Banff 2022 Vascularized Composite Allotransplantation Meeting Report: Diagnostic criteria for vascular changes.

As more data become available, the Banff 2007 working classification of skin-containing vascularized composite allograft (VCA) pathology is expected to evolve and develop. This report represents the Banff VCA Working Group's consensus on the first revision of the 2007 scoring system. Prior to the 2022 Banff-CanXadian Society of Transplantation Joint Meeting, 83 clinicians and/or researchers were invited to a virtual meeting to discuss whether the 2007 Banff VCA system called for a revision. Unanimously, it was determined that the vascular changes were to be included in the first revision. Subsequently, 2 international online surveys, each followed by virtual discussions, were launched. The goals were (1) to identify which changes define severe rejection, (2) to grade their importance in the evaluation of severe rejection, and (3) to identify emerging criteria to diagnose rejection. A final hybrid (in-person and virtual) discussion at the Banff/Canadian Society of Transplantation Joint Meeting finalized the terminology, the definition, a scoring system, and a reporting system of the vascular changes. This proposal represents an international consensus on this topic and establishes the first revision of the Banff 2007 working classification of skin-containing vascularized composite allograft pathology.

Two Head and Neck Carcinomas With Squamous and Mucinous Components and Human Papillomavirus Associations: Maxillary Mucoepidermoid Carcinoma ex Sinonasal Schneiderian Papilloma and Tonsillar Invasive Stratified Mucin Producing Carcinoma (ISMC).

Carcinomas of the head-and-neck region with squamous and glandular/mucinous features constitute a heterogeneous group, with a significant minority of tumors showing an human papillomavirus (HPV) association. The differential diagnosis is usually between mucoepidermoid carcinoma (MEC) and adenosquamous carcinoma. We present here two tumors that exemplify both the challenges of diagnostic classification, as well as the complex relationship to HPV: (a) a low risk HPV positive/p16 negative carcinoma that is most consistent with a relatively typical intermediate grade mucoepidermoid type carcinoma with complete MEC phenotype (three cell types), originating from intranasal sinonasal papillomas with exophytic and inverted patterns, and invading surrounding maxillary compartments, and (b) a p16 and keratin 7 (KRT7) positive carcinoma of the right tonsil, characterized by stratified squamous and mucinous cell (mucocyte) features. Whereas the first tumor represents a typical MEC ex-Schneiderian papilloma, the second is morphologically most consistent with the, novel for this anatomic location, diagnosis of "invasive stratified mucin producing carcinoma" (ISMC), pointing to an analogy to similar, high-risk HPV-driven malignancies recently described in the gynecologic (GYN) and genitourinary (GU) areas. Both tumors, despite their mucoepidermoid-like features had no connection to salivary glands and lacked the MAML2 translocation typical of salivary gland MEC, pointing to a mucosal/non-salivary gland origin. Using these two carcinomas as examples, we attempt to address questions related to: (a) the histological distinction between MEC, adenosquamous carcinoma, and ISMC, (b) similarities and differences between these histological entities in mucosal sites versus morphologically similar salivary gland tumors, and (c) the role of HPV in these tumors.

The Banff 2022 Kidney Meeting Report: Reappraisal of microvascular inflammation and the role of biopsy-based transcript diagnostics.

The XVI-th Banff Meeting for Allograft Pathology was held at Banff, Alberta, Canada, from 19th to 23rd September 2022, as a joint meeting with the Canadian Society of Transplantation. To mark the 30th anniversary of the first Banff Classification, premeeting discussions were held on the past, present, and future of the Banff Classification. This report is a summary of the meeting highlights that were most important in terms of their effect on the Classification, including discussions around microvascular inflammation and biopsy-based transcript analysis for diagnosis. In a postmeeting survey, agreement was reached on the delineation of the following phenotypes: (1) "Probable antibody-mediated rejection (AMR)," which represents donor-specific antibodies (DSA)-positive cases with some histologic features of AMR but below current thresholds for a definitive AMR diagnosis; and (2) "Microvascular inflammation, DSA-negative and C4d-negative," a phenotype of unclear cause requiring further study, which represents cases with microvascular inflammation not explained by DSA. Although biopsy-based transcript diagnostics are considered promising and remain an integral part of the Banff Classification (limited to diagnosis of AMR), further work needs to be done to agree on the exact classifiers, thresholds, and clinical context of use.

