Reuben Sarwal | University of California, Berkeley (original) (raw)

Papers by Reuben Sarwal

Journal of Clinical Medicine, 2022

Sub-optimal sensitivity and specificity in current allograft monitoring methodologies underscore ... more Sub-optimal sensitivity and specificity in current allograft monitoring methodologies underscore the need for more accurate and reflexive immunosurveillance to uncover the flux in alloimmunity between allograft health and the onset and progression of rejection. QSant—a urine based multi-analyte diagnostic test—was developed to profile renal transplant health and prognosticate injury, risk of evolution, and resolution of acute rejection. Q-Score—the composite score, across measurements of DNA, protein and metabolic biomarkers in the QSant assay—enables this risk prognostication. The domain of immune quiescence—below a Q-Score threshold of 32—is well established, based on published AUC of 98% for QSant. However, the trajectory of rejection is variable, given that causality is multi-factorial. Injury and subtypes of rejection are captured by the progression of Q-Score. This publication explores the clinical utility of QSant across the alloimmunity gradient of 32–100 for the early diagn...

Transplantation, 2022

The current standard of serum creatinine and biopsy to monitor allograft health has many limitati... more The current standard of serum creatinine and biopsy to monitor allograft health has many limitations. The most significant drawback of the current standard is the lack of sensitivity and specificity to allograft injuries, which are diagnosed only after significant damage to the allograft. Thus, it is of critical need to identify a biomarker that is sensitive and specific to the early detection of allograft injuries. Urine, as the direct renal ultrafiltrate that can be obtained noninvasively, directly reflects intrarenal processes in the allograft at greater accuracy than analysis of peripheral blood. We review transcriptomic, metabolomic, genomic, and proteomic discovery-based approaches to identifying urinary biomarkers for the noninvasive detection of allograft injuries, as well as the use of urine cell-free DNA in the QSant urine assay as a sensitive surrogate for the renal allograft biopsy for rejection diagnosis.

Journal of Clinical Medicine, 2018

Standard noninvasive methods for detecting renal allograft rejection and injury have poor sensiti... more Standard noninvasive methods for detecting renal allograft rejection and injury have poor sensitivity and specificity. Plasma donor-derived cell-free DNA (dd-cfDNA) has been reported to accurately detect allograft rejection and injury in transplant recipients and shown to discriminate rejection from stable organ function in kidney transplant recipients. This study used a novel single nucleotide polymorphism (SNP)-based massively multiplexed PCR (mmPCR) methodology to measure dd-cfDNA in various types of renal transplant recipients for the detection of allograft rejection/injury without prior knowledge of donor genotypes. A total of 300 plasma samples (217 biopsy-matched: 38 with active rejection (AR), 72 borderline rejection (BL), 82 with stable allografts (STA), and 25 with other injury (OI)) were collected from 193 unique renal transplant patients; dd- cfDNA was processed by mmPCR targeting 13,392 SNPs. Median dd-cfDNA was significantly higher in samples with biopsy-proven AR (2.3...

