Joshua D. Cohen

Detection and localization of surgically resectable cancers with a multi-analyte blood test

SEEK and you may find cancer earlier Many cancers can be cured by surgery and/or systemic therapies when detected before they have metastasized. This clinical reality, coupled with the growing appreciation that cancers rapid genetic evolution limits its response to drugs, have fueled interest in methodologies for earlier detection of the disease. Cohen et al. developed a noninvasive blood test, called CancerSEEK that can detect eight common human cancer types (see the Perspective by Kalinich and Haber). The test assesses eight circulating protein biomarkers and tumor-specific mutations in circulating DNA. In a study of 1000 patients previously diagnosed with cancer and 850 healthy control individuals, CancerSEEK detected cancer with a sensitivity of 69 to 98% (depending on cancer type) and 99% specificity. Science, this issue p. 926; see also p. 866 A blood test that combines protein and DNA markers may allow earlier detection of eight common cancer types. Earlier detection is key to reducing cancer deaths. Here, we describe a blood test that can detect eight common cancer types through assessment of the levels of circulating proteins and mutations in cell-free DNA. We applied this test, called CancerSEEK, to 1005 patients with nonmetastatic, clinically detected cancers of the ovary, liver, stomach, pancreas, esophagus, colorectum, lung, or breast. CancerSEEK tests were positive in a median of 70% of the eight cancer types. The sensitivities ranged from 69 to 98% for the detection of five cancer types (ovary, liver, stomach, pancreas, and esophagus) for which there are no screening tests available for average-risk individuals. The specificity of CancerSEEK was greater than 99%: only 7 of 812 healthy controls scored positive. In addition, CancerSEEK localized the cancer to a small number of anatomic sites in a median of 83% of the patients.

Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters

Significance Conventional models of cancer progression propose that single cells leave the primary tumor, enter the circulation, and seed clonal metastases. However, metastases can contain multiple clones, raising the question: How do polyclonal metastases form? We demonstrate that cancer cells seed distant organs as cohesive clusters, composed of two molecularly distinct subpopulations, whose proportions vary systematically during metastasis. We establish that collective dissemination is a frequent mechanism for metastasis and identify a molecular program in the most invasive, keratin 14+ (K14+) cancer cells, regulating cell–cell adhesion, cell–matrix adhesion, and immune evasion. We demonstrate that this metastatic phenotype is dependent upon K14 expression. Understanding the molecular basis of collective dissemination may therefore enable novel prognostics and therapies to improve patient outcomes. Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14+ cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14+ epithelial tumor cell clusters disseminate collectively to colonize distant organs.

Cancer-Associated Mutations in Endometriosis without Cancer

Background Endometriosis, defined as the presence of ectopic endometrial stroma and epithelium, affects approximately 10% of reproductive‐age women and can cause pelvic pain and infertility. Endometriotic lesions are considered to be benign inflammatory lesions but have cancerlike features such as local invasion and resistance to apoptosis. Methods We analyzed deeply infiltrating endometriotic lesions from 27 patients by means of exomewide sequencing (24 patients) or cancer‐driver targeted sequencing (3 patients). Mutations were validated with the use of digital genomic methods in microdissected epithelium and stroma. Epithelial and stromal components of lesions from an additional 12 patients were analyzed by means of a droplet digital polymerase‐chain‐reaction (PCR) assay for recurrent activating KRAS mutations. Results Exome sequencing revealed somatic mutations in 19 of 24 patients (79%). Five patients harbored known cancer driver mutations in ARID1A, PIK3CA, KRAS, or PPP2R1A, which were validated by Safe‐Sequencing System or immunohistochemical analysis. The likelihood of driver genes being affected at this rate in the absence of selection was estimated at P=0.001 (binomial test). Targeted sequencing and a droplet digital PCR assay identified KRAS mutations in 2 of 3 patients and 3 of 12 patients, respectively, with mutations in the epithelium but not the stroma. One patient harbored two different KRAS mutations, c.35G→T and c.35G→C, and another carried identical KRAS c.35G→A mutations in three distinct lesions. Conclusions We found that lesions in deep infiltrating endometriosis, which are associated with virtually no risk of malignant transformation, harbor somatic cancer driver mutations. Ten of 39 deep infiltrating lesions (26%) carried driver mutations; all the tested somatic mutations appeared to be confined to the epithelial compartment of endometriotic lesions.

Combined circulating tumor DNA and protein biomarker-based liquid biopsy for the earlier detection of pancreatic cancers

Significance Few patients with pancreatic cancer survive longer than 5 y, in part because most patients are identified only after their disease has progressed to an advanced stage. In this study, we show how combining mutations in circulating tumor DNA (ctDNA) with protein markers can result in a screening test with improved sensitivity while retaining specificity. The combination of the ctDNA and protein markers was superior to any single marker. Moreover, the combination detected nearly two-thirds of pancreatic cancers that had no evidence of distant metastasis at the time of surgical resection. The strategy may represent an approach to detect cancers of many types at an earlier stage. The earlier diagnosis of cancer is one of the keys to reducing cancer deaths in the future. Here we describe our efforts to develop a noninvasive blood test for the detection of pancreatic ductal adenocarcinoma. We combined blood tests for KRAS gene mutations with carefully thresholded protein biomarkers to determine whether the combination of these markers was superior to any single marker. The cohort tested included 221 patients with resectable pancreatic ductal adenocarcinomas and 182 control patients without known cancer. KRAS mutations were detected in the plasma of 66 patients (30%), and every mutation found in the plasma was identical to that subsequently found in the patient’s primary tumor (100% concordance). The use of KRAS in conjunction with four thresholded protein biomarkers increased the sensitivity to 64%. Only one of the 182 plasma samples from the control cohort was positive for any of the DNA or protein biomarkers (99.5% specificity). This combinatorial approach may prove useful for the earlier detection of many cancer types.

