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Supplementary Figure S1-S6 and Supplementary Tables S1-S8

PURPOSE: Therapeutic efficacy of hormonal therapies to target estrogen receptor (ER)-positive breast cancer is limited by the acquisition of ligand-independent ESR1 mutations, which confer treatment resistance to aromatase inhibitors (AIs). Monitoring for the emergence of such mutations may enable individualized therapy. We thus assessed CTC- and ctDNA-based detection of ESR1 mutations with the aim of evaluating non-invasive approaches for the determination of endocrine resistance. PATIENTS AND METHODS: In a prospective cohort of 55 women with hormone receptor-positive metastatic breast cancer, we isolated circulating tumor cells (CTCs) and developed a high-sensitivity method for the detection of ESR1 mutations in these CTCs. In patients with sufficient plasma for the simultaneous extraction of circulating tumor DNA (ctDNA), we performed a parallel analysis of ESR1 mutations using multiplex droplet digital PCR (ddPCR) and examined the agreement between these two platforms. Finally, we isolated single CTCs from a subset of these patients and reviewed RNA expression to explore alternate methods of evaluating endocrine responsiveness. RESULTS: High-sensitivity ESR1 sequencing from CTCs revealed mono- and oligoclonal mutations in 22% of patients. These were concordant with plasma DNA sequencing in 95% of cases. Emergence of ESR1 mutations was correlated both with time to metastatic relapse and duration of AI therapy following such recurrence. The Presence of an ESR1 mutation, compared to ESR1 wild type, was associated with markedly shorter Progression-Free Survival on AI-based therapies (p = 0.0006), but unaltered to other non-AI-based therapies (p = 0.73). Compared with ESR1 mutant cases, AI-resistant CTCs with wild-type ESR1 showed an elevated ER-coactivator RNA signature, consistent with their predicted response to second-line hormonal therapies. CONCLUSION: Blood-based serial monitoring may guide the selection of precision therapeutics for women with AI-resistant ER-positive breast cancer.

Early cancer detection requires high-performing biomarkers and effective tools to enable early intervention. Telomeres, located at the terminal of linear chromosomes, affect genome integrity and cell immortalization. At the very end of the telomere is a 3’ overhanging guanine-rich tail (G-tail) of which shortening is closely related to cell divisions. However, G-tail DNA cannot be detected with conventional sequencing technologies. Here we develop a high-preservation technology to quantify telomeres in circulating cell-free DNA and show that short G-tail DNA was nearly 20-fold higher in hepatocellular carcinoma patients with blood collected at diagnosis than controls and was also strongly correlated with poorer survival. We then used an independent prospective population cohort to show that the abundance of circulating short telomere G-tail increased with time approaching cancer diagnosis. These results indicate that circulating short-telomere G-tail, measured using our novel bilateral single-strand sequencing method, can effectively detect early and aggressive hepatocellular carcinoma.

Background: Genome-wide cell-free DNA fragmentation profiling was suggested to be useful for early cancer detection. However, it still needed to be evaluated using sample before cancer diagnosis. Methods: A prospective cohort with hepatitis B virus (HBV)-seropositive participants were enrolled in 2012 and followed-up biannually with blood sample collections till 31 December 2019. A case-control study with hospital hepatocellular carcinoma (HCC) cases were conducted to identify potential biomarkers for HCC detection (Discovery). We adapted the DELFI method (Cristiano et al, Nature, 2019) and evaluated cfDNA fragmentation profile (AdaptDelfi) for detecting HCC at the Discovery phase and validated AdaptDelfi in an independent prospective population cohort (Validation). Results: Of 18,373 participants, 2,893 were HBV-seropositive and developed 81 incident HCC cases. In Discovery, the AdaptDelfi model distinguished HCC cases from controls excellently (AUC [95% confidence interval] = 0.999 [0.997 - 1.0]), while the serum alpha-fetoprotein (AFP) had an AUC of 0.851 (95% CI = 0.796 - 0.906). High AdaptDelfi was associated with advanced tumor stage (P = 0.038). The hazard of death increased in high AdaptDelfi (hazard ratio 2.43, 95% CI = 1.13 - 5.2) after adjustment for tumor stage. In Validation, AdaptDelfi showed slightly increasing performance using pre-HCC samples collected ≥4 years (AUC = 0.530), 3-4 years (0.615), 2-3 years (0.781), 1-2 years (0.711), and 0-1 year (0.770) before diagnosis. These were comparable with the traditional biomarker - AFP that showed corresponding AUCs of 0.521, 0.525, 0.517, 0.575 and 0.727, respectively. Using a fixed cutoff value from Discovery, AdaptDelfi had a specificity of 88.0% and a sensitivity of 32.5% within one year before diagnosis in detecting early HCC, yielding an estimated positive predict value (PPV) of 1.4% (95% CI = 0.8% - 2.4%) among HBV-seropositive male population. Conclusions: The genome-wide cell-free DNA fragmentation is correlated with HCC stage and may be a useful biomarker for diagnosing clinical HCC and predict patient survival. However, the low PPV suggests its utility in HCC screening in high-risk populations so far may be limited. Citation Format: Shifeng Lian, Fang Fang, Zhiwei Liu, Mingfang Ji, Zongli Zheng. Genome-wide cell-free DNA fragmentation in early detection and disease monitoring of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 759.

