Atezolizumab in Combination With Carboplatin and Survival Outcomes in Patients With Metastatic Triple-Negative Breast Cancer: The TBCRC 043 Phase 2 Randomized Clinical Trial.

Importance: Agents targeting programmed death ligand 1 (PD-L1) have demonstrated efficacy in triple-negative breast cancer (TNBC) when combined with chemotherapy and are now the standard of care in patients with PD-L1-positive metastatic disease. In contrast to microtubule-targeting agents, the effect of combining platinum compounds with programmed cell death 1 (PD-1)/PD-L1 immunotherapy has not been extensively determined. Objective: To evaluate the efficacy of atezolizumab with carboplatin in patients with metastatic TNBC. Design, Setting, and Participants: This phase 2 randomized clinical trial was conducted in 6 centers from August 2017 to June 2021. Interventions: Patients with metastatic TNBC were randomized to receive carboplatin area under the curve (AUC) 6 alone or with atezolizumab, 1200 mg, every 3 weeks until disease progression or unacceptable toxic effects with a 3-year duration of follow-up. Main Outcome and Measures: The primary end point was investigator-assessed progression-free survival (PFS). Secondary end points included overall response rate (ORR), clinical benefit rate (CBR), and overall survival (OS). Other objectives included correlation of response with tumor PD-L1 levels, tumor-infiltrating lymphocytes (TILs), tumor DNA- and RNA-sequenced biomarkers, TNBC subtyping, and multiplex analyses of immune markers. Results: All 106 patients with metastatic TNBC who were enrolled were female with a mean (range) age of 55 (27-79) years, of which 12 (19%) identified as African American/Black, 1 (1%) as Asian, 73 (69%) as White, and 11 (10%) as unknown. Patients were randomized and received either carboplatin (n = 50) or carboplatin and atezolizumab (n = 56). The combination improved PFS (hazard ratio [HR], 0.66; 95% CI, 0.44-1.01; P = .05) from a median of 2.2 to 4.1 months, increased ORR from 8.0% (95% CI, 3.2%-18.8%) to 30.4% (95% CI, 19.9%-43.3%), increased CBR at 6 months from 18.0% (95% CI, 9.8%-30.1%) to 37.5% (95% CI, 26.0%-50.6%), and improved OS (HR, 0.60; 95% CI, 0.37-0.96; P = .03) from a median of 8.6 to 12.6 months. Subgroup analysis showed PD-L1-positive tumors did not benefit more from adding atezolizumab (HR, 0.62; 95% CI, 0.23-1.65; P = .35). Patients with high TILs (HR, 0.12; 95% CI, 0.30-0.50), high mutation burden (HR, 0.50; 95% CI, 0.23-1.06), and prior chemotherapy (HR, 0.59; 95% CI, 0.36-0.95) received greater benefit on the combination. Patients with obesity and patients with more than 125 mg/dL on-treatment blood glucose levels were associated with better PFS (HR, 0.35; 95% CI, 0.10-1.80) on the combination. TNBC subtypes benefited from adding atezolizumab, except the luminal androgen receptor subtype. Conclusions and Relevance: In this randomized clinical trial, the addition of atezolizumab to carboplatin significantly improved survival of patients with metastatic TNBC regardless of PD-L1 status. Further, lower risk of disease progression was associated with increased TILs, higher mutation burden, obesity, and uncontrolled blood glucose levels. Trial Registration: ClinicalTrials.gov Identifier: NCT03206203.

Automated HL7v2 LRI informatics framework for streamlining genomics-EHR data integration.