eLS, 2015

Organ failure results in major human morbidity and economic burden. Quality of life and life expe... more Organ failure results in major human morbidity and economic burden. Quality of life and life expectancy are optimised with organ transplantation, usually occurring across histocompatibility barriers. The molecular mechanisms of graft rejection are based on recognition of foreign transplanted cells or tissues by the expression of polymorphic, codominant genes coded by the major histocompatibility complex (MHC) of human leukocyte antigens (HLA) or genes on chromosome 6 by recipient T cells and by the generation of donor-specific antibodies to HLA and other donor-specific immunogenic antigens. Advances in histocompatibility and immunosuppression have improved short-term graft and patient survival rates, but rates of accelerated graft loss due to humoral alloimmunity have remained largely unchanged. Key Concepts In the ‘direct’ pathway of antigen presentation, MHC molecules on donor APCs from the graft tissue present graft-derived peptides to host T cells. In the ‘indirect’ pathway of antigen presentation, host APCs take up graft antigens and present donor-derived processed peptides on host MHC molecules to host T cells. HLA antibodies and endothelial cell activation are important regulators of immunogenicity, recruiting leukocytes to the site of inflammation. Acute rejection can be classified by presentation into hyperacute (occurring within minutes), acute (occurring within days to weeks), late acute (occurring after 3 months) or chronic (occurring months to years after transplantation). Acute rejection can be classified according to pathophysiological changes (cellular-interstitial, vascular, antibody-endothelial) or underlying immunologic mechanisms (adaptive or innate immune injury) or severity (histologic injury graded by the Banff schema). Acute rejection can be clinical or subclinical (based on the presence or absence of associated renal dysfunction). Acute rejection can be treatment resistant or sensitive (based on clinical and histologic resolution of diagnostic histology 4–6 weeks after treatment intensification). Keywords: alloantibodies; allograft; antigen-presenting cells; autograft; chronic rejection; cytokines; endothelial cells; hyperacute rejection; immunosuppression; inflammation; major histocompatibility complex; T cells; transplantation; xenograft

Frontiers in Immunology

Long-term kidney transplant (KT) allograft outcomes have not improved as expected despite a bette... more Long-term kidney transplant (KT) allograft outcomes have not improved as expected despite a better understanding of rejection and improved immunosuppression. Previous work had validated a computed rejection score, the tissue common rejection module (tCRM), measured by amplification-based assessment of 11 genes from formalin-fixed paraffin-embedded (FFPE) biopsy specimens, which allows for quantitative, unbiased assessment of immune injury. We applied tCRM in a prospective trial of 124 KT recipients, and contrasted assessment by tCRM and histology reads from 2 independent pathologists on protocol and cause biopsies post-transplant. Four 10-μm shaves from FFPE biopsy specimens were used for RNA extraction and amplification by qPCR of the 11 tCRM genes, from which the tCRM score was calculated. Biopsy diagnoses of either acute rejection (AR) or borderline rejection (BL) were considered to have inflammation present, while stable biopsies had no inflammation. Of the 77 biopsies that were...

Journal of Clinical Medicine

In this clinical validation study, we developed and validated a urinary Q-Score generated from th... more In this clinical validation study, we developed and validated a urinary Q-Score generated from the quantitative test QSant, formerly known as QiSant, for the detection of biopsy-confirmed acute rejection in kidney transplants. Using a cohort of 223 distinct urine samples collected from three independent sites and from both adult and pediatric renal transplant patients, we examined the diagnostic utility of the urinary Q-Score for detection of acute rejection in renal allografts. Statistical models based upon the measurements of the six QSant biomarkers (cell-free DNA, methylated-cell-free DNA, clusterin, CXCL10, creatinine, and total protein) generated a renal transplant Q-Score that reliably differentiated stable allografts from acute rejections in both adult and pediatric renal transplant patients. The composite Q-Score was able to detect both T cell-mediated rejection and antibody-mediated rejection patients and differentiate them from stable non-rejecting patients with a receive...

Journal of Clinical Medicine

Despite new advancements in surgical tools and therapies, exposure to immunosuppressive drugs rel... more Despite new advancements in surgical tools and therapies, exposure to immunosuppressive drugs related to non-immune and immune injuries can cause slow deterioration and premature failure of organ transplants. Diagnosis of these injuries by non-invasive urine monitoring would be a significant clinical advancement for patient management, especially in pediatric cohorts. We investigated the metabolomic profiles of biopsy matched urine samples from 310 unique kidney transplant recipients using gas chromatography–mass spectrometry (GC-MS). Focused metabolite panels were identified that could detect biopsy confirmed acute rejection with 92.9% sensitivity and 96.3% specificity (11 metabolites) and could differentiate BK viral nephritis (BKVN) from acute rejection with 88.9% sensitivity and 94.8% specificity (4 metabolites). Overall, targeted metabolomic analyses of biopsy-matched urine samples enabled the generation of refined metabolite panels that non-invasively detect graft injury pheno...