The Cas9 nuclease from Staphylococcus aureus (SaCas9) holds great potential for use in gene therapy, and variants with increased fidelity have been engineered. However, we find that existing variants have not reached the greatest accuracy to discriminate base mismatches and exhibited much reduced activity when their mutations were grafted onto the KKH mutant of SaCas9 for editing an expanded set of DNA targets. We performed structure-guided combinatorial mutagenesis to re-engineer KKH-SaCas9 with enhanced accuracy. We uncover that introducing a Y239H mutation on KKH-SaCas9’s REC domain substantially reduces off-target edits while retaining high on-target activity when added to a set of mutations on REC and RuvC domains that lessen its interactions with the target DNA strand. The Y239H mutation is modelled to have removed an interaction from the REC domain with the guide RNA backbone in the guide RNA-DNA heteroduplex structure. We further confirmed the greatly improved genome-wide editing accuracy and single-base mismatch discrimination of our engineered variants, named KKH-SaCas9-SAV1 and SAV2, in human cells. In addition to generating broadly useful KKH-SaCas9 variants with unprecedented accuracy, our findings demonstrate the feasibility for multi-domain combinatorial mutagenesis on SaCas9’s DNA- and guide RNA- interacting residues to optimize its editing fidelity.

RNA-guided CRISPR-Cas9 proteins have been widely used for genome editing, but their off-target activities limit broad application. The minimal Cas9 ortholog from Staphylococcus aureus (SaCas9) is commonly used for in vivo genome editing; however, no variant conferring high genome-wide specificity is available. Here, we report rationally engineered SaCas9 variants with highly specific genome-wide activity in human cells without compromising on-target efficiency. One engineered variant, referred to as SaCas9-HF, dramatically improved genome-wide targeting accuracy based on the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method and targeted deep sequencing analyses. Among 15 tested human endogenous sites with the canonical NNGRRT protospacer adjacent motif (PAM), SaCas9-HF rendered no detectable off-target activities at 9 sites, minimal off-target activities at 6 sites, and comparable on-target efficiencies to those of wild-type SaCas9. Furthermore, among 4 known promiscuous targeting sites, SaCas9-HF profoundly reduced off-target activities compared with wild type. When delivered by an adeno-associated virus vector, SaCas9-HF also showed reduced off-target effects when targeting VEGFA in a human retinal pigmented epithelium cell line compared with wild type. Then, we further altered a previously described variant named KKH-SaCas9 that has a wider PAM recognition range. Similarly, the resulting KKH-HF remarkably reduced off-target activities and increased on- to off-target editing ratios. Our finding provides an alternative to wild-type SaCas9 for genome editing applications requiring exceptional genome-wide precision.

The combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering proteins through combinatorial modifications en masse.

The genome-editing Cas9 protein uses multiple amino-acid residues to bind the target DNA. Considering only the residues in proximity to the target DNA as potential sites to optimise Cas9’s activity, the number of combinatorial variants to screen through is too massive for a wet-lab experiment. Here we generate and cross-validate ten in silico and experimental datasets of multi-domain combinatorial mutagenesis libraries for Cas9 engineering, and demonstrate that a machine learning-coupled engineering approach reduces the experimental screening burden by as high as 95% while enriching top-performing variants by ∼7.5-fold in comparison to the null model. Using this approach and followed by structure-guided engineering, we identify the N888R/A889Q variant conferring increased editing activity on the protospacer adjacent motif-relaxed KKH variant of Cas9 nuclease from Staphylococcus aureus (KKH-SaCas9) and its derived base editor in human cells. Our work validates a readily applicable workflow to enable resource-efficient high-throughput engineering of genome editor’s activity.

Introduction Gene fusion variants in ALK-rearranged NSCLC may predict patient outcomes, but previous results have been inconclusive. Fusion isoforms coexisting in the same tumor may affect the efficacy of targeted therapy, but they have not been investigated. Methods Patients with ALK-rearranged NSCLC who received crizotinib treatments were recruited. Precrizotinib tumor tissues were analyzed by the anchored multiplex polymerase chain reaction for targeted RNA sequencing. Kaplan-Meier and Cox regression were used to compare overall and progression-free survivals. Results Of the 51 studied subjects, EML4-ALK variant types v1, v2, v3, and others were detected in 23 (45.1%), five (9.8%), 19 (37.3%), and four patients (7.8%), respectively. Multiple EML4-ALK RNA isoforms were detected in 24 tumors (47.1%), and single isoform in 27 (52.9%). Most of the v3 tumors (16 of 19) harbored both v3a and v3b RNA isoforms. Multiple isoforms were also detected in eight non-v3 tumors (33.3% of all 24 multiple isoforms; five v1, two v5′, and one v2). Compared with patients with single isoform, those with multiple isoforms had worse progression-free (hazard ratio and 95% confidence interval: 2.45 [1.06–5.69]) and overall (hazard ratio [95% confidence interval]: 3.74 [1.26–11.13]) survivals after adjusting for potential confounders including variant type. Using the patient-derived H2228 cells known to express v3a and v3b, our single-cell polymerase chain reaction detected either v3a or v3b in most single cells. Treatment of H2228 cells by three ALK inhibitors revealed increased ratios of v3a-to-v3b expression over time. Conclusions Intratumoral EML4-ALK isoforms may predict the efficacy of targeted therapy in ALK-rearranged NSCLC. Temporal changes of intratumoral fusion isoforms may result from differential selection pressures that a drug might have on one isoform over another. Larger studies on fusion heterogeneity using RNA sequencing are warranted.

