Human dermal microvascular endothelial cell morphological response to fluid shear stress.

As the cells that line the vasculature, endothelial cells are continually exposed to fluid shear stress by blood flow. Recent studies suggest that the morphological response of endothelial cells to fluid shear stress depends on the endothelial cell type. Thus, the present study characterizes the morphological response of human dermal microvascular endothelial cells (HMEC-1) and nuclei to steady, laminar, and unidirectional fluid shear stress. Cultured HMEC-1 monolayers were exposed to shear stress of 0.3 dyn/cm2, 16 dyn/cm2, or 32 dyn/cm2 for 72 h with hourly live-cell imaging capturing both the nuclear and cellular morphology. Despite changes in elongation and alignment occurring with increasing fluid shear stress, there was a lack of elongation and alignment over time under each fluid shear stress condition. Conversely, changes in cellular and nuclear area exhibited dependence on both time and fluid shear stress magnitude. The trends in cellular morphology differed at shear stress levels above and below 16 dyn/cm2, whereas the nuclear orientation was independent of fluid shear stress magnitude. These findings show the complex morphological response of HMEC-1 to fluid shear stress.

Multiethnic PDX models predict a possible immune signature associated with TNBC of African ancestry.

PURPOSE: Triple-negative breast cancer (TNBC) is an aggressive subtype most prevalent among women of Western Sub-Saharan African ancestry. It accounts for 15-25% of African American (AA) breast cancers (BC) and up to 80% of Ghanaian breast cancers, thus contributing to outcome disparities in BC for black women. The aggressive biology of TNBC has been shown to be regulated partially by breast cancer stem cells (BCSC) which mediate tumor recurrence and metastasis and are more abundant in African breast tumors. METHODS: We studied the biological differences between TNBC in women with African ancestry and those of Caucasian women by comparing the gene expression of the BCSC. From low-passage patient derived xenografts (PDX) from Ghanaian (GH), AA, and Caucasian American (CA) TNBCs, we sorted for and sequenced the stem cell populations and analyzed for differential gene enrichment. RESULTS: In our cohort of TNBC tumors, we observed that the ALDH expressing stem cells display distinct ethnic specific gene expression patterns, with the largest difference existing between the GH and AA ALDH+ cells. Furthermore, the tumors from the women of African ancestry [GH/AA] had ALDH stem cell (SC) enrichment for expression of immune related genes and processes. Among the significantly upregulated genes were CD274 (PD-L1), CXCR9, CXCR10 and IFI27, which could serve as potential drug targets. CONCLUSIONS: Further exploration of the role of immune regulated genes and biological processes in BCSC may offer insight into developing novel approaches to treating TNBC to help ameliorate survival disparities in women with African ancestry.

Somatic inactivating PTPRJ mutations and dysregulated pathways identified in canine malignant melanoma by integrated comparative genomic analysis.

Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology. We mapped the genomic landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses. We identified novel predominantly truncating mutations in the putative tumor suppressor gene PTPRJ in 19% of cases. No BRAF mutations were detected, but activating RAS mutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes. MDM2 amplifications (24%) and TP53 mutations (19%) were mutually exclusive. Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations. Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition. This mutational landscape resembles that seen in BRAF wild-type and sun-shielded human melanoma subtypes. Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species.

Comprehensive molecular profiling of 718 Multiple Myelomas reveals significant differences in mutation frequencies between African and European descent cases.

Multiple Myeloma (MM) is a plasma cell malignancy with significantly greater incidence and mortality rates among African Americans (AA) compared to Caucasians (CA). The overall goal of this study is to elucidate differences in molecular alterations in MM as a function of self-reported race and genetic ancestry. Our study utilized somatic whole exome, RNA-sequencing, and correlated clinical data from 718 MM patients from the Multiple Myeloma Research Foundation CoMMpass study Interim Analysis 9. Somatic mutational analyses based upon self-reported race corrected for ancestry revealed significant differences in mutation frequency between groups. Of interest, BCL7A, BRWD3, and AUTS2 demonstrate significantly higher mutation frequencies among AA cases. These genes are all involved in translocations in B-cell malignancies. Moreover, we detected a significant difference in mutation frequency of TP53 and IRF4 with frequencies higher among CA cases. Our study provides rationale for interrogating diverse tumor cohorts to best understand tumor genomics across populations.

