Severus: accurate detection and characterization of somatic structural variation in tumor genomes using long reads.

Most current studies rely on short-read sequencing to detect somatic structural variation (SV) in cancer genomes. Long-read sequencing offers the advantage of better mappability and long-range phasing, which results in substantial improvements in germline SV detection. However, current long-read SV detection methods do not generalize well to the analysis of somatic SVs in tumor genomes with complex rearrangements, heterogeneity, and aneuploidy. Here, we present Severus: a method for the accurate detection of different types of somatic SVs using a phased breakpoint graph approach. To benchmark various short- and long-read SV detection methods, we sequenced five tumor/normal cell line pairs with Illumina, Nanopore, and PacBio sequencing platforms; on this benchmark Severus showed the highest F1 scores (harmonic mean of the precision and recall) as compared to long-read and short-read methods. We then applied Severus to three clinical cases of pediatric cancer, demonstrating concordance with known genetic findings as well as revealing clinically relevant cryptic rearrangements missed by standard genomic panels.

Improved detection of low-frequency within-host variants from deep sequencing: A case study with human papillomavirus.

High-coverage sequencing allows the study of variants occurring at low frequencies within samples, but is susceptible to false-positives caused by sequencing error. Ion Torrent has a very low single nucleotide variant (SNV) error rate and has been employed for the majority of human papillomavirus (HPV) whole genome sequences. However, benchmarking of intrahost SNVs (iSNVs) has been challenging, partly due to limitations imposed by the HPV life cycle. We address this problem by deep sequencing three replicates for each of 31 samples of HPV type 18 (HPV18). Errors, defined as iSNVs observed in only one of three replicates, are dominated by C→T (G→A) changes, independently of trinucleotide context. True iSNVs, defined as those observed in all three replicates, instead show a more diverse SNV type distribution, with particularly elevated C→T rates in CCG context (CCG→CTG; CGG→CAG) and C→A rates in ACG context (ACG→AAG; CGT→CTT). Characterization of true iSNVs allowed us to develop two methods for detecting true variants: (1) VCFgenie, a dynamic binomial filtering tool which uses each variant's allele count and coverage instead of fixed frequency cut-offs; and (2) a machine learning binary classifier which trains eXtreme Gradient Boosting models on variant features such as quality and trinucleotide context. Each approach outperforms fixed-cut-off filtering of iSNVs, and performance is enhanced when both are used together. Our results provide improved methods for identifying true iSNVs in within-host applications across sequencing platforms, specifically using HPV18 as a case study.

Association Between Chondrolabral Junction Breakdown and Conversion to Total Hip Arthroplasty After Hip Arthroscopy for Symptomatic Labral Tears: Minimum 8-Year Follow-up.

BACKGROUND: Arthroscopic treatment of femoroacetabular impingement (FAI) and symptomatic labral tears confers short- to midterm benefits, yet further long-term evidence is needed. Moreover, despite the physiological and biomechanical significance of the chondrolabral junction (CLJ), the clinical implications of damage to this transition zone remain understudied. PURPOSE: To (1) report minimum 8-year survivorship and patient-reported outcome measures after hip arthroscopy for FAI and (2) characterize associations between outcomes and patient characteristics (age, body mass index, sex), pathological parameters (Tönnis angle, alpha angle, type of FAI, CLJ breakdown), and procedures performed (labral management, FAI treatment, microfracture). STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: This retrospective cohort study included patients who underwent primary hip arthroscopy for symptomatic labral tears secondary to FAI by a single surgeon between 2002 and 2013. All patients were ≥18 years of age with minimum 8-year follow-up and available preoperative radiographs. The primary outcome was conversion to total hip arthroplasty (THA), and secondary outcomes included revision arthroscopy, patient-reported outcome measures, and patient satisfaction. CLJ breakdown was assessed using the Beck classification. Kaplan-Meier estimates and weighted Cox regression were used to estimate 10-year survivorship (no conversion to THA) and identify risk factors associated with THA conversion. RESULTS: In this study of 174 hips (50.6% female; mean age, 37.8 ± 11.2 years) with mean follow-up of 11.1 ± 2.5 years, the 10-year survivorship rate was 81.6% (95% CI, 75.9%-87.7%). Conversion to THA occurred at a mean 4.7 ± 3.8 years postoperatively. Unadjusted analyses revealed several variables significantly associated with THA conversion, including older age; higher body mass index; higher Tönnis grade; labral debridement; and advanced breakdown of the CLJ, labrum, or articular cartilage. Survivorship at 10 years was inferior in patients exhibiting severe (43.6%; 95% CI, 31.9%-59.7%) versus mild (97.9%; 95% CI, 95.1%-100%) breakdown of the CLJ (P < .001). Multivariable analysis identified worsening CLJ breakdown (weighted hazard ratio per 1-unit increase, 6.41; 95% CI, 3.11-13.24), older age (1.09; 95% CI, 1.04-1.14), and higher Tönnis grade (4.59; 95% CI, 2.13-9.90) as independent negative prognosticators (P < .001 for all). CONCLUSION: Although most patients achieved favorable minimum 8-year outcomes, several pre- and intraoperative factors were associated with THA conversion; of these, worse CLJ breakdown, higher Tönnis grade, and older age were the strongest predictors.