Science Translational Medicine

Accurate and noninvasive monitoring of renal allograft posttransplant is essential for early dete... more Accurate and noninvasive monitoring of renal allograft posttransplant is essential for early detection of acute rejection (AR) and to affect the long-term survival of the transplant. We present the development and validation of a noninvasive, spot urine–based diagnostic assay based on measurements of six urinary DNA, protein, and metabolic biomarkers. The performance of this assay for detecting kidney injury in both native kidneys and renal allografts is presented on a cohort of 601 distinct urine samples. The urinary composite score enables diagnosis of AR, with a receiver-operator characteristic curve area under the curve of 0.99 and an accuracy of 96%. In addition, we demonstrate the clinical utility of this assay for predicting AR before a rise in the serum creatinine, enabling earlier detection of rejection than currently possible by standard of care tests. This noninvasive, sensitive, and quantitative approach is a robust and informative method for the rapid and routine monito...

International Journal of Molecular Sciences

Standard methods for detecting and monitoring of IgA nephropathy (IgAN) have conventionally requi... more Standard methods for detecting and monitoring of IgA nephropathy (IgAN) have conventionally required kidney biopsies or suffer from poor sensitivity and specificity. The Kidney Injury Test (KIT) Assay of urinary biomarkers has previously been shown to distinguish between various kidney pathologies, including chronic kidney disease, nephrolithiasis, and transplant rejection. This validation study uses the KIT Assay to investigate the clinical utility of the non-invasive detection of IgAN and predicting the progression of renal damage over time. The study design benefits from longitudinally collected urine samples from an investigator-initiated, multicenter, prospective study, evaluating the efficacy of corticosteroids versus Rituximab for preventing progressive IgAN. A total of 131 urine samples were processed for this study; 64 urine samples were collected from 34 IgAN patients, and urine samples from 64 demographically matched healthy controls were also collected; multiple urinary ...

Journal of Clinical Medicine

The current standard of care measures for kidney function, proteinuria, and serum creatinine (SCr... more The current standard of care measures for kidney function, proteinuria, and serum creatinine (SCr) are poor predictors of early-stage kidney disease. Measures that can detect chronic kidney disease in its earlier stages are needed to enable therapeutic intervention and reduce adverse outcomes of chronic kidney disease. We have developed the Kidney Injury Test (KIT) and a novel KIT Score based on the composite measurement and validation of multiple biomarkers across a unique set of 397 urine samples. The test is performed on urine samples that require no processing at the site of collection and without target sequencing or amplification. We sought to verify that the pre-defined KIT test, KIT Score, and clinical thresholds correlate with established chronic kidney disease (CKD) and may provide predictive information on early kidney injury status above and beyond proteinuria and renal function measurements alone. Statistical analyses across six DNA, protein, and metabolite markers were...

Journal of Clinical Medicine

Standard methods for detecting chronic lung allograft dysfunction (CLAD) and rejection have poor ... more Standard methods for detecting chronic lung allograft dysfunction (CLAD) and rejection have poor sensitivity and specificity and have conventionally required bronchoscopies and biopsies. Plasma cell-free DNA (cfDNA) has been shown to be increased in various types of allograft injury in transplant recipients and CXCL10 has been reported to be increased in the lung tissue of patients undergoing CLAD. This study used a novel cfDNA and CXCL10 assay to evaluate the noninvasive assessment of CLAD phenotype and prediction of survival from bronchoalveolar lavage (BAL) fluid. A total of 60 BAL samples (20 with bronchiolitis obliterans (BOS), 20 with restrictive allograft syndrome (RAS), and 20 with stable allografts (STA)) were collected from 60 unique lung transplant patients; cfDNA and CXCL10 were measured by the ELISA-based KIT assay. Median cfDNA was significantly higher in BOS patients (6739 genomic equivalents (GE)/mL) versus STA (2920 GE/mL) and RAS (4174 GE/mL) (p < 0.01 all compa...

