Advanced adenomas (AAs), precursors to colorectal cancer (CRC), affect 5% of individuals under 50 and 8% of those 50 and older. Current blood-based tests have limited AA sensitivity (12-13%) compared to FIT (23.8%), highlighting the need for improved detection methods. The FirstSight assay combines circulating tumor DNA (ctDNA) and circulating epithelial cells (CECs) to enhance AA detection. Initial results show that FirstSight achieves higher sensitivity and specificity than existing blood-based tests. Here we analyze the contributions of ctDNA and CEC in the detection of AA. FirstSight was validated in a study of 1, 038 prospectively collected, diverse subjects from a majority screening cohort. CEC data were analyzed using a multi-dye machine learning algorithm. The FirstSight algorithm incorporated patient age, sex, and CRC incidence from SEER statistics to adjust baseline risk and refine probability distributions for improved predictive performance. We estimated the contribution of ctDNA and CEC in the detection of AA, and as an example, the cumulative effect of ctDNA and a single CEC-derived marker GM1. CEC (p < 0.01) and ctDNA (p < 0.0001) biomarkers were significant predictors of AA. A CEC marker, GM1, improved specificity for AA. At a prespecified 90% specificity for the detection of advanced neoplasia (CRC+AA), the best single ctDNA marker and the CEC marker GM1 individually had a sensitivity of 20% and 16%, respectively, for AA. The combined use of all ctDNA markers had a sensitivity of 39% for AA and the further addition of GM1 raised the sensitivity to 49% for AA. Finally, the integration of ctDNA, CEC markers, their interaction, and demographics improved FirstSight sensitivity to 54.5% for AA.The contribution of GM1 was significant (ꭓ2 = 9.7272, p = 0.02103); removing GM1 reduced the AUC for AA from 0.8165 to 0.745. Consistent with the CRC dysplasia-malignancy continuum, FirstSight demonstrated higher sensitivity for stage I CRC compared to first-generation blood-based tests (92.3% vs. 54.5%). The multimodal FirstSight test, combining ctDNA and CEC biomarkers, achieved 54.5% sensitivity and 90.6% specificity for AA detection in a 1, 038-subject study. CEC signals are found to be most contributory to the detection of AA and the interaction of a single CEC marker, GM1, with ctDNA further improves AA sensitivity, making the performance not simply additive. FirstSight demonstrates the potential for superior AA detection and effective colorectal cancer prevention strategies. This minimally invasive test offers a patient-friendly alternative, with ongoing validation to confirm its clinical utility. Shai Friedland, Drew Watson, Zhen Zhang, Alexander Atkins, Huangpin Ben Hsieh, Jr-Ming Lai, Stephen Su, Hung-Jen Shao, Jen-chia Wu, John Sninsky, Rui Mei. Minimally invasive detection of advanced adenomas in colorectal cancer screening through circulating epithelial cells and somatic DNA aberrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7064.