While VCF formatted files are the lingua franca of next-generation sequencing, most EHRs do not provide native VCF support. As a result, labs often must send non-structured PDF reports to the EHR. On the other hand, while FHIR adoption is growing, most EHRs support HL7 interoperability standards, particularly those based on the HL7 Version 2 (HL7v2) standard. The HL7 Version 2 genomics component of the HL7 Laboratory Results Interface (HL7v2 LRI) standard specifies a formalism for the structured communication of genomic data from lab to EHR. We previously described an open-source tool (vcf2fhir) that converts VCF files into HL7 FHIR format. In this report, we describe how the utility has been extended to output HL7v2 LRI data that contains both variants and variant annotations (e.g., predicted phenotypes and therapeutic implications). Using this HL7v2 converter, we implemented an automated pipeline for moving structured genomic data from the clinical laboratory to EHR. We developed an open source hl7v2GenomicsExtractor that converts genomic interpretation report files into a series of HL7v2 observations conformant to HL7v2 LRI. We further enhanced the converter to produce output conformant to Epic's genomic import specification and to support alternative input formats. An automated pipeline for pushing standards-based structured genomic data directly into the EHR was successfully implemented, where genetic variant data and the clinical annotations are now both available to be viewed in the EHR through Epic's genomics module. Issues encountered in the development and deployment of the HL7v2 converter primarily revolved around data variability issues, primarily lack of a standardized representation of data elements within various genomic interpretation report files. The technical implementation of a HL7v2 message transformation to feed genomic variant and clinical annotation data into an EHR has been successful. In addition to genetic variant data, the implementation described here releases the valuable asset of clinically relevant genomic annotations provided by labs from static PDFs to calculable, structured data in EHR systems.

Current clinical practices and challenges in molecular testing: a GOAL Consortium Hematopathology Working Group report.

While molecular testing of hematologic malignancies is now standard of care, there is variability in practice and testing capabilities between different academic laboratories, with common questions arising on how to best meet clinical expectations. A survey was sent to hematopathology subgroup members of the Genomics Organization for Academic Laboratories consortium to assess current and future practice and potentially establish a reference for peer institutions. Responses were received from 18 academic tertiary-care laboratories regarding next-generation sequencing (NGS) panel design, sequencing protocols and metrics, assay characteristics, laboratory operations, case reimbursement, and development plans. Differences in NGS panel size, use, and gene content were reported. Gene content for myeloid processes was reported to be generally excellent, while genes for lymphoid processes were less well covered. The turnaround time (TAT) for acute cases, including acute myeloid leukemia, was reported to range from 2 to 7 calendar days to 15 to 21 calendar days, with different approaches to achieving rapid TAT described. To help guide NGS panel design and standardize gene content, consensus gene lists based on current and future NGS panels in development were generated. Most survey respondents expected molecular testing at academic laboratories to continue to be viable in the future, with rapid TAT for acute cases likely to remain an important factor. Molecular testing reimbursement was reported to be a major concern. The results of this survey and subsequent discussions improve the shared understanding of differences in testing practices for hematologic malignancies between institutions and will help provide a more consistent level of patient care.

Deleterious mutations in esophageal carcinoma cuniculatum detected by next generation sequencing.

Esophageal carcinoma cuniculatum (ECC) is a rare form of extremely well-differentiated squamous cell carcinoma of esophagus that is often misdiagnosed preoperatively. The molecular changes underlying ECC remain unknown. This study aimed to explore the molecular signature of ECC using next-generation sequencing (NGS). Five cases of ECC were collected from our pathology database from 2014 to 2019. One patient received chemoradiation and the remaining four patients were treatment-naïve. Areas of normal squamous mucosa, non-invasive component, and invasive component of ECC were circled and macrodissected. Genomic DNA extracted from the macrodissected tissue was sequenced using GatorSeq NGS Panel. Deleterious mutations, predicted by Sorting Intolerant from Tolerant (SIFT), were identified using tumor/normal pairs and annotated by amino acid change. The normal-appearing squamous mucosa in the ECC harbored recurrent deleterious somatic mutations in ROS1 and POLE genes. ECC tumor-specific deleterious mutations were identified on TP53, NOTCH1, and PIK3CA genes. Our results support a mutually exclusive pattern in NOTCH1 and PIK3CA mutation. Non-invasive and invasive components in ECC had identical mutation profiles. Chemoradiation therapy led to disappearance of NOTCH1 mutation in one ECC case. Our results suggest molecular testing may help pre-operative diagnosis, and provide therapeutic targets in patients with advanced or unresectable ECC.

GATM-Mediated Creatine Biosynthesis Enables Maintenance of FLT3-ITD-Mutant Acute Myeloid Leukemia.

FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in acute myeloid leukemia (AML), with the most common mutation being internal tandem duplications (ITD). The presence of FLT3-ITD in AML carries a particularly poor prognosis and renders therapeutic resistance. New druggable targets are thus needed in this disease. In this study, we demonstrate the effects of de novo creatine biosynthesis upregulation by FLT3-ITD on AML sustainability. Our data show that FLT3-ITD constitutively activates the STAT5 signaling pathway, which upregulates the expression of glycine amidinotransferase (GATM), the first rate-limiting enzyme of de novo creatine biosynthesis. Pharmacologic FLT3-ITD inhibition reduces intracellular creatinine levels through transcriptional downregulation of genes in the de novo creatine biosynthesis pathway. The same reduction can be achieved by cyclocreatine or genetic GATM knockdown with shRNA and is reflected in significant decrease of cell proliferation and moderate increase of cell apoptosis in FLT3-ITD-mutant cell lines. Those effects are at least partially mediated through the AMPK/mTOR signaling pathway. This study uncovers a previously uncharacterized role of creatine metabolic pathway in the maintenance of FLT3-ITD-mutant AML and suggests that targeting this pathway may serve as a promising therapeutic strategy for FLT3-ITD-positive AML. IMPLICATIONS: FLT3-ITD mutation in AML upregulates de novo creatine biosynthesis that we show can be suppressed to diminish the proliferation and survival of blast cells.

The Hologic Aptima SARS-CoV-2 assay enables high ratio pooling saving reagents and improving turnaround time.

BACKGROUND: The Hologic Aptima™ TMA SARS-CoV-2 assay was employed to test pooled nasopharyngeal (NP) samples to evaluate the performance of pooled sample testing and characterize variables influencing results. METHODS: Results on 1033 previously tested NP samples were retrieved to characterize the relative light units (RLU) of SARS-CoV-2-positive samples in the tested population. The pooling strategy of combining 10 SARS-CoV-2 samples into one pool (10/1) was used in this study. The results were compared with neat sample testing using the same Aptima™ TMA SARS-CoV-2 assay and also the CDC RT-PCR and the Cepheid SARS-CoV-2 assays. RESULTS: The Aptima assay compares favorably with both CDC RT-PCR and the Cepheid SARS-CoV-2 assays. Once samples are pooled 10 to 1 as in our experiments, the resulting signal strength of the assay suffers. A divide opens between pools assembled from strong-positive versus only weak-positive samples. Pools of the former can be reliably detected with positive percent agreement (PPA) of 95.2%, while pools of the latter are frequently misclassified as negative with PPA of 40%. When the weak-positive samples with kRLU value lower than 1012 constitute 3.4% of the total sample profile, the assay PPA approaches 93.4% suggesting that 10/1 pooled sample testing by the Aptima assay is an effective screening tool for SARS-CoV-2. CONCLUSION: Performing pooled testing, one should monitor the weak positives with kRLU lower than 1012 or quantification cycle (Cq) value higher than 35 on an ongoing basis and adjust pooling approaches to avoid reporting false negatives.

Pathology Informatics and Robotics Strategies for Improving Efficiency of COVID-19 Pooled Testing.

The global rise of the coronavirus disease 2019 pandemic resulted in an exponentially increasing demand for severe acute respiratory syndrome coronavirus 2 testing, which resulted in shortage of reagents worldwide. This shortage has been further worsened by screening of asymptomatic populations such as returning employees, students, and so on, as part of plans to reopen the economy. To optimize the utilization of testing reagents and human resources, pool testing of populations with low prevalence has emerged as a promising strategy. Although pooling is an effective solution to reduce the number of reagents used for testing, the process of pooling samples together and tracking them throughout the entire workflow is challenging. To be effective, samples must be tracked into each pool, pool-tested and reported individually. In this article, we address these challenges using robotics and informatics.

How to Transition from Single-Gene Pharmacogenetic Testing to Preemptive Panel-Based Testing: A Tutorial.

