Detection of aneuploidy in patients with cancer through amplification of long interspersed nucleotide elements (LINEs).

Aneuploidy is a feature of most cancer cells, and a myriad of approaches have been developed to detect it in clinical samples. We previously described primers that could be used to amplify ∼38,000 unique long interspersed nucleotide elements (LINEs) from throughout the genome. Here we have developed an approach to evaluate the sequencing data obtained from these amplicons. This approach, called Within-Sample AneupLoidy DetectiOn (WALDO), employs supervised machine learning to detect the small changes in multiple chromosome arms that are often present in cancers. We used WALDO to search for chromosome arm gains and losses in 1,677 tumors and in 1,522 liquid biopsies of blood from cancer patients or normal individuals. Aneuploidy was detected in 95% of cancer biopsies and in 22% of liquid biopsies. Using single-nucleotide polymorphisms within the amplified LINEs, WALDO concomitantly assesses allelic imbalances, microsatellite instability, and sample identification. WALDO can be used on samples containing only a few nanograms of DNA and as little as 1% neoplastic content and has a variety of applications in cancer diagnostics and forensic science.

Intraductal Papillary Mucinous Neoplasms Arise From Multiple Independent Clones, Each With Distinct Mutations.

BACKGROUND & AIMS: Intraductal papillary mucinous neoplasms (IPMNs) are lesions that can progress to invasive pancreatic cancer and constitute an important system for studies of pancreatic tumorigenesis. We performed comprehensive genomic analyses of entire IPMNs to determine the diversity of somatic mutations in genes that promote tumorigenesis. METHODS: We microdissected neoplastic tissues from 6-24 regions each of 20 resected IPMNs, resulting in 227 neoplastic samples that were analyzed by capture-based targeted sequencing. Somatic mutations in genes associated with pancreatic tumorigenesis were assessed across entire IPMN lesions, and the resulting data were supported by evolutionary modeling, whole-exome sequencing, and in situ detection of mutations. RESULTS: We found a high prevalence of heterogeneity among mutations in IPMNs. Heterogeneity in mutations in KRAS and GNAS was significantly more prevalent in IPMNs with low-grade dysplasia than in IPMNs with high-grade dysplasia (P < .02). Whole-exome sequencing confirmed that IPMNs contained multiple independent clones, each with distinct mutations, as originally indicated by targeted sequencing and evolutionary modeling. We also found evidence for convergent evolution of mutations in RNF43 and TP53, which are acquired during later stages of tumorigenesis. CONCLUSIONS: In an analysis of the heterogeneity of mutations throughout IPMNs, we found that early-stage IPMNs contain multiple independent clones, each with distinct mutations, indicating their polyclonal origin. These findings challenge the model in which pancreatic neoplasms arise from a single clone. Increasing our understanding of the mechanisms of IPMN polyclonality could lead to strategies to identify patients at increased risk for pancreatic cancer.

Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations.

The identification of mutations that are present at low frequencies in clinical samples is an essential component of precision medicine. The development of molecular barcoding for next-generation sequencing has greatly enhanced the sensitivity of detecting such mutations by massively parallel sequencing. However, further improvements in specificity would be useful for a variety of applications. We herein describe a technology (BiSeqS) that can increase the specificity of sequencing by at least two orders of magnitude over and above that achieved with molecular barcoding and can be applied to any massively parallel sequencing instrument. BiSeqS employs bisulfite treatment to distinguish the two strands of molecularly barcoded DNA; its specificity arises from the requirement for the same mutation to be identified in both strands. Because no library preparation is required, the technology permits very efficient use of the template DNA as well as sequence reads, which are nearly all confined to the amplicons of interest. Such efficiency is critical for clinical samples, such as plasma, in which only tiny amounts of DNA are often available. We show here that BiSeqS can be applied to evaluate transversions, as well as small insertions or deletions, and can reliably detect one mutation among >10,000 wild-type molecules.

Incidence and distribution of UroSEEK gene panel in a multi-institutional cohort of bladder urothelial carcinoma.

