at baseline did not affect the survival of patients receiving immune checkpoint blockade for advanced NSCLC. Patients with an increased fT3/fT4 ratio experienced a significantly decreased risk of disease progression and mortality. The longitudinal assessment of fT3/fT4 ratio may play a predictive role in this specific therapeutic setting.

Gastric cancer remains a major global health challenge with high morbidity and mortality rates. Recent advancements in immunology and inflammation research have highlighted the crucial roles that these biological processes play in tumor progression and patient outcomes. This has sparked new interest in developing prognostic biomarkers that integrate these two key biological processes. In this letter, we discuss the recent study by Ba et al, which proposed a novel prognostic immunoinflammatory index for patients with gastric cancer. We underscore the importance of this research, its potential impact on medical practice, and the prospective avenues for further investigation in this rapidly emerging area of study.

Esophageal cancer (EC), a common type of malignant tumor, ranks as the sixth highest contributor to cancer-related mortality worldwide. Due to the condition that most patients with EC are diagnosed at advanced or metastatic status, the efficacy of conventional treatments including surgery, chemotherapy and radiotherapy is limited, resulting in a dismal 5-year overall survival rate. In recent years, the application of immune checkpoint inhibitors (ICIs) has presented a novel therapeutic avenue for EC patients. Both ICIs monotherapy and immunotherapy combined with chemotherapy or chemoradiotherapy (CRT) have demonstrated marked benefits for patients with advanced EC. Adjuvant or neoadjuvant therapy incorporating immunotherapy has also demonstrated promising prospects in the context of perioperative treatment. Nonetheless, due to the variable response observed among patients undergoing immunotherapy, it is of vital importance to identify predictive biomarkers for patient stratification, to facilitate identification of subgroups who may derive greater benefits from immunotherapy. In this review, we summarize validated or potential biomarkers for immunotherapy in EC in three dimensions: tumor-cell-associated biomarkers, tumor-immune microenvironment (TIME)-associated factors, and host-associated biomarkers, so as to provide a theoretical foundation to inform tailored therapy for individuals diagnosed with EC.

Predictive biomarkers for nivolumab in recurrent or metastatic head and neck squamous cell carcinoma (RMHNSCC) have not yet been established.

The tumor proportion score (TPS), combined positive score (CPS), and soluble forms of programmed cell death ligand-1 (PD-L1) and programmed cell death ligand-2 (PD-L2) were retrospectively analyzed in patients with RMHNSCC treated with nivolumab.

The positivity rates for TPS (PD-L1), CPS (PD-L1), TPS (PD-L2), and CPS (PD-L2) were 73.8%, 78.2%, 56.4%, and 78.2%, respectively. Patients with high TPS (PD-L1), CPS (PD-L1), or CPS (PD-L1 and PD-L2) showed significantly prolonged progression-free survival. Favorable overall survival was associated with high CPS (PD-L1 and PD-L2) and low soluble PD-L1 and PD-L2 levels. The expressions of tissue and soluble PD-L1/2 were not correlated.

Our study revealed that compared to PD-L1 expression alone, dual expression of PD-L1 and PD-L2 in tissue or soluble form could be feasible biomarkers in patients with RMHNSCC who received nivolumab.

Patients with non-small cell lung cancer (NSCLC) have been shown to exhibit elevated levels of soluble programmed death-ligand 1 (sPD-L1) in the blood, associated with poor survival in NSCLC. The bronchoalveolar lavage fluid (BALF) composition reflects the tumor microenvironment of lung cancer. In this study, we investigated sPD-L1 levels in BALF and its role as a prognostic and predictive marker in patients with stage IV NSCLC.

We prospectively obtained BALF from lung cancer patients who underwent bronchoscopy between January 2020 and September 2022 at Chungnam National University Hospital (CNUH). Finally, 94 NSCLC stage IV patients were included in this study. Soluble PD-L1 levels in BALF were measured using a human PD-L1 Quantikine ELISA kit.

The correlation between PD-L1 expression in tumor cells and sPD-L1 in BALF was weakly positive (rho =0.314, P=0.002). The median overall survival (OS) of the low sPD-L1 in BALF group was 16.47 months [95% confidence interval (CI): 11.15-21.79 months], which is significantly longer than 8.87 months (95% CI: 0.0-19.88 months, P=0.001) in the high sPD-L1 in BALF group. In 64 patients treated with or without immune checkpoint inhibitors (ICIs), sPD-L1 in BALF was significantly associated with progression-free survival (PFS) and OS. In the subgroup analysis of 31 patients treated with ICI, the objective response rate (ORR) in the low sPD-L1 BALF group was significantly higher than in high sPD-L1 in BALF group (ORR: 60.9% vs. 12.5%, P=0.02).

Soluble PD-L1 in BALF is a potential prognostic indicator for patients with stage IV NSCLC and a predictive marker for ICI treatment response.

