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Eculizumab is effective for complement-mediated thrombotic microangiopathy (CM-TMA), also known as atypical hemolytic uremic syndrome. Although lifelong therapy had been suggested, discontinuation does not universally lead to relapse. Comprehensive data evaluating risk factors for recurrence following discontinuation are limited. Our aim was to systematically review available literature assessing the role of complement genetic variants in this setting. Reports on CM-TMA and eculizumab withdrawal published before 1 January 2021, were included. Key reasons for patient exclusion were no follow-up after drug withdrawal and patients lacking complement genetic testing. Two-hundred eighty patients from 40 publications were included. Median age was 28 years, and 25 patients had a known history of renal transplant. Complement genetic variants were identified in 60%, most commonly in CFH (n = 59) and MCP/CD46 (n = 38). Of patients with a complement gene variant, 51.3% had ≥1 likely pathogenic/pathogenic variant whereas the remaining had variants of uncertain significance (VUS). Overall relapse rate after therapy discontinuation was 29.6%. Relapse rate was highest among patients with CFH variants and MCP/CD46 variants in canonical splice regions. VUS (P < .001) and likely pathogenic/pathogenic variants (P < .001) were associated with increased relapse. Presence of a renal allograft (P = .009); decreasing age (P = .029); and detection of variants in CFH (P < .001), MCP/CD46 (P < .001), or C3 (P < .001) were all independently associated with relapse after eculizumab discontinuation. Eculizumab discontinuation is appropriate in specific patients with CM-TMA. Caution should be exerted when attempting such a strategy in patients with high risk of recurrence, including a subgroup of patients with MCP/CD46 variants.

The literature regarding the neuropathological findings in cases of SARS-CoV-2 infection, which causes coronavirus disease 2019 (COVID-19), is expanding. We identified 72 patients who died of COVID-19 (n = 48) or had recovered shortly before death (n = 24) and had autopsies performed at our institution (49 males, 23 females; median age at death 76.4 years, range: 0.0-95.0 years). Droplet digital polymerase chain reaction (ddPCR) for the detection of SARS-CoV-2 was performed (n = 58) in multiple brain regions. In cases the assay was successfully completed (n = 50), 98.0% were negative (n = 49) and 2% were indeterminate (n = 1). Most histologic findings were typical of the patient age demographic, such as neurodegenerative disease and arteriolosclerosis. A subset of cases demonstrated findings which may be associated with sequelae of critical illness. We identified 3 cases with destructive perivascular lesions with axonal injury, one of which also harbored perivascular demyelinating lesions. These rare cases may represent a parainfectious process versus sequelae of vascular injury. The lack of detectable SARS-CoV-2 by ddPCR or significant histologic evidence of direct infection suggests that active encephalitis is not a feature of COVID-19.

Purpose: Inherited variability in the prognosis of lung cancer patients treated with platinum-based chemotherapy has been widely investigated. However, the overall contribution of genetic variation to platinum response is not well established. To identify novel candidate single nucleotide polymorphisms (SNP)/genes, we carried out a genome-wide association study (GWAS) for cisplatin cytotoxicity by using lymphoblastoid cell lines (LCL), followed by an association study of selected SNPs from the GWAS with overall survival (OS) in lung cancer patients.Experimental Design: A GWAS for cisplatin was conducted with 283 ethnically diverse LCLs. A total of 168 top SNPs were genotyped in 222 small cell lung cancer (SCLC) and 961 non-SCLC (NSCLC) patients treated with platinum-based therapy. Association of the SNPs with OS was determined by using the Cox regression model. Selected candidate genes were functionally validated by siRNA knockdown in human lung cancer cells.Results: Among 157 successfully genotyped SNPs, 9 and 10 SNPs were top SNPs associated with OS for patients with NSCLC and SCLC, respectively, although they were not significant after adjusting for multiple testing. Fifteen genes, including 7 located within 200 kb up or downstream of the 4 top SNPs and 8 genes for which expression was correlated with 3 SNPs in LCLs were selected for siRNA screening. Knockdown of DAPK3 and METTL6, for which expression levels were correlated with the rs11169748 and rs2440915 SNPs, significantly decreased cisplatin sensitivity in lung cancer cells.Conclusions: This series of clinical and complementary laboratory-based functional studies identified several candidate genes/SNPs that might help predict treatment outcomes for platinum-based therapy of lung cancer. Clin Cancer Res; 17(17); 5801–11. ©2011 AACR.

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Supplementary Table 1 from Glutathione Pathway Genetic Polymorphisms and Lung Cancer Survival After Platinum-Based Chemotherapy

Supplementary Tables S1-S6 from Glutathione S-Transferase T1 and M1: Gene Sequence Variation and Functional Genomics