Noninvasive approaches for early detection of bladder cancer are actively being investigated. We recently developed a urine- based molecular assay for the detection and surveillance of bladder neoplasms (UroSEEK). UroSEEK is designed to detect alterations in 11 genes that include most common genetic alterations in bladder cancer. In this study, we analyzed 527 cases, including 373 noninvasive and 154 invasive urothelial carcinomas of bladder from transurethral resections or cystectomies performed at four institutions (1991-2016). Two different mutational analysis assays of a representative tumor area were performed: first, a singleplex PCR assay for evaluation of the TERT promoter region (TERTSeqS) and second, a multiplex PCR assay using primers designed to amplify regions of interest of 10 (FGFR3, PIK3CA, TP53, HRAS, KRAS, ERBB2, CDKN2A, MET, MLL, and VHL) genes (UroSeqS). Overall, 92% of all bladder tumors were positive for at least one genetic alteration in the UroSEEK panel. We found TERT promoter mutations in 77% of low-grade noninvasive papillary carcinomas, with a relatively lower incidence of 65% in high-grade noninvasive papillary carcinomas and carcinomas in situ; p = 0.017. Seventy-two percent of pT1 and 63% of muscle-invasive bladder tumors harbored TERT promoter mutations with g.1295228C>T alteration being the most common in all groups. FGFR3 and PIK3CA mutations were more frequent in low-grade noninvasive papillary carcinomas compared with high-grade noninvasive papillary carcinomas and carcinomas in situ (p < 0.0001), while the opposite was true for TP53 (p < 0.0001). Significantly higher rates of TP53 and CDKN2A mutation rates (p = 0.005 and 0.035, respectively) were encountered in muscle-invasive bladder tumors compared with those of pT1 stage. The overwhelming majority of all investigated tumors showed at least one mutation among UroSEEK assay genes, confirming the comprehensive coverage of the panel and supporting its potential utility as a noninvasive urine-based assay.

Detection of tumor-derived DNA in cerebrospinal fluid of patients with primary tumors of the brain and spinal cord.

Cell-free DNA shed by cancer cells has been shown to be a rich source of putative tumor-specific biomarkers. Because cell-free DNA from brain and spinal cord tumors cannot usually be detected in the blood, we studied whether the cerebrospinal fluid (CSF) that bathes the CNS is enriched for tumor DNA, here termed CSF-tDNA. We analyzed 35 primary CNS malignancies and found at least one mutation in each tumor using targeted or genome-wide sequencing. Using these patient-specific mutations as biomarkers, we identified detectable levels of CSF-tDNA in 74% [95% confidence interval (95% CI) = 57-88%] of cases. All medulloblastomas, ependymomas, and high-grade gliomas that abutted a CSF space were detectable (100% of 21 cases; 95% CI = 88-100%), whereas no CSF-tDNA was detected in patients whose tumors were not directly adjacent to a CSF reservoir (P < 0.0001, Fisher's exact test). These results suggest that CSF-tDNA could be useful for the management of patients with primary tumors of the brain or spinal cord.

A combination of molecular markers and clinical features improve the classification of pancreatic cysts.

BACKGROUND & AIMS: The management of pancreatic cysts poses challenges to both patients and their physicians. We investigated whether a combination of molecular markers and clinical information could improve the classification of pancreatic cysts and management of patients. METHODS: We performed a multi-center, retrospective study of 130 patients with resected pancreatic cystic neoplasms (12 serous cystadenomas, 10 solid pseudopapillary neoplasms, 12 mucinous cystic neoplasms, and 96 intraductal papillary mucinous neoplasms). Cyst fluid was analyzed to identify subtle mutations in genes known to be mutated in pancreatic cysts (BRAF, CDKN2A, CTNNB1, GNAS, KRAS, NRAS, PIK3CA, RNF43, SMAD4, TP53, and VHL); to identify loss of heterozygozity at CDKN2A, RNF43, SMAD4, TP53, and VHL tumor suppressor loci; and to identify aneuploidy. The analyses were performed using specialized technologies for implementing and interpreting massively parallel sequencing data acquisition. An algorithm was used to select markers that could classify cyst type and grade. The accuracy of the molecular markers was compared with that of clinical markers and a combination of molecular and clinical markers. RESULTS: We identified molecular markers and clinical features that classified cyst type with 90%-100% sensitivity and 92%-98% specificity. The molecular marker panel correctly identified 67 of the 74 patients who did not require surgery and could, therefore, reduce the number of unnecessary operations by 91%. CONCLUSIONS: We identified a panel of molecular markers and clinical features that show promise for the accurate classification of cystic neoplasms of the pancreas and identification of cysts that require surgery.