Banff 2022 pancreas transplantation multidisciplinary report: Refinement of guidelines for T cell-mediated rejection, antibody-mediated rejection and islet pathology. Assessment of duodenal cuff biopsies and noninvasive diagnostic methods.

The Banff pancreas working schema for diagnosis and grading of rejection is widely used for treatment guidance and risk stratification in centers that perform pancreas allograft biopsies. Since the last update, various studies have provided additional insight regarding the application of the schema and enhanced our understanding of additional clinicopathologic entities. This update aims to clarify terminology and lesion description for T cell-mediated and antibody-mediated allograft rejections, in both active and chronic forms. In addition, morphologic and immunohistochemical tools are described to help distinguish rejection from nonrejection pathologies. For the first time, a clinicopathologic approach to islet pathology in the early and late posttransplant periods is discussed. This update also includes a discussion and recommendations on the utilization of endoscopic duodenal donor cuff biopsies as surrogates for pancreas biopsies in various clinical settings. Finally, an analysis and recommendations on the use of donor-derived cell-free DNA for monitoring pancreas graft recipients are provided. This multidisciplinary effort assesses the current role of pancreas allograft biopsies and offers practical guidelines that can be helpful to pancreas transplant practitioners as well as experienced pathologists and pathologists in training.

Muscarinic receptor agonist-induced ßPix binding to ß-catenin promotes colon neoplasia.

M3 muscarinic receptors (M3R) modulate ß-catenin signaling and colon neoplasia. CDC42/RAC guanine nucleotide exchange factor, ßPix, binds to ß-catenin in colon cancer cells, augmenting ß-catenin transcriptional activity. Using in silico, in vitro, and in vivo approaches, we explored whether these actions are regulated by M3R. At the invasive fronts of murine and human colon cancers, we detected co-localized nuclear expression of ßPix and ß-catenin in stem cells overexpressing M3R. Using immunohistochemistry, immunoprecipitation, proximity ligand, and fluorescent cell sorting assays in human tissues and established and primary human colon cancer cell cultures, we detected time-dependent M3R agonist-induced cytoplasmic and nuclear association of ßPix with ß-catenin. ßPix knockdown attenuated M3R agonist-induced human colon cancer cell proliferation, migration, invasion, and expression of PTGS2, the gene encoding cyclooxygenase-2, a key player in colon neoplasia. Overexpressing ßPix dose-dependently augmented ß-catenin binding to the transcription factor TCF4. In a murine model of sporadic colon cancer, advanced neoplasia was attenuated in conditional knockout mice with intestinal epithelial cell deficiency of ßPix. Expression levels of ß-catenin target genes and proteins relevant to colon neoplasia, including c-Myc and Ptgs2, were reduced in colon tumors from ßPix-deficient conditional knockout mice. Targeting the M3R/ßPix/ß-catenin axis may have therapeutic potential.

Selective Activation of M1 Muscarinic Receptors Attenuates Human Colon Cancer Cell Proliferation.

M3 muscarinic receptor (M3R) activation stimulates colon cancer cell proliferation, migration, and invasion; M3R expression is augmented in colon cancer and ablating M3R expression in mice attenuates colon neoplasia. Several lines of investigation suggest that in contrast to these pro-neoplastic effects of M3R, M1R plays an opposite role, protecting colon epithelial cells against neoplastic transformation. To pursue these intriguing findings, we examined the relative expression of M1R versus M3R in progressive stages of colon neoplasia and the effect of treating colon cancer cells with selective M1R agonists. We detected divergent expression of M1R and M3R in progressive colon neoplasia, from aberrant crypt foci to adenomas, primary colon cancers, and colon cancer metastases. Treating three human colon cancer cell lines with two selective M1R agonists, we found that in contrast to the effects of M3R activation, selective activation of M1R reversibly inhibited cell proliferation. Moreover, these effects were diminished by pre-incubating cells with a selective M1R inhibitor. Mechanistic insights were gained using selective chemical inhibitors of post-muscarinic receptor signaling molecules and immunoblotting to demonstrate M1R-dependent changes in the activation (phosphorylation) of key downstream kinases, EGFR, ERK1/2, and p38 MAPK. We did not detect a role for drug toxicity, cellular senescence, or apoptosis in mediating M1R agonist-induced attenuated cell proliferation. Lastly, adding M1R-selective agonists to colon cancer cells augmented the anti-proliferative effects of conventional chemotherapeutic agents. Collectively, these results suggest that selective M1R agonism for advanced colon cancer, alone or in combination with conventional chemotherapy, is a therapeutic strategy worth exploring.