Gene fusions are common drivers and therapeutic targets in cancers, but clinical-grade bioinformatics callers are lacking. Here we introduce a novel method SplitFusion, which is fast by leveraging BWA-MEM split alignments, can detect cryptic splice site fusions, and can infer frame-ness and exon-boundary alignments for functional prediction and minimizing false-positives. SplitFusion demonstrates superior sensitivity, specificity, accuracy and consumes minimal computing resources. In our study of 1,076 formalin-fixed paraffin-embedded lung cancer samples, SplitFusion detected not only common fusions (EML4 4.7%, ROS1 2.0% and RET 1.1%) with various partners, but also rare (KLC1-ALK, CD74-NRG1, and TPR-NTRK1) and novel (FGFR3-JAKMP1, CLIP2-BRAF, and ITPR2-ETV6) fusions. In 35 glioblastoma samples, SplitFusion-Target detected six (17%) EGFR vIII (exons 2-7 deletion) cases. Furthermore, we find that the EML4-ALK variant 3 is significantly associated with occurrence of multiple breakpoint-defined subclones, namely high intratumor heterogeneity. In conclusion, SplitFusion is well-suited for clinical use and for studying fusion-defined tumor heterogeneity.

Summary: We present a CRISPR-based multi-gene knockout screening system and toolkits for extensible assembly of barcoded high-order combinatorial guide RNA libraries en masse. We apply this system for systematically identifying not only pairwise but also three-way synergistic therapeutic target combinations and successfully validate double- and triple-combination regimens for suppression of cancer cell growth and protection against Parkinson’s disease-associated toxicity. This system overcomes the practical challenges of experimenting on a large number of high-order genetic and drug combinations and can be applied to uncover the rare synergistic interactions between druggable targets.

Ewing sarcomas are typified by EWSR1 fusion to ETS gene family members. Tumors with fusion partners other than ETS family members and atypical histologic features pose significant diagnostic challenges and controversies as to their classification. In this article, we report a tumor with EWSR1-NFATC2 fusion in the left femur of a 43-year-old man and with unusual morphologic features that resemble undifferentiated high-grade sarcoma. Analysis together with reported cases in the literature shows that tumors with EWSR1-NFATC2 exhibit distinctive clinicopathologic features, including predilection for young male adults, highly variable histology that varies from round cell tumors frequently associated with nuclear irregularity, short spindle cells with nuclear pleomorphism, to myoepithelial tumor-like with or without myxohyaline matrix. They show variable positivity to CD99, frequent expression of cytokeratins, and consistent high-level amplification of EWSR1-NFATC2 fusion gene with distinctive gene expression profile. These tumors thus deserve classification separate from Ewing sarcoma.

We sought to uncover genetic drivers of hormone receptor–positive (HR+) breast cancer, using a targeted next-generation sequencing approach for detecting expressed gene rearrangements without prior knowledge of the fusion partners. We identified intergenic fusions involving driver genes, including PIK3CA, AKT3, RAF1, and ESR1, in 14% (24/173) of unselected patients with advanced HR+ breast cancer. FISH confirmed the corresponding chromosomal rearrangements in both primary and metastatic tumors. Expression of novel kinase fusions in nontransformed cells deregulates phosphoprotein signaling, cell proliferation, and survival in three-dimensional culture, whereas expression in HR+ breast cancer models modulates estrogen-dependent growth and confers hormonal therapy resistance in vitro and in vivo. Strikingly, shorter overall survival was observed in patients with rearrangement-positive versus rearrangement-negative tumors. Correspondingly, fusions were uncommon ( Significance: By using a powerful clinical molecular diagnostic assay, we identified expressed intergenic fusions as frequent contributors to treatment resistance and poor survival in advanced HR+ breast cancer. The prevalence and biological and prognostic significance of these alterations suggests that their detection may alter clinical management and bring to light new therapeutic opportunities. Cancer Discov; 8(3); 336–53. ©2017 AACR. See related commentary by Natrajan et al., p. 272. See related article by Liu et al., p. 354. This article is highlighted in the In This Issue feature, p. 253

