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The analysis of cell-free DNA (cfDNA) from plasma offers great promise for the earlier detection of cancer. At present, changes in DNA sequence, methylation, or copy number are the most sensitive ways to detect the presence of cancer. To further increase the sensitivity of such assays with limited amounts of sample, it would be useful to be able to evaluate the same template molecules for all these changes. Here, we report an approach, called MethylSaferSeqS, that achieves this goal, and can be applied to any standard library preparation method suitable for massively parallel sequencing. The innovative step was to copy both strands of each DNA-barcoded molecule with a primer that allows the subsequent separation of the original strands (retaining their 5-methylcytosine residues) from the copied strands (in which the 5-methylcytosine residues are replaced with unmodified cytosine residues). The epigenetic and genetic alterations present in the DNA molecules can then be obtained from the original and copied strands, respectively. We applied this approach to plasma from 265 individuals, including 198 with cancers of the pancreas, ovary, lung, and colon, and found the expected patterns of mutations, copy number alterations, and methylation. Furthermore, we could determine which original template DNA molecules were methylated and/or mutated. MethylSaferSeqS should be useful for addressing a variety of questions relating genetics and epigenetics.

The role of adjuvant chemotherapy in stage II colon cancer continues to be debated. The presence of circulating tumor DNA (ctDNA) after surgery predicts very poor recurrence-free survival, whereas its absence predicts a low risk of recurrence. The benefit of adjuvant chemotherapy for ctDNA-positive patients is not well understood.We conducted a trial to assess whether a ctDNA-guided approach could reduce the use of adjuvant chemotherapy without compromising recurrence risk. Patients with stage II colon cancer were randomly assigned in a 2:1 ratio to have treatment decisions guided by either ctDNA results or standard clinicopathological features. For ctDNA-guided management, a ctDNA-positive result at 4 or 7 weeks after surgery prompted oxaliplatin-based or fluoropyrimidine chemotherapy. Patients who were ctDNA-negative were not treated. The primary efficacy end point was recurrence-free survival at 2 years. A key secondary end point was adjuvant chemotherapy use.Of the 455 patients who underwent randomization, 302 were assigned to ctDNA-guided management and 153 to standard management. The median follow-up was 37 months. A lower percentage of patients in the ctDNA-guided group than in the standard-management group received adjuvant chemotherapy (15% vs. 28%; relative risk, 1.82; 95% confidence interval [CI], 1.25 to 2.65). In the evaluation of 2-year recurrence-free survival, ctDNA-guided management was noninferior to standard management (93.5% and 92.4%, respectively; absolute difference, 1.1 percentage points; 95% CI, -4.1 to 6.2 [noninferiority margin, -8.5 percentage points]). Three-year recurrence-free survival was 86.4% among ctDNA-positive patients who received adjuvant chemotherapy and 92.5% among ctDNA-negative patients who did not.A ctDNA-guided approach to the treatment of stage II colon cancer reduced adjuvant chemotherapy use without compromising recurrence-free survival. (Supported by the Australian National Health and Medical Research Council and others; DYNAMIC Australian New Zealand Clinical Trials Registry number, ACTRN12615000381583.).

The analysis of cell-free DNA (cfDNA) from plasma offers great promise for the earlier detection of cancer. At present, changes in DNA sequence, methylation, or copy number are the most sensitive ways to detect the presence of cancer. To further increase the sensitivity of such assays with limited amounts of sample, it would be useful to be able to evaluate the same template molecules for all these changes. Here we report an approach, called MethylSaferSeqS, that achieves this goal, and can be applied to any standard library preparation method suitable for massively parallel sequencing. The innovative step was to copy both strands of each DNA-barcoded molecule with a primer that allows the subsequent separation of the original strands (retaining their 5-methylcytosine residues) from the copied strands (in which the 5-methylcytosine residues are replaced with unmodified cytosine residues). The epigenetic and genetic alterations present in the DNA molecules can then be obtained from the original and copied strands, respectively. We applied this approach to plasma from 265 individuals, including 198 with cancers of the pancreas, ovary, lung and colon, and found the expected patterns of mutations, copy number alterations, and methylation. Furthermore, we could determine which original template DNA molecules were methylated and/or mutated. MethylSaferSeqS should be useful for addressing a variety of questions relating genetics and epigenetics in the future.

One of the most intriguing characteristics of cell-free DNA (cfDNA) from plasma is the sequence at the ends of the fragments. Previous studies have shown that these end-sequences are somewhat different in cancer patients than in healthy individuals. While investigating this characteristic, we noticed that the bases at the 5’-ends of a double-stranded fragment were highly correlated with the GC content of that particular fragment. This led us to develop a method, called MendSeqS (Modified End-based sequencing System), that incorporates the correlation between end-motifs and GC content into the analysis of shallow (0.5x) whole genome sequencing (WGS). When applied to plasma samples, MendSeqS was able to classify patients with a sensitivity of 96% at 98% specificity in a cohort comprised of 107 individuals evaluated in our laboratory (43 with cancer and 64 without). In cohorts evaluated in three other laboratories, comprising a total of 401 individuals (193 with cancer and 208 without), MendSeqS achieved a sensitivity of 87% at 98% specificity. MendSeqS could in principle be combined with other methods of cfDNA analysis to enhance cancer detection.