Secretory-IgA binding to intestinal microbiota attenuates inflammatory reactions as the intestinal barrier of preterm infants matures.

Previous work has shown that Secretory-IgA (SIgA) binding to the intestinal microbiota is variable and may regulate host inflammatory bowel responses. Nevertheless, the impact of the SIgA functional binding to the microbiota remains largely unknown in preterm infants whose immature epithelial barriers make them particularly susceptible to inflammation. Here, we investigated SIgA binding to intestinal microbiota isolated from stools of preterm infants <33 weeks gestation with various levels of intestinal permeability. We found that SIgA binding to intestinal microbiota attenuates inflammatory reactions in preterm infants. We also observed a significant correlation between SIgA affinity to the microbiota and the infant's intestinal barrier maturation. Still, SIgA affinity was not associated with developing host defenses, such as the production of mucus and inflammatory calprotectin protein, but it depended on the microbiota shifts as the intestinal barrier matures. In conclusion, we reported an association between the SIgA functional binding to the microbiota and the maturity of the preterm infant's intestinal barrier, indicating that the pattern of SIgA coating is altered as the intestinal barrier matures.

An Initial Analysis of the Baseline Levels of Dd-cfDNA After Pancreas Transplantation: A Prospective Study From High-volume Centers in the United States.

Pancreas transplantation offers patients with diabetes an opportunity for glucose homeostasis. Current blood tests to surveil for rejection have poor sensitivity and specificity for identifying rejection, and pancreas biopsies are challenging and associated with morbidity and graft loss. Donor-derived cell-free DNA (dd-cfDNA) is shed from transplanted organs and detectable in peripheral blood. Thus, a potential dd-cfDNA blood test assessing rejection would be clinically advantageous. Methods: One hundred eighty-one dd-cfDNA samples (n) were collected from 77 patients (N) up to 132 mo posttransplant. Results: The median dd-cfDNA level among all subjects was 0.28% (0.13%, 0.71%). In simultaneous pancreas-kidney (SPK) transplant recipients, the median dd-cfDNA level was 0.29% (0.13%, 0.71%), and it was 0.23% (0.08%, 0.71%) in pancreas transplant alone (PTA) recipients. When isolating for when without infection or rejection, the median dd-cfDNA level was 0.28% (0.13%, 0.64%) for SPK and 0.20% (0.00%, 0.32%) for PTA. Both transplant types approached 1.0% ≤1 mo posttransplant followed by a decrease in median dd-cfDNA. During episodes of rejection or infection, median dd-cfDNA levels were greater among all transplant types. Conclusions: The mean dd-cfDNA level for all pancreas transplant recipients is <1.0%, consistent with the published kidney transplant rejection threshold (>1.0%), regardless of SPK or PTA. Early posttransplant dd-cfDNA levels are transiently higher than later measurements. Dd-cfDNA elevation also correlates with rejection and infection and thus is a promising biomarker for surveilling pancreas transplant dysfunction.

Zinc finger protein 277 is an intestinal transit-amplifying cell marker and colon cancer oncogene.

Sustained proliferative signaling and resisting cell death are hallmarks of cancer. Zinc finger protein 277 (ZNF277; murine Zfp277), a transcription factor regulating cellular senescence, is overexpressed in colon cancer, but its actions in intestinal homeostasis and neoplasia are unclear. Using human and murine intestine, human colon cancer cells, and ApcMin/+ mice with dysregulated ß-catenin signaling and exuberant intestinal neoplasia, we explored the actions of ZNF277/Zfp277 and defined the underlying mechanisms. In normal human and murine intestine, ZNF277/Zfp277 was expressed uniquely in early stem cell progenitors, undifferentiated transit-amplifying cells (TACs). Zfp277 was overexpressed in the ApcMin/+ mouse colon, implicating ZNF277/Zfp277 as a transcriptional target of ß-catenin signaling. We confirmed this by showing ß-catenin knockdown reduced ZNF277 expression and, using chromatin IP, identified 2 ß-catenin binding sites in the ZNF277 promoter. Zfp277 deficiency attenuated intestinal epithelial cell proliferation and tumor formation, and it strikingly prolonged ApcMin/+ mouse survival. RNA-Seq and PCR analyses revealed that Zfp277 modulates expression of genes in key cancer pathways, including ß-catenin signaling, the HOXD family that regulates development, and p21WAF1, a cell cycle inhibitor and tumor suppressor. In both human colon cancer cells and the murine colon, ZNF277/Zfp277 deficiency induced p21WAF1 expression and promoted senescence. Our findings identify ZNF277/Zfp277 as both a TAC marker and colon cancer oncogene that regulates cellular proliferation and senescence, in part by repressing p21WAF1 expression.