E6201, an intravenous MEK1 inhibitor, achieves an exceptional response in BRAF V600E-mutated metastatic malignant melanoma with brain metastases.

Malignant melanoma (MM) exhibits a high propensity for central nervous system dissemination with ~50% of metastatic MM patients developing brain metastases (BM). Targeted therapies and immune checkpoint inhibitors have improved overall survival for MM patients with BM. However, responses are usually of short duration and new agents that effectively penetrate the blood brain barrier (BBB) are needed. Here, we report a MM patient with BM who experienced an exceptional response to E6201, an ATP-competitive MEK1 inhibitor, on a Phase 1 study, with ongoing near-complete response and overall survival extending beyond 8 years. Whole exome and transcriptome sequencing revealed a high mutational burden tumor (22 mutations/Megabase) with homozygous BRAF V600E mutation. Correlative preclinical studies demonstrated broad activity for E6201 across BRAF V600E mutant melanoma cell lines and effective BBB penetration in vivo. Together, these results suggest that E6201 may represent a potential new treatment option for BRAF-mutant MM patients with BM.

Non-Invasive Electromagnetic Skin Patch Sensor to Measure Intracranial Fluid-Volume Shifts.

Elevated intracranial fluid volume can drive intracranial pressure increases, which can potentially result in numerous neurological complications or death. This study's focus was to develop a passive skin patch sensor for the head that would non-invasively measure cranial fluid volume shifts. The sensor consists of a single baseline component configured into a rectangular planar spiral with a self-resonant frequency response when impinged upon by external radio frequency sweeps. Fluid volume changes (10 mL increments) were detected through cranial bone using the sensor on a dry human skull model. Preliminary human tests utilized two sensors to determine feasibility of detecting fluid volume shifts in the complex environment of the human body. The correlation between fluid volume changes and shifts in the first resonance frequency using the dry human skull was classified as a second order polynomial with R² = 0.97. During preliminary and secondary human tests, a ≈24 MHz and an average of ≈45.07 MHz shifts in the principal resonant frequency were measured respectively, corresponding to the induced cephalad bio-fluid shifts. This electromagnetic resonant sensor may provide a non-invasive method to monitor shifts in fluid volume and assist with medical scenarios including stroke, cerebral hemorrhage, concussion, or monitoring intracranial pressure.

Case report: whole exome sequencing of primary cardiac angiosarcoma highlights potential for targeted therapies.

BACKGROUND: Primary cardiac angiosarcomas are rare, but they are the most aggressive type of primary cardiac neoplasms. When patients do present, it is with advanced pulmonary and/or cardiac symptoms. Therefore, many times the correct diagnosis is not made at the time of initial presentation. These patients have metastatic disease and the vast majority of these patients die within a few months after diagnosis. Currently the treatment choices are limited and there are no targeted therapies available. CASE PRESENTATION: A 56-year-old male presented with shortness of breath, night sweats, and productive cough for a month. Workup revealed pericardial effusion and multiple bilateral pulmonary nodules suspicious for metastatic disease. Transthoracic echocardiogram showed a large pericardial effusion and a large mass in the base of the right atrium. Results of biopsy of bilateral lung nodules established a diagnosis of primary cardiac angiosarcoma. Aggressive pulmonary disease caused rapid deterioration; the patient went on hospice and subsequently died. Whole exome sequencing of the patient's postmortem tumor revealed a novel KDR (G681R) mutation, and focal high-level amplification at chromosome 1q encompassing MDM4, a negative regulator of TP53. CONCLUSION: Mutations in KDR have been reported previously in angiosarcomas. Previous studies also demonstrated that KDR mutants with constitutive KDR activation could be inhibited with specific KDR inhibitors in vitro. Thus, patients harboring activating KDR mutations could be candidates for treatment with KDR-specific inhibitors.