Purpose: The glutathione S-transferases (GSTs) catalyze the glutathione conjugation of reactive electrophiles, including carcinogens and many antineoplastic drugs. GSTT1 and GSTM1 are polymorphically deleted, but the full range of genetic variation in these two genes has not yet been explored. We set out to systematically identify common polymorphisms in GSTT1 and GSTM1, followed by functional genomic studies.Experimental Design: First, multiplex PCR was used to determine GSTT1 and GSTM1 copy number in 400 DNA samples (100 each from 4 ethnic groups). Exons, splice junctions, and 5′-flanking regions (5′-FR) were then resequenced using DNA samples that contained at least one copy of GSTT1 or GSTM1.Results: Gene deletion frequencies among ethnic groups were from 33.5% to 73.5% for GSTT1 and from 50.5% to 78.0% for GSTM1. GSTT1 deletion data correlated with the results of mRNA microarray expression studies. The 18 single nucleotide polymorphisms (SNP) observed in GSTT1 included three nonsynonymous coding SNPs (cSNPs) and one single-nucleotide deletion, whereas the 51 GSTM1 SNPs included two nonsynonymous cSNPs. Two of the GSTT1 nonsynonymous cSNPs resulted in decreases in levels of immunoreactive protein to 56% and 12% of wild type (WT), whereas those in GSTM1 resulted in modest increases in protein levels. Reporter gene assays showed that one GSTT1 5′-FR haplotype, with a frequency of 32% in African-American subjects, resulted in an increase in transcription in JEG-3 cells to 351% of that for the WT sequence, and one GSTM1 5′-FR haplotype resulted in an increase in transcription in JEG-3 cells to 129% of WT.Conclusions: These observations suggest that functionally significant pharmacogenomic variation beyond GSTT1 and GSTM1 gene deletion may contribute to carcinogenesis or individual variation in antineoplastic drug therapy response.

Glutathione S-transferase P1 (GSTP1) is of importance for cancer research because of its role in detoxifying carcinogens, activating antineoplastic prodrugs, metabolizing chemotherapeutic agents, and its involvement in cell cycle and apoptosis regulation. Two common GSTP1 genetic polymorphisms have been studied extensively. However, the full range of GSTP1 genetic variation has not been systematically characterized in the absence of disease pathology. We set out to identify common GSTP1 polymorphisms in four ethnic groups, followed by functional genomic studies. All exons, splice junctions, and the 5′-flanking region of GSTP1 were resequenced using 60 DNA samples each from four ethnic groups. The 35 single-nucleotide polymorphisms (SNP) identified included six nonsynonymous SNPs and 17 previously unreported polymorphisms. GSTP1 variant allozymes were then expressed in COS-1 cells, and five displayed significantly altered levels of enzyme activity. One decreased to 22% of the wild-type (WT) activity. Four variant allozymes had Km values that differed significantly from that of the WT, and five showed altered levels of immunoreactive protein compared with WT, with a significant correlation (r = 0.79, P < 0.007) between levels of immunoreactive protein and enzyme activity in these samples. In the Mexican American population, five linked SNPs were significantly associated with GSTP1 mRNA expression, one of which was found by electrophoretic mobility shift assay to alter protein binding. These studies have identified functionally significant genetic variation, in addition to the two frequently studied GSTP1 nonsynonymous SNPs, that may influence GSTP1's contribution to carcinogen and drug metabolism, and possibly disease pathogenesis and/or drug response. [Cancer Res 2008;68(12):4791–801]

Supplementary Data from Glutathione S-Transferase P1: Gene Sequence Variation and Functional Genomic Studies

Context.— Studies of lungs in patients with COVID-19 have focused on early findings. Objective.— To systematically study histopathologic and imaging features and presence of SARS-CoV-2 RNA in lung tissue from patients in later stages of COVID-19. Design.— Autopsies, explants, surgical lung biopsies, transbronchial biopsies, cryobiopsies, and needle biopsies from patients with COVID-19 whose onset of symptoms/confirmed diagnosis was more than 28 days before the procedure were studied. Available images were reviewed. Reverse transcription droplet digital polymerase chain reaction for SARS-CoV-2 RNA was performed on lung tissue. Results.— Of 44 specimens (43 patients; median age, 59.3 years; 26 [60.5%] male) features of acute lung injury (ALI) were seen in 39 (88.6%), predominantly organizing pneumonia and diffuse alveolar damage, up to 298 days after onset of COVID-19. Fibrotic changes were found in 33 specimens (75%), most commonly fibrotic diffuse alveolar damage (n = 22) and cicatricial organizing pneumonia (n = 12). Time between acquiring COVID-19 and specimen was shorter in patients with diffuse ALI (median, 61.5 days) compared with patients with focal (140 days) or no ALI (130 days) (P = .009). Sixteen (of 20; 80%) SARS-CoV-2 reverse transcription droplet digital polymerase chain reaction tests were positive, up to 174 days after COVID-19 onset. Time between COVID-19 onset and most recent computed tomography in patients with consolidation on imaging was shorter (median, 43.0 days) versus in patients without consolidation (87.5 days; P = .02). Reticulations were associated with longer time to computed tomography after COVID-19 onset (median, 82 versus 23.5 days; P = .006). Conclusions.— ALI and SARS-CoV-2 RNA can be detected in patients with COVID-19 for many months. ALI may evolve into fibrotic interstitial lung disease.