Germline pathogenic variants associated with triple-negative breast cancer in US Hispanic and Guatemalan women using hospital and community-based recruitment strategies.

PURPOSE: Recruit and sequence breast cancer subjects in Guatemalan and US Hispanic populations. Identify optimum strategies to recruit Latin American and Hispanic women into genetic studies of breast cancer. METHODS: We used targeted gene sequencing to identify pathogenic variants in 19 familial breast cancer susceptibility genes in DNA from unselected Hispanic breast cancer cases in the US and Guatemala. Recruitment across the US was achieved through community-based strategies. In addition, we obtained patients receiving cancer treatment at major hospitals in Texas and Guatemala. RESULTS: We recruited 287 Hispanic US women, 38 (13%) from community-based and 249 (87%) from hospital-based strategies. In addition, we ascertained 801 Guatemalan women using hospital-based recruitment. In our experience, a hospital-based approach was more efficient than community-based recruitment. In this study, we sequenced 103 US and 137 Guatemalan women and found 11 and 10 pathogenic variants, respectively. The most frequently mutated genes were BRCA1, BRCA2, CHEK2, and ATM. In addition, an analysis of 287 US Hispanic patients with pathology reports showed a significantly higher percentage of triple-negative disease in patients with pathogenic variants (41% vs. 15%). Finally, an analysis of mammography usage in 801 Guatemalan patients found reduced screening in women with a lower socioeconomic status (p < 0.001). CONCLUSION: Guatemalan and US Hispanic women have rates of hereditary breast cancer pathogenic variants similar to other populations and are more likely to have early age at diagnosis, a family history, and a more aggressive disease. Patient recruitment was higher using hospital-based versus community enrollment. This data supports genetic testing in breast cancer patients to reduce breast cancer mortality in Hispanic women.

Insights into the mechanisms and structure of breakage-fusion-bridge cycles in cervical cancer using long-read sequencing.

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cancer cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and our study applies long-read sequencing to this important chromosomal rearrangement type. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes, and only one BFB breakpoint showed chromothripsis. Five cell lines have a chromosomal region 11q BFB event, with YAP1-BIRC3-BIRC2 amplification. Indeed, YAP1 amplification is associated with a 10-year-earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that individuals with cervical cancer and YAP1-BIRC3-BIRC2 amplification, especially those of African ancestry, might benefit from targeted therapy. In summary, we uncovered valuable insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

The Effect of Pelvic Incidence on Outcomes After Hip Arthroscopy for Femoroacetabular Impingement and Acetabular Labral Tears.