Identification and quantification of low-frequency mutations remain challenging despite improvements in the baseline error rate of next-generation sequencing technologies. Here, we describe a method, termed SaferSeqS, that addresses these challenges by (1) efficiently introducing identical molecular barcodes in the Watson and Crick strands of template molecules and (2) enriching target sequences with strand-specific PCR. The method achieves high sensitivity and specificity and detects variants at frequencies below 1 in 100,000 DNA template molecules with a background mutation rate of 100-fold.

Studies in multiple solid tumor types have demonstrated the prognostic significance of ctDNA analysis after curative intent surgery. A combined analysis of data across completed studies could further our understanding of circulating tumor DNA (ctDNA) as a prognostic marker and inform future trial design. We combined individual patient data from three independent cohort studies of nonmetastatic colorectal cancer (CRC). Plasma samples were collected 4 to 10 weeks after surgery. Mutations in ctDNA were assayed using a massively parallel sequencing technique called SafeSeqS. We analyzed 485 CRC patients (230 Stage II colon, 96 Stage III colon, and 159 locally advanced rectum). ctDNA was detected after surgery in 59 (12%) patients overall (11.0%, 12.5% and 13.8% for samples taken at 4–6, 6–8 and 8–10 weeks; P = .740). ctDNA detection was associated with poorer 5-year recurrence-free (38.6% vs 85.5%; P < .001) and overall survival (64.6% vs 89.4%; P < .001). The predictive accuracy of postsurgery ctDNA for recurrence was higher than that of individual clinicopathologic risk features. Recurrence risk increased exponentially with increasing ctDNA mutant allele frequency (MAF) (hazard ratio, 1.2, 2.5 and 5.8 for MAF of 0.1%, 0.5% and 1%). Postsurgery ctDNA was detected in 3 of 20 (15%) patients with locoregional and 27 of 60 (45%) with distant recurrence (P = .018). This analysis demonstrates a consistent long-term impact of ctDNA as a prognostic marker across nonmetastatic CRC, where ctDNA outperforms other clinicopathologic risk factors and MAF further stratifies recurrence risk. ctDNA is a better predictor of distant vs locoregional recurrence.

BackgroundDetection of circulating tumour DNA (ctDNA) after curative intent treatment reflects the presence of minimal residual disease and predicts for recurrence. DYNAMIC trial is a randomised phase II investigating the clinical utility of ctDNA-guided adjuvant treatment strategy compared with the standard of care (non-ctDNA based) approach in patients with stage II colon cancer. The primary trial endpoint is recurrence-free survival estimated from Kaplan Meier Method at 2 years. Secondary endpoints include proportion of patients treated with adjuvant chemotherapy, time-to-recurrence, overall survival, ctDNA clearance rate (investigational arm) and quality-adjusted life-years.ObjectiveTo outline and publish the pre-determined statistical analysis plan (SAP) before the database lock and the start of analysis.MethodsThe SAP describes basic analysis principles, methods for dealing with a range of commonly encountered data analysis issues and the specific statistical procedures for analysing efficacy and safety outcomes. The SAP was approved after closure of recruitment and before completion of patient follow-up. It outlines the planned primary analyses and a range of subgroup and sensitivity analyses regarding the clinical outcomes. Health economic outcomes are not included in this plan but will be analysed separately. The SAP will be adhered to for the final data analysis of this trial to avoid analysis bias arising from knowledge of the data.Trial RegistrationANZ Clinical Trials Registry, ACTRN12615000381583. Registered on 27th April 2015.

In early-stage pancreatic cancer, there are currently no biomarkers to guide selection of therapeutic options. This prospective biomarker trial evaluated the feasibility and potential clinical utility of circulating tumor DNA (ctDNA) analysis to inform adjuvant therapy decision making.Patients considered by the multidisciplinary team to have resectable pancreatic adenocarcinoma were enrolled. Pre- and post-operative samples for ctDNA analysis were collected. PCR-based-SafeSeqS assays were used to identify mutations at codon 12, 13 and 61 of KRAS in the primary pancreatic tumor and to detect ctDNA. Results of ctDNA analysis were correlated with CA19-9, recurrence-free and overall survival (OS). Patient management was per standard of care, blinded to ctDNA data.Of 112 patients consented pre-operatively, 81 (72%) underwent resection. KRAS mutations were identified in 91% (38/42) of available tumor samples. Of available plasma samples (N = 42), KRAS mutated ctDNA was detected in 62% (23/37) pre-operative and 37% (13/35) post-operative cases. At a median follow-up of 38.4 months, ctDNA detection in the pre-operative setting was associated with inferior recurrence-free survival (RFS) [hazard ratio (HR) 4.1; P = 0.002)] and OS (HR 4.1; P = 0.015). Detectable ctDNA following curative intent resection was associated with inferior RFS (HR 5.4; P 0.0001) and OS (HR 4.0; P = 0.003). Recurrence occurred in 13/13 (100%) patients with detectable ctDNA post-operatively, including in seven that received gemcitabine-based adjuvant chemotherapy.ctDNA studies in localized pancreatic cancer are challenging, with a substantial number of patients not able to undergo resection, not having sufficient tumor tissue for analysis or not completing per protocol sample collection. ctDNA analysis, pre- and/or post-surgery, is a promising prognostic marker. Studies of ctDNA guided therapy are justified, including of treatment intensification strategies for patients with detectable ctDNA post-operatively who appear at very high risk of recurrence despite gemcitabine-based adjuvant therapy.