Background: Cyclin dependent 4/6 kinase inhibitors (CDK4/6i) and endocrine therapy (ET) have improved progression-free survival (PFS) and overall survival in HR+ MBC, but progression of disease ultimately occurs. Apart from HR+ status, there are no clinically available biomarkers that enable oncologists to determine prognosis and predict response to CDK4/6i. An emerging biomarker is serum thymidine kinase 1 (TK1), a secreted marker of proliferation that is prognostic in pts with HR+ HER2- MBC. High levels of TKa are associated with inferior PFS, whereas pts with low TKa levels pretreatment, or TKa levels that decrease on ET and a CDK4/6i, have superior PFS. Notably, TKa levels rebound ≥ 5 days off Pb, with resumption of cell cycling. PROMISE (NCT0281902) is a prospective study that enrolled women with HR+ MBC starting Pb + letrozole (L) in the 1st line [FL] or Pb + fulvestrant in the 2nd line [SL] setting. The trial includes a comprehensive “omic” assessment of blood, tumor, urine and the fecal microbiome to identify novel genomic variants and pathways associated with an early decline in TKa (measured after 2 months or end of cycle [C]2) and PFS. Here, we report the association between i) pre-treatment TKa (pre-TKa) levels and PFS (i.e. from registration to the 1st disease event) and ii) TKa levels at the end of C2 (C2-TKa) and PFS-2 (i.e. from the start of C3 to the 1st disease event).Methods: TKa testing was performed using the DiviTum assay (Biovica). TKa+ disease was defined as ≥ 200 Du/L and TKa- disease as below limit of detection to 200 Du/L. Log-rank test and univariate Cox modeling were used to assess the association between pre-TKa levels and PFS and between end of C2-TKa levels and PFS-2. The database was locked on June 28, 2021. Results: Of 68 pts enrolled, 4 were ineligible and pre-TKa data was unavailable for 4. Of the remaining 60 pts (45 FL, 15 SL), the percentage of pts with pre-TKa+ disease was 33.3% in FL (15/45, 95% CI: 20.0-49.0%), and 46.7% (7/15, 95% CI: 21.4-71.9%) in the SL. The median follow-up time for pts on study was 24 months (range: 2-42 months). There were 22 disease events (13 in FL, 9 in SL). In the FL setting, PFS was significantly shorter for preTKa+ pts compared to preTKa- pts (HR: 4.15, 95% CI:1.35-12.74; p=0.007), but not for SL pts (HR: 1.11, 95% CI: 0.30-4.18, p=0.875). End of C2 TKa data was obtained for pts while on Pb (n=5), or after stopping Pb as follows: 1-4 days (n=9), 5-8 days (n=28) and 9-36 days (n=11). PFS-2 was not associated with C2-TKa in the FL (p=0.834) or SL (p=0.454) settings. An analysis of TKa levels by metastatic site will be presented at the meeting.Conclusions: A secreted biomarker of proliferation (TK1) obtained prior to initiating CDK4/6i and ET for the treatment of HR+ MBC is associated with PFS in pts receiving 1st line Pb + L, but not in those receiving 2nd line Pb + fulvestrant. While the end of C2 TKa levels were not associated with PFS, the interpretability of these data are limited, given treatment delays (0-36 days) prior to starting C3 that may result in TKa rebound. Future studies evaluating the predictive nature of TKa and Pb response should focus on earlier timepoints while on drug. Citation Format: Ciara C O'Sullivan, Jun He, Jason Sinnwell, Vera J Suman, Krishna R Kalari, Peter T Vedell, Ann M Moyer, Xiaojia Tang, Kevin J Thompson, Abe Eyman Casey, Alvaro Moreno-Aspitia, Donald W Northfelt, Minetta C Liu, Tufia C Haddad, Saranya Chumsri, Brendan McMenomy, Prema Peethambaram, Kathryn J Ruddy, Karthik V Giridhar, Roberto A Leon-Ferre, Mattias Bergqvist, Adrian Nordmark, Richard M Weinshilboum, Liewei Wang, Matthew P Goetz. Serum thymidine kinase 1 activity (TKa) levels and progression-free survival (PFS) in patients (pts) with hormone receptor positive (HR+) HER2-negative metastatic breast cancer (MBC) on palbociclib (Pb) and endocrine therapy (ET) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-22.

The Pharmacogenomics (PGx) Profile Service was a proof-of-concept project to implement PGx in patient care at Mayo Clinic. Eighty-two healthy individuals aged 18 and older underwent genotyping of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, SLCO1B1, HLA-B*58:01, and VKORC1. A PGx pharmacist was involved in ordering, meeting with patients, interpreting, reviewing, and documenting results. Ninety three percent were CYP1A2 rapid metabolizers, 92% CYP3A4 normal metabolizers, and 88% CYP3A5 poor metabolizers; phenotype frequencies for CYP2C19 and CYP2D6 varied. Seventy-three percent had normal functioning SLCO1B1 transporter, 4% carried the HLA-B*58:01 risk variant, and 35% carried VKORC1 and CYP2C9 variants that increased warfarin sensitivity. Pre-emptive PGx testing offered medication improvement opportunity in 56% of participants for commonly used medications. A collaborative approach involving a PGx pharmacist integrated within a clinical practice with regards to utility of PGx results allowed for implementation of the PGx Profile Service.