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

Endometrial and ovarian cancers can be detected through the analysis of DNA from Pap test fluids, intrauterine samples, and plasma. Brushing up on early cancer detection Despite the many recent advances in cancer diagnosis and treatment, ovarian cancer remains one of the most lethal malignancies, in part because there are no accurate screening methods for this disease and it is often diagnosed at a late stage. To develop a screening tool for ovarian and endometrial cancers, Wang et al. combined genetic analysis of fluids obtained through routine Papanicolau testing, normally done for cervical cancer, with analysis of tumor DNA circulating in the blood. The authors also used intrauterine sampling with Tao brushes to further increase the sensitivity of detection for the less accessible tumors. 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.

Serial circulating tumour DNA analysis during multimodality treatment of locally advanced rectal cancer: a prospective biomarker study

Objective For patients with locally advanced rectal cancer (LARC), adjuvant chemotherapy selection following surgery remains a major clinical dilemma. Here, we investigated the ability of circulating tumour DNA (ctDNA) to improve risk stratification in patients with LARC. Design We enrolled patients with LARC (T3/T4 and/or N+) planned for neoadjuvant chemoradiotherapy. Plasma samples were collected pretreatment, postchemoradiotherapy and 4–10 weeks after surgery. Somatic mutations in individual patient’s tumour were identified via massively parallel sequencing of 15 genes commonly mutated in colorectal cancer. We then designed personalised assays to quantify ctDNA in plasma samples. Patients received adjuvant therapy at clinician discretion, blinded to the ctDNA results. Results We analysed 462 serial plasma samples from 159 patients. ctDNA was detectable in 77%, 8.3% and 12% of pretreatment, postchemoradiotherapy and postsurgery plasma samples. Significantly worse recurrence-free survival was seen if ctDNA was detectable after chemoradiotherapy (HR 6.6; P<0.001) or after surgery (HR 13.0; P<0.001). The estimated 3-year recurrence-free survival was 33% for the postoperative ctDNA-positive patients and 87% for the postoperative ctDNA-negative patients. Postoperative ctDNA detection was predictive of recurrence irrespective of adjuvant chemotherapy use (chemotherapy: HR 10.0; P<0.001; without chemotherapy: HR 22.0; P<0.001). Postoperative ctDNA status remained an independent predictor of recurrence-free survival after adjusting for known clinicopathological risk factors (HR 6.0; P<0.001). Conclusion Postoperative ctDNA analysis stratifies patients with LARC into subsets that are either at very high or at low risk of recurrence, independent of conventional clinicopathological risk factors. ctDNA analysis could potentially be used to guide patient selection for adjuvant chemotherapy.

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

Significance The detection of aneuploidy in clinical samples can be critical for various diagnostic applications in cancer and can also inform cancer genetics. Next-generation sequencing protocols such as whole-genome and exome sequencing are typically used to detect aneuploidy in cancer samples, but amplicon-based protocols achieve high coverage depth at relatively low cost and can be used when only tiny amounts of DNA are available. In this paper, we describe new bioinformatic tools to detect aneuploidy using data generated from amplification with a single primer pair. This approach can be applied to 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. 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.

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

Significance The detection of rare mutations in clinical samples is essential to the screening, diagnosis, and treatment of cancer. Although next-generation sequencing has greatly enhanced the sensitivity of detecting mutations, the relatively high error rate of these platforms limits their overall clinical utility. The elimination of sequencing artifacts could facilitate the detection of early-stage cancers and provide improved treatment recommendations tailored to the genetic profile of a tumor. Here, we report the development of BiSeqS, a bisulfite conversion-based sequencing approach that allows for the strand-specific detection and quantification of rare mutations. We demonstrate that BiSeqS eliminates nearly all sequencing artifacts in three common types of mutations and thereby considerably increases the signal-to-noise ratio for diagnostic analyses. 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.

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.

Anomalously Diffusing and Persistently Migrating Cells in 2D and 3D Culture Environments

Appropriately chosen descriptive models of cell migration in biomaterials will allow researchers to characterize and ultimately predict the movement of cells in engineered systems for a variety of applications in tissue engineering. The persistent random walk (PRW) model accurately describes cell migration on two-dimensional (2D) substrates. However, this model inherently cannot describe subdiffusive cell movement, i.e., migration paths in which the root mean square displacement increases more slowly than the square root of the time interval. Subdiffusivity is a common characteristic of cells moving in confined environments, such as three-dimensional (3D) porous scaffolds, hydrogel networks, and in vivo tissues. We demonstrate that a generalized anomalous diffusion (AD) model, which uses a simple power law to relate the mean square displacement to time, more accurately captures individual cell migration paths across a range of engineered 2D and 3D environments than does the more commonly used PRW model. We used the AD model parameters to distinguish cell movement profiles on substrates with different chemokinetic factors, geometries (2D vs 3D), substrate adhesivities, and compliances. Although the two models performed with equal precision for superdiffusive cells, we suggest a simple AD model, in lieu of PRW, to describe cell trajectories in populations with a significant subdiffusive fraction, such as cells in confined, 3D environments.