Background In patients with resectable colorectal liver metastases (CRLM), the role of pre- and postoperative systemic therapy continues to be debated. Previous studies have shown that circulating tumor DNA (ctDNA) analysis, as a marker of minimal residual disease, is a powerful prognostic factor in patients with nonmetastatic colorectal cancer (CRC). Serial analysis of ctDNA in patients with resectable CRLM could inform the optimal use of perioperative chemotherapy. Here, we performed a validation study to confirm the prognostic impact of postoperative ctDNA in resectable CRLM observed in a previous discovery study. Methods and findings We prospectively collected plasma samples from patients with resectable CRLM, including presurgical and postsurgical samples, serial samples during any pre- or postoperative chemotherapy, and serial samples in follow-up. Via targeted sequencing of 15 genes commonly mutated in CRC, we identified at least 1 somatic mutation in each patient’s tumor. We then designed a personalized assay to assess 1 mutation in plasma samples using the Safe-SeqS assay. A total of 380 plasma samples from 54 patients recruited from July 2011 to Dec 2014 were included in our analysis. Twenty-three (43%) patients received neoadjuvant chemotherapy, and 42 patients (78%) received adjuvant chemotherapy after surgery. Median follow-up was 51 months (interquartile range, 31 to 60 months). At least 1 somatic mutation was identified in all patients’ tumor tissue. ctDNA was detectable in 46/54 (85%) patients prior to any treatment and 12/49 (24%) patients after surgery. There was a median 40.93-fold (19.10 to 87.73, P < 0.001) decrease in ctDNA mutant allele fraction with neoadjuvant chemotherapy, but ctDNA clearance during neoadjuvant chemotherapy was not associated with a better recurrence-free survival (RFS). Patients with detectable postoperative ctDNA experienced a significantly lower RFS (HR 6.3; 95% CI 2.58 to 15.2; P < 0.001) and overall survival (HR 4.2; 95% CI 1.5 to 11.8; P < 0.001) compared to patients with undetectable ctDNA. For the 11 patients with detectable postoperative ctDNA who had serial ctDNA sampling during adjuvant chemotherapy, ctDNA clearance was observed in 3 patients, 2 of whom remained disease-free. All 8 patients with persistently detectable ctDNA after adjuvant chemotherapy have recurred. End-of-treatment (surgery +/− adjuvant chemotherapy) ctDNA detection was associated with a 5-year RFS of 0% compared to 75.6% for patients with an undetectable end-of-treatment ctDNA (HR 14.9; 95% CI 4.94 to 44.7; P < 0.001). Key limitations of the study include the small sample size and the potential for false-positive findings with multiple hypothesis testing. Conclusions We confirmed the prognostic impact of postsurgery and posttreatment ctDNA in patients with resected CRLM. The potential utility of serial ctDNA analysis during adjuvant chemotherapy as an early marker of treatment efficacy was also demonstrated. Further studies are required to define how to optimally integrate ctDNA analyses into decision-making regarding the use and timing of adjuvant therapy for resectable CRLM. Trial registration ACTRN12612000345886., Author summary Why was this study done? Recurrence risk remains high in patients who underwent curative-intent surgical resection of colorectal cancer liver metastases (CRLM), with limited additional benefit from pre- or postoperative chemotherapy. There is currently no validated biomarker of recurrence for resected CRLM that could help guide the use of chemotherapy for the individual patient. Circulating tumor DNA (ctDNA), representing DNA shed from tumor cells into the circulation, is a versatile blood-based biomarker that can provide real-time assessment of cancer burden. It has been shown in a previous small study of 18 patients that ctDNA detection after surgery for CRLM is highly predictive of eventual cancer relapse. What did the researchers do and find? We performed a larger study involving 54 patients with resectable CRLM to confirm the ability of postoperative ctDNA to detect microscopic residual disease and predict relapse. We also analysed serial ctDNA during and after chemotherapy. ctDNA was detected in 24% of patients immediately after surgery, and these patients had a very high recurrence risk of 83% compared to only 31% in those with undetectable ctDNA after surgery. All patients with detectable postoperative ctDNA who failed to clear their ctDNA following adjuvant chemotherapy experienced recurrence, while 67% of patients whose ctDNA became undetectable after chemotherapy remained disease-free. What do these findings mean? ctDNA detection after surgery or after completion of adjuvant chemotherapy is associated with a very high risk of recurrence and death in patients with resectable CRLM; ctDNA dynamics before and after adjuvant chemotherapy reflected adjuvant treatment efficacy. Future studies should aim to assess how best to incorporate ctDNA testing into clinical practice to guide adjuvant treatment decision.