Objective To compare coronavirus disease 2019 (COVID-19) acute kidney injury (AKI) to sepsis-AKI (S-AKI). The morphology and transcriptomic and proteomic characteristics of autopsy kidneys were analyzed. Patients and Methods Individuals 18 years of age and older who died from COVID-19 and had an autopsy performed at Mayo Clinic between April 2020 to October 2020 were included. Morphological evaluation of the kidneys of 17 individuals with COVID-19 was performed. In a subset of seven COVID-19 cases with postmortem interval of less than or equal to 20 hours, ultrastructural and molecular characteristics (targeted transcriptome and proteomics analyses of tubulointerstitium) were evaluated. Molecular characteristics were compared with archived cases of S-AKI and nonsepsis causes of AKI. Results The spectrum of COVID-19 renal pathology included macrophage-dominant microvascular inflammation (glomerulitis and peritubular capillaritis), vascular dysfunction (peritubular capillary congestion and endothelial injury), and tubular injury with ultrastructural evidence of mitochondrial damage. Investigation of the spatial architecture using a novel imaging mass cytometry revealed enrichment of CD3+CD4+ T cells in close proximity to antigen-presenting cells, and macrophage-enriched glomerular and interstitial infiltrates, suggesting an innate and adaptive immune tissue response. Coronavirus disease 2019 AKI and S-AKI, as compared to nonseptic AKI, had an enrichment of transcriptional pathways involved in inflammation (apoptosis, autophagy, major histocompatibility complex class I and II, and type 1 T helper cell differentiation). Proteomic pathway analysis showed that COVID-19 AKI and to a lesser extent S-AKI were enriched in necroptosis and sirtuin-signaling pathways, both involved in regulatory response to inflammation. Upregulation of the ceramide-signaling pathway and downregulation of oxidative phosphorylation in COVID-19 AKI were noted. Conclusion This data highlights the similarities between S-AKI and COVID-19 AKI and suggests that mitochondrial dysfunction may play a pivotal role in COVID-19 AKI. This data may allow the development of novel diagnostic and therapeutic targets.

BACKGROUND: Preeclampsia and hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome share many clinical and biologic features with thrombotic microangiopathy syndromes caused by complement abnormalities. Our hypothesis was that similar functional and genetic alterations in the complement alternative pathway (CAP) are present in these disorders of pregnancy. METHODS: We conducted quantitative analysis of proteins involved in CAP using ELISA and nephelometry on prospectively collected blood samples from patients with severe phenotype preeclampsia (defined as delivery ≤34 weeks due to preeclampsia), HELLP syndrome, or eclampsia, and matched normotensive controls (n=25 in each arm) between 2011 and 2016. Sequencing was performed to interrogate 14 genes encoding CAP components. RESULTS: Both groups were similar in age, gravidity, parity, marital status, and race. The study group had a higher BMI (mean±SD, 32±8 versus 25±4 kg/m(2); P=0.002) and earlier gestational age at delivery (32.5±3.6 versus 40.3±1 weeks; P

The goals of the Association for Molecular Pathology Clinical Practice Committee's Pharmacogenomics (PGx) Working Group are to define the key attributes of pharmacogenetic alleles recommended for clinical testing, and to determine a minimal set of variants that should be included in clinical PGx genotyping assays. This document series provides recommendations on a minimal panel of variant alleles (Tier 1) and an extended panel of variant alleles (Tier 2) that will aid clinical laboratories in designing assays for PGx testing. When developing these recommendations, the Association for Molecular Pathology PGx Working Group considered the functional impact of the variant alleles, allele frequencies in multiethnic populations, the availability of reference materials, as well as other technical considerations with regard to PGx testing. The ultimate goal of this Working Group is to promote standardization of PGx gene/allele testing across clinical laboratories. This document is focused on clinical CYP2D6 PGx testing that may be applied to all cytochrome P450 2D6–metabolized medications. These recommendations are not meant to be interpreted as prescriptive but to provide a reference guide for clinical laboratories that may be either implementing PGx testing or reviewing and updating their existing platform.

To evaluate the impact of killer immunoglobulin-like receptor (KIR) genotyping in allogeneic hematopoietic stem cell transplantation for myeloid disorders at our institution, retrospective KIR genotyping was performed on 77 patients and their 10/10 matched unrelated donors. In a multivariate model including donor age

The goals of the Association for Molecular Pathology Clinical Practice Committee's Pharmacogenomics (PGx) Working Group are to define the key attributes of pharmacogenetic alleles recommended for clinical testing and a minimum set of variants that should be included in clinical PGx genotyping assays. This article provides recommendations for a minimum panel of variant alleles (Tier 1) and an extended panel of variant alleles (Tier 2) that will aid clinical laboratories when designing assays for PGx testing. The Association for Molecular Pathology PGx Working Group considered the functional impact of the variant alleles, allele frequencies in multiethnic populations, the availability of reference materials, as well as other technical considerations for PGx testing when developing these recommendations. The ultimate goal of this Working Group is to promote standardization of PGx gene/allele testing across clinical laboratories. This article focuses on clinical TPMT and NUDT15 PGx testing, which may be applied to all thiopurine S-methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15)-related medications. These recommendations are not to be interpreted as prescriptive, but to provide a reference guide.

The genetic region on the short arm of chromosome 6 where the human leukocyte antigen (HLA) genes are located is the major histocompatibility complex. The genes in this region are highly polymorphic, and some loci have a high degree of homology with other genes and pseudogenes. Histocompatibility testing has traditionally been performed in the setting of transplantation and involves determining which specific alleles are present. Several HLA alleles have been associated with disease risk or increased risk of adverse drug reaction (ADR) when treated with certain medications. Testing for these applications differs from traditional histocompatibility in that the desired result is simply presence or absence of the allele of interest, rather than determining which allele is present. At present, the majority of HLA typing is done by molecular methods using commercially available kits. A subset of pharmacogenomics laboratories has developed their own methods, and in some cases, query single nucleotide variants associated with certain HLA alleles rather than directly testing for the allele. In this chapter, a brief introduction to the HLA system is provided, followed by an overview of a variety of testing technologies including those specifically used in pharmacogenomics, and the chapter concludes with details regarding specific HLA alleles associated with ADR.