High prevalence of TERT promoter mutations in primary squamous cell carcinoma of the urinary bladder.

TERT promoter mutations (TERT-mut) are detectable in the majority of urothelial carcinomas. The detection of TERT-mut in urine is under investigation as a potential urine-based molecular-screening assay for bladder cancer. A small but significant number of bladder carcinomas are pure squamous cell carcinoma. We sought to assess the incidence of TERT-mut in squamous cell carcinoma of the urinary bladder. A retrospective search of the institutional pathology archives yielded 15 cystectomy specimens performed for squamous cell carcinoma (2000-2014). Histologic slides were reviewed by a senior urologic pathologist to confirm the diagnosis and select a representative formalin-fixed paraffin-embedded tissue block for mutational analysis. All cases yielded adequate material for DNA analysis. Sequencing for TERT-mut was performed using previously described SafeSeq technique. We detected TERT-mut in 12/15 (80%) of bladder squamous cell carcinomas. TERT promoter mutations, commonly found in conventional urothelial carcinoma, are also highly prevalent in urinary bladder squamous cell carcinoma suggesting a common tumorigenesis and potential utility as a molecular urine-based-screening assay.

Engineering targeted chromosomal amplifications in human breast epithelial cells.

Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including "sawtooth" patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

Isolation of circulating tumor cells using a microvortex-generating herringbone-chip.

Rare circulating tumor cells (CTCs) present in the bloodstream of patients with cancer provide a potentially accessible source for detection, characterization, and monitoring of nonhematological cancers. We previously demonstrated the effectiveness of a microfluidic device, the CTC-Chip, in capturing these epithelial cell adhesion molecule (EpCAM)-expressing cells using antibody-coated microposts. Here, we describe a high-throughput microfluidic mixing device, the herringbone-chip, or "HB-Chip," which provides an enhanced platform for CTC isolation. The HB-Chip design applies passive mixing of blood cells through the generation of microvortices to significantly increase the number of interactions between target CTCs and the antibody-coated chip surface. Efficient cell capture was validated using defined numbers of cancer cells spiked into control blood, and clinical utility was demonstrated in specimens from patients with prostate cancer. CTCs were detected in 14 of 15 (93%) patients with metastatic disease (median = 63 CTCs/mL, mean = 386 ± 238 CTCs/mL), and the tumor-specific TMPRSS2-ERG translocation was readily identified following RNA isolation and RT-PCR analysis. The use of transparent materials allowed for imaging of the captured CTCs using standard clinical histopathological stains, in addition to immunofluorescence-conjugated antibodies. In a subset of patient samples, the low shear design of the HB-Chip revealed microclusters of CTCs, previously unappreciated tumor cell aggregates that may contribute to the hematogenous dissemination of cancer.

Global genome analysis reveals a vast and dynamic anellovirus landscape within the human virome.

Anelloviruses are a ubiquitous component of healthy human viromes and remain highly prevalent after being acquired early in life. The full extent of "anellome" diversity and its evolutionary dynamics remain unexplored. We employed in-depth sequencing of blood-transfusion donor(s)-recipient pairs coupled with public genomic resources for a large-scale assembly of anellovirus genomes and used the data to characterize global and personal anellovirus diversity through time. The breadth of the anellome is much greater than previously appreciated, and individuals harbor unique anellomes and transmit lineages that can persist for several months within a diverse milieu of endemic host lineages. Anellovirus sequence diversity is shaped by extensive recombination at all levels of divergence, hindering traditional phylogenetic analyses. Our findings illuminate the transmission dynamics and vast diversity of anelloviruses and set the foundation for future studies to characterize their biology.