BACKGROUND: In the setting of femoroacetabular impingement (FAI), decompression osteoplasties reconcile deleterious loading patterns caused by cam and pincer lesions. However, native variations of spinopelvic sagittal alignment may continue to perpetuate detrimental effects on the labrum, chondrolabral junction, and articular cartilage after hip arthroscopy. PURPOSE: To evaluate the effect of pelvic incidence (PI) on postoperative outcomes after hip arthroscopy for acetabular labral tears in the setting of FAI. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A retrospective query of prospectively collected data identified patients ≥18 years of age who underwent primary hip arthroscopy for FAI and acetabular labral tears between February 2014 and January 2022, with 3-, 6-, 12-, and 24-month follow-ups. Measurements for PI, pelvic tilt (PT), sacral slope (SS), and acetabular version were obtained via advanced diagnostic imaging. Patients were stratified into low-PI (<45°), moderate-PI (45°≤ PI ≤ 60°), and high-PI (>60°) cohorts. Patient-reported outcome measures (PROMs), clinically meaningful outcomes (ie, minimal clinically important difference, Patient Acceptable Symptom State, substantial clinical benefit, and maximal outcome improvement), visual analog scale (VAS) pain scores, and patient satisfaction were compared across cohorts. RESULTS: A total of 74 patients met eligibility criteria and were stratified into low-PI (n = 28), moderate-PI (n = 31), and high-PI (n = 15) cohorts. Correspondingly, patients with high PI displayed significantly greater values for PT (P = .001), SS (P < .001), acetabular version (P < .001), and acetabular inclination (P = .049). By the 12- and 24-month follow-ups, the high-PI cohort was found to have significantly inferior PROMs, VAS pain scores, rates of clinically meaningful outcome achievement, and satisfaction relative to patients with moderate and/or low PI. No significant differences were found between cohorts regarding rates of revision arthroscopy, subsequent spine surgery, or conversion to total hip arthroplasty. CONCLUSION: After hip arthroscopy, patients with a high PI (>60°) exhibited inferior PROMs, rates of achieving clinically meaningful thresholds, and satisfaction at 12 and 24 months relative to patients with low or moderate PI. Conversely, the outcomes of patients with low PI (<45°) were found to match the trajectory of those with a neutral spinopelvic alignment (45°≤ PI ≤ 60°). These findings highlight the importance of analyzing spinopelvic parameters preoperatively to prognosticate outcomes before hip arthroscopy for acetabular labral tears and FAI.

Preoperative Paralabral Cysts Predict More Significant Chondral Damage but Not 2-Year Functional Outcomes Following Arthroscopic Labral Repair.

PURPOSE: To investigate whether paralabral cysts identified incidentally on preoperative magnetic resonance imaging (MRI/MRA) predict 2-year functional outcomes after arthroscopic acetabular labral repair. METHODS: Prospectively collected data for patients undergoing primary hip arthroscopy by a single surgeon from 2014 to 2020 were retrospectively reviewed. Included patients were ≥18 years and completed baseline patient-reported outcome measures (PROMs) with additional follow-up at 3, 6, 12, and 24 months. Exclusion criteria were labral debridement, hip dysplasia, advanced hip osteoarthritis (Tönnis >1), or previous ipsilateral hip surgery. Patients were stratified based on the presence of paralabral cysts identified on MRI/MRA. Primary outcomes were International Hip Outcome Tool (iHOT-33) and modified Harris Hip Score (mHHS). Secondary outcomes included other PROMs and the visual analog pain scale. Outcomes were compared between cohorts using linear mixed-effects models and Fisher's exact tests. Sensitivity analyses accounted for preoperative PROMs, nonlinear improvement trajectories, and relevant baseline characteristics. RESULTS: Of the 182 included hips (47.8% female; mean ± standard deviation age, 36.9 ± 11.4), 30 (16.4%) had paralabral cysts. During the 2-year study period, there were no significant differences between patients with and without paralabral cysts in terms of iHOT-33 scores (weighted difference = 1.60; 95% confidence interval [CI], -5.09, 8.28; P = .64), mHHS scores (weighted difference = 0.56; 95% CI, -4.16, 5.28; P = .82), or any secondary outcomes (except for HOS-Sports Subscale at 3 months [mean difference = -11.85; 95% CI, -22.85, -0.84; P = .035]). Furthermore, there were no significant differences in clinically meaningful outcomes (P > .05 for all), revision rates (P = 1.00), or conversion to total hip arthroplasty between cohorts (P = 1.00). These results held across all sensitivity analyses. CONCLUSIONS: Although preoperative paralabral cysts were associated with worse cam impingement and more severe chondral damage observed intraoperatively, they did not predict 2-year functional outcomes or clinically meaningful improvements, suggesting that incidentally discovered paralabral cysts are not a contraindication for arthroscopic labral repair. LEVEL OF EVIDENCE: Level III, retrospective cohort study.