e15527 Background: Multimodal diagnostic classifiers survey signals from multiple biological compartments and provide iterative, independent and interactive information. Detection of both CRC and AA is critical for noninvasive colorectal screening to improve overall survival and prevent the 2.5-5% annual transition of AA to CRC. FirstSight integrates clinically validated somatic variants from cfDNA and circulating epithelial cells (CECs) adjusting for age and sex. CECs provide information from both intrinsic factors of the adenoma and extrinsic factors such as adenoma microenvironment which facilitates early systemic entry. We sought to assess differential information from CEC signals for the detection of colorectal cancer and/or advanced adenoma (AA). Methods: Blood samples and colonoscopy pathology results were obtained from 438 asymptomatic screening subjects enriched for CRC/AA obtained from 15 US medical centers. The cohort included 18 CRCs and 64 AAs. Somatic variants from cfDNA were identified using NGS and qPCR. CECs were captured by the CellMax biomimetic platform (CMx) using high-avidity EpCAM antibody embedded in the CMx biochip and confirmed with immunostaining (DAPI: nucleus, CK20: epithelial cell and CD45: WBC). CMx platform’s AI/ML analyses of CEC images quantify stain intensities and cellular features. CEC derived signal GM1was evaluated for its predictive capability beyond somatic variants from cfDNA. Results: Genetic or epigenetic variants were not detected in 52% (33/64) of AA cases, limiting the sensitivity of these markers for early disease. However, distinct CEC signals have been identified that aid in the detection of subjects with CRC or AA, or conversely subjects with negative colonoscopies or non-advanced adenomas (nAA). Among them, a novel feature (referred to as GM1) derived from CEC signals was able to differentiate with statistical significance AA from negative/non-neoplastic findings or nAAs in study subjects with negative results in corresponding somatic variants from cfDNA (p < 0.0001). While targeted somatic variants from cfDNA performed well on CRC (17/18), they provided no predictive information for detection of AA for the 353 subjects which were negative for variants, GM1 provided 100.0% (33/33) sensitivity at 35.1% (112/319) specificity, showing its ability to rule out AA with high negative predictive value. Conclusions: Somatic variant detection modes of the First Sight multi-modal assay have high sensitivity for CRC and modest sensitivity for AA. We demonstrate that CEC signal GM1 of FirstSight provides significant independent information for the detection of CRC and AA beyond somatic variants from cfDNA. Additional CEC signals may further improve the sensitivity and specificity for detection of early-stage colorectal neoplasia. The GM1 CEC signal marker will need to be validated further in future studies.

Background Elevated levels of donor-derived cell-free DNA (dd-cfDNA) in the plasma of renal allograft recipients indicates organ injury and an increased probability of active rejection. Donor-specific antibodies (DSA) to HLA antigens are associated with risk of antibody-mediated rejection (ABMR). This study assessed the combined use of dd-cfDNA and DSA testing to diagnose active ABMR. Methods Donor-derived cell-free DNA was assayed in 90 blood samples with paired DSA and clinically indicated biopsies from 87 kidney transplant patients. Sixteen cases met criteria for active ABMR. Performance characteristics of dd-cfDNA for diagnosis of active ABMR were determined for samples with prior or current positive DSA (DSA+, n = 33). Results The median level of dd-cfDNA (2.9%) in DSA+ patients with active ABMR was significantly higher than the median level (0.34%) in DSA+ patients without ABMR (P < 0.001). The median level of dd-cfDNA in DSA- patients was 0.29%. The positive predictive value of dd-cfDNA (at 1%) to detect active ABMR in DSA+ patients was 81%, whereas the negative predictive value was 83%. The positive predictive value for DSA+ alone was 48%. Conclusions The combined use of dd-cfDNA and DSA testing may improve the noninvasive diagnosis of active ABMR in kidney transplant patients. Patients with dd-cfDNA+/ DSA+ results have a high probability of active ABMR.

Background: Previous studies have demonstrated that donor-derived cell-free DNA (dd-cfDNA) found in circulating blood of transplant recipients may serve as a noninvasive biomarker of allograft rejection. To better interpret the clinical meaning of dd-cfDNA, it is essential to understand the biological variation of this biomarker in stable healthy recipients. This report establishes the biological variation and clinical reference intervals of dd-cfDNA in renal transplant recipients by using an analytically validated assay that has a CV of 6.8%. Methods: We sampled venous blood at patient surveillance visits (typically at posttransplant months 1-4, 6, 9, and 12) in a 14-center observational study. Patients with stable renal allograft function spanning >= 3 serial visits were selected. We used AlloSure (R), a targeted next-generation sequencing-based approach, to measure dd-cfDNA in the plasma and computed the intraindividual CV (CVI) and interindividual CV (CVG), the index of individuality (II), and reference change value (RCV). Results: Of 93 patients, 61% were men, 56% were Caucasian, mean age was 49 years, and 63% were deceased donor kidney recipients. Of 380 blood samples, the dd-cfDNA median value was 0.21% (interquartile range 0.12%-0.39%) and the 97.5th percentile was 1.20%. In 18 patients with an average of 4.1 tests, the CVI was 21%, CVG was 37%, II was 0.57, and RCV was 61%. Conclusions: In a renal transplant recipient, a dd-cfDNA level above 1.2% is out of range and potentially abnormal. A serial increase of up to 61% in level of dd-cfDNA in a patient may be attributable to biological variation.