There have been significant advancements in precision medicine and approaches to medication selection based on pharmacogenetic results. With the availability of direct-to-consumer genetic testing and growing awareness of genetic interindividual variability, patient demand for more precise, individually tailored drug regimens is increasing. The University of Florida (UF) Health Precision Medicine Program (PMP) was established in 2011 to improve integration of genomic data into clinical practice. In the ensuing years, the UF Health PMP has successfully implemented several single-gene tests to optimize the precision of medication prescribing across a variety of clinical settings. Most recently, the UF Health PMP launched a custom-designed pharmacogenetic panel, including pharmacogenes relevant to supportive care medications commonly prescribed to patients undergoing chemotherapy treatment, referred to as "GatorPGx." This tutorial provides guidance and information to institutions on how to transition from the implementation of single-gene pharmacogenetic testing to a preemptive panel-based testing approach. Here, we demonstrate application of the preemptive panel in the setting of an adult solid tumor oncology clinic. Importantly, the information included herein can be applied to other clinical practice settings.

Optimizing COVID-19 testing capabilities and clinical management using pathology informatics.

Coronavirus disease 2019, first reported in China in late 2019, has quickly spread across the world. The outbreak was declared a pandemic by the World Health Organization on March 11, 2020. Here, we describe our initial efforts at the University of Florida Health for processing of large numbers of tests, streamlining data collection, and reporting data for optimizing testing capabilities and superior clinical management. Specifically, we discuss clinical and pathology informatics workflows and informatics instruments which we designed to meet the unique challenges of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing. We hope these results benefit institutions preparing to implement SARS-CoV-2 testing.

Co-occurring Alterations in the RAS-MAPK Pathway Limit Response to MET Inhibitor Treatment in MET Exon 14 Skipping Mutation-Positive Lung Cancer.

PURPOSE: Although patients with advanced-stage non-small cell lung cancers (NSCLC) harboring MET exon 14 skipping mutations (METex14) often benefit from MET tyrosine kinase inhibitor (TKI) treatment, clinical benefit is limited by primary and acquired drug resistance. The molecular basis for this resistance remains incompletely understood. EXPERIMENTAL DESIGN: Targeted sequencing analysis was performed on cell-free circulating tumor DNA obtained from 289 patients with advanced-stage METex14-mutated NSCLC. RESULTS: Prominent co-occurring RAS-MAPK pathway gene alterations (e.g., in KRAS, NF1) were detected in NSCLCs with METex14 skipping alterations as compared with EGFR-mutated NSCLCs. There was an association between decreased MET TKI treatment response and RAS-MAPK pathway co-occurring alterations. In a preclinical model expressing a canonical METex14 mutation, KRAS overexpression or NF1 downregulation hyperactivated MAPK signaling to promote MET TKI resistance. This resistance was overcome by cotreatment with crizotinib and the MEK inhibitor trametinib. CONCLUSIONS: Our study provides a genomic landscape of co-occurring alterations in advanced-stage METex14-mutated NSCLC and suggests a potential combination therapy strategy targeting MAPK pathway signaling to enhance clinical outcomes.

SMARCA4-Deficient Thoracic Sarcoma: A Case Report and Review of Literature.

SMARCA4-deficient thoracic sarcoma (SMARCA4-DTS) is a recently described entity with a poor prognosis that is defined by certain genetic alterations in the BAF chromatin remodeling complex, specifically SMARCA4 and SMARCA2. We present a case of a SMARCA4-DTS in a 59 year-old male with a heavy smoking history who was found to have an unexpected right upper lobe lung mass on routine chest radiograph after a visit to his primary care physician. This led to a biopsy with a diagnosis of poorly differentiated carcinoma at an outside institution. The patient was subsequently seen at our facility for surgical intervention. The right upper lobectomy contained a 7.2-cm poorly differentiated malignancy with slightly discohesive cells arranged in sheets and nests, abundant geographic necrosis, and with many areas showing rhabdoid morphology. The tumor was focally reactive for CK7, AE1/3, Cam5.2, and SALL4 and showed scattered reactivity for CD34 and SOX2. There was complete loss of reactivity for both SMARCA4 and SMARCA2. The histology and immunophenotype were all consistent with the diagnosis of a SMARCA4-DTS. Next-generation sequencing showed a frameshift mutation in the SMARCA4 gene and no abnormality with the SMARCA2 gene. Interestingly, this tumor was confined to the pulmonary parenchyma with no invasion of the visceral pleura nor the mediastinum and with no clinically apparent metastases at the time of presentation. This case is presented to add to the cohort of cases described to date and to discuss the immunohistochemical and molecular findings with regard to SMARCA2.