Biomarkers have a wide range of applications in the clinical management of cancer, including screening and therapeutic management. Tumor DNA released from neoplastic cells has become a particularly active area of cancer biomarker development due to the critical role somatic alterations play in the pathophysiology of cancer and the ability to assess released tumor DNA in accessible clinical samples, in particular blood (i.e., liquid biopsy). Many of the early applications of tumor DNA as a biomarker were pioneered in colorectal cancer due to its well-defined genetics and common occurrence, the effectiveness of early detection, and the availability of effective therapeutic options. Herein, in the context of colorectal cancer, we describe how the intended clinical application dictates desired biomarker test performance, how features of tumor DNA provide unique challenges and opportunities for biomarker development, and conclude with specific examples of clinical application of tumor DNA as a biomarker with particular emphasis on early detection. See all articles in this CEBP Focus section, “NCI Early Detection Research Network: Making Cancer Detection Possible.”

ObjectiveIntraductal papillary mucinous neoplasms (IPMNs) are non-invasive precursor lesions that can progress to invasive pancreatic cancer and are classified as low-grade or high-grade based on the morphology of the neoplastic epithelium. We aimed to compare genetic alterations in low-grade and high-grade regions of the same IPMN in order to identify molecular alterations underlying neoplastic progression.DesignWe performed multiregion whole exome sequencing on tissue samples from 17 IPMNs with both low-grade and high-grade dysplasia (76 IPMN regions, including 49 from low-grade dysplasia and 27 from high-grade dysplasia). We reconstructed the phylogeny for each case, and we assessed mutations in a novel driver gene in an independent cohort of 63 IPMN cyst fluid samples.ResultsOur multiregion whole exome sequencing identified KLF4, a previously unreported genetic driver of IPMN tumorigenesis, with hotspot mutations in one of two codons identified in >50% of the analyzed IPMNs. Mutations in KLF4 were significantly more prevalent in low-grade regions in our sequenced cases. Phylogenetic analyses of whole exome sequencing data demonstrated diverse patterns of IPMN initiation and progression. Hotspot mutations in KLF4 were also identified in an independent cohort of IPMN cyst fluid samples, again with a significantly higher prevalence in low-grade IPMNs.ConclusionHotspot mutations in KLF4 occur at high prevalence in IPMNs. Unique among pancreatic driver genes, KLF4 mutations are enriched in low-grade IPMNs. These data highlight distinct molecular features of low-grade and high-grade dysplasia and suggest diverse pathways to high-grade dysplasia via the IPMN pathway.

BACKGROUND: Sodium phenylbutyrate and taurursodiol have been found to reduce neuronal death in experimental models. The efficacy and safety of a combination of the two compounds in persons with amyotrophic lateral sclerosis (ALS) are not known. METHODS: In this multicenter, randomized, double-blind trial, we enrolled participants with definite ALS who had had an onset of symptoms within the previous 18 months. Participants were randomly assigned in a 2:1 ratio to receive sodium phenylbutyrate–taurursodiol (3 g of sodium phenylbutyrate and 1 g of taurursodiol, administered once a day for 3 weeks and then twice a day) or placebo. The primary outcome was the rate of decline in the total score on the Amyotrophic Lateral Sclerosis Functional Rating Scale–Revised (ALSFRS-R; range, 0 to 48, with higher scores indicating better function) through 24 weeks. Secondary outcomes were the rates of decline in isometric muscle strength, plasma phosphorylated axonal neurofilament H subunit levels, and the slow vital capacity; the time to death, tracheostomy, or permanent ventilation; and the time to death, tracheostomy, permanent ventilation, or hospitalization. RESULTS: A total of 177 persons with ALS were screened for eligibility, and 137 were randomly assigned to receive sodium phenylbutyrate–taurursodiol (89 participants) or placebo (48 participants). In a modified intention-to-treat analysis, the mean rate of change in the ALSFRS-R score was −1.24 points per month with the active drug and −1.66 points per month with placebo (difference, 0.42 points per month; 95% confidence interval, 0.03 to 0.81; P = 0.03). Secondary outcomes did not differ significantly between the two groups. Adverse events with the active drug were mainly gastrointestinal. CONCLUSIONS: Sodium phenylbutyrate–taurursodiol resulted in slower functional decline than placebo as measured by the ALSFRS-R score over a period of 24 weeks. Secondary outcomes were not significantly different between the two groups. Longer and larger trials are necessary to evaluate the efficacy and safety of sodium phenylbutyrate–taurursodiol in persons with ALS. (Funded by Amylyx Pharmaceuticals and others; CENTAUR Clinicaltrials.gov number, NCT03127514.)