Context.— Next-generation sequencing–based assays are increasingly used in clinical molecular laboratories to detect somatic variants in solid tumors and hematologic malignancies and to detect constitutional variants. Proficiency testing data are potential sources of information about challenges in performing these assays. Objective.— To examine the most common sources of unacceptable results from the College of American Pathologists Next-Generation Sequencing Bioinformatics, Hematological Malignancies, Solid Tumor, and Germline surveys and provide recommendations on how to avoid these pitfalls and improve performance. Design.— The College of American Pathologists next-generation sequencing somatic and germline proficiency testing survey results from 2016 to 2019 were analyzed to identify the most common causes of unacceptable results. Results.— On somatic and germline proficiency testing surveys, 95.9% (18 815/19 623) and 97.8% (33 890/34 641) of all variants were correctly identified, respectively. The most common causes of unacceptable results related to sequencing were false-negative errors in genomic regions that were difficult to sequence because of high GC content. False-positive errors occurred in the context of homopolymers and pseudogenes. Recurrent errors in variant annotation were seen for dinucleotide and duplication variants and included unacceptable transcript selection and outdated variant nomenclature. A small percentage of preanalytic or postanalytic errors were attributed to specimen swaps and transcription errors. Conclusions.— Laboratories demonstrate overall excellent performance for detecting variants in both somatic and germline proficiency testing surveys. Proficiency testing survey results highlight infrequent, but recurrent, analytic and nonanalytic challenges in performing next- generation sequencing–based assays and point to remedies to help laboratories improve performance.

Pharmacogenetic testing is increasingly available from clinical and research laboratories. However, only a limited number of quality control and other reference materials are currently available for many of the variants that are tested. The Association for Molecular Pathology Pharmacogenetic Work Group has published a series of papers recommending alleles for inclusion in clinical testing. Several of the alleles were not considered for tier 1 because of a lack of reference materials. To address this need, the Division of Laboratory Systems, Centers for Disease Control and Prevention–based Genetic Testing Reference Material (GeT-RM) program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 18 DNA samples derived from Coriell cell lines. DNA samples were distributed to five volunteer testing laboratories for genotyping using three commercially available and laboratory developed tests. Several tier 2 variants, including CYP2C9∗13, CYP2C19∗35, the CYP2C cluster variant (rs12777823), two variants in VKORC1 (rs61742245 and rs72547529) related to warfarin resistance, and two variants in GGCX (rs12714145 and rs11676382) related to clotting factor activation, were identified among these samples. These publicly available materials complement the pharmacogenetic reference materials previously characterized by the GeT-RM program and will support the quality assurance and quality control programs of clinical laboratories that perform pharmacogenetic testing.

Several healthcare organizations across Minnesota have developed formal pharmacogenomic (PGx) clinical programs to increase drug safety and effectiveness. Healthcare professional and student education is strong and there are multiple opportunities in the state for learners to gain workforce skills and develop advanced competency in PGx. Implementation planning is occurring at several organizations and others have incorporated structured utilization of PGx into routine workflows. Laboratory-based and translational PGx research in Minnesota has driven important discoveries in several therapeutic areas. This article reviews the state of PGx activities in Minnesota including educational programs, research, national consortia involvement, technology, clinical implementation and utilization and reimbursement, and outlines the challenges and opportunities in equitable implementation of these advances.

Contains fulltext : 234032.pdf (Publisher’s version ) (Closed access) Recurrence of primary membranous nephropathy after transplantation occurs in up to 44% of patients and is driven by PLA2R antibody. Here, we asked whether genetic determinants could improve risk prediction. First, we sequenced PLA2R1 and HLA-D loci in 248 patients with primary membranous nephropathy and identified two independent single nucleotide polymorphisms (SNPs) at risk for primary membranous nephropathy at each locus. These were rs9271188 (intergenic between HLA-DRB1 and HLA-DQA1,) and rs9275086 (intergenic between HLA-DQB1 and HLA-DQA2) at the HLA-D locus along with rs6726925 and rs13018963 at the PLA2R1 locus. Then we investigated whether primary membranous nephropathy at-risk variants were associated with recurrence in a retrospective cohort of 105 donor-recipient pairs and a replication cohort of 40 pairs. Seven SNPs located between HLA-DRB1 and HLA-DQA1 in linkage disequilibrium with rs9271188, and three SNPs in the PLA2R1 region predicted recurrence when presented by the donor, but not when presented by the recipient. The two SNPs in the HLA-D region most strongly associated with recurrence (rs9271705 and rs9271550) were confirmed in the replication cohort. A genetic risk score based on the two best predictors at each locus (rs9271705, rs9271550, rs17830558, and rs3828323) identified a group of patients with high risk of recurrence. Thus, our results suggest that the graft contributes to recurrence of primary membranous nephropathy through the disease susceptibility HLA-D and PLA2R1 SNPs in an autoimmune milieu. Further studies are needed before implementation of genetic testing for these in donor selection.