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.

Long insert whole genome sequencing for copy number variant and translocation detection.

As next-generation sequencing continues to have an expanding presence in the clinic, the identification of the most cost-effective and robust strategy for identifying copy number changes and translocations in tumor genomes is needed. We hypothesized that performing shallow whole genome sequencing (WGS) of 900-1000-bp inserts (long insert WGS, LI-WGS) improves our ability to detect these events, compared with shallow WGS of 300-400-bp inserts. A priori analyses show that LI-WGS requires less sequencing compared with short insert WGS to achieve a target physical coverage, and that LI-WGS requires less sequence coverage to detect a heterozygous event with a power of 0.99. We thus developed an LI-WGS library preparation protocol based off of Illumina's WGS library preparation protocol and illustrate the feasibility of performing LI-WGS. We additionally applied LI-WGS to three separate tumor/normal DNA pairs collected from patients diagnosed with different cancers to demonstrate our application of LI-WGS on actual patient samples for identification of somatic copy number alterations and translocations. With the evolution of sequencing technologies and bioinformatics analyses, we show that modifications to current approaches may improve our ability to interrogate cancer genomes.

Simultaneous characterization of somatic events and HPV-18 integration in a metastatic cervical carcinoma patient using DNA and RNA sequencing.

OBJECTIVE: Integration of carcinogenic human papillomaviruses (HPVs) into the host genome is a significant tumorigenic factor in specific cancers including cervical carcinoma. Although major strides have been made with respect to HPV diagnosis and prevention, identification and development of efficacious treatments for cervical cancer patients remains a goal and thus requires additional detailed characterization of both somatic events and HPV integration. Given this need, the goal of this study was to use the next generation sequencing to simultaneously evaluate somatic alterations and expression changes in a patient's cervical squamous carcinoma lesion metastatic to the lung and to detect and analyze HPV infection in the same sample. MATERIALS AND METHODS: We performed tumor and normal exome, tumor and normal shallow whole-genome sequencing, and RNA sequencing of the patient's lung metastasis. RESULTS: We generated over 1.2 billion mapped reads and identified 130 somatic point mutations and indels, 21 genic translocations, 16 coding regions demonstrating copy number changes, and over 36 genes demonstrating altered expression in the tumor (corrected P < 0.05). Sequencing also revealed the HPV type 18 (HPV-18) integration in the metastasis. Using both DNA and RNA reads, we pinpointed 3 major events indicating HPV-18 integration into an intronic region of chromosome 6p25.1 in the patient's tumor and validated these events with Sanger sequencing. This integration site has not been reported for HPV-18. CONCLUSIONS: We demonstrate that DNA and RNA sequencing can be used to concurrently characterize somatic alterations and expression changes in a biopsy and delineate HPV integration at base resolution in cervical cancer. Further sequencing will allow us to better understand the molecular basis of cervical cancer pathogenesis.

Drug Delivery Strategies and Nanozyme Technologies to Overcome Limitations for Targeting Oxidative Stress in Osteoarthritis.

Oxidative stress is an important, but elusive, therapeutic target for osteoarthritis (OA). Antioxidant strategies that target oxidative stress through the elimination of reactive oxygen species (ROS) have been widely evaluated for OA but are limited by the physiological characteristics of the joint. Current hallmarks in antioxidant treatment strategies include poor bioavailability, poor stability, and poor retention in the joint. For example, oral intake of exogenous antioxidants has limited access to the joint space, and intra-articular injections require frequent dosing to provide therapeutic effects. Advancements in ROS-scavenging nanomaterials, also known as nanozymes, leverage bioactive material properties to improve delivery and retention. Material properties of nanozymes can be tuned to overcome physiological barriers in the knee. However, the clinical application of these nanozymes is still limited, and studies to understand their utility in treating OA are still in their infancy. The objective of this review is to evaluate current antioxidant treatment strategies and the development of nanozymes as a potential alternative to conventional small molecules and enzymes.