Performance of novel non-invasive urine assay UroSEEK in cohorts of equivocal urine cytology.

Urine cytology is an essential element of the diagnostic work up of hematuria. A significant proportion of cases continue to be placed in the "atypical" or "suspicious" categories of the Paris system for urine cytology, posing difficulty in patient management. We report on the performance of our recently described urine-based assay "UroSEEK" in cases with equivocal diagnosis in patients who are investigated for bladder cancer. Urine samples were collected from two cohorts. The first consisted of patients who presented with hematuria or lower urinary tract symptoms (early detection cohort) and the second of patients that are in follow-up for prior bladder cancer (surveillance cohort). Urine samples were analyzed for mutations in 11 genes and aneuploidy. In the early detection setting, we found high sensitivity and specificity (96% and 88%, respectively) and a strong negative predictive value of 99%. The assay performance was less robust in the surveillance cohort (sensitivity of 74%, specificity of 72%, and negative predictive value of 53%). UroSEEK demonstrated a notable lead time to cancer diagnosis. Seven cases in the early detection cohort and 71 surveillance cases were detected at least 6 months prior to clinical diagnosis. Our results suggest a potential role for UroSEEK assay in guiding management of patients with atypical urine cytology if confirmed in future prospective trials.

Targeted sequencing of plasmacytoid urothelial carcinoma reveals frequent TERT promoter mutations.

Activating mutations in the promoter of the telomerase reverse transcriptase (TERT) gene are the most common genetic alterations in urothelial carcinoma (UC) of the bladder and upper urinary tract. Although the cadherin 1 (CDH1) gene is commonly mutated in the clinically aggressive plasmacytoid variant of urothelial carcinoma (PUC), little is known about their TERT promoter mutation status. A retrospective search of our archives for PUC and UC with plasmacytoid and/or signet ring cell features (2007-2014) was performed. Ten specimens from 10 patients had archived material available for DNA analysis and were included in the study. Intratumoral areas of nonplasmacytoid histology were also evaluated when present. Samples were analyzed for TERT promoter mutations with Safe-SeqS, a sequencing error-reduction technology, and sequenced using a targeted panel of the 10 most commonly mutated genes in bladder cancer on the Illumina MiSeq platform. TERT promoter mutations were detected in specimens with pure and focal plasmacytoid features (6/10). Similar to conventional UC, the predominant mutation identified was g.1295228C>T. In heterogeneous tumors with focal variant histology, concordant mutations were found in plasmacytoid and corresponding conventional, glandular, or sarcomatoid areas. Co-occurring mutations in tumor protein p53 (TP53, 2 cases) and kirsten rat sarcoma (KRAS) viral proto-oncogene (1 case) were also detected. TERT promoter mutations are frequently present in PUC, which provides further evidence that TERT promoter mutations are common events in bladder cancer, regardless of histologic subtype, and supports their inclusion in any liquid biopsy assay for bladder cancer.

A multimodality test to guide the management of patients with a pancreatic cyst.

Pancreatic cysts are common and often pose a management dilemma, because some cysts are precancerous, whereas others have little risk of developing into invasive cancers. We used supervised machine learning techniques to develop a comprehensive test, CompCyst, to guide the management of patients with pancreatic cysts. The test is based on selected clinical features, imaging characteristics, and cyst fluid genetic and biochemical markers. Using data from 436 patients with pancreatic cysts, we trained CompCyst to classify patients as those who required surgery, those who should be routinely monitored, and those who did not require further surveillance. We then tested CompCyst in an independent cohort of 426 patients, with histopathology used as the gold standard. We found that clinical management informed by the CompCyst test was more accurate than the management dictated by conventional clinical and imaging criteria alone. Application of the CompCyst test would have spared surgery in more than half of the patients who underwent unnecessary resection of their cysts. CompCyst therefore has the potential to reduce the patient morbidity and economic costs associated with current standard-of-care pancreatic cyst management practices.