A real-time trial of a cancer blood test Cancers diagnosed early are often more responsive to treatment. Blood tests that detect molecular markers of cancer have successfully identified individuals already known to have the disease. Lennon et al. conducted an exploratory study that more closely reflects the way in which such blood tests would be used in the future. They evaluated the feasibility and safety of incorporating a multicancer blood test into the routine clinical care of 10,000 women with no history of cancer. Over a 12-month period, the blood test detected 26 cancers of different types. A combination of the blood test and positron emission tomography–computed tomography (PET-CT) imaging led to surgical removal of nine of these cancers. Use of the blood test did not result in a large number of futile follow-up procedures. Science , this issue p. eabb9601

We report a sensitive PCR-based assay called Repetitive Element AneupLoidy Sequencing System (RealSeqS) that can detect aneuploidy in samples containing as little as 3 pg of DNA. Using a single primer pair, we amplified ∼350,000 amplicons distributed throughout the genome. Aneuploidy was detected in 49% of liquid biopsies from a total of 883 nonmetastatic, clinically detected cancers of the colorectum, esophagus, liver, lung, ovary, pancreas, breast, or stomach. Combining aneuploidy with somatic mutation detection and eight standard protein biomarkers yielded a median sensitivity of 80% in these eight cancer types, while only 1% of 812 healthy controls scored positive.

Adjuvant chemotherapy in patients with stage III colon cancer prevents recurrence by eradicating minimal residual disease. However, which patients remain at high risk of recurrence after completing standard adjuvant treatment cannot currently be determined. Postsurgical circulating tumor DNA (ctDNA) analysis can detect minimal residual disease and is associated with recurrence in colorectal cancers.To determine whether serial postsurgical and postchemotherapy ctDNA analysis could provide a real-time indication of adjuvant therapy efficacy in stage III colon cancer.This multicenter, Australian, population-based cohort biomarker study recruited 100 consecutive patients with newly diagnosed stage III colon cancer planned for 24 weeks of adjuvant chemotherapy from November 1, 2014, through May 31, 2017. Patients with another malignant neoplasm diagnosed within the last 3 years were excluded. Median duration of follow-up was 28.9 months (range, 11.6-46.4 months). Physicians were blinded to ctDNA results. Data were analyzed from December 10, 2018, through June 23, 2019.Serial plasma samples were collected after surgery and after chemotherapy. Somatic mutations in individual patients' tumors were identified via massively parallel sequencing of 15 genes commonly mutated in colorectal cancer. Personalized assays were designed to quantify ctDNA.Detection of ctDNA and recurrence-free interval (RFI).After 4 exclusions, 96 eligible patients were eligible; median patient age was 64 years (range, 26-82 years); 49 (51%) were men. At least 1 somatic mutation was identified in the tumor tissue of all 96 evaluable patients. Circulating tumor DNA was detectable in 20 of 96 (21%) postsurgical samples and was associated with inferior recurrence-free survival (hazard ratio [HR], 3.8; 95% CI, 2.4-21.0; P .001). Circulating tumor DNA was detectable in 15 of 88 (17%) postchemotherapy samples. The estimated 3-year RFI was 30% when ctDNA was detectable after chemotherapy and 77% when ctDNA was undetectable (HR, 6.8; 95% CI, 11.0-157.0; P .001). Postsurgical ctDNA status remained independently associated with RFI after adjusting for known clinicopathologic risk factors (HR, 7.5; 95% CI, 3.5-16.1; P .001).Results suggest that ctDNA analysis after surgery is a promising prognostic marker in stage III colon cancer. Postchemotherapy ctDNA analysis may define a patient subset that remains at high risk of recurrence despite completing standard adjuvant treatment. This high-risk population presents a unique opportunity to explore additional therapeutic approaches.

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.

ObjectiveFor 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.DesignWe 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.ResultsWe 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; PConclusionPostoperative 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.

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.

Early detection of metastatic colorectal cancer, at initial diagnosis or during routine surveillance, can improve survival outcomes. Current routine investigations, including CEA and CT, have limited sensitivity and specificity. Recent studies of colorectal cancer cohorts under post surgery surveillance indicate circulating tumor DNA (ctDNA) evidence of recurrence can occur many months before clinical detection. Another possible role for ctDNA is in the further assessment of indeterminate findings on standard CEA or CT investigations. To further explore this potential, we undertook a prospective study. Further investigation, including FDG-PET imaging, was at clinician discretion, blinded to ctDNA analysis. Forty-nine patients were enrolled. Analyzed here are the 45 patients with an evaluable blood sample of whom 6 had an isolated elevated CEA, 30 had indeterminate CT findings, and 9 had both. FDG-PET scans were performed in 30 patients. Fourteen of 45 patients (31%) had detectable ctDNA. At completion of the planned 2 year follow-up, recurrence has occurred in 21 (47%) patients. Detectable ctDNA at study entry was associated with inferior relapse free survival (HR 4.85, p < 0.0001). Where FDG-PET scan was normal/equivocal (n = 15, 50%) 1 of 1 with detectable ctDNA versus 3 of 14 with undetectable ctDNA ultimately had recurrence confirmed. In summary, for colorectal cancer patients with indeterminate findings on routine investigations, ctDNA detection increases the probability that the findings indicate metastatic disease, including in a nonpredefined subset that also underwent FDG-PET imaging. Further studies of the value of ctDNA analysis during patient surveillance are warranted.