Histologic analysis of the allograft biopsy specimen is the standard method used to differentiate rejection from other injury in kidney transplants. Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive test of allograft injury that may enable more frequent, quantitative, and safer assessment of allograft rejection and injury status. To investigate this possibility, we prospectively collected blood specimens at scheduled intervals and at the time of clinically indicated biopsies. In 102 kidney recipients, we measured plasma levels of dd-cfDNA and correlated the levels with allograft rejection status ascertained by histology in 107 biopsy specimens. The dd-cfDNA level discriminated between biopsy specimens showing any rejection (T cell-mediated rejection or antibody-mediated rejection [ABMR]) and controls (no rejection histologically), P<0.001 (receiver operating characteristic area under the curve [AUC], 0.74; 95% confidence interval [95% CI], 0.61 to 0.86). Positive and negative predictive values for active rejection at a cutoff of 1.0% dd-cfDNA were 61% and 84%, respectively. The AUC for discriminating ABMR from samples without ABMR was 0.87 (95% CI, 0.75 to 0.97). Positive and negative predictive values for ABMR at a cutoff of 1.0% dd-cfDNA were 44% and 96%, respectively. Median dd-cfDNA was 2.9% (ABMR), 1.2% (T cell-mediated types ≥IB), 0.2% (T cell-mediated type IA), and 0.3% in controls (P=0.05 for T cell-mediated rejection types ≥IB versus controls). Thus, dd-cfDNA may be used to assess allograft rejection and injury; dd-cfDNA levels <1% reflect the absence of active rejection (T cell-mediated type ≥IB or ABMR) and levels >1% indicate a probability of active rejection.

The urgent need for donor lungs and a shortage of suitable organs requires transplantation of HLA mismatched donor:recipient pairs. Post-transplant high resolution HLA typing of the donor and recipient, if available, presents a post hoc opportunity to investigate comparative allelic and epitope mismatch strategies to predict post-transplant events and optimize immunosuppressive therapy. Thirty donor-recipient lung transplant pairs with available extracted DNA were studied (De Vlaminck et al. (2015)). High resolution NGS HLA typing required targeted sequencing and was performed according to Moonsamy et al. (2013). Respective two-region HLA types were assigned using Conexio ASSIGN ATF 454 software and compared to the catalog of common and well-documented HLA alleles. Epitope mismatches were determined using HLAMatchmaker. Prior transplant studies noted the importance of HLA-A, HLA-B and DRB1 for short and long term survival and therefore were selected for this study. Thirty lung transplant pairs had 3 (n = 1), 4 (5), 5 (10) and 6 (14) HLA-A, HLA-B and HLA DRB1 allelic mismatches. Epitope mismatches ranged from 16–51. The epitope mismatch extremes each had 6 allelic mismatches: 16: HLA-A,-B 9; DRB1 7 and 51: HLA-A,-B 19; DRB1 32. The single 3 allelic mismatch pair had 31 epitope mismatches: HLA-A,-B 10; DRB1 21. DRB1 epitope mismatch contribution to total epitope mismatches ranged from 19% to 63%. Allelic (antigenic) mismatches have a more limited range than epitope (immunogenic) mismatches, and mismatch burdens using these two models varied considerably in lung transplants in this study. The relative contribution of epitope mismatches in HLA-A, HLA-B and DRB1 varies in transplant donor recipient pairs. Prognostic estimates of antibody mediated rejection to inform surveillance and increased risk of post-transplant malignancies and infection to inform long term immunosuppressive therapy merit investigation using epitope mismatch burden.