Insights into the Mechanisms and Structure of Breakage-Fusion-Bridge Cycles in Cervical Cancer using Long-Read Sequencing.

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and this is one of the first analyses of these events using long-read sequencing. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes and only one BFB breakpoint showed chromothripsis. Five cell lines have a Chr11q BFB event, with YAP1/BIRC2/BIRC3 gene amplification. Indeed, YAP1 amplification is associated with a 10-year earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that cervical cancer patients with YAP1/BIRC2/BIRC3-amplification, especially those of African American ancestry, might benefit from targeted therapy. In summary, we uncovered new insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

Outcomes of Hip Arthroscopy in the Setting of Concomitant Symptomatic Lumbosacral Spine Pathology: A Matched Control Study With Minimum 24-Month Follow-up.

BACKGROUND: The overlapping biomechanical relationship between the lumbosacral spine and pelvis poses unique challenges to patients with concomitant pathologies limiting spinopelvic range of motion. PURPOSE: To assess the influence of concomitant, symptomatic lumbosacral spine pathology on patient-reported outcome measures (PROMs) after hip arthroscopy for the treatment of femoroacetabular impingement (FAI) and symptomatic labral tears. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A retrospective query of prospectively collected data identified patients aged ≥18 years with a minimum 24-month follow-up who underwent hip arthroscopy by a single surgeon for the treatment of symptomatic labral tears secondary to FAI. Patients were stratified into cohorts based on the presence (hip-spine [HS]) or absence (matched control [MC]) of symptomatic lumbosacral spine pathology. Inclusion within the HS cohort required confirmation of lower back pain/symptoms on preoperative surveys plus a diagnosis of lumbosacral spine pathology verified by radiology reports and correlating clinical documentation. Patients with previous spine surgery were excluded. PROMs were compared between groups, along with rates of achieving minimal clinically important difference (MCID) thresholds, Patient Acceptable Symptom State (PASS) thresholds, revision arthroscopy, and conversion to total hip arthroplasty (THA). RESULTS: A total of 70 patients with lumbosacral pathology were coarsened exact matched to 87 control patients without spinal pathology. The HS cohort had preoperative baseline scores that were significantly worse for nearly all PROMs. Follow-ups at 3, 6, 12, and 24 months displayed similar trends, with the HS cohort demonstrating significantly worse scores for most collected outcomes. However, at every time point, HS and MC patients exhibited similar magnitudes of improvement across all PROM and pain metrics. Furthermore, while significantly fewer HS patients achieved PASS for nearly all PROMs at 12- and 24-month follow-ups, MCID thresholds were reached at similar or greater rates across all PROMs relative to the MC cohort. Finally, there were no significant differences in rates of revision or THA between cohorts at maximum available follow-up. CONCLUSION: After hip arthroscopy to address labral tears in the setting of FAI, patients with symptomatic lumbosacral pathologies and no history of spine surgery were found to exhibit inferior pre- and postoperative PROMs but achieved statistically similar clinical benefit and rates of PROM improvement through 24-month follow-up compared with the MC cohort with isolated hip disease. These findings aid in providing a realistic recovery timeline and evidence that coexisting hip and spine disorders are not a contraindication for arthroscopic hip preservation surgery.

Labral Reconstruction via Capsular Augmentation Maintains Perfusion to the Acetabular Labrum and Locally Transferred Autograft: An in Vivo Laser Doppler Flowmetry Analysis.