CYP2D6 duplication has important pharmacogenomic implications. Reflex testing with long-range PCR (LR-PCR) can resolve the genotype when a duplication and alleles with differing activity scores are detected. We evaluated whether visual inspection of plots from real-time-PCR-based targeted genotyping with copy number variation (CNV) detection could reliably determine the duplicated CYP2D6 allele. Six reviewers evaluated QuantStudio OpenArray CYP2D6 genotyping results and the TaqMan Genotyper plots for seventy-three well-characterized cases with three copies of CYP2D6 and two different alleles. Reviewers blinded to the final genotype visually assessed the plots to determine the duplicated allele or opt for reflex sequencing. Reviewers achieved 100% accuracy for cases with three CYP2D6 copies that they opted to report. Reviewers did not request reflex sequencing in 49–67 (67–92%) cases (and correctly identified the duplicated allele in each case); all remaining cases (6–24) were marked by at least one reviewer for reflex sequencing. In most cases with three copies of CYP2D6, the duplicated allele can be determined using a combination of targeted genotyping using real-time PCR with CNV detection without need for reflex sequencing. In ambiguous cases and those with >3 copies, LR-PCR and Sanger sequencing may still be necessary for determination of the duplicated allele.

Background: Cyclin dependent 4/6 kinase inhibitors (CDK4/6i) and endocrine therapy (ET) have improved progression-free survival (PFS) and overall survival in hormone-receptor (HR)-positive metastatic breast cancer (MBC), but progression of disease is inevitable. Serum thymidine kinase-1 (TK1) is a secreted marker of proliferation that is prognostic in patients (pts) with HR-positive, HER2-negative MBC and may be predictive of ET and CDK 4/6i response. PROMISE (NCT0281902) is a prospective study enrolling women with HR-positive MBC starting palbociclib (Pb) + letrozole (L) (1st line) or Pb + fulvestrant (2nd line). We undertook a comprehensive “omic” assessment of blood, tumor, urine and the fecal microbiome in order to identify novel genomic variants and pathways associated with an early decline in TK1 (measured after 2 months) and PFS. Additionally, patient derived xenografts/organoids were generated at baseline and progression to test new therapeutic approaches to overcome resistance to CDK4/6i and ET. We report the initial association between the baseline genomic landscape and baseline TK1 levels. Methods: FFPE tumor biopsies were obtained for DNA/RNA sequencing (TempusTM) and blood samples for TK1 (Divitum® assay, Biovica) were collected pretreatment (pre-Pb) and after 2 cycles of Pb + ET (post-Pb2). Both whole-exome (exome capture) sequencing (WES) and RNA-Seq used the Integrated DNA Technologies xGen Exome Research Panel v1.0 capture kit. TK1+ disease was defined as > 200 Du/L and TK1- disease as below limit of detection up to 200 Du/L. We tested the association between genomic and transcriptomic characteristics with baseline TK1 data in pretreatment samples where both WES and RNA-seq and TK1 was available. The data were analyzed using bioinformatics pipelines for somatic and germline mutations and copy number alterations. The current analysis focuses on baseline 1st-line pre-Pb omics data in conjunction with baseline TK1 levels. The database was locked for analysis on 5/29/2020. Results: Thirty-three pts (median age: 59 yrs.) were evaluable, with paired samples for TK1 in 32. Six pts had TK1+ disease pre-Pb and post-Pb2. Twenty-two pts had TK1- disease pre-Pb and post-Pb2. Four pts had a decrease in TK1 after 2 cycles of treatment that altered the classification from TK1+ to TK1-. Both baseline RNA seq and serum TK1 (n=16) were available for 4 TK1+ and 12 TK1- pts. In this group, 476 genes were differentially regulated (398 upregulated; 78 downregulated). Pathway analysis demonstrated enrichment in complement and coagulation cascade pathway, PPAR signaling pathway, and metabolism-related pathways related to up-regulation of CYP and UGT gene families. Further testing for the association of WES data with baseline TK1+ (n=8) and TK1- (n=16) disease demonstrated somatic copy number variations on chromosomes 6, 11, 12 and 15. CDK4 copy number gains were observed in 3/8 TK1+ pts and 0/16 TK1- pts. We also observed that somatic mutations (LOH, copy number and/or SNV/INDELs) were more prevalent in the TK1+ compared to the TK1- pre-Pb group in several cancer-associated genes (FAS [p=0.06] PTEN, PIK3CB, NAB2, SOX9 and FAT1 [p=0.08], TP53, and MAP2K4 [p=0.22]). Conversely, we also noted that 6/7 pts with GATA3 mutations had TK1- disease (p=0.23). Conclusions: Using a comprehensive “omics” approach, our data suggest that a secreted biomarker of proliferation (TK1) obtained prior to initiating CDK4/6i and ET for the first line treatment of HR+ MBC is associated with established and novel genes and pathways associated with prognosis of pts receiving ET and CDK 4/6i. Analysis of on-treatment (after 2 cycles) tumor RNA seq and its association with change in TK1 as well as data from the 2nd-line cohort will be presented at the meeting. Citation Format: Ciara C O Sullivan, Krishna R Kalari, Vera J Suman, Peter T Vedell, Ann Moyer, Abraham D Eyman Casey, Jason Sinnwell, Xiaojia Tang, Kevin Thompson, Alvaro Moreno-Aspitia, Donald W Northfelt, Minetta C Liu, Tufia C Haddad, Saranya Chumsri, Prema Peethambaram, Kathryn J Ruddy, Karthik V Giridhar, Roberto A Leon-Ferre, Adrian Nordmark, Mattias Bergqvist, Brendan P McMenomy, Richard M Weinshilboum, Liewei Wang, Matthew P Goetz. Novel genomic variants and pathways associated with baseline serum thymidine kinase 1 levels in HR-positive HER2-negative MBC patients commencing palbociclib and letrozole [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-24.