Several targeted therapies have been approved for treatment of solid tumors. Identification of gene mutations that indicate response to these therapies is rapidly progressing. A 34-gene next-generation sequencing (NGS) panel, developed and validated by us, was evaluated to detect additional mutations in community-based cancer specimens initially sent to our reference laboratory for routine molecular testing.

The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.

AIMS:To investigate the clinical and genetic risk factors associated with hepatocellular carcinoma (HCC) in cirrhotic patients with chronic hepatitis B (CHB). METHODS:Nine hundred forty-nine Chinese Han patients with CHB were studied, including noncirrhotic patients without HCC (N = 234), cirrhotic patients without (N = 281) and with HCC (N = 434). Patients were genotyped for 10 candidate single nucleotide polymorphisms (SNPs) by the polymerase chain reaction (PCR)-ligase detection reaction (LDR) method. RESULTS:By multivariate logistic regression analysis adjusted for Child-Pugh scores, noneffective antiviral treatment, drinking history, family history of HCC, and age ≥50 years old were associated with HCC risk (odds ratio [OR] = 5.923, 2.456, 2.241, 1.955, respectively). Sixty-two of 170 cirrhotic patients who achieved sustained virological suppression by antiviral treatment developed HCC, with fatty liver disease, family history of HCC, and family history of hepatitis B virus (HBV) infection as the risk factors (OR = 11.646, 3.339, 2.537, respectively). The SNPs associated with HCC risk in patients with cirrhosis and CHB were rs11536889 in TLR4 and rs2853744 in SPP1. Polymorphisms of TLR4 rs2149356, AP3S2 rs2290351, STXBP5L rs2169302, MLEC rs7976497, and SOCS3 rs4969168 were associated with HCC risk in specific stratified analyses with gender, age, and drinking history in the cirrhotic patients. CONCLUSIONS:Inadequate antiviral treatment, family history of HCC, drinking history, and age ≥50 years old are risk factors for HCC. Sustained suppression of HBV does not eliminate the risk of HCC. Specific host genetic factors may impact HCC development in Han Chinese cirrhotic patients with CHB, including SNPs in TLR4, SPP1, AP3S2, STXBP5L, MLEC, and SOCS3, which warrant further validation in additional cohorts.

OBJECTIVE:To determine whether a next-generation sequencing (NGS) panel of 34 cancer-associated genes would cost-effectively aid in the treatment selection for patients with metastatic melanoma, compared with a single-site BRAF V600 mutation test.METHODS:A decision model was developed to estimate the costs and health outcomes of the two test strategies. The cost effectiveness of these two strategies was analyzed from a payer perspective over a 2-year time horizon with model parameters taken from the literature.RESULTS:In the base case, the gene sequencing panel strategy resulted in a cost of US$120,022 and 0.721 quality-adjusted life years (QALYs) per patient, whereas the single-site mutation test strategy resulted in a cost of US$128,965 and 0.704 QALYs. Thus, the gene sequencing panel strategy cost US$8943 less per patient and increased QALYs by 0.0174 per patient. Sensitivity analyses showed that, compared with the single-site mutation test strategy, the gene sequencing panel strategy had a 90.9% chance of having reduced costs and increased QALYs, with the cost of the gene sequencing panel test having minimal effect on the incremental cost.CONCLUSION:Compared with the single-site mutation test, the use of an NGS panel of 34 cancer-associated genes as an aid in selecting therapy for patients with metastatic melanoma reduced costs and increased QALYs. If the base-case results were applied to the 8900 patients diagnosed with metastatic melanoma in the USA each year, the gene sequencing panel strategy could result in an annual savings of US$79.6 million and a gain of 155 QALYs.