Journal of Clinical Medicine, 2022

Sub-optimal sensitivity and specificity in current allograft monitoring methodologies underscore ... more Sub-optimal sensitivity and specificity in current allograft monitoring methodologies underscore the need for more accurate and reflexive immunosurveillance to uncover the flux in alloimmunity between allograft health and the onset and progression of rejection. QSant—a urine based multi-analyte diagnostic test—was developed to profile renal transplant health and prognosticate injury, risk of evolution, and resolution of acute rejection. Q-Score—the composite score, across measurements of DNA, protein and metabolic biomarkers in the QSant assay—enables this risk prognostication. The domain of immune quiescence—below a Q-Score threshold of 32—is well established, based on published AUC of 98% for QSant. However, the trajectory of rejection is variable, given that causality is multi-factorial. Injury and subtypes of rejection are captured by the progression of Q-Score. This publication explores the clinical utility of QSant across the alloimmunity gradient of 32–100 for the early diagn...

Transplantation, 2022

The current standard of serum creatinine and biopsy to monitor allograft health has many limitati... more The current standard of serum creatinine and biopsy to monitor allograft health has many limitations. The most significant drawback of the current standard is the lack of sensitivity and specificity to allograft injuries, which are diagnosed only after significant damage to the allograft. Thus, it is of critical need to identify a biomarker that is sensitive and specific to the early detection of allograft injuries. Urine, as the direct renal ultrafiltrate that can be obtained noninvasively, directly reflects intrarenal processes in the allograft at greater accuracy than analysis of peripheral blood. We review transcriptomic, metabolomic, genomic, and proteomic discovery-based approaches to identifying urinary biomarkers for the noninvasive detection of allograft injuries, as well as the use of urine cell-free DNA in the QSant urine assay as a sensitive surrogate for the renal allograft biopsy for rejection diagnosis.

Journal of Clinical Medicine, 2018

Standard noninvasive methods for detecting renal allograft rejection and injury have poor sensiti... more Standard noninvasive methods for detecting renal allograft rejection and injury have poor sensitivity and specificity. Plasma donor-derived cell-free DNA (dd-cfDNA) has been reported to accurately detect allograft rejection and injury in transplant recipients and shown to discriminate rejection from stable organ function in kidney transplant recipients. This study used a novel single nucleotide polymorphism (SNP)-based massively multiplexed PCR (mmPCR) methodology to measure dd-cfDNA in various types of renal transplant recipients for the detection of allograft rejection/injury without prior knowledge of donor genotypes. A total of 300 plasma samples (217 biopsy-matched: 38 with active rejection (AR), 72 borderline rejection (BL), 82 with stable allografts (STA), and 25 with other injury (OI)) were collected from 193 unique renal transplant patients; dd- cfDNA was processed by mmPCR targeting 13,392 SNPs. Median dd-cfDNA was significantly higher in samples with biopsy-proven AR (2.3...