Prescribing the right medication, at the right dose, to the right patient is the goal of every physician. Pharmacogenomic information is an emerging tool that can be used to deliver precision medicine. In this review, we discuss the pharmacogenomics of available PPIs, racial differences of CYP2C19 and how PPI pharmacogenomics affects the treatment of common gastrointestinal diseases. We also provide practical guidance on when to order pharmacogenomic testing, which test to order, and how to modify treatment based on published guidelines.

Objective To assess the potential impact of Pharmacogenomic (PGx) variation in cytochrome P450 2D6 (CYP2D6) enzyme function, using loss in quality-adjusted life years (QALYs) associated with treatment problems, and the willingness to pay to avoid treatment problems from patients’ and payers’ perspectives. Patients and Methods The study included patients prescribed tramadol or codeine, or both, between January 1, 2005, and December 31, 2017. Demographic information and adverse drug events, including adverse drug events and poor pain control, were collected from the electronic health records using natural language processing techniques and review by trained abstractors. Patients’ willingness to pay and QALY estimates were based on comprehensive literature review. The CYP2D6 phenotypes were divided into 4 groups: ultra-rapid metabolizers, normal metabolizers, intermediate metabolizers, and poor metabolizers. Results Among the 2860 identified patients, 63 (2%) were ultrarapid metabolizers, 1449 (50%) were normal metabolizers, 1155 (40%) were intermediate metabolizers, and 193 (7%) were poor metabolizers. The patients’ average estimated willingness-to-pay value to avoid treatment problems was $23 per month; poor metabolizers developed problems with the highest estimated willingness-to-pay value ($32 per month). The mean QALY loss among all patients was 0.024 QALYs (8.8 healthy days); poor metabolizers had the highest loss (0.027 QALYs, 9.9 healthy days). Conclusion Patients with various phenotypes developed different treatment problem profiles. Poor CYP2D6 metabolizers developed problems with highest willingness to pay, and they might potentially benefit most from PGx-guided treatment and problem prevention.

SARS-CoV-2 is an RNA virus that primarily causes respiratory disease; however, infection of other tissue has been reported. Evaluation of SARS-CoV-2 in tissue specimens may increase understanding of SARS-CoV-2 pathobiology.A qualitative test for detection of SARS-CoV-2 in formalin-fixed paraffin-embedded (FFPE) tissues was developed and validated using droplet digital PCR (ddPCR), which has a lower limit of detection than reverse transcription (RT)-qPCR. After extraction of total RNA from unstained FFPE tissue, SARS-CoV-2 nucleocapsid (N1, N2) target sequences were amplified and quantified, along with human RPP30 as a control using the Bio-Rad SARS-CoV-2 ddPCR kit.SARS-CoV-2 was detected in all 21 known positive samples and none of the 16 negative samples. As few as approximately 5 viral copies were reliably detected. Since January 2021, many tissue types have been clinically tested. Of the 195 clinical specimens, the positivity rate was 35% with placenta and fetal tissue showing the highest percentage of positive cases.This sensitive FFPE-based assay has broad clinical utility with applications as diverse as pregnancy loss and evaluation of liver transplant rejection. This assay will aid in understanding atypical presentations of COVID-19 as well as long-term sequelae.

The goal of the Association for Molecular Pathology (AMP) Clinical Practice Committee's AMP Pharmacogenomics (PGx) Working Group is to define the key attributes of PGx alleles recommended for clinical testing and a minimum set of variants that should be included in clinical PGx genotyping assays. This document series provides recommendations for a minimum panel of variant alleles (tier 1) and an extended panel of variant alleles (tier 2) that will aid clinical laboratories when designing assays for PGx testing. The AMP PGx Working Group considered functional impact of the variants, allele frequencies in multiethnic populations, the availability of reference materials, as well as other technical considerations for PGx testing when developing these recommendations. The ultimate goal is to promote standardization of PGx gene/allele testing across clinical laboratories. These recommendations are not to be interpreted as prescriptive but to provide a reference guide. Of note, a separate article with recommendations for CYP2C9 allele selection was previously developed by the PGx Working Group that can be applied broadly to CYP2C9-related medications. The warfarin allele recommendations in this report incorporate the previous CYP2C9 allele recommendations and additional genes and alleles that are specific to warfarin testing.

Programmed death ligand-1 (PD-L1 or B7-H1) is well known for its role in immune checkpoint regulation, but its function inside the tumor cells has rarely been explored. Here we report that nuclear PD-L1 is important for cancer cell sister chromatid cohesion. We found that depletion of PD-L1 suppresses cancer cell proliferation, colony formation in vitro, and tumor growth in vivo in immune-deficient NSG mice independent of its role in immune checkpoint. Specifically, PD-L1 functions as a subunit of the cohesin complex, and its deficiency leads to formation of multinucleated cells and causes a defect in sister chromatid cohesion. Mechanistically, PD-L1 compensates for the loss of Sororin, whose expression is suppressed in cancer cells overexpressing PD-L1. PD-L1 competes with Wing Apart-Like (WAPL) for binding to PDS5B, and secures proper sister chromatid cohesion and segregation. Our findings suggest an important role for nuclear PD-L1 in cancer cells independent of its function in immune checkpoint.