The present study sought to examine the relationships between systemic inflammatory composite ratios/cumulative scores, magnitude of systemic inflammatory response (SIR) and survival in good performance status patients (ECOG-PS 0/1) with advanced NSCLC receiving palliative radiotherapy.

Systemic inflammatory composite ratios/cumulative scores included the neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), lymphocyte-monocyte ratio (LMR), C-reactive protein, (CRP)-albumin ratio (CAR), neutrophil- lymphocyte score (NLS), platelet-lymphocyte score (PLS), lymphocyte-monocyte score (LMS), neutrophil-platelet score (NPS), modified Glasgow prognostic score (mGPS). The magnitude of SIR was determined by serum CRP concentration, with a median CRP concentration of >10 m mg/L considered to be systemically inflamed. Relationships between systemic inflammatory composite ratios/ cumulative scores and clinicopathological characteristics were examined using chi-square analysis. Relationships between overall survival (OS) and systemic inflammatory composite ratios/ cumulative scores were examined using cox regression analysis.

479 patients were included. 48% (n = 231) of patients were male and 70% (n = 338) were ≥65 years of age. 29% (n = 140) patients were ECOG-PS 0 and 71% (n = 339) were ECOG-PS 1. 98% (n = 469) of patients died during follow-up. The median survival was 5 months (2-11). A similar prevalence of systemic inflammation was noted across the various ratios/scores (NLR >3 68%; LMR <2.4 65%; PLR >150 70%; CAR >0.20 83%; NLS ≥1 66%; LMS ≥1 71%; NPS≥1 50%; PLS≥1 60% and mGPS≥1 75%). Despite not considered to be systemically inflamed, an NLR <3, LMR ≥2.4, PLR ≤150, NLS 0, LMS 0, NPS 0 and PLS 0 all had a median CRP concentration of >10 mg/L. When adjusted for ECOG-PS, CAR>0.40 (p < 0.001) and mGPS 2 (p < 0.05) remained significantly associated with OS.

Liver-based measures of systemic inflammation (CAR and mGPS) appear more reliable for the quantification of the magnitude of SIR and have prognostic value in patients with advanced NSCLC.

Lung cancer is one of the most common malignant tumors in the world, of which non-small cell lung cancer (NSCLC) is the majority. The emergence of immune checkpoint inhibitors (ICIs) has greatly changed the treatment strategy of NSCLC and improved the prognosis of patients. However, in reality, only a small number of patients can achieve long-term benefit. Therefore, the identification of reliable predictive biomarkers is essential for the selection of treatment modalities. With the development of molecular biology and genome sequencing technology in recent years, as well as the in-depth understanding of tumor and its host immune microenvironment, research on biomarkers has emerged in an endless stream. This review focuses on the predictive biomarkers of immunotherapy efficacy in NSCLC, in order to provide some guidance for precision immunotherapy.
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Many studies have focused on the prognostic role of soluble programmed death ligand 1 (sPD-L1) in non-small cell lung cancer (NSCLC), but outcomes are ambiguous and further investigations are needed. We addressed the matter by studying sPD-L1 in baseline samples and in longitudinal samples taken prior to three subsequent cycles of anti-PD-1/anti-PD-L1 treatments. Eighty patients with NSCLC were enrolled. Median sPD-L1 level at baseline was 52 pg/mL [95% confidence interval (CI) 49-57]. In patients treated with pembrolizumab and nivolumab, the concentration of sPD-L1 remained rather stable throughout treatment. In contrast, sPD-L1 rose by 50-fold following the first cycle of atezolizumab therapy. We found the baseline level of sPD-L1 to be related to overall survival (OS) after two years of follow-up in simple Cox analysis (p = 0.006) and multiple Cox Regression, hazard ratio 1.02 (95% CI 1.00-1.03) (p = 0.033). There was no association between sPD-L1 and tissue PD-L1 expression, overall response rate, or progression free survival. In conclusion, sPD-L1 measured in baseline serum samples may be associated with OS in NSCLC patients receiving anti-PD1/anti-PD-L1 treatment. Importantly, the results signify that further research is warranted to explore the clinical utility of sPD-L1 in patients treated with anti-PD-L1.