RNA-guided CRISPR-Cas9 proteins have been widely used for genome editing, but their off-target activities limit broad application. The minimal Cas9 ortholog from Staphylococcus aureus (SaCas9) is commonly used for in vivo genome editing; however, no variant conferring high genome-wide specificity is available. Here, we report rationally engineered SaCas9 variants with highly specific genome-wide activity in human cells without compromising on-target efficiency. One engineered variant, referred to as SaCas9-HF, dramatically improved genome-wide targeting accuracy based on the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method and targeted deep sequencing analyses. Among 15 tested human endogenous sites with the canonical NNGRRT protospacer adjacent motif (PAM), SaCas9-HF rendered no detectable off-target activities at 9 sites, minimal off-target activities at 6 sites, and comparable on-target efficiencies to those of wild-type SaCas9. Furthermore, among 4 known promiscuous targeting sites, SaCas9-HF profoundly reduced off-target activities compared with wild type. When delivered by an adeno-associated virus vector, SaCas9-HF also showed reduced off-target effects when targeting VEGFA in a human retinal pigmented epithelium cell line compared with wild type. Then, we further altered a previously described variant named KKH-SaCas9 that has a wider PAM recognition range. Similarly, the resulting KKH-HF remarkably reduced off-target activities and increased on- to off-target editing ratios. Our finding provides an alternative to wild-type SaCas9 for genome editing applications requiring exceptional genome-wide precision.

The quantification of changes in gene copy number is critical to our understanding of tumor biology and for the clinical management of cancer patients. DNA fluorescence in situ hybridization is the gold standard method to detect copy number alterations, but it is limited by the number of genes one can quantify simultaneously. To increase the throughput of this informative technique, a fluorescent bar-code system for the unique labeling of dozens of genes and an automated image analysis algorithm that enabled their simultaneous hybridization for the quantification of gene copy numbers were devised. We demonstrate the reliability of this multiplex approach on normal human lymphocytes, metaphase spreads of transformed cell lines, and cultured circulating tumor cells. It also opens the door to the development of gene panels for more comprehensive analysis of copy number changes in tissue, including the study of heterogeneity and of high-throughput clinical assays that could provide rapid quantification of gene copy numbers in samples with limited cellularity, such as circulating tumor cells.

Detection of circulating tumor DNA is a promising approach for the diagnosis and monitor of solid tumors. However, sensitive detection of ctDNA remains challenging when minor allele frequency (MAF) is ultra-low. Numerous noise reduction data algorithms have been developed to reduce next-generation sequencing (NGS) errors, but variant callers that can take advantage of ultra-deep sequencing data are lacking. Here we develop a bin-based genotyping (BinGo) method for canceling sequencing noise using ultra-deep (> 100,000 X depth) targeted sequencing data. A mutation reference sample with 44 mutations was diluted to different MAFs at 1% and 0.5%. One blood sample for proficiency test was included. We constructed targeted sequencing libraries by anchored multiplex enrichment for a panel of targets that cover the 44 mutations, and sequenced to a very high depth (Mean X ranged: 71K to 350K).After raw NGS reads were mapped by BWA MEM, BinGo tags unique molecular index (UMI) to each alignment (BAM) to preserve alignment signal of all bases in all raw reads. BinGo then bins all reads with the same UMI into one bin, generating millions of bins. Millions of parallel Samtools mpileup was enabled by the GNU parallel tool. Typical mpileup analyses use the human genome (requires ~4GB RAM) as a reference, making millions of such analyses essentially impossible in a regular computing server. To overcome this hurdle, BinGo uses tailor-made reference sequences comprising the targets only (< 100KB), drastically reduces computation memory consumption and enables millions of mpileup analyses. BinGo recompiles Samtools to increase its default max depth from 8,000 to 8,000,000 to accommodate the ultra-deep data. Variants derived from the same bin are effectively phased, thereby enabling calling of di-nuclei mutations such as BRAF V600K (GT>TT) and V600R (GT>AG). This binning also allows for sensitive calling of insertions and deletions even down to a single read. To profile noise patterns, BinGo uses Samtools tview to acquire every base in every alignment position of the target sequences in all raw reads. Finally, BinGo uses multiple statistical testing to call variants, including calling nucleotide changes over the background noise model that is defined by nucleotide change and sequence context; filtering out strand imbalance likely due to DNA template damage, and a low number of supporting ligation starting sites. With 10 ng DNA as input and at an expected MAF of 0.5%, BinGo demonstrates a 100% (44/44) analytical sensitivity and 100% specificity. When using 1 ng DNA as input and at an expected MAF of 0.5%, only one mutation was missed, leading to a sensitivity of 97.73% (43/44) and 100% specificity. Furthermore, BinGo detected in the blood DNA sample a 15-bp EGFR exon 19 deletion at 0.05%.Bingo integrates UMI-bin derived variants and raw sequencing background noise modeling, enabling analysis of ultra-deep targeted NGS data for rare variant detection. Citation Format: Weiwei Bian, Firaol Kebede, Zongli Zheng. A computation method for noise reduction based on ultra-deep targeted sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 244.