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.

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.

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.

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.

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 cancer's 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

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 (MSD) 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.

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.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.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.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.

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.

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.

Importance For patients with resected, nonmetastatic colorectal cancer (CRC), the optimal surveillance protocol remains unclear. Objective To evaluate whether serial circulating tumor DNA (ctDNA) levels detected disease recurrence earlier, compared with conventional postoperative surveillance, in patients with resected CRC. Design, Setting, and Participants This study included patients (n = 58) with stage I, II, or III CRC who underwent radical surgical resection at 4 Swedish hospitals from February 2, 2007, to May 8, 2013. Eighteen patients received adjuvant chemotherapy at the discretion of their clinicians, who were blinded to the ctDNA results. Blood samples were collected at 1 month after the surgical procedure and every 3 to 6 months thereafter for ctDNA analysis. Patients were followed up until metachronous metastases were detected, or for a median of 49 months. Data analysis was performed from March 1, 2009, to June 23, 2018. Main Outcomes and Measures Sensitivity and timing of ctDNA positivity were compared with those of conventional surveillance modalities (computed tomographic scans and serum carcinoembryonic antigen tests) for the detection of disease recurrence. Results This study included 319 blood samples from 58 patients, with a median (range) age of 69 (47-83) years and 34 males (59%). The recurrence rate among patients with positive ctDNA levels was 77% (10 of 13 patients). Positive ctDNA preceded radiologic and clinical evidence of recurrence by a median of 3 months. Of the 45 patients with negative ctDNA throughout follow-up, none (0%; 95% CI, 0%-7.9%) experienced a relapse, with a median follow-up of 49 months. However, 3 (6%; 95% CI, 1.3%-17%) of the 48 patients without relapse had a positive ctDNA result, which subsequently fell to undetectable levels during follow-up. Conclusion and Relevance Although these findings need to be validated in a larger, prospective trial, they suggest that ctDNA analysis could complement conventional surveillance strategies as a triage test to stratify patients with resected CRC on the basis of risk of disease recurrence.

3518 Background: Studies in multiple tumor types have demonstrated the prognostic impact of ctDNA analysis after curative intent surgery. Emerging data suggest that conversion of ctDNA from detectable to undetectable after adjuvant chemotherapy may reflect treatment efficacy. Further review of existing study data could increase the precision of ctDNA guided adjuvant therapy decision making. Methods: We combined individual patient (pt) data from three independent prospective cohort studies that enrolled 485 pts with stage II or III CRC. Clinicians were blinded to ctDNA results. We evaluated pt outcomes over a 5-year follow-up period (median, 47.2 months). Plasma samples were collected 4 to 10 weeks after surgery. Mutations in ctDNA were assayed using Safe-SeqS. Results: ctDNA was detected after surgery in 59 (12%) pts overall (11.0%, 12.5% and 13.8% respectively for samples taken at 4-6, 6-8 and 8-10 weeks; P = 0.740). ctDNA detection was associated with nodal status; 8.7%, 16.7% and 32.4% in N0, N1 and N2 disease (P < 0.001), but remained an independent adverse prognostic factor in multivariable analysis. ctDNA detection was associated with poor overall survival for pts treated (mortality ratio, 3.0; P = 0.026) or not treated with adjuvant chemotherapy (mortality ratio, 5.17; P < 0.001). The median MAF (mutant allele frequency) in pts with detectable ctDNA was 0.046%. For pts not treated with adjuvant chemotherapy, 3 year recurrence free survival (RFS) was 9% in pts with a MAF > 0.046% vs 33% with a MAF ≤ 0.046% (HR, 2.7; P = 0.032). For chemotherapy treated pts, 3 year RFS was 25% in pts with a MAF > 0.046% vs 70% with a MAF ≤ 0.046 (HR, 3.1; P = 0.025). In 90 pts with recurrence, ctDNA had been detected post surgery in 3 of 20 (15%) with locoregional recurrence, 27 of 60 (45%) with distant recurrence and 5 of 10 (50%) with both (P = 0.044). Conclusions: Where samples for ctDNA analysis were collected 4 to 10 weeks post surgery, sampling timing may not significantly impact detection rates. The prognostic significance of ctDNA detection can be further stratified by MAF level, but MAF level may not impact adjuvant treatment benefit. ctDNA analysis is most sensitive for detecting minimal residual disease at distant sites.