CONTEXTPatients with nonalcoholic fatty liver disease (NAFLD) are at increased risk of cardiovascular disease, and atherogenic lipoproteins may play an important role.OBJECTIVEThe objective of the study was to determine the contribution of the severity of steatohepatitis to atherogenic dyslipidemia in patients with NAFLD.DESIGNThis was a cross-sectional study.SETTINGThe study was conducted at a university hospital.PATIENTSPatients were recruited from outpatient clinics or from the general population (n = 188).INTERVENTIONSMeasurement of hepatic triglyceride content by magnetic resonance spectroscopy, histology (liver biopsy), metabolic profile by means of an oral glucose tolerance test, and lipoprotein analyses were performed.OUTCOMESOutcomes measured included standard lipids, lipoprotein subfraction analysis (apolipoprotein B/A1 levels, low-density lipoprotein (LDL) particle size/phenotype, and LDL/high-density lipoprotein subfractions), and insulin resistance.RESULTSPatients with NAFLD had severe insulin resistance, especially at the level of the adipose tissue, when compared with patients without NAFLD. Despite small differences in triglycerides and high-density lipoprotein-cholesterol, patients with NAFLD had a significantly higher plasma apolipoprotein B to apolipoprotein A1 ratio (0.66 ± 0.02 vs 0.58 ± 0.02, P = .01) and smaller LDL particle size (216.2 ± 0.7 vs 219.4 ± 1.1 Å, P = .01). Of note, these differences between patients with/without NAFLD were independent of the presence of obesity. Severity of steatohepatitis did not significantly influence the lipoprotein profile. Worse atherogenic dyslipidemia was best predicted by the degree of liver fat accumulation and adipose tissue and systemic insulin resistance.CONCLUSIONSNAFLD was associated with a worse atherogenic lipoprotein profile, regardless of similar body mass index and other clinical parameters. We speculate that this lipoprotein profile is driven mostly by liver fat content and insulin resistance and appears not to be worsened by obesity or the severity of liver disease (nonalcoholic steatohepatitis).

e22132 Background: Several targeted therapies have been approved for treatment of solid tumors. Identification of gene mutations associated with response to these targeted therapies is rapidly progressing. We developed and validated a 34-gene next-generation sequencing (NGS) panel and used this to test community-based samples sent for routine molecular testing for actionable gene mutations. Methods: A laboratory-developed NGS test on an Ion Torrent PGM sequencer was used to determine mutation profiles for 121 consecutive, de-identified FFPE tissue samples collected from patients diagnosed with melanoma (n = 31), lung (n = 27), colorectal (n = 33), and breast cancer (n = 30). NGS results were compared to those of the routine single-gene molecular tests. Results: Twenty-four (20%) samples had mutations detected by the originally requested tests, all of which were also detected by NGS. However, NGS detected at least 1 additional mutation not detected by the original test in 92 (76%) of the 121 samples. Of the additional mutations detected, 16 were actionable according to NCCN guidelines (3 BRAF [2 V600E and K483Q] in melanoma specimens; 2 KRAS [A146T and L19F], 2 BRAF [2 V600E] and 1 NRAS [Q61K] in colorectal cancer; and 2 EGFR [E746-A750 and R680Q] and 6 KRAS [A146T, G12C, G12V, Q22K, G12A and G13C] in lung cancer). Most of the additional mutations detected by NGS were in regions not covered by the routine molecular test(s); in the other cases, NGS may have provided greater sensitivity than the routine test. Overall, mutations were detected in 21 of the 34 genes included in the NGS panel, 17 of which were detected in multiple tumor types. The most frequently mutated gene was TP53 (64/121), but 83% (100/121) of the specimens harbored mutations in at least 1 gene other than TP53. Conclusions: This NGS assay frequently detected actionable mutations not identified by routine single-gene tests. These findings suggest that broader mutation profiling of solid tumors, as with the 34-gene NGS mutation panel, could provide additional information for treatment selection in a substantial proportion of cases.