Non-invasive detection of urothelial cancer through the analysis of driver gene mutations and aneuploidy.

Current non-invasive approaches for detection of urothelial cancers are suboptimal. We developed a test to detect urothelial neoplasms using DNA recovered from cells shed into urine. UroSEEK incorporates massive parallel sequencing assays for mutations in 11 genes and copy number changes on 39 chromosome arms. In 570 patients at risk for bladder cancer (BC), UroSEEK was positive in 83% of those who developed BC. Combined with cytology, UroSEEK detected 95% of patients who developed BC. Of 56 patients with upper tract urothelial cancer, 75% tested positive by UroSEEK, including 79% of those with non-invasive tumors. UroSEEK detected genetic abnormalities in 68% of urines obtained from BC patients under surveillance who demonstrated clinical evidence of recurrence. The advantages of UroSEEK over cytology were evident in low-grade BCs; UroSEEK detected 67% of cases whereas cytology detected none. These results establish the foundation for a new non-invasive approach for detection of urothelial cancer.

Evaluation of liquid from the Papanicolaou test and other liquid biopsies for the detection of endometrial and ovarian cancers.

We report the detection of endometrial and ovarian cancers based on genetic analyses of DNA recovered from the fluids obtained during a routine Papanicolaou (Pap) test. The new test, called PapSEEK, incorporates assays for mutations in 18 genes as well as an assay for aneuploidy. In Pap brush samples from 382 endometrial cancer patients, 81% [95% confidence interval (CI), 77 to 85%] were positive, including 78% of patients with early-stage disease. The sensitivity in 245 ovarian cancer patients was 33% (95% CI, 27 to 39%), including 34% of patients with early-stage disease. In contrast, only 1.4% of 714 women without cancer had positive Pap brush samples (specificity, ~99%). Next, we showed that intrauterine sampling with a Tao brush increased the detection of malignancy over endocervical sampling with a Pap brush: 93% of 123 (95% CI, 87 to 97%) patients with endometrial cancer and 45% of 51 (95% CI, 31 to 60%) patients with ovarian cancer were positive, whereas none of the samples from 125 women without cancer were positive (specificity, 100%). Finally, in 83 ovarian cancer patients in whom plasma was available, circulating tumor DNA was found in 43% of patients (95% CI, 33 to 55%). When plasma and Pap brush samples were both tested, the sensitivity for ovarian cancer increased to 63% (95% CI, 51 to 73%). These results demonstrate the potential of mutation-based diagnostics to detect gynecologic cancers at a stage when they are more likely to be curable.

Spectrum of genetic mutations in de novo PUNLMP of the urinary bladder.

Our group and others have previously demonstrated the presence of TERT promoter mutations (TERT-mut) in 60-80% of urothelial carcinomas and some of their histologic variants. Five other genes have been frequently implicated in bladder cancer: FGRF3, TP53, PIK3CA, HRAS, and CDKN2A. In the current study, we sought to determine the prevalence of mutations in TERT and these five other genes in de novo papillary urothelial neoplasms of low malignant potential (PUNLMP) of the urinary bladder. A retrospective search of our archives for PUNLMP was performed and 30 de novo cases were identified and included in the study. We found mutations in TERT (TERT-mut) and FGFR3 (FGFR3-mut) to be the most common alterations in the cohort (63 and 60%, respectively). The majority of the TERT-mut-positive tumors (84%) had a g.1295228C > T alteration with the remaining tumors demonstrating g.1295250C > T. Approximately one fourth of tumors had TP53 mutations. These findings support the potential utility of a uniform genetic mutation panel to detect bladder cancers of various subtypes.