Background Lung cancer is one of the leading cause of cancer death worldwide. Non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancer. The discovery of immune checkpoints and the development of immune checkpoint inhibitors (ICIs) is revolutionary in cancer immunology. However, only about 20-30% of cancer patients could benefit from ICIs. Recently, secreted and 5' UTR truncated PD-L1 isoforms were shown to mediate resistance to ICI therapies. Immunohistochemistry (IHC) is the current standard PD-L1 detection method to triage patients for ICI therapies but has limitations, including various antibodies with various detection thresholds, challenge to distinguish PD-L1 isoforms, and inability in multiplex analysis of other immune related factors and genetic alterations. Here, we developed a new method to detect the mRNA expression of CD274 (encoding PD-L1), its various splicing variants and multiplex with T-cell activity factors and other genetic drivers by next-generation sequencing (NGS). Patients and Methods We recruited two patient cohorts. Cohort I included 182 NSCLC (number of stage I, II, III and IV was 73, 48, 59, and 2, respectively) patients with surgery resections. Cohort II included 33 patients who failed first-line therapies and further received anti-PD-1 therapy, prior to which needle biopsies were obtained. Tumor tissues were used for total nucleic acids (DNA and RNA) extraction and subjected to an anchored multiplex PCR NGS panel for one-tube enrichment of 63 genes relevant in NSCLC, including EGFR, KRAS, BRAF, ALK, ROS1, RET, MET, NTRK1/2/3, NRG1 and immune related genes CD274, GZMA (encoding granzyme A) and PRF1 (encoding perforin). We calculated exon-level mRNA expression per copy genomic DNA of the same target, expressed as RNA-to-DNA ratio, without relying on the conventional housekeeping gene normalization. Gene fusions and isoforms were analyzed using our bioinformatics pipeline designed for a clinically established Anchored Multiplex PCR NGS platform (Zheng, et. al, Nat Med, 2014). Kaplan-Meier curve and Cox regression were used for analyses of overall survival (OS) in Cohort I and progression free survival (PSF) in Cohort II. Results In Cohort I, the mRNA expression of CD274 was correlated with its protein expression assayed by a clinical IHC assay (antibody 22C3; R2 = 0.51, P value < 0.01). ALK mRNA expression were highest among those with ALK fusions. Interestingly, patients with high expression of CD274 exon3 and low expression of exon5 (presumably resembling those secreted and 5-UTR-trancated PD-L1 isoforms) had worst overall survival when compared to patients with other CD274 splicing isoforms and patients with low CD274 expression (P value < 0.01). In Cohort II, the analysis of pre-ICI treatment needle biopsies showed that a high expression of CD274 was associated with response to ICIs and with a better PFS compared to patients with low CD274 expression (P value < 0.01). Further, among patients with high CD274 expression, a high GZMA expression was associated with a trend towards better PFS (P value = 0.06), whereas a high PRF1 expression level was not. Conclusion The data suggests that PD-L1and GZMA mRNA expression may predict the prognosis and response to immune checkpoint blockade therapies in NSCLC patients, and larger studies are warranted. Citation Format: Zhengbo Song, Chenyu Lu, Hongxiang Wang, Chunwei Xu, Baifeng Zhang, Wenxian Wang, Juxiang Chen, Zongli Zheng. PD-L1 and granzyme A mRNA expression associated with prognosis and response to immune checkpoint inhibitors in non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6490.

Ewing sarcomas are typified by

Purpose Next-generation sequencing technologies are actively applied in clinical oncology. Bioinformatics pipeline analysis is an integral part of this process; however, humans cannot yet realize the full potential of the highly complex pipeline output. As a result, the decision to include a variant in the final report during routine clinical sign-out remains challenging. Methods We used an artificial intelligence approach to capture the collective clinical sign-out experience of six board-certified molecular pathologists to build and validate a decision support tool for variant reporting. We extracted all reviewed and reported variants from our clinical database and tested several machine learning models. We used 10-fold cross-validation for our variant call prediction model, which derives a contiguous prediction score from 0 to 1 (no to yes) for clinical reporting. Results For each of the 19,594 initial training variants, our pipeline generates approximately 500 features, which results in a matrix of > 9 million data points. From a comparison of naive Bayes, decision trees, random forests, and logistic regression models, we selected models that allow human interpretability of the prediction score. The logistic regression model demonstrated 1% false negativity and 2% false positivity. The final models’ Youden indices were 0.87 and 0.77 for screening and confirmatory cutoffs, respectively. Retraining on a new assay and performance assessment in 16,123 independent variants validated our approach (Youden index, 0.93). We also derived individual pathologist-centric models (virtual consensus conference function), and a visual drill-down functionality allows assessment of how underlying features contributed to a particular score or decision branch for clinical implementation. Conclusion Our decision support tool for variant reporting is a practically relevant artificial intelligence approach to harness the next-generation sequencing bioinformatics pipeline output when the complexity of data interpretation exceeds human capabilities.

CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.