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.

e16206Background: While adjuvant therapy provides a survival benefit for patients (pts) with early-stage pancreatic cancer there are currently no biomarkers that define pts that are more or less li...

3516Background: Adjuvant CT in stage III CC prevents recurrence by eradicating minimal residual disease (MRD) not visible on imaging. However, many patients (pts) will not have MRD and not all pts with MRD will benefit from standard CT. In this study, we determined (i) if the presence of ctDNA following surgery was predictive of recurrence following CT; (ii) if ctDNA could be used to determine the effectiveness of CT during treatment and (iii) if the presence of ctDNA following CT completion was predictive for later recurrence. Methods: Serial plasma samples from stage III CC pts planned for adjuvant CT were collected post-surgery, during CT and at treatment completion. Somatic mutations in individual tumors were identified via massively parallel sequencing of 15 genes commonly mutated in colorectal cancer. Personalized Safe-SeqS assays to quantify ctDNA in plasma samples were designed. Clinicians were blinded to ctDNA results. Results: 95 pts were enrolled from Nov-2014 to May-2017, median age was 64 yea...

3521 Background: The optimal approach to adjuvant chemotherapy for rectal cancer is keenly debated. Routine practice and clinical guidelines vary widely. After pre-operative chemoradiation (CRT), a pathologic complete response (pCR) or nodal involvement (pN+) are prognostic markers that can guide clinical decision-making, but markers that better define the patients (pts) that are likely or unlikely to benefit from chemotherapy are urgently needed. We investigated the potential role of ctDNA as a biomarker to guide therapy. Methods: We conducted a prospective, multi-centre study in pts with LARC (T3/T4 and/or N+) planned for CRT and curative resection. Serial plasma samples were collected pre-CRT, post-CRT, and 4-10 weeks after surgery. Somatic mutations in individual pts’ tumor were identified via sequencing of 15 genes commonly mutated in colorectal cancers. We then designed personalized assays to quantify ctDNA in plasma samples. Pts received adjuvant therapy at clinician discretion. Results: 200 pts were enrolled between Apr-2012 and Dec-2015. Median age was 62 years (range 28-86), 67% were male and 159 pts had pre-CRT and post-op ctDNA samples available for analysis. Of these, 122 (77%) pts had detectable ctDNA prior to therapy. After surgery, 19 pts had detectable ctDNA and 11 of these 19 (58%) have recurred during a median follow up of 22 months. Recurrence occurred in only 12 of 140 (8.6%) with negative ctDNA (HR 12, p < 0.001). One hundred and two (64%) pts received adjuvant chemotherapy. Post-op ctDNA detection was predictive of recurrence irrespective of adjuvant chemotherapy (chemo: HR 10, p < 0.001; no chemo: HR 16, p < 0.001). Thirty-four pts (21%) achieved a pCR, 43 (27%) had pN+ disease. pCR (vs non-pCR) was associated with a trend for lower recurrence risk (HR 0.31, p = 0.089) and pN+ (vs pN0) with a higher recurrence risk (HR 4.2, p < 0.001). ctDNA detection remained predictive of recurrence among pts with a pCR (HR 14, p = 0.014) or with pN+ disease (HR 11, p < 0.001). Conclusions: Post-op ctDNA analysis stratifies pts with LARC into very high and low risk groups. ctDNA analysis remains strongly predictive of recurrence among pts with both lower risk (pCR) and higher risk (pN+) disease.

Somatic mutations are exquisitely specific cancer biomarkers that are not merely associated with cancer, rather they are the root cause of cancer. The realization that this markers are present in bodily fluids of individuals with cancer and the development of digital methods for their detection ushered a new era in the management of cancer patients providing opportunities for the development of noninvasive methods for early detection, detection of minimal residual disease, monitoring of response to therapy, early detection of resistance and diagnosis. There are both technical and biological challenges for the development of noninvasive tests for the detection of somatic mutations. The number of circulating tumor DNA (ctDNA) molecules is very low compared to that of DNA molecules with wild-type sequence and requires very sensitive methods for their detection. To this end, we have developed a method called SafeSeqS that is able to reliably resolve mutations at 0.01% in background of wild-type DNA across multiple regions of interest. To determine the spectrum of cancers in which ctDNA measurements could prove clinically useful, we evaluated the detection of ctDNA in plasma (liquid biopsy) from patients with a large number of tumor types. ctDNA was detectable in the plasma of most patients with advanced tumor, but in less than 50% in patients with certain tumor types, like primary brain cancers. More than half of the patients with localized colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, had detectable ctDNA in their plasma. The sensitivity of detection of advanced colorectal cancer (CRC) was close to 100% and greater than 75% for localized cancers indicating that liquid biopsy could be utilized for a number of clinical applications. One such application is the detection of minimal residual disease after surgery or therapy with curative intent. Currently, there are not efficient ways to determine which patients are cured and which have residual disease that will result in recurrence. In a prospective cohort of 230 patients with resected stage II colon cancer, ctDNA detection after surgery provided direct evidence of residual disease and identified patients at very high risk of recurrence. Another application of liquid biopsy in CRC is monitoring resistance to therapies, like EGFR, providing an early indication that resistance ensues. The holy grail of non-invasive detection of cancer is early detection. Most tumors are incurable because they are detected at a late stage. Early detection of cancer, before symptoms appear, at a stage curable by surgery, holds great promise for decreasing the rate of deaths due to cancer. We will share our efforts on the development of tests for the early detection of cancer. Our overall vision is that non-invasive, somatic mutation based tests become routine in the clinical management of cancer patients. Citation Format: Nickolas Papadopoulos, Yuxuan Wang, Joshua Cohen, Ralph Hruban, Peter Gibbs, Jeanne Tie, Luis Diaz, Kenneth Kinzler, Bert Vogelstein. Non-invasive detection of somatic mutations in the management of CRC. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr IA17.