Hypertension contributes to exacerbated osteoarthritis pathophysiology in rats in a sex-dependent manner.

BACKGROUND: Hypertension is a common comorbidity of osteoarthritis (OA) with known autonomic dysregulation; thus, the autonomic nervous system may provide a shared underlying mechanism. The objective of this study was to examine the role of the autonomic nervous system in a preclinical model of OA and hypertension. METHODS: Experiments were conducted in spontaneously hypertensive rats and a normotensive control strain, including male and female rats. OA was surgically induced via medial meniscus transection with skin incision used as a sham control (n = 7-8/strain/sex/surgery). Tactile sensitivity, anxiety-related behavior, and serum corticosterone were measured at baseline then bi-weekly across 8 weeks. At weeks 9-10, cardiovascular responses to a chemical vagal nerve agonist were determined to indirectly evaluate vagus nerve function. The joint structure was assessed via grading of histological sections. RESULTS: In males, OA resulted in thinner cartilage in both hypertensive (OA vs. non-OA p < 0.001) and normotensive (OA vs. non-OA p < 0.001). Only females with comorbid hypertension and OA displayed thinner cartilage (p = 0.013). Male hypertensive OA animals had increased calcified subchondral bone compared to normotensive OA animals (p = 0.043) while female hypertensive OA animals had increased calcified subchondral bone compared to hypertensive sham animals (p < 0.001). All MCLT+MMT groups developed low-grade synovitis; interestingly, hypertensive OA females had higher synovitis scores than normotensive OA females (p = 0.046). Additionally, hypertension led to larger drops in blood pressure with vagal activation in both OA (hypertensive vs. normotensive p = 0.018) and sham (hypertensive vs. normotensive p < 0.001) male animals. In females, this trend held true only in OA animals (normotensive vs. hypertensive p = 0.005). CONCLUSION: These data provide preliminary evidence that hypertension influences OA progression and encourages further study into the autonomic nervous system as a possible mechanism.

Multi-omic molecular profiling guide's efficacious treatment selection in refractory metastatic breast cancer: a prospective phase II clinical trial.

This prospective phase II clinical trial (Side Out 2) explored the clinical benefits of treatment selection informed by multi-omic molecular profiling (MoMP) in refractory metastatic breast cancers (MBCs). Core needle biopsies were collected from 32 patients with MBC at trial enrollment. Patients had received an average of 3.94 previous lines of treatment in the metastatic setting before enrollment in this study. Samples underwent MoMP, including exome sequencing, RNA sequencing (RNA-Seq), immunohistochemistry, and quantitative protein pathway activation mapping by Reverse Phase Protein Microarray (RPPA). Clinical benefit was assessed using the previously published growth modulation index (GMI) under the hypothesis that MoMP-selected therapy would warrant further investigation for GMI ≥ 1.3 in ≥ 35% of the patients. Of the 32 patients enrolled, 29 received treatment based on their MoMP and 25 met the follow-up criteria established by the trial protocol. Molecular information was delivered to the tumor board in a median time frame of 14 days (11-22 days), and targetable alterations for commercially available agents were found in 23/25 patients (92%). Of the 25 patients, 14 (56%) reached GMI ≥ 1.3. A high level of DNA topoisomerase I (TOPO1) led to the selection of irinotecan-based treatments in 48% (12/25) of the patients. A pooled analysis suggested clinical benefit in patients with high TOPO1 expression receiving irinotecan-based regimens (GMI ≥ 1.3 in 66.7% of cases). These results confirmed previous observations that MoMP increases the frequency of identifiable actionable alterations (92% of patients). The MoMP proposed allows the identification of biomarkers that are frequently expressed in MBCs and the evaluation of their role as predictors of response to commercially available agents. Lastly, this study confirmed the role of MoMP for informing treatment selection in refractory MBC patients: more than half of the enrolled patients reached a GMI ≥ 1.3 even after multiple lines of previous therapies for metastatic disease.

Analytic validation and clinical utilization of the comprehensive genomic profiling test, GEM ExTra®.