Acute cholecystitis is a common complication to gallstone disease. The relation between the severity of acute cholecystitis and risk of bile duct injury during cholecystectomy has not yet been addressed and is the main focus of this study. All cases with iatrogenic bile duct injury during cholecystectomy, within the Lake Malaren region, Sweden, were identified through ICD procedure codes for biliary reconstruction within the Swedish Inpatient Register and matched to non-injured cholecystectomized controls. Information regarding perioperative variables was collected through medical record review. After review, 158 cases and 623 controls remained for analyses. Adjusted risk of bile duct injury was doubled among patients with acute cholecystitis (OR 1.97 95 % CI 1.05–3.72), whereas a mild acute cholecystitis (Tokyo grade I) did not affect the risk of bile duct injury (OR 0.96 95 % CI 0.41–2.25), a moderate (Tokyo grade II) more than doubled the risk (OR 2.41 95 % CI 1.21–4.80). Severe cholecystitis (Tokyo grade III) had a close to significant eightfold increase in risk (OR 8.43 95 % CI 0.97–72.9). The intention to use intraoperative cholangiography reduced injury risk by 52 % (OR 0.48, 95 % CI 0.29–0.81). Patients with on-going acute cholecystitis had twice the risk of sustaining a biliary lesion compared to patients without acute cholecystitis. There was a relation between the Tokyo guidelines severity grading of acute cholecystitis and injury risk and the intention to use intraoperative cholangiography halved the risk of reconstructed bile duct injury during cholecystectomy.

Introduction Chromosomal rearrangements involving neurotrophic tyrosine kinase 1 ( NTRK1 ) occur in a subset of non-small cell lung cancers (NSCLCs) and other solid tumor malignancies, leading to expression of an oncogenic TrkA fusion protein. Entrectinib (RXDX-101) is an orally available tyrosine kinase inhibitor, including TrkA. We sought to determine the frequency of NTRK1 rearrangements in NSCLC and to assess the clinical activity of entrectinib. Methods We screened 1378 cases of NSCLC using anchored multiplex polymerase chain reaction (AMP). A patient with an NTRK1 gene rearrangement was enrolled onto a Phase 1 dose escalation study of entrectinib in adult patients with locally advanced or metastatic tumors (NCT02097810). We assessed safety and response to treatment. Results We identified NTRK1 gene rearrangements at a frequency of 0.1% in this cohort. A patient with stage IV lung adenocrcinoma with an SQSTM1-NTRK1 fusion transcript expression was treated with entrectinib. Entrectinib was well tolerated, with no grade 3–4 adverse events. Within three weeks of starting on treatment, the patient reported resolution of prior dyspnea and pain. Restaging CT scans demonstrated a RECIST partial response (PR) and complete resolution of all brain metastases. This patient has continued on treatment for over 6 months with an ongoing PR. Conclusions Entrectinib demonstrated significant anti-tumor activity in a patient with NSCLC harboring an SQSTM1-NTRK1 gene rearrangement, indicating that entrectinib may be an effective therapy for tumors with NTRK gene rearrangements, including those with central nervous system metastases.

CRISPR-Cas9 nucleases target specific DNA sequences using a guide RNA but also require recognition of a protospacer adjacent motif (PAM) by the Cas9 protein. Although longer PAMs can potentially improve the specificity of genome editing, they limit the range of sequences that Cas9 orthologs can target. One potential strategy to relieve this restriction is to relax the PAM recognition specificity of Cas9. Here we used molecular evolution to modify the NNGRRT PAM of Staphylococcus aureus Cas9 (SaCas9). One variant we identified, referred to as KKH SaCas9, showed robust genome editing activities at endogenous human target sites with NNNRRT PAMs, thereby increasing SaCas9 targeting range by two- to fourfold. Using GUIDE-seq, we show that wild-type and KKH SaCas9 induce comparable numbers of off-target effects in human cells. Our strategy for evolving PAM specificity does not require structural information and therefore should be applicable to a wide range of Cas9 orthologs.

BACKGROUNDPancreatic ductal adenocarcinoma (PDAC) has a poor prognosis. Variant profiling is crucial for developing personalized treatment and elucidating the etiology of this disease.METHODSPatients with PDAC undergoing surgery from 2007 to 2012 (n = 73) were followed from diagnosis until death or the end of the study. We applied an anchored multiplex PCR (AMP)-based next-generation sequencing (NGS) method to a panel of 65 selected genes and assessed analytical performance by sequencing a quantitative multiplex DNA reference standard. In clinical PDAC samples, detection of low-level KRAS (Kirsten rat sarcoma viral oncogene homolog) mutations was validated by allele-specific PCR and digital PCR. We compared overall survival of patients according to KRAS mutation status by log-rank test and applied logistic regression to evaluate the association between smoking and tumor variant types.RESULTSThe AMP-based NGS method could detect variants with allele frequencies as low as 1% given sufficient sequencing depth (>1500×). Low-frequency KRAS G12 mutations (allele frequency 1%–5%) were all confirmed by allele-specific PCR and digital PCR. The most prevalent genetic alterations were in KRAS (78% of patients), TP53 (tumor protein p53) (25%), and SMAD4 (SMAD family member 4) (8%). Overall survival in T3-stage PDAC patients differed among KRAS mutation subtypes (P = 0.019). Transversion variants were more common in ever-smokers than in never-smokers (odds ratio 5.7; 95% CI 1.2–27.8).CONCLUSIONSThe AMP-based NGS method is applicable for profiling tumor variants. Using this approach, we demonstrated that in PDAC patients, KRAS mutant subtype G12V is associated with poorer survival, and that transversion variants are more common among smokers.