Translation of results from genetic findings to inform medical practice is a highly anticipated goal of human genetics. The aim of this paper is to review and discuss the role of genetics in medically-relevant prediction. Germline genetics presages disease onset and therefore can contribute prognostic signals that augment laboratory tests and clinical features. As such, the impact of genetic-based predictive models on clinical decisions and therapy choice could be profound. However, given that (i) medical traits result from a complex interplay between genetic and environmental factors, (ii) the underlying genetic architectures for susceptibility to common diseases are not well-understood, and (iii) replicable susceptibility alleles, in combination, account for only a moderate amount of disease heritability, there are substantial challenges to constructing and implementing genetic risk prediction models with high utility. In spite of these challenges, concerted progress has continued in this area with an ongoing accumulation of studies that identify disease predisposing genotypes. Several statistical approaches with the aim of predicting disease have been published. Here we summarize the current state of disease susceptibility mapping and pharmacogenetics efforts for risk prediction, describe methods used to construct and evaluate genetic-based predictive models, and discuss applications.

Dr. Superko is the Chief Medical Officer for Celera.

BACKGROUND:Low-density lipoprotein cholesterol (LDL-C) lowering is the primary objective of patient management for cardiovascular disease. However, large numbers of patients who have achieved their LDL-C goal remain at risk for cardiovascular events. Low-density lipoprotein subfractions may provide insight into this residual risk. Thus, LDL subfraction standardization and consistency are critical to these efforts.AIM:This study aimed to determine the agreement of the analytical results among 4 methods commonly used for LDL subfractionation, namely, segmented gradient gel electrophoresis (sGGE), ultracentrifugation-vertical auto profile (VAP), nuclear magnetic resonance (NMR), and ion mobility (IM).METHODS:Blood samples were collected from 228 apparently healthy adults and sent to 4 clinical reference laboratories for analysis. The LDL phenotype was reported as pattern A (larger, less dense particles) or pattern B (smaller, more dense particles), respectively, and was the primary measure of comparison. An intermediate pattern (A/B) was also reported for sGGE and VAP.RESULTS:We observed complete agreement in the LDL phenotype among the 4 methods in 64% of subjects and agreement among at least 3 of the 4 methods in 87% of subjects. Agreement among pairs of methods ranged from 73% to 98% depending on how differences in reporting of subjects with intermediate results were considered. When subjects having intermediate A/B pattern were excluded, sGGE and IM had the highest agreement (98%) of any pair of methods.CONCLUSIONS:We found substantial agreement in the reported LDL phenotype among 4 LDL subfraction measurement technologies as performed by different clinical reference laboratories.

BACKGROUND AND AIMS:Recently, genetic association studies have linked a number of single nucleotide polymorphisms (SNPs) with liver fibrosis risk of hepatitis C. The present study was designed to validate the association of emerging SNPs with development of liver cirrhosis and chronicity in a Chinese population infected with hepatitis B virus (HBV).METHODS:714 Chinese subjects with persistent HBV infection (429 with evident liver cirrhosis and 285 without cirrhosis clinically or pathologically) and 280 subjects with spontaneous HBV clearance were studied. Six SNPs in five candidate genes were detected with the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method. The distribution of each polymorphism was compared between the age-matched cirrhotic and noncirrhotic subjects, and between subjects with persistent infection and spontaneous HBV clearance.RESULTS:The rs2679757 polymorphism of antizyme inhibitor 1 (AZIN1) gene was associated with the risk of cirrhosis (odds ratio [OR] for GG+AG versus AA=1.47, 95% confidence interval [CI]=1.08-2.01, p=0.01). So was rs886277 in the transient receptor potential cation channel subfamily M, member 5 (TRPM5) gene (OR for CC versus CT+TT=1.63, 95% CI=1.20-2.22, p=0.002). The frequencies of these two SNPs were also associated with the severity of decompensated cirrhosis based on the Child-Pugh classification. Genotype frequencies of other SNPs were not different between the cirrhotic and noncirrhotic groups. No SNPs were associated with the outcome of spontaneous HBV clearance.CONCLUSIONS:AZIN1 rs2679757 and TRPM5 rs886277 are associated with the risk of HBV-related liver cirrhosis in Chinese. The emerging SNPs warrant further clinical validation in other cohorts or ethnic groups, and could lead to mechanistic studies to reveal their contributions to fibrosis progression.