The paradigm of non-small cell lung cancer (NSCLC) treatment has been profoundly influenced by the development of immune checkpoint inhibitors (ICI), but the range of clinical responses observed among patients poses significant challenges. To date, analyses of tumor biopsies are the only parameter used to guide prognosis to ICI therapy. Tumor biopsies, however, are often difficult to obtain and tissue-based biomarkers are limited by intratumoral heterogeneity and temporal variability. In response, there has been a growing emphasis on the development of "liquid biopsy"‒ derived biomarkers, which offer a minimally invasive means to dynamically monitor the immune status of NSCLC patients either before and/or during the course of treatment. Here we review studies in which multiple blood-based biomarkers encompassing circulating soluble analytes, immune cell subsets, circulating tumor DNA, blood-based tumor mutational burden, and circulating tumor cells have shown promising associations with the clinical response of NSCLC patients to ICI therapy. These investigations have unveiled compelling correlations between the peripheral immune status of patients both before and during ICI therapy and patient outcomes, which include response rates, progression-free survival, and overall survival. There is need for rigorous validation and standardization of these blood-based assays for broader clinical application. Integration of multiple blood-based biomarkers into comprehensive panels or algorithms also has the potential to enhance predictive accuracy. Further research aimed at longitudinal monitoring of circulating biomarkers is also crucial to comprehend immune dynamics and resistance mechanisms and should be used alongside tissue-based methods that interrogate the tumor microenvironment to guide treatment decisions and may inform on the development of novel therapeutic strategies. The data reviewed here reinforce the opportunity to refine patient stratification, optimize treatments, and improve outcomes not only in NSCLC but also in the wider spectrum of solid tumors undergoing immunotherapy.

Recent studies have revealed that neutrophils play a crucial role in cancer progression. This study aimed to explore the diagnostic value of neutrophil-related biomarkers for non-small-cell lung cancer (NSCLC).

We initially assessed the associations between classic neutrophil-related biomarkers (neutrophil-to-lymphocyte ratio (NLR), absolute neutrophil counts (NEU), absolute lymphocyte counts (LYM)) and NSCLC in 3942 cases and 6791 controls. Then, we measured 11 novel neutrophil-related biomarkers via Luminex Assays in 132 cases and 66 controls, individually matching on sex and age (±5 years), and evaluated their associations with NSCLC risk. We also developed the predictive models by sequentially adding variables of interest and assessed model improvement.

Interleukin-6 (IL-6) (odds ratio (OR) = 10.687, 95% confidence interval (CI): 3.875, 29.473) and Interleukin 1 Receptor Antagonist (IL-1RA) (OR = 8.113, 95% CI: 3.182, 20.689) shows strong associations with NSCLC risk after adjusting for body mass index, smoking status, NLR, and carcinoembryonic antigen. Adding the two identified biomarkers to the predictive model significantly elevated the model performance from an area under the receiver operating characteristic curve of 0.716 to 0.851 with a net reclassification improvement of 97.73%.

IL-6 and IL-1RA were recognized as independent risk factors for NSCLC, improving the predictive performance of the model in identifying disease.

Background and objective Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer type that affects the mucosal lining of the upper aerodigestive tract. Soluble programmed death-ligand 1 (sPD-L1) is a significant factor in hindering T cells' function, which prevents cancer cells from being detected by the immune system. This means that sPD-L1 is an essential component in the immune evasion of cancer. This study aimed to explore the potential of sPD-L1 as a prognostic biomarker for patients with HNSCC undergoing concurrent chemotherapy and radiation therapy. Methodology The study included 106 patients with locally advanced HNSCC who received three courses of induction chemotherapy followed by concurrent chemoradiation and 60 healthy subjects as controls. sPD-L1 levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit, and the cutoff value was determined based on receiver operating characteristic (ROC) curve analysis. Results The results showed that sPD-L1 levels were significantly higher in HNSCC patients compared to healthy controls, with a cutoff value of 31.51 pg/mL. Higher sPD-L1 levels were associated with poorer overall survival rates. Conclusions These findings suggest that sPD-L1 may serve as a valuable prognostic biomarker for HNSCC patients undergoing concurrent chemotherapy and radiation therapy. The study highlights the importance of exploring new biomarkers and therapeutic strategies for HNSCC to improve patient outcomes and reduce morbidity and mortality rates associated with this disease.

In order to develop a biomarker predicting the efficacy of treatments for patients with esophageal squamous cell carcinoma (ESCC), we evaluated the subpopulation of T cells in ESCC patients treated with chemotherapy (CT), chemoradiotherapy (CRT), and nivolumab therapy (NT). Fifty-five ESCC patients were enrolled in this study, and peripheral blood samples were collected before and after CT or CRT and during NT. Frequencies of memory, differentiated, and exhausted T cells were evaluated using flow cytometry among cStages, treatment strategies, pathological responses of CT/CRT, and during NT. The frequencies of PD-1⁺ or TIM-3⁺CD4⁺ T cells were significantly higher in patients with cStage IV. PD-1⁺CD4⁺ and TIM-3⁺CD8⁺ T-cell populations were significantly higher in patients treated with CRT but were not associated with treatment response. The frequencies of both CD4⁺ and CD8⁺ CD45RA^-CD27⁺CD127⁺ central memory T cells (T_CM) were significantly decreased during the course of NT in the progressive disease group. Taken together, the alteration in frequency of CD45RA^-CD27⁺CD127⁺ T_CM during NT may be a biomarker to predict its therapeutic response in ESCC patients.