Although CRISPR-Cas9 nucleases are widely used for genome editing1, 2, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM)3–6. As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-Seq analysis7. In addition, we identified and characterized another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also found that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

Identification of p53-positive cells by immunohistochemistry in bone marrow from primary myelodysplastic syndrome patients correlates with the presence of TP53 mutations and poor prognosis. Mutations in the tumor suppressor gene TP53 are more frequent in therapy-related acute myeloid leukemia and myelodysplastic syndrome than in de novo disease, but the role of p53 immunohistochemistry in the therapy-related setting has not been specifically investigated. We studied p53 protein immunoreactivity in bone marrow biopsies of therapy-related myeloid neoplasms and correlated protein expression with TP53 mutation status, clinicopathologic features and outcome. We first studied 32 patients with therapy-related acute myeloid leukemia and 63 patients with therapy-related myelodysplastic syndrome/chronic myelomonocytic leukemia from one institution and then validated our results in a separate group of 32 patients with therapy-related acute myeloid leukemia and 56 patients with therapy-related myelodysplastic syndrome from a different institution. Strong p53 immunostaining in ≥1% of bone marrow cells was highly predictive of a TP53 gene mutation (P

To determine the proportion and clinical features of programmed death ligand 1 (PD-L1) expression in East Asian non-small cell lung cancer (NSCLC).PD-L1 expression was assessed by immunohistochemistry (IHC) and tumor proportion score (TPS) with the use of PD-L1 IHC 22C3 antibody (Dako North America) in 108 surgically resected lung squamous cell carcinomas (SCC) and 221 lung adenocarcinomas (LUADs), and was correlated with clinical variables, histologic subtypes, and common driver mutations.Positive PD-L1 expression was found in 37 lung SCC (37/108, 34.3%), including 15 cases with TPS ≥50% (15/108, 13.9%) and 22 cases with TPS50% (22/108, 20.4%). In adenocarcinoma cohort, 9 cases were found PD-L1 expression positive (9/221, 4.1%), including 1 case with TPS ≥50% (1/221, 0.5%) and 8 cases with TPS50% (8/221, 3.9%). Totally, high PD-L1 expression (TPS ≥50%) was significantly associated with male sex (P=0.026), current/ever smoking history (P=0.008) and SCC subtype (P0.001). Positive PD-L1 expression (including TPS ≥50% and TPS50%) in LUAD cohort was significantly associated with male sex (P=0.046), current/ever smoking history (P=0.002), mutation pan-negative status (P=0.038), solid-predominant subtype (P0.001), large tumor size (P=0.027) and lymph node metastasis (P=0.019). No significant difference was found between PD-L1 high expression group (TPS ≥50%) and low/negative expression group in SCC cohort.This study revealed the unique distribution of PD-L1 expression in East Asian NSCLCs, which is largely different from Western populations. Since the high response rate of pembrolizumab in the treatment of lung cancer patients with PD-L1 TPS ≥50%, this result indicates that prospective PD-L1 expression testing in specific East Asian patients could facilitate decision making for immunotherapy.

Genomic analysis of tumours has led to the identification of hundreds of cancer genes on the basis of the presence of mutations in protein-coding regions. By contrast, much less is known about cancer-causing mutations in non-coding regions. Here we perform deep sequencing in 360 primary breast cancers and develop computational methods to identify significantly mutated promoters. Clear signals are found in the promoters of three genes. FOXA1, a known driver of hormone-receptor positive breast cancer, harbours a mutational hotspot in its promoter leading to overexpression through increased E2F binding. RMRP and NEAT1, two non-coding RNA genes, carry mutations that affect protein binding to their promoters and alter expression levels. Our study shows that promoter regions harbour recurrent mutations in cancer with functional consequences and that the mutations occur at similar frequencies as in coding regions. Power analyses indicate that more such regions remain to be discovered through deep sequencing of adequately sized cohorts of patients.

CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide, off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint 'hotspots'. Finally, GUIDE-seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced, off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases before clinical use.

Chromosomal rearrangements of the gene encoding ROS1 proto-oncogene receptor tyrosine kinase (ROS1) define a distinct molecular subgroup of non-small-cell lung cancers (NSCLCs) that may be susceptible to therapeutic ROS1 kinase inhibition. Crizotinib is a small-molecule tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK), ROS1, and another proto-oncogene receptor tyrosine kinase, MET.We enrolled 50 patients with advanced NSCLC who tested positive for ROS1 rearrangement in an expansion cohort of the phase 1 study of crizotinib. Patients were treated with crizotinib at the standard oral dose of 250 mg twice daily and assessed for safety, pharmacokinetics, and response to therapy. ROS1 fusion partners were identified with the use of next-generation sequencing or reverse-transcriptase-polymerase-chain-reaction assays.The objective response rate was 72% (95% confidence interval [CI], 58 to 84), with 3 complete responses and 33 partial responses. The median duration of response was 17.6 months (95% CI, 14.5 to not reached). Median progression-free survival was 19.2 months (95% CI, 14.4 to not reached), with 25 patients (50%) still in follow-up for progression. Among 30 tumors that were tested, we identified 7 ROS1 fusion partners: 5 known and 2 novel partner genes. No correlation was observed between the type of ROS1 rearrangement and the clinical response to crizotinib. The safety profile of crizotinib was similar to that seen in patients with ALK-rearranged NSCLC.In this study, crizotinib showed marked antitumor activity in patients with advanced ROS1-rearranged NSCLC. ROS1 rearrangement defines a second molecular subgroup of NSCLC for which crizotinib is highly active. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