eLS, 2015

Organ failure results in major human morbidity and economic burden. Quality of life and life expe... more Organ failure results in major human morbidity and economic burden. Quality of life and life expectancy are optimised with organ transplantation, usually occurring across histocompatibility barriers. The molecular mechanisms of graft rejection are based on recognition of foreign transplanted cells or tissues by the expression of polymorphic, codominant genes coded by the major histocompatibility complex (MHC) of human leukocyte antigens (HLA) or genes on chromosome 6 by recipient T cells and by the generation of donor-specific antibodies to HLA and other donor-specific immunogenic antigens. Advances in histocompatibility and immunosuppression have improved short-term graft and patient survival rates, but rates of accelerated graft loss due to humoral alloimmunity have remained largely unchanged. Key Concepts In the ‘direct’ pathway of antigen presentation, MHC molecules on donor APCs from the graft tissue present graft-derived peptides to host T cells. In the ‘indirect’ pathway of antigen presentation, host APCs take up graft antigens and present donor-derived processed peptides on host MHC molecules to host T cells. HLA antibodies and endothelial cell activation are important regulators of immunogenicity, recruiting leukocytes to the site of inflammation. Acute rejection can be classified by presentation into hyperacute (occurring within minutes), acute (occurring within days to weeks), late acute (occurring after 3 months) or chronic (occurring months to years after transplantation). Acute rejection can be classified according to pathophysiological changes (cellular-interstitial, vascular, antibody-endothelial) or underlying immunologic mechanisms (adaptive or innate immune injury) or severity (histologic injury graded by the Banff schema). Acute rejection can be clinical or subclinical (based on the presence or absence of associated renal dysfunction). Acute rejection can be treatment resistant or sensitive (based on clinical and histologic resolution of diagnostic histology 4–6 weeks after treatment intensification). Keywords: alloantibodies; allograft; antigen-presenting cells; autograft; chronic rejection; cytokines; endothelial cells; hyperacute rejection; immunosuppression; inflammation; major histocompatibility complex; T cells; transplantation; xenograft

Frontiers in Immunology

Long-term kidney transplant (KT) allograft outcomes have not improved as expected despite a bette... more Long-term kidney transplant (KT) allograft outcomes have not improved as expected despite a better understanding of rejection and improved immunosuppression. Previous work had validated a computed rejection score, the tissue common rejection module (tCRM), measured by amplification-based assessment of 11 genes from formalin-fixed paraffin-embedded (FFPE) biopsy specimens, which allows for quantitative, unbiased assessment of immune injury. We applied tCRM in a prospective trial of 124 KT recipients, and contrasted assessment by tCRM and histology reads from 2 independent pathologists on protocol and cause biopsies post-transplant. Four 10-μm shaves from FFPE biopsy specimens were used for RNA extraction and amplification by qPCR of the 11 tCRM genes, from which the tCRM score was calculated. Biopsy diagnoses of either acute rejection (AR) or borderline rejection (BL) were considered to have inflammation present, while stable biopsies had no inflammation. Of the 77 biopsies that were...

Journal of Clinical Medicine

In this clinical validation study, we developed and validated a urinary Q-Score generated from th... more In this clinical validation study, we developed and validated a urinary Q-Score generated from the quantitative test QSant, formerly known as QiSant, for the detection of biopsy-confirmed acute rejection in kidney transplants. Using a cohort of 223 distinct urine samples collected from three independent sites and from both adult and pediatric renal transplant patients, we examined the diagnostic utility of the urinary Q-Score for detection of acute rejection in renal allografts. Statistical models based upon the measurements of the six QSant biomarkers (cell-free DNA, methylated-cell-free DNA, clusterin, CXCL10, creatinine, and total protein) generated a renal transplant Q-Score that reliably differentiated stable allografts from acute rejections in both adult and pediatric renal transplant patients. The composite Q-Score was able to detect both T cell-mediated rejection and antibody-mediated rejection patients and differentiate them from stable non-rejecting patients with a receive...

Journal of Clinical Medicine

Despite new advancements in surgical tools and therapies, exposure to immunosuppressive drugs rel... more Despite new advancements in surgical tools and therapies, exposure to immunosuppressive drugs related to non-immune and immune injuries can cause slow deterioration and premature failure of organ transplants. Diagnosis of these injuries by non-invasive urine monitoring would be a significant clinical advancement for patient management, especially in pediatric cohorts. We investigated the metabolomic profiles of biopsy matched urine samples from 310 unique kidney transplant recipients using gas chromatography–mass spectrometry (GC-MS). Focused metabolite panels were identified that could detect biopsy confirmed acute rejection with 92.9% sensitivity and 96.3% specificity (11 metabolites) and could differentiate BK viral nephritis (BKVN) from acute rejection with 88.9% sensitivity and 94.8% specificity (4 metabolites). Overall, targeted metabolomic analyses of biopsy-matched urine samples enabled the generation of refined metabolite panels that non-invasively detect graft injury pheno...