Causes of Renal Allograft Injury in Recipients With Normal Donor-derived Cell-free DNA.

BACKGROUND: Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive biomarker for the early detection of organ transplant rejection and other causes of graft injury. For nonrejection renal injuries, there is little information about the performance characteristics of this biomarker. We highlight some of the possible causes of kidney injury that may arise in patients with normal dd-cfDNA levels. METHODS: We performed a retrospective analysis of solitary renal transplant cases between January 2017 and November 2019. Those who had an abnormal laboratory or pathological finding within 1 mo of a normal dd-cfDNA test were selected. Subgroups were stratified for those who had normal or abnormal/rising serum creatinine, and differences between the groups were analyzed. RESULTS: Of 414 individuals who received a kidney transplant, 24 (7.5%) had a total of 41 normal dd-cfDNA values and 51 abnormal laboratory tests or histologic findings. The most common graft-injuring event was BK virus viremia (24 of 51). Other abnormal findings included urinary traction infections (n = 4), CMV viremia (n = 4), and biopsies demonstrating antibody-mediated rejection (AMR) (n = 2), T cell-mediated rejection (n = 1), focal segmental glomerulosclerosis (n = 2), nondonor-specific antibody chronic AMR (n = 1), and interstitial fibrosis and tubular atrophy (n = 7). Subgroup analysis of those with normal dd-cfDNA and normal/stable versus abnormal/rising creatinine showed that BK virus viremia was the most common abnormal finding in both groups at 53% and 38% respectively. On biopsy, 1 case of acute T cell-mediated rejection (1B and 2B) was seen with normal/stable creatinine, whereas 1 of nonspecific C4d focally positive and 1 of nondonor-specific antibody AMR were seen with abnormal/rising creatinine. CONCLUSIONS: Low levels of serum dd-cfDNA do not preclude detection of active graft-injuring events and that subclinical injuries may be developing. Context is important in the interpretation of dd-cfDNA, so renal biopsy remains a part of the diagnostic pathway for allograft dysfunction and maintenance of allograft health.

Parotid Salivary Duct Carcinoma With a Prominent Squamous Component: Immunohistochemical Profile, Diagnostic Pitfalls, and Therapeutic Implications.

Salivary duct carcinoma of the parotid gland is a highly aggressive epithelial malignancy morphologically resembling high-grade, invasive, and in situ breast carcinoma. It can occasionally present with variable morphology making it diagnostically challenging in cases with unusual morphological components. Ancillary testing, particularly androgen receptor (AR) positivity on immunohistochemistry, can be very helpful in cases that demonstrate extensive squamous morphology, since AR positivity is uncommon in both the primary salivary gland and metastatic squamous cell carcinomas to the parotid. In this report, we describe a case of salivary duct carcinoma that showed only a squamous cell carcinoma component on the initial primary tumor site biopsy, as well as in subsequent contralateral neck lymph node and skin metastases. Apart from the variable morphology, the typical salivary duct and squamous cell carcinoma tumor components also showed significant immunohistochemical differences, including differential staining of human epidermal growth factor receptor 2/neu. The associated diagnostic pitfalls, distinct immunoprofiles of the tumor components, helpful adjuncts for making the correct diagnosis, and associated therapeutic implications are discussed.

Air-Evacuation-Relevant Hypobaria Following Traumatic Brain Injury Plus Hemorrhagic Shock in Rats Increases Mortality and Injury to the Gut, Lungs, and Kidneys.