The diagnosis of the follicular variant of papillary thyroid carcinoma (FVPTC) is increasingly common. Recent studies have suggested that FVPTC is heterogeneous and comprises multiple tumor types with distinct biological behaviors and underlying genetics.The purpose of this work was to identify the prevalence of mutations and gene fusions in known oncogenes in a panel representative of the common spectrum of FVPTC diagnosed at an academic medical center and correlate the clinical and pathological features obtained at the initial diagnosis with the tumor genotype.We performed SNaPshot genotyping on a panel of 129 FVPTCs of ≥1 cm for 90 point mutations or small deletions in known oncogenes and tumor suppressors and identified gene fusions using an anchored multiplex PCR assay targeting a panel of rearranged oncogenes.We identified a mutation or gene fusion in 70% (89 of 127) of cases. Mutations targeting the RAS family of oncogenes were the most frequently observed class of alterations, present in 36% (46 of 127) of cases, followed by BRAF mutation, present in 30% (38 of 127). We also detected oncogenic rearrangements not previously associated with FVPTC, including TFG-ALK and CREB3L2-PPARγ. BRAF mutation was significantly associated with unencapsulated tumor status.These data support the hypothesis that FVPTC is composed of distinct biological entities, with one class being identified by BRAF mutation and support the use of clinical genotyping assays that detect a diverse array of rearrangements involving ALK and PPARγ. Additional studies are necessary to identify genetic drivers in the 30% of FVPTCs with no known oncogenic alteration and to better predict behavior in tumors with known genotypes.

Staying one step ahead of tumors Cancer treatments require continual adjustment. A drug that works initially will lose its potency as the tumor acquires new mutations that allow it to bypass the drug's lethal effects. To stay ahead of the tumor, oncologists need a noninvasive way to collect tumor cells from patients over the course of their treatment. Analyzing the mutations in these samples may help them choose the right drugs as the tumors change. In a small study of breast cancer patients, Yu et al. show that rare tumor cells circulating in the blood can be captured in viable form and used for this purpose. Science , this issue p. 216

Introduction: Anaplastic lymphoma kinase ( ALK ) fusion is the most common mechanism for overexpression and activation in non–small-cell lung carcinoma. Several fusion partners of ALK have been reported, including echinoderm microtubule-associated protein-like 4, TRK-fused gene, kinesin family member 5B , kinesin light chain 1 (KLC1), protein tyrosine phosphatase and nonreceptor type 3 , and huntingtin interacting protein 1 ( HIP1 ). Methods and Results: A 60-year-old Korean man had a lung mass which was a poorly differentiated adenocarcinoma with ALK overexpression. By using an Anchored Multiplex polymerase chain reaction assay and sequencing, we found that tumor had a novel translocated promoter region ( TPR)-ALK fusion. The fusion transcript was generated from an intact, in-frame fusion of TPR exon 15 and ALK exon 20 (t(1;2)(q31.1;p23)). The TPR-ALK fusion encodes a predicted protein of 1192 amino acids with a coiled-coil domain encoded by the 5'-2 nd of the TPR and juxtamembrane and kinase domains encoded by the 3'-end of the ALK . Conclusions: The novel fusion gene and its protein TRP-ALK, harboring coiled-coil and kinase domains, could possess transforming potential and responses to treatment with ALK inhibitors. This case is the first report of TPR-ALK fusion transcript in clinical tumor samples and could provide a novel diagnostic and therapeutic candidate target for patients with cancer, including non–small-cell lung carcinoma.

Circulating tumor cells (CTCs) are present at low concentrations in the peripheral blood of patients with solid tumors. It has been proposed that the isolation, ex vivo culture, and characterization of CTCs may provide an opportunity to noninvasively monitor the changing patterns of drug susceptibility in individual patients as their tumors acquire new mutations. In a proof-of-concept study, we established CTC cultures from six patients with estrogen receptor-positive breast cancer. Three of five CTC lines tested were tumorigenic in mice. Genome sequencing of the CTC lines revealed preexisting mutations in the PIK3CA gene and newly acquired mutations in the estrogen receptor gene (ESR1), PIK3CA gene, and fibroblast growth factor receptor gene (FGFR2), among others. Drug sensitivity testing of CTC lines with multiple mutations revealed potential new therapeutic targets. With optimization of CTC culture conditions, this strategy may help identify the best therapies for individual cancer patients over the course of their disease.