CYP2D6-guided opioid therapy improves pain control in CYP2D6 intermediate and poor metabolizers: a pragmatic clinical trial.

PURPOSE: CYP2D6 bioactivates codeine and tramadol, with intermediate and poor metabolizers (IMs and PMs) expected to have impaired analgesia. This pragmatic proof-of-concept trial tested the effects of CYP2D6-guided opioid prescribing on pain control. METHODS: Participants with chronic pain (94% on an opioid) from seven clinics were enrolled into CYP2D6-guided (n = 235) or usual care (n = 135) arms using a cluster design. CYP2D6 phenotypes were assigned based on genotype and CYP2D6 inhibitor use, with recommendations for opioid prescribing made in the CYP2D6-guided arm. Pain was assessed at baseline and 3 months using PROMIS® measures. RESULTS: On stepwise multiple linear regression, the primary outcome of composite pain intensity (composite of current pain and worst and average pain in the past week) among IM/PMs initially prescribed tramadol/codeine (n = 45) had greater improvement in the CYP2D6-guided versus usual care arm (-1.01 ± 1.59 vs. -0.40 ± 1.20; adj P = 0.016); 24% of CYP2D6-guided versus 0% of usual care participants reported ≥30% (clinically meaningful) reduction in the composite outcome. In contrast, among normal metabolizers prescribed tramadol or codeine at baseline, there was no difference in the change in composite pain intensity at 3 months between CYP2D6-guided (-0.61 ± 1.39) and usual care (-0.54 ± 1.69) groups (adj P = 0.540). CONCLUSION: These data support the potential benefits of CYP2D6-guided pain management.

Clinical implementation of rapid CYP2C19 genotyping to guide antiplatelet therapy after percutaneous coronary intervention.

BACKGROUND: The CYP2C19 nonfunctional genotype reduces clopidogrel effectiveness after percutaneous coronary intervention (PCI). Following clinical implementation of CYP2C19 genotyping at University Florida (UF) Health Shands Hospital in 2012, where genotype results are available approximately 3 days after PCI, testing was expanded to UF Health Jacksonville in 2016 utilizing a rapid genotyping approach. We describe metrics with this latter implementation. METHODS: Patients at UF Health Jacksonville undergoing left heart catheterization with intent to undergo PCI were targeted for genotyping using the Spartan RX™ system. Testing metrics and provider acceptance of testing and response to genotype results were examined, as was antiplatelet therapy over the 6 months following genotyping. RESULTS: In the first year, 931 patients, including 392/505 (78%) total patients undergoing PCI, were genotyped. The median genotype test turnaround time was 96 min. Genotype results were available for 388 (99%) PCI patients prior to discharge. Of 336 genotyped PCI patients alive at discharge and not enrolled in an antiplatelet therapy trial, 1/6 (17%) poor metabolizers (PMs, with two nonfunctional alleles), 38/93 (41%) intermediate metabolizers (IMs, with one nonfunctional allele), and 119/237 (50%) patients without a nonfunctional allele were prescribed clopidogrel (p = 0.110). Clopidogrel use was higher among non-ACS versus ACS patients (78.6% vs. 42.2%, p < 0.001). Six months later, among patients with follow-up data, clopidogrel was prescribed in 0/4 (0%) PMs, 33/65 (51%) IMs, and 115/182 (63%) patients without a nonfunctional allele (p = 0.008 across groups; p = 0.020 for PMs versus those without a nonfunctional allele). CONCLUSION: These data demonstrate that rapid genotyping is clinically feasible at a high volume cardiac catheterization facility and allows informed chronic antiplatelet prescribing, with lower clopidogrel use in PMs at 6 months. Trial registration ClinicalTrials.gov Identifier: NCT02724319; registered March 31, 2016; https://www.clinicaltrials.gov/ct2/show/NCT02724319?term=angiolillo&rank=7.

A TEAD1/p65 complex regulates the eutherian-conserved MnSOD intronic enhancer, eRNA transcription and the innate immune response.