ABSTRACT: Rats exposed to hypobaria equivalent to what occurs during aeromedical evacuation within a few days after isolated traumatic brain injury exhibit greater neurologic injury than those remaining at sea level. Moreover, administration of excessive supplemental O2 during hypobaria further exacerbates brain injury. This study tested the hypothesis that exposure of rats to hypobaria following controlled cortical impact (CCI)-induced brain injury plus mild hemorrhagic shock worsens multiple organ inflammation and associated mortality. In this study, at 24 h after CCI plus hemorrhagic shock, rats were exposed to either normobaria (sea level) or hypobaria (=8,000 ft altitude) for 6 h under normoxic or hyperoxic conditions. Injured rats exhibited mortality ranging from 30% for those maintained under normobaria and normoxia to 60% for those exposed to 6 h under hypobaric and hyperoxia. Lung histopathology and neutrophil infiltration at 2 days postinjury were exacerbated by hypobaria and hyperoxia. Gut and kidney inflammation at 30 days postinjury were also worsened by hypobaric hyperoxia. In conclusion, exposure of rats after brain injury and hemorrhagic shock to hypobaria or hyperoxia results in increased mortality. Based on gut, lung, and kidney histopathology at 2 to 30 days postinjury, increased mortality is consistent with multi-organ inflammation. These findings support epidemiological studies indicating that increasing aircraft cabin pressures to 4,000 ft altitude (compared with standard 8,000 ft) and limiting excessive oxygen administration will decrease critical complications during and following aeromedical transport.

Targeted intestinal deletion of Rho guanine nucleotide exchange factor 7, ßPIX, impairs enterocyte proliferation, villus maturation, and mucosal defenses in mice.

Rho guanine nucleotide exchange factors (RhoGEFs) regulate Rho GTPase activity and cytoskeletal and cell adhesion dynamics. ßPix, a CDC42/RAC family RhoGEF encoded by ARHGEF7, is reported to modulate human colon cancer cell proliferation and postwounding restitution of rat intestinal epithelial monolayers. We hypothesized that ßPix plays a role in maintaining intestinal epithelial homeostasis. To test this hypothesis, we examined ßPix distribution in the human and murine intestine and created mice with intestinal epithelial-selective ßPix deletion [ßPixflox/flox/Tg(villin-Cre); Arhgef7 CKO mice]. Using Arhgef7 conditional knockout (CKO) and control mice, we investigated the consequences of ßPix deficiency in vivo on intestinal epithelial and enteroid development, dextran sodium sulfate-induced mucosal injury, and gut permeability. In normal human and murine intestines, we observed diffuse cytoplasmic and moderate nuclear ßPix immunostaining in enterocytes. Arhgef7 CKO mice were viable and fertile, with normal gross intestinal architecture but reduced small intestinal villus height, villus-to-crypt ratio, and goblet cells; small intestinal crypt cells had reduced Ki67 staining, compatible with impaired cell proliferation. Enteroids derived from control mouse small intestine were viable for more than 20 passages, but those from Arhgef7 CKO mice did not survive beyond 24 h despite addition of Wnt proteins or conditioned media from normal enteroids. Adding a Rho kinase (ROCK) inhibitor partially rescued CKO enteroid development. Compared with littermate control mice, dextran sodium sulfate-treated ßPix-deficient mice lost more weight and had greater impairment of intestinal barrier function, and more severe colonic mucosal injury. These findings reveal ßPix expression is important for enterocyte development, intestinal homeostasis, and resistance to toxic injury.NEW & NOTEWORTHY To explore the role of ßPix, a guanine nucleotide exchange factor encoded by ARHGEF7, in intestinal development and physiology, we created mice with intestinal epithelial cell Arhgef7/ßPix deficiency. We found ßPix essential for normal small intestinal epithelial cell proliferation, villus development, and mucosal resistance to injury. Moreover, Rho kinase signaling mediated developmental arrest observed in enteroids derived from ßPix-deficient small intestinal crypts. Our studies provide insights into the role Arhgef7/ßPix plays in intestinal epithelial homeostasis.

Emphysematous gastritis due to Sarcina ventriculi infection in a diabetic liver-kidney transplant recipient.

Emphysematous gastritis (EG) is a rare and potentially lethal process caused by invasive, gas-producing bacteria leading to inflammation and gas dissection of the stomach. The most common etiologic agents are Clostridium infections, but other organisms, including enterobacteria, staphylococcus, and fungi have also been identified. We report the first case of EG due to Sarcina ventriculi in a solid organ transplant recipient, who presented with epigastric pain and vomiting. The patient had a history of type 1 diabetes mellitus (DM) with recurrent episodes of ketoacidosis and systemic diabetic complications, including severe gastroparesis. CT scan studies demonstrated EG with venous air, and endoscopy showed severe gastritis and ulcerations. In the gastric biopsies, abundant Sarcina ventriculi were noted in areas of mucosal/submucosal necrosis. Antibiotic treatment was instituted at admission, and subsequent endoscopy demonstrated the disappearance of Sarcina, with some improvement of the gastric inflammation; however, the patient developed septic shock with multiorgan failure and expired. This case highlights the need to consider other infectious etiologies in transplant patients, in addition to the well-known opportunistic infections.