Science Translational Medicine

Accurate and noninvasive monitoring of renal allograft posttransplant is essential for early dete... more Accurate and noninvasive monitoring of renal allograft posttransplant is essential for early detection of acute rejection (AR) and to affect the long-term survival of the transplant. We present the development and validation of a noninvasive, spot urine–based diagnostic assay based on measurements of six urinary DNA, protein, and metabolic biomarkers. The performance of this assay for detecting kidney injury in both native kidneys and renal allografts is presented on a cohort of 601 distinct urine samples. The urinary composite score enables diagnosis of AR, with a receiver-operator characteristic curve area under the curve of 0.99 and an accuracy of 96%. In addition, we demonstrate the clinical utility of this assay for predicting AR before a rise in the serum creatinine, enabling earlier detection of rejection than currently possible by standard of care tests. This noninvasive, sensitive, and quantitative approach is a robust and informative method for the rapid and routine monito...

International Journal of Molecular Sciences

Standard methods for detecting and monitoring of IgA nephropathy (IgAN) have conventionally requi... more Standard methods for detecting and monitoring of IgA nephropathy (IgAN) have conventionally required kidney biopsies or suffer from poor sensitivity and specificity. The Kidney Injury Test (KIT) Assay of urinary biomarkers has previously been shown to distinguish between various kidney pathologies, including chronic kidney disease, nephrolithiasis, and transplant rejection. This validation study uses the KIT Assay to investigate the clinical utility of the non-invasive detection of IgAN and predicting the progression of renal damage over time. The study design benefits from longitudinally collected urine samples from an investigator-initiated, multicenter, prospective study, evaluating the efficacy of corticosteroids versus Rituximab for preventing progressive IgAN. A total of 131 urine samples were processed for this study; 64 urine samples were collected from 34 IgAN patients, and urine samples from 64 demographically matched healthy controls were also collected; multiple urinary ...

Journal of Clinical Medicine

The current standard of care measures for kidney function, proteinuria, and serum creatinine (SCr... more The current standard of care measures for kidney function, proteinuria, and serum creatinine (SCr) are poor predictors of early-stage kidney disease. Measures that can detect chronic kidney disease in its earlier stages are needed to enable therapeutic intervention and reduce adverse outcomes of chronic kidney disease. We have developed the Kidney Injury Test (KIT) and a novel KIT Score based on the composite measurement and validation of multiple biomarkers across a unique set of 397 urine samples. The test is performed on urine samples that require no processing at the site of collection and without target sequencing or amplification. We sought to verify that the pre-defined KIT test, KIT Score, and clinical thresholds correlate with established chronic kidney disease (CKD) and may provide predictive information on early kidney injury status above and beyond proteinuria and renal function measurements alone. Statistical analyses across six DNA, protein, and metabolite markers were...

Journal of Clinical Medicine

Standard methods for detecting chronic lung allograft dysfunction (CLAD) and rejection have poor ... more Standard methods for detecting chronic lung allograft dysfunction (CLAD) and rejection have poor sensitivity and specificity and have conventionally required bronchoscopies and biopsies. Plasma cell-free DNA (cfDNA) has been shown to be increased in various types of allograft injury in transplant recipients and CXCL10 has been reported to be increased in the lung tissue of patients undergoing CLAD. This study used a novel cfDNA and CXCL10 assay to evaluate the noninvasive assessment of CLAD phenotype and prediction of survival from bronchoalveolar lavage (BAL) fluid. A total of 60 BAL samples (20 with bronchiolitis obliterans (BOS), 20 with restrictive allograft syndrome (RAS), and 20 with stable allografts (STA)) were collected from 60 unique lung transplant patients; cfDNA and CXCL10 were measured by the ELISA-based KIT assay. Median cfDNA was significantly higher in BOS patients (6739 genomic equivalents (GE)/mL) versus STA (2920 GE/mL) and RAS (4174 GE/mL) (p < 0.01 all compa...