Manganese superoxide dismutase (MnSOD), a critical anti-oxidant enzyme, detoxifies the mitochondrial-derived reactive oxygen species, superoxide, elicited through normal respiration or the inflammatory response. Proinflammatory stimuli induce MnSOD gene expression through a eutherian-conserved, intronic enhancer element. We identified two prototypic enhancer binding proteins, TEAD1 and p65, that when co-expressed induce MnSOD expression comparable to pro-inflammatory stimuli. TEAD1 causes the nuclear sequestration of p65 leading to a novel TEAD1/p65 complex that associates with the intronic enhancer and is necessary for cytokine induction of MnSOD. Unlike typical NF-κB-responsive genes, the induction of MnSOD does not involve p50. Beyond MnSOD, the TEAD1/p65 complex regulates a subset of genes controlling the innate immune response that were previously viewed as solely NF-κB-dependent. We also identified an enhancer-derived RNA (eRNA) that is induced by either proinflammatory stimuli or the TEAD1/p65 complex, potentially linking the intronic enhancer to intra- and interchromosomal gene regulation through the inducible eRNA.

Human traumatic brain injury induces autoantibody response against glial fibrillary acidic protein and its breakdown products.

The role of systemic autoimmunity in human traumatic brain injury (TBI) and other forms of brain injuries is recognized but not well understood. In this study, a systematic investigation was performed to identify serum autoantibody responses to brain-specific proteins after TBI in humans. TBI autoantibodies showed predominant immunoreactivity against a cluster of bands from 38-50 kDa on human brain immunoblots, which were identified as GFAP and GFAP breakdown products. GFAP autoantibody levels increased by 7 days after injury, and were of the IgG subtype predominantly. Results from in vitro tests and rat TBI experiments also indicated that calpain was responsible for removing the amino and carboxyl termini of GFAP to yield a 38 kDa fragment. Additionally, TBI autoantibody staining co-localized with GFAP in injured rat brain and in primary rat astrocytes. These results suggest that GFAP breakdown products persist within degenerating astrocytes in the brain. Anti-GFAP autoantibody also can enter living astroglia cells in culture and its presence appears to compromise glial cell health. TBI patients showed an average 3.77 fold increase in anti-GFAP autoantibody levels from early (0-1 days) to late (7-10 days) times post injury. Changes in autoantibody levels were negatively correlated with outcome as measured by GOS-E score at 6 months, suggesting that TBI patients with greater anti-GFAP immune-responses had worse outcomes. Due to the long lasting nature of IgG, a test to detect anti-GFAP autoantibodies is likely to prolong the temporal window for assessment of brain damage in human patients.

Characterization of a mechanism to inhibit ovarian follicle activation.

OBJECTIVE: To demonstrate that a small molecule can induce the transcription factor Foxo3 in the ovary and lead to inhibition of follicle activation. DESIGN: Cell culture, organ culture, and animal studies. SETTING: University-based laboratory. ANIMAL(S): 23 female C57BL/6 mice. INTERVENTION(S): Human ovary cells and mouse ovaries in culture treated with 2-deoxyglucose (2-DG) to mimic glucose deprivation, and mice intraperitoneally injected with 100 mg/kg, 300 mg/kg, or 600 mg/kg 2-DG daily for 2 weeks. MAIN OUTCOME MEASURE(S): In cell and organ culture, Foxo3 expression analyzed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR); in treated animals, expression of genes regulated by nutrient deprivation (Foxo3, ATF4, GRP78, CHOP, ASNS, c-Myc) measured in brain, kidney, and ovary by qRT-PCR; and ovarian follicles histologically classified and counted. RESULT(S): Foxo3 expression is induced by 2-DG at both the mRNA and protein level in human ovarian cell culture, possibly through ATF4-dependent gene regulation. Foxo3 expression is also induced by 2-DG in ovarian organ culture. Treatment of mice with 100 mg/kg 2-DG resulted in a 2.6 fold induction of Foxo3 in the ovary and a 58% decrease in type 3a primary follicles. CONCLUSION(S): Expression of Foxo3 is induced by nutrient deprivation in cell culture, organ culture, and in vivo. In mice, 2-DG treatment results in an inhibition of primordial follicle activation. These data indicate that Foxo3 induction by 2-DG may be useful for fertility preservation.