A novel approach for selecting combination clinical markers of pathology applied to a large retrospective cohort of surgically resected pancreatic cysts.

OBJECTIVE: Our objective was to develop an approach for selecting combinatorial markers of pathology from diverse clinical data types. We demonstrate this approach on the problem of pancreatic cyst classification. MATERIALS AND METHODS: We analyzed 1026 patients with surgically resected pancreatic cysts, comprising 584 intraductal papillary mucinous neoplasms, 332 serous cystadenomas, 78 mucinous cystic neoplasms, and 42 solid-pseudopapillary neoplasms. To derive optimal markers for cyst classification from the preoperative clinical and radiological data, we developed a statistical approach for combining any number of categorical, dichotomous, or continuous-valued clinical parameters into individual predictors of pathology. The approach is unbiased and statistically rigorous. Millions of feature combinations were tested using 10-fold cross-validation, and the most informative features were validated in an independent cohort of 130 patients with surgically resected pancreatic cysts. RESULTS: We identified combinatorial clinical markers that classified serous cystadenomas with 95% sensitivity and 83% specificity; solid-pseudopapillary neoplasms with 89% sensitivity and 86% specificity; mucinous cystic neoplasms with 91% sensitivity and 83% specificity; and intraductal papillary mucinous neoplasms with 94% sensitivity and 90% specificity. No individual features were as accurate as the combination markers. We further validated these combinatorial markers on an independent cohort of 130 pancreatic cysts, and achieved high and well-balanced accuracies. Overall sensitivity and specificity for identifying patients requiring surgical resection was 84% and 81%, respectively. CONCLUSIONS: Our approach identified combinatorial markers for pancreatic cyst classification that had improved performance relative to the individual features they comprise. In principle, this approach can be applied to any clinical dataset comprising dichotomous, categorical, and continuous-valued parameters.

Diagnostic potential of tumor DNA from ovarian cyst fluid.

We determined whether the mutations found in ovarian cancers could be identified in the patients' ovarian cyst fluids. Tumor-specific mutations were detectable in the cyst fluids of 19 of 23 (83%) borderline tumors, 10 of 13 (77%) type I cancers, and 18 of 18 (100%) type II cancers. In contrast, no mutations were found in the cyst fluids of 18 patients with benign tumors or non-neoplastic cysts. Though large, prospective studies are needed to demonstrate the safety and clinical utility of this approach, our results suggest that the genetic evaluation of cyst fluids might be able to inform the management of the large number of women with these lesions.

High prevalence of TERT promoter mutations in micropapillary urothelial carcinoma.

Somatic activating mutations in the promoter of the telomerase reverse transcriptase (TERT) gene are the most common genetic alterations in urothelial carcinoma (UC) of the bladder and upper urinary tract. Little is known, however, about TERT-mutation status in the relatively uncommon but clinically aggressive micropapillary (MPC) variant. We evaluated the presence of TERT promoter mutations in MPC of the bladder and upper urinary tract. A retrospective search of our archives for MPC and UC with micropapillary features (2005-2014) was performed. All slides were reviewed to confirm the histologic diagnosis. Thirty-three specimens from 31 patients had FFPE blocks available for DNA analysis and were included in the study. Intratumoral areas of non-micropapillary histology were also evaluated when present. Samples were analyzed with Safe-SeqS, a sequencing error reduction technology, and sequenced using the Illumina MiSeq platform. TERT promoter mutations were detected in all specimens with pure MPC (18 of 18) and UC with focal micropapillary features (15 of 15). Similar to conventional UC, the predominant mutations identified occurred at positions -124 (C228T) (85 %) and -146 (C250T) (12 %) bp upstream of the TERT ATG start site. In heterogeneous tumors with focal variant histology, intratumoral concordant mutations were found in variant (MPC and non-MPC) and corresponding conventional UC. We found TERT promoter mutations, commonly found in conventional UC, to be frequently present in MPC. Our finding of concordant intratumoral mutational alterations in cases with focal variant histology lends support to the common oncogenesis origin of UC and its variant histology.