Background Several small studies have previously investigated associations between the cytochrome P450 2D6 (CYP2D6) metabolism and response to opioids. We used a large sample of patients to study associations between CYP2D6 phenotypes and estimated CYP2D6 enzymatic activity scores with pain control and adverse reactions related to codeine and tramadol use. We conducted additional analyses to determine whether our results were consistent among men and women. Methods We used data from 2,877 participants in the RIGHT Protocol who were prescribed codeine and/or tramadol between 01/01/2005 and 12/31/2017 and who were not prescribed CYP2D6 inhibitors within 1 year prior to the opioid prescription. CYP2D6 phenotype categories were condensed into four groups: (1) Ultra-rapid and Rapid (n = 61), (2) Normal and Intermediate to Normal (n = 1,448), (3) Intermediate and Intermediate to Poor (n = 1,175), and (4) Poor metabolizer status (n = 193). Opioid-related outcomes included indications of poor pain control or adverse reactions related to medication use. We modeled the risk of each outcome using logistic regression, adjusting for age, sex, race, and ethnicity. Results The results revealed a trend from poor to ultra-rapid and rapid CYP2D6 phenotypes in which the risk of adverse reactions incrementally increased and the risk of poor pain control incrementally decreased. This trend reached statistical significance among female (but not male) participants. Among normal and intermediate to normal metabolizers, a larger proportion of women experienced adverse reactions relative to men. Discussion We replicated and extended the findings of previous research indicating associations between CYP2D6 phenotypes and response to opioids. In addition, the observed associations were stronger in women than in men. We recommend sex differences to be factored in future research investigating associations between pharmacogenomics and response to medications.

Background: The combination of cyclin dependent 4/6 kinase inhibitors (CDK4/6i) with endocrine therapy (ET) has resulted in clinically significant improvements in progression-free survival (PFS) and overall survival (OS) in hormone-receptor (HR)-positive metastatic breast cancer (MBC). However, most patients’ disease ultimately progresses on CDK4/6i and ET. Therefore, further research is necessary to understand the mechanisms driving primary and secondary resistance. PROMISE is a multicenter prospective cohort study enrolling women with HR-positive MBC commencing treatment with palbociclib + letrozole (1st line) or palbociclib + fulvestrant (2nd Line). The study provides a comprehensive “omic” assessment of blood, tumor, urine and the fecal microbiome to identify molecular or cellular features associated with primary endocrine resistance (e.g. disease progression ≤ 12 months on treatment) and acquired resistance to CDK 4/6i. Additionally, patient derived xenografts and organoids are created to test new drug strategies designed to overcome resistance to CDK 4/6i and ET. Here, we present initial sequencing results from pretreatment biospecimens collected from PROMISE study participants. Methods: On-study tumor biopsies and blood samples were collected for DNA/RNA sequencing (TempusTM). The analyzed biospecimens were all obtained prior to initiation of palbociclib and ET. We correlated patient clinical characteristics (phenotypes) with molecular data and responses to protocol treatment. The data were analyzed using a series of cutting-edge bioinformatics pipelines for somatic and germline mutations in addition to copy number alterations (CNAs). The study database was locked for analysis on 06/20/2019. Results: We analyzed the somatic single nucleotide variants/INDELs (sSNV/INDEL) profiles across the tumor samples to determine the genes that were least likely to occur as a result of background mutation processes. Twenty-six patients had somatic copy number alterations (sCNA) and/or sSNV/INDEL in at least one of 18 genes with the most significant sSNV/INDEL profiles (p < 0.03) which included clinically and biologically relevant genes. The genes with the most statistically significant sSNV/INDEL mutation profiles were GATA3, PIK3CA, CDH1, and ESR1 (p < 0.0009). We observed a high percentage of tumors with somatic alterations in GATA3 (23% sSNV/INDEL, 15% sCNA), PIK3CA (38%, 12%), CDH1 (19%, 50%) and ESR1 (19%, 58%). ESR1 mutations were more frequent in patients receiving 2nd line treatment. Other frequently altered genes included TP53 (15%, 46%), MAP2K4 (8%, 50%), DNAAF1 (8%, 50%), and CDKN1B (8%, 35%). Further, ZNF317 and F3 were altered in 9 and 7 patients, respectively. Twenty-four samples had alterations in at least one of the CDK4/6 pathway genes (RB1, CCNE2, CCND1, CDK6, ESR1, CDKN2A, CCND3, CDK4, CDK2 and CCNE1). Four patients progressed on therapy; three of the four patients had mutations in PIK3CA, and one had a mutation in ESR1. Results of the RNA sequencing data (N=26) will be presented at the SABCS meeting. Conclusions: This is the first report of a prospective study designed to characterize the genomic landscape of ER+/HER2- MBC prior to palbociclib treatment. We observed high frequencies of known targetable alterations in PIK3CA and ESR1, including in patients that progress, which is consistent with previous reports. RNA sequencing data will be presented at the meeting. Citation Format: Ciara C O Sullivan, Krishna R Kalari, Vera J Suman, Peter T Vedell, Ann Moyer, Erin Carlson, Jason Sinnwell, Tejaswi Alaparthi, Xiaojia Tang, Kevin Thompson, Jaeyun Sung, Alvaro Moreno-Aspitia, Donald Northfelt, Minetta C Liu, Tufia C Haddad, Prema Peethambaram, Saranya Chumsri, Kathryn J Ruddy, Karthik V Giridhar, Roberto A Leon-Ferre, Paula Gill, Mohammad Ranginwala, Asad Javed, Sameer Batoo, Brendan P. McMenomy, Richard Weinshilboum, Liewei Wang, Matthew P Goetz. Comprehensive tumor sequencing to identify biomarkers of palbociclib response: First report of the PROMISE study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-11-07.