We developed and analytically validated a comprehensive genomic profiling (CGP) assay, GEM ExTra, for patients with advanced solid tumors that uses Next Generation Sequencing (NGS) to characterize whole exomes employing a paired tumor-normal subtraction methodology. The assay detects single nucleotide variants (SNV), indels, focal copy number alterations (CNA), TERT promoter region, as well as tumor mutation burden (TMB) and microsatellite instability (MSI) status. Additionally, the assay incorporates whole transcriptome sequencing of the tumor sample that allows for the detection of gene fusions and select special transcripts, including AR-V7, EGFR vIII, EGFRvIV, and MET exon 14 skipping events. The assay has a mean target coverage of 180X for the normal (germline) and 400X for tumor DNA including enhanced probe design to facilitate the sequencing of difficult regions. Proprietary bioinformatics, paired with comprehensive clinical curation results in reporting that defines clinically actionable, FDA-approved, and clinical trial drug options for the management of the patient's cancer. GEM ExTra demonstrated analytic specificity (PPV) of > 99.9% and analytic sensitivity of 98.8%. Application of GEM ExTra to 1,435 patient samples revealed clinically actionable alterations in 83.9% of reports, including 31 (2.5%) where therapeutic recommendations were based on RNA fusion findings only.

Identifying treatment options for BRAFV600 wild-type metastatic melanoma: A SU2C/MRA genomics-enabled clinical trial.

Although combination BRAF and MEK inhibitors are highly effective for the 40-50% of cutaneous metastatic melanomas harboring BRAFV600 mutations, targeted agents have been ineffective for BRAFV600wild-type (wt) metastatic melanomas. The SU2C Genomics-Enabled Medicine for Melanoma Trial utilized a Simon two-stage optimal design to assess whether comprehensive genomic profiling improves selection of molecular-based therapies for BRAFV600wt metastatic melanoma patients who had progressed on standard-of-care therapy, which may include immunotherapy. Of the response-evaluable patients, binimetinib was selected for 20 patients randomized to the genomics-enabled arm, and nine were treated on the alternate treatment arm. Response rates for 27 patients treated with targeted recommendations included one (4%) partial response, 18 (67%) with stable disease, and eight (30%) with progressive disease. Post-trial genomic and protein pathway activation mapping identified additional drug classes that may be considered for future studies. Our results highlight the complexity and heterogeneity of metastatic melanomas, as well as how the lack of response in this trial may be associated with limitations including monotherapy drug selection and the dearth of available single and combination molecularly-driven therapies to treat BRAFV600wt metastatic melanomas.

In situ fixation and subsequent collection of cultured endothelial cells in a shear flow.

In situ fixation of adherent cells is a necessary process for downstream assays. Current methods to dissociate adherent endothelial cells require the use of a cell scraper that may introduce variability in nuclear morphology. Also, a cell scraper is not an option for experiments using sealed flow chambers. HMEC-1 cells were sheared at 5 dyn/cm2 for 24 h and then fixed in situ, quenched, and dissociated at the same shear rate. Analysis revealed no statistically significant change in nuclear shape between the steps of fixation and dissociation. This method outlines an alternative for the dissociation of adherent sheared endothelial cells after being fixed in situ in a micro-scale channel without causing a change in the nuclear morphology. •This method can be used with any commercially available, or custom-made, flow chamber and flow system.•Allows for downstream experimentation with adherent cells fixed in situ, such as Hi-C analysis, without impacting nuclear morphology or chromatin organization.•Cells are cultured, fixed, and dissociated at the same shear rate. Using the same shear rate for each step yields results that are not influenced by variable forces.

Canine osteosarcoma genome sequencing identifies recurrent mutations in DMD and the histone methyltransferase gene SETD2.