Background: The purpose of the present study was to examine the effects of arthroscopic labral repair with capsular augmentation on blood flow in vivo with use of laser Doppler flowmetry (LDF) to measure microvascular perfusion of the labrum and autograft tissue. Methods: The present prospective case series included patients ≥18 years old who underwent arthroscopic acetabular labral repair with capsular augmentation; all procedures were performed by a single surgeon between 2018 and 2022. The LDF probe measured microvascular blood flow flux within 1 mm3 of the surrounding labral and capsular tissue of interest. Mean baseline measurements of flux were compared with readings immediately following capsular elevation and after completing labral augmentation. Blood flux changes were expressed as the percent change from the baseline measurements. Results: The present study included 41 patients (24 men [58.5%] and 17 women [41.5%]) with a mean age (and standard deviation) of 31.3 ± 8.4 years, a mean BMI of 24.6 ± 3.4 kg/m2, a mean lateral center-edge of angle 35.3° ± 4.9°, a mean Tönnis angle of 5.8° ± 5.8°, and a mean arterial pressure of 93.7 ± 10.9 mm Hg. Following capsular elevation, the mean percent change in capsular blood flow flux was significantly different from baseline (-9.24% [95% confidence interval (CI), -18.1% to -0.04%]; p < 0.001). Following labral augmentation, the mean percent change in labral blood flow flux was significantly different from baseline both medially (-22.3% [95% CI, -32.7% to -11.9%]; p < 0.001) and laterally (-32.5% [95% CI, -41.5% to -23.6%]; p = 0.041). There was no significant difference between the changes in medial and lateral perfusion following repair (p = 0.136). Conclusions: Labral repair with capsular augmentation sustains a reduced blood flow to the native labrum and capsular tissue at the time of fixation. The biological importance of this reduction is unknown, but these findings may serve as a benchmark for other labral preservation techniques and support future correlations with clinical outcomes. Level of Evidence: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Analysis of Several Common APOBEC-type Mutations in Bladder Tumors Suggests Links to Viral Infection.

FGFR3 and PIK3CA are among the most frequently mutated genes in bladder tumors. We hypothesized that recurrent mutations in these genes might be caused by common carcinogenic exposures such as smoking and other factors. We analyzed 2,816 bladder tumors with available data on FGFR3 and/or PIK3CA mutations, focusing on the most recurrent mutations detected in ≥10% of tumors. Compared to tumors with other FGFR3/PIK3CA mutations, FGFR3-Y375C was more common in tumors from smokers than never-smokers (P = 0.009), while several APOBEC-type driver mutations were enriched in never-smokers: FGFR3-S249C (P = 0.013) and PIK3CA-E542K/PIK3CA-E545K (P = 0.009). To explore possible causes of these APOBEC-type mutations, we analyzed RNA sequencing (RNA-seq) data from 798 bladder tumors and detected several viruses, with BK polyomavirus (BKPyV) being the most common. We then performed IHC staining for polyomavirus (PyV) Large T-antigen (LTAg) in an independent set of 211 bladder tumors. Overall, by RNA-seq or IHC-LTAg, we detected PyV in 26 out of 1,010 bladder tumors with significantly higher detection (P = 4.4 × 10-5), 25 of 554 (4.5%) in non-muscle-invasive bladder cancers (NMIBC) versus 1 of 456 (0.2%) of muscle-invasive bladder cancers (MIBC). In the NMIBC subset, the FGFR3/PIK3CA APOBEC-type driver mutations were detected in 94.7% (18/19) of PyV-positive versus 68.3% (259/379) of PyV-negative tumors (P = 0.011). BKPyV tumor positivity in the NMIBC subset with FGFR3- or PIK3CA-mutated tumors was also associated with a higher risk of progression to MIBC (P = 0.019). In conclusion, our results support smoking and BKPyV infection as risk factors contributing to bladder tumorigenesis in the general patient population through distinct molecular mechanisms. PREVENTION RELEVANCE: Tobacco smoking likely causes one of the most common mutations in bladder tumors (FGFR3-Y375C), while viral infections might contribute to three others (FGFR3-S249C, PIK3CA-E542K, and PIK3CA-E545K). Understanding the causes of these mutations may lead to new prevention and treatment strategies, such as viral screening and vaccination.