ChIP-exonuclease (ChIP-exo) is a modified ChIP-seq approach for high resolution mapping of transcription factor DNA sites. We describe an Illumina-based ChIP-exo method which provides a global improvement of the data quality of estrogen receptor (ER) ChIP and insights into the motif structure for key ER-associated factors. ChIP-exo of the ER pioneer factor FoxA1 identifies protected DNA with a predictable 8 bp overhang from the Forkhead motif, which we term mesas. We show that mesas occur in multiple cellular contexts and exist as single or overlapping motifs. Our Illumina-based ChIP-exo provides high resolution mapping of transcription factor binding sites.

After injury, regeneration of the central nervous system (CNS) is inhibited by the secretion of repulsive guidance cues that mediate axon growth and guidance. These repulsive cues are perceived as spatial gradients of neurons. Microfluidic systems can enable studies of neuron response to spatial gradients, by generating precise and reproducible surface gradients of guidance cues. Understanding neuron response to these gradients could provide valuable information for therapies seeking to promote regeneration in the CNS. Here we report on the successful use of a microfluidic gradient generator to deposit spatially-extended and uniform surface gradients of one repulsive guidance cue, chondroitin sulfate proteoglycans (CSPGs). The patterned substrates are compatible with high-quality culture of large numbers of primary neurons, and initial findings indicate that neuron growth and guidance are responsive to the deposited CSPGs.

Despite recent clinical trials of percutaneous transluminal coronary angioplasty (PTCA) in acute myocardial infarction, specific groups of patients that may benefit from adjunctive or alternative therapy have yet to be adequately characterized. The in-hospital outcome of 151 consecutive patients treated for acute myocardial infarction with urgent PTCA of the infarct-related artery was studied to identify a subgroup of patients at high risk. Patients were divided into two groups based on the angiographic presence of either single-vessel (n = 86) or multivessel (n = 65) coronary artery disease. Despite PTCA of only the infarct-related artery and similar baseline clinical characteristics such as age, peak serum creatine kinase concentration, left ventricular ejection fraction, and time from the onset of chest pain to arrival at the hospital, the group with multivessel disease had a lower rate of successful angioplasty (75% vs 92%, p0.005), with higher incidences of persistent total occlusion of the infarct-related artery (14% vs 3%, p0.02) and procedural complications during PTCA (28% vs 13%, por = 0.02), and were more likely to have multiple complications (12% vs 1%, p0.004). In addition, the group with multivessel disease had a higher rate of urgent (or = 24 hours) coronary artery bypass graft surgery (13% vs 2%, p0.05) and a trend toward a higher in-hospital mortality rate (6% vs 1%, por = 0.17). By stepwise logistic regression, only the presence of single-vessel versus multivessel disease was predictive of PTCA success (p0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Cathepsin K, the most potent mammalian collagenase, has been implicated in osteoporosis, cancer metastasis, atherosclerosis, and arthritis. Although procathepsin K is stable and readily detected, the active mature cathepsin K eludes detection by in vitro methods due to its shorter half-life and inactivation at neutral pH. We describe, for the first time, reliable detection, visualization, and quantification of mature cathepsin K to femtomole resolution using gelatin zymography. The specificity of the method was validated with cathepsin K knockdown using small interfering RNA (siRNA) transfection of human monocyte-derived macrophages, and enzymatic activity confirmed with benzyloxycarbonyl-glycine-proline-arginine-7-amino-4-methylcoumarin (Z-GPR-AMC) substrate hydrolysis was fit to a computational model of enzyme kinetics. Furthermore, cathepsin K zymography was used to show that murine osteoclasts secrete more cathepsin K than is stored intracellularly, and this was opposite to the behavior of the macrophages from which they were differentiated. In summary, this inexpensive, species-independent, antibody-free protocol describes a sensitive method with broad potential to elucidate previously undetectable cathepsin K activity.