Osteosarcoma (OS) is a rare, metastatic, human adolescent cancer that also occurs in pet dogs. To define the genomic underpinnings of canine OS, we performed multi-platform analysis of OS tumors from 59 dogs, including whole genome sequencing (n = 24) and whole exome sequencing (WES; n = 13) of primary tumors and matched normal tissue, WES (n = 10) of matched primary/metastatic/normal samples and RNA sequencing (n = 54) of primary tumors. We found that canine OS recapitulates features of human OS including low point mutation burden (median 1.98 per Mb) with a trend towards higher burden in metastases, high structural complexity, frequent TP53 (71%), PI3K pathway (37%), and MAPK pathway mutations (17%), and low expression of immune-associated genes. We also identified novel features of canine OS including putatively inactivating somatic SETD2 (42%) and DMD (50%) aberrations. These findings set the stage for understanding OS development in dogs and humans, and establish genomic contexts for future comparative analyses.

A prospective pilot study of genome-wide exome and transcriptome profiling in patients with small cell lung cancer progressing after first-line therapy.

BACKGROUND: Small cell lung cancer (SCLC) that has progressed after first-line therapy is an aggressive disease with few effective therapeutic strategies. In this prospective study, we employed next-generation sequencing (NGS) to identify therapeutically actionable alterations to guide treatment for advanced SCLC patients. METHODS: Twelve patients with SCLC were enrolled after failing platinum-based chemotherapy. Following informed consent, genome-wide exome and RNA-sequencing was performed in a CLIA-certified, CAP-accredited environment. Actionable targets were identified and therapeutic recommendations made from a pharmacopeia of FDA-approved drugs. Clinical response to genomically-guided treatment was evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. RESULTS: The study completed its accrual goal of 12 evaluable patients. The minimum tumor content for successful NGS was 20%, with a median turnaround time from sample collection to genomics-based treatment recommendation of 27 days. At least two clinically actionable targets were identified in each patient, and six patients (50%) received treatment identified by NGS. Two had partial responses by RECIST 1.1 on a clinical trial involving a PD-1 inhibitor + irinotecan (indicated by MLH1 alteration). The remaining patients had clinical deterioration before NGS recommended therapy could be initiated. CONCLUSIONS: Comprehensive genomic profiling using NGS identified clinically-actionable alterations in SCLC patients who progressed on initial therapy. Recommended PD-1 therapy generated partial responses in two patients. Earlier access to NGS guided therapy, along with improved understanding of those SCLC patients likely to respond to immune-based therapies, should help to extend survival in these cases with poor outcomes.

A method to reduce ancestry related germline false positives in tumor only somatic variant calling.

BACKGROUND: Significant clinical and research applications are driving large scale adoption of individualized tumor sequencing in cancer in order to identify tumors-specific mutations. When a matched germline sample is available, somatic mutations may be identified using comparative callers. However, matched germline samples are frequently not available such as with archival tissues, which makes it difficult to distinguish somatic from germline variants. While population databases may be used to filter out known germline variants, recent studies have shown private germline variants result in an inflated false positive rate in unmatched tumor samples, and the number germline false positives in an individual may be related to ancestry. METHODS: First, we examined the relationship between the germline false positives and ancestry. Then we developed and implemented a tumor only caller (LumosVar) that leverages differences in allelic frequency between somatic and germline variants in impure tumors. We used simulated data to systematically examine how copy number alterations, tumor purity, and sequencing depth should affect the sensitivity of our caller. Finally, we evaluated the caller on real data. RESULTS: We find the germline false-positive rate is significantly higher for individuals of non-European Ancestry largely due to the limited diversity in public polymorphism databases and due to population-specific characteristics such as admixture or recent expansions. Our Bayesian tumor only caller (LumosVar) is able to greatly reduce false positives from private germline variants, and our sensitivity is similar to predictions based on simulated data. CONCLUSIONS: Taken together, our results suggest that studies of individuals of non-European ancestry would most benefit from our approach. However, high sensitivity requires sufficiently impure tumors and adequate sequencing depth. Even in impure tumors, there are copy number alterations that result in germline and somatic variants having similar allele frequencies, limiting the sensitivity of the approach. We believe our approach could greatly improve the analysis of archival samples in a research setting where the normal is not available.