Operative management of rotator cuff tears: identifying disparities in access on a national level.

BACKGROUND: The purpose of this study was to identify nationwide disparities in the rates of operative management of rotator cuff tears based on race, ethnicity, insurance type, and socioeconomic status. METHODS: Patients diagnosed with a full or partial rotator cuff tear from 2006 to 2014 were identified in the Healthcare Cost and Utilization Project's National Inpatient Sample database using International Classification of Diseases, Ninth Revision diagnosis codes. Bivariate analysis using chi-square tests and adjusted, multivariable logistic regression models were used to evaluate differences in the rates of operative vs. nonoperative management for rotator cuff tears. RESULTS: This study included 46,167 patients. When compared with white patients, adjusted analysis showed that minority race and ethnicity were associated with lower rates of operative management for Black (adjusted odds ratio [AOR]: 0.31, 95% confidence interval [CI]: 0.29-0.33; P < .001), Hispanic (AOR: 0.49, 95% CI: 0.45-0.52; P < .001), Asian or Pacific Islander (AOR: 0.72, 95% CI: 0.61-0.84; P < .001), and Native American patients (AOR: 0.65, 95% CI: 0.50-0.86; P = .002). In comparison to privately insured patients, our analysis also found that self-payers (AOR: 0.08, 95% CI: 0.07-0.10; P < .001), Medicare beneficiaries (AOR: 0.76, 95% CI: 0.72-0.81; P < .001), and Medicaid beneficiaries (AOR: 0.33, 95% CI: 0.30-0.36; P < .001) had lower odds of receiving surgical intervention. Additionally, relative to those in the bottom income quartile, patients in all other quartiles experienced nominally higher rates of operative repair; these differences were statistically significant for the second quartile (AOR: 1.09, 95% CI: 1.03-1.16; P = .004). CONCLUSION: There are significant nationwide disparities in the likelihood of receiving operative management for rotator cuff tear patients of differing race/ethnicity, payer status, and socioeconomic status. Further investigation is needed to fully understand and address causes of these discrepancies to optimize care pathways.

Nitrated Polycyclic Aromatic Hydrocarbon (Nitro-PAH) Signatures and Somatic Mutations in Diesel Exhaust-Exposed Bladder Tumors.

BACKGROUND: Diesel exhaust is a complex mixture, including polycyclic aromatic hydrocarbons (PAH) and nitrated PAHs (nitro-PAH), many of which are potent mutagens and possible bladder carcinogens. To explore the association between diesel exposure and bladder carcinogenesis, we examined the relationship between exposure and somatic mutations and mutational signatures in bladder tumors. METHODS: Targeted sequencing was conducted in bladder tumors from the New England Bladder Cancer Study. Using data on 797 cases and 1,418 controls, two-stage polytomous logistic regression was used to evaluate etiologic heterogeneity between bladder cancer subtypes and quantitative, lifetime estimates of respirable elemental carbon (REC), a surrogate for diesel exposure. Poisson regression was used to evaluate associations between REC and mutational signatures. RESULTS: We observed significant heterogeneity in the diesel-bladder cancer risk relationship, with a strong positive association among cases with high-grade, nonmuscle invasive TP53-mutated tumors compared with controls [ORTop Tertile vs.Unexposed, 4.8; 95% confidence interval (CI), 2.2-10.5; Ptrend < 0.001; Pheterogeneity = 0.002]. In muscle-invasive tumors, we observed a positive association between diesel exposure and the nitro-PAH signatures of 1,6-dintropyrene (RR, 1.93; 95% CI, 1.28-2.92) and 3-nitrobenzoic acid (RR, 1.97; 95% CI, 1.33-2.92). CONCLUSIONS: The relationship between diesel exhaust and bladder cancer was heterogeneous based on the presence of TP53 mutations in tumors, further supporting the link between PAH exposure and TP53 mutations in carcinogenesis. Future studies that can identify nitro-PAH signatures in exposed tumors are warranted to add human data supporting the link between diesel and bladder cancer. IMPACT: This study provides additional insight into the etiology and possible mechanisms related to diesel exhaust-induced bladder cancer.