BK Polyomavirus Evades Innate Immune Sensing by Disrupting the Mitochondrial Network and Promotes Mitophagy.

Immune escape contributes to viral persistence, yet little is known about human polyomaviruses. BK-polyomavirus (BKPyV) asymptomatically infects 90% of humans but causes premature allograft failure in kidney transplant patients. Despite virus-specific T cells and neutralizing antibodies, BKPyV persists in kidneys and evades immune control as evidenced by urinary shedding in immunocompetent individuals. Here, we report that BKPyV disrupts the mitochondrial network and membrane potential when expressing the 66aa-long agnoprotein during late replication. Agnoprotein is necessary and sufficient, using its amino-terminal and central domain for mitochondrial targeting and network disruption, respectively. Agnoprotein impairs nuclear IRF3-translocation, interferon-beta expression, and promotes p62/SQSTM1-mitophagy. Agnoprotein-mutant viruses unable to disrupt mitochondria show reduced replication and increased interferon-beta expression but can be rescued by type-I interferon blockade, TBK1-inhibition, or CoCl2-treatment. Mitochondrial fragmentation and p62/SQSTM1-autophagy occur in allograft biopsies of kidney transplant patients with BKPyV nephropathy. JCPyV and SV40 infection similarly disrupt mitochondrial networks, indicating a conserved mechanism facilitating polyomavirus persistence and post-transplant disease.

Emphysematous gastritis due to Sarcina ventriculi infection in a diabetic liver-kidney transplant recipient

Emphysematous gastritis (EG) is a rare and potentially lethal process caused by invasive, gas-producing bacteria leading to inflammation and gas dissection of the stomach. The most common etiologic agents are Clostridium infections, but other organisms, including enterobacteria, staphylococcus, and fungi have also been identified. We report the first case of EG due to Sarcina ventriculi in a solid organ transplant recipient, who presented with epigastric pain and vomiting. The patient had a history of type 1 diabetes mellitus (DM) with recurrent episodes of ketoacidosis and systemic diabetic complications, including severe gastroparesis. CT scan studies demonstrated EG with venous air, and endoscopy showed severe gastritis and ulcerations. In the gastric biopsies, abundant Sarcina ventriculi were noted in areas of mucosal/submucosal necrosis. Antibiotic treatment was instituted at admission, and subsequent endoscopy demonstrated the disappearance of Sarcina, with some improvement of the gastric inflammation; however, the patient developed septic shock with multiorgan failure and expired. This case highlights the need to consider other infectious etiologies in transplant patients, in addition to the well-known opportunistic infections.

Intragraft gene expression in native kidney BK virus nephropathy versus T cell-mediated rejection: Prospects for molecular diagnosis and risk prediction.

Novel tools are needed to improve diagnostic accuracy and risk prediction in BK virus nephropathy (BKVN). We assessed the utility of intragraft gene expression testing for these purposes. Eight hundred genes were measured in 110 archival samples, including a discovery cohort of native kidney BKVN (n = 5) vs pure T cell-mediated rejection (TCMR; n = 10). Five polyomavirus genes and seven immune-related genes (five associated with BKVN and two associated with TCMR) were significantly differentially expressed between these entities (FDR < 0.05). These three sets of genes were further evaluated in samples representing a spectrum of BK infection (n = 25), followed by a multicenter validation cohort of allograft BKVN (n = 60) vs TCMR (n = 10). Polyomavirus 5-gene set expression reliably distinguished BKVN from TCMR (validation cohort AUC = 0.992), but the immune gene sets demonstrated suboptimal diagnostic performance (AUC ≤ 0.720). Within the validation cohort, no significant differences in index biopsy gene expression were identified between BKVN patients demonstrating resolution (n = 35), persistent infection (n = 14) or de novo rejection (n = 11) 6 months following a standardized reduction in immunosuppression. These results suggest that, while intragraft polyomavirus gene expression may be useful as an ancillary diagnostic for BKVN, assessment for concurrent TCMR and prediction of clinical outcome may not be feasible with current molecular tools.