Evidence for virus-mediated oncogenesis in bladder cancers arising in solid organ transplant recipients.

A small percentage of bladder cancers in the general population have been found to harbor DNA viruses. In contrast, up to 25% of tumors of solid organ transplant recipients, who are at an increased risk of developing bladder cancer and have an overall poorer outcomes, harbor BK polyomavirus (BKPyV). To better understand the biology of the tumors and the mechanisms of carcinogenesis from potential oncoviruses, we performed whole genome and transcriptome sequencing on bladder cancer specimens from 43 transplant patients. Nearly half of the tumors from this patient population contained viral sequences. The most common were from BKPyV (N=9, 21%), JC polyomavirus (N=7, 16%), carcinogenic human papillomaviruses (N=3, 7%), and torque teno viruses (N=5, 12%). Immunohistochemistry revealed variable Large T antigen expression in BKPyV-positive tumors ranging from 100% positive staining of tumor tissue to less than 1%. In most cases of BKPyV-positive tumors, the viral genome appeared to be clonally integrated into the host chromosome consistent with microhomology-mediated end joining and coincided with focal amplifications of the tumor genome similar to other virus-mediated cancers. Significant changes in host gene expression consistent with the functions of BKPyV Large T antigen were also observed in these tumors. Lastly, we identified four mutation signatures in our cases, with those attributable to APOBEC3 and SBS5 being the most abundant. Mutation signatures associated with an antiviral drug, ganciclovir, and aristolochic acid, a nephrotoxic compound found in some herbal medicines, were also observed. The results suggest multiple pathways to carcinogenesis in solid organ transplant recipients with a large fraction being virus-associated.

Extrachromosomal Amplification of Human Papillomavirus Episomes Is a Mechanism of Cervical Carcinogenesis.

SIGNIFICANCE: Multimers of the HPV genome are generated in cervical tumors replicating as extrachromosomal episomes, which is associated with deletion and rearrangement of the HPV genome and provides a mechanism for oncogenesis without integration.

Spatially resolved gene regulatory and disease-related vulnerability map of the adult Macaque cortex.

Single cell approaches have increased our knowledge about the cell type composition of the non-human primate (NHP), but a detailed characterization of area-specific regulatory features remains outstanding. We generated single-cell transcriptomic and chromatin accessibility (single-cell ATAC) data of 358,237 cells from prefrontal cortex (PFC), primary motor cortex (M1) and primary visual cortex (V1) of adult female cynomolgus monkey brain, and integrated this dataset with Stereo-seq (spatial enhanced resolution omics-sequencing) of the corresponding cortical areas to assign topographic information to molecular states. We identified area-specific chromatin accessible sites and their targeted genes, including the cell type-specific transcriptional regulatory network associated with excitatory neurons heterogeneity. We reveal calcium ion transport and axon guidance genes related to specialized functions of PFC and M1, identified the similarities and differences between adult macaque and human oligodendrocyte trajectories, and mapped the genetic variants and gene perturbations of human diseases to NHP cortical cells. This resource establishes a transcriptomic and chromatin accessibility combinatory regulatory landscape at a single-cell and spatially resolved resolution in NHP cortex.

HPV16 E7 Nucleotide Variants Found in Cancer-Free Subjects Affect E7 Protein Expression and Transformation.

The human papillomavirus (HPV) type 16 E7 oncogene is critical to carcinogenesis and highly conserved. Previous studies identified a preponderance of non-synonymous E7 variants amongst HPV16-positive cancer-free controls compared to those with cervical cancer. To investigate the function of E7 variants, we constructed full-length HPV16 E7 genes and tested variants at positions H9R, D21N, N29S, E33K, T56I, D62N, S63F, S63P, T64M, E80K, D81N, P92L, and P92S (found only in controls); D14E, N29H cervical intraepithelial neoplasia (CIN2), and P6L, H51N, R77S (CIN3). We determined the steady-state level of cytoplasmic and nuclear HPV16 E7 protein. All variants from controls showed a reduced level of E7 protein, with 7/13 variants having lower protein levels. In contrast, 2/3 variants from the CIN3 precancer group had near-wild type E7 levels. We assayed the activity of representative variants in stably transfected NIH3T3 cells. The H9R, E33K, P92L, and P92S variants found in control subjects had lower transforming activity than D14E and N29H variants (CIN2), and the R77S (CIN3) had activity only slightly reduced from wild-type E7. In addition, R77S and WT E7 caused increased migration of NIH3T3 cells in a wound-healing assay compared with H9R, E33K, P92L, and P92S (controls) and D14E (CIN2). These data provide evidence that the E7 variants found in HPV16-positive cancer-free women are partially defective for transformation and cell migration, further demonstrating the importance of fully active E7 in cancer development.

Characterization of Philadelphia-like Pre-B Acute Lymphoblastic Leukemia: Experiences in Mexican Pediatric Patients.

Ph-like subtypes with CRLF2 abnormalities are frequent among Hispano-Latino children with pre-B ALL. Therefore, there is solid ground to suggest that this subtype is frequent in Mexican patients. The genomic complexity of Ph-like subtype constitutes a challenge for diagnosis, as it requires diverse genomic methodologies that are not widely available in diagnostic centers in Mexico. Here, we propose a diagnostic strategy for Ph-like ALL in accordance with our local capacity. Pre-B ALL patients without recurrent gene fusions (104) were classified using a gene-expression profile based on Ph-like signature genes analyzed by qRT-PCR. The expressions of the CRLF2 transcript and protein were determined by qRT-PCR and flow cytometry. The P2RY8::CRLF2, IGH::CRLF2, ABL1/2 rearrangements, and Ik6 isoform were screened using RT-PCR and FISH. Surrogate markers of Jak2-Stat5/Abl/Ras pathways were analyzed by phosphoflow. Mutations in relevant kinases/transcription factors genes in Ph-like were assessed by target-specific NGS. A total of 40 patients (38.5%) were classified as Ph-like; of these, 36 had abnormalities associated with Jak2-Stat5 and 4 had Abl. The rearrangements IGH::CRLF2,P2RY8::CRLF2, and iAMP21 were particularly frequent. We propose a strategy for the detection of Ph-like patients, by analyzing the overexpression/genetic lesions of CRLF2, the Abl phosphorylation of surrogate markers confirmed by gene rearrangements, and Sanger sequencing.

The human ATP-binding cassette (ABC) transporter superfamily.

The ATP-binding cassette (ABC) transporter superfamily comprises membrane proteins that efflux various substrates across extra- and intracellular membranes. Mutations in ABC genes cause 21 human disorders or phenotypes with Mendelian inheritance, including cystic fibrosis, adrenoleukodystrophy, retinal degeneration, cholesterol, and bile transport defects. To provide tools to study the function of human ABC transporters we compiled data from multiple genomics databases. We analyzed ABC gene conservation within human populations and across vertebrates and surveyed phenotypes of ABC gene mutations in mice. Most mouse ABC gene disruption mutations have a phenotype that mimics human disease, indicating they are applicable models. Interestingly, several ABCA family genes, whose human function is unknown, have cholesterol level phenotypes in the mouse. Genome-wide association studies confirm and extend ABC traits and suggest several new functions to investigate. Whole-exome sequencing of tumors from diverse cancer types demonstrates that mutations in ABC genes are not common in cancer, but specific genes are overexpressed in select tumor types. Finally, an analysis of the frequency of loss-of-function mutations demonstrates that many human ABC genes are essential with a low level of variants, while others have a higher level of genetic diversity.