cfDNA methylome profiling for detection and subtyping of small cell lung cancers.

Small cell lung cancer (SCLC) is characterized by morphologic, epigenetic and transcriptomic heterogeneity. Subtypes based upon predominant transcription factor expression have been defined that, in mouse models and cell lines, exhibit potential differential therapeutic vulnerabilities, with epigenetically distinct SCLC subtypes also described. The clinical relevance of these subtypes is unclear, due in part to challenges in obtaining tumor biopsies for reliable profiling. Here we describe a robust workflow for genome-wide DNA methylation profiling applied to both patient-derived models and to patients' circulating cell-free DNA (cfDNA). Tumor-specific methylation patterns were readily detected in cfDNA samples from patients with SCLC and were correlated with survival outcomes. cfDNA methylation also discriminated between the transcription factor SCLC subtypes, a precedent for a liquid biopsy cfDNA-methylation approach to molecularly subtype SCLC. Our data reveal the potential clinical utility of cfDNA methylation profiling as a universally applicable liquid biopsy approach for the sensitive detection, monitoring and molecular subtyping of patients with SCLC.

Early Dissemination of Circulating Tumor Cells: Biological and Clinical Insights.

Circulating tumor cells (CTCs) play a causal role in the development of metastasis, the major cause of cancer-associated mortality worldwide. In the past decade, the development of powerful cellular and molecular technologies has led to a better understanding of the molecular characteristics and timing of dissemination of CTCs during cancer progression. For instance, genotypic and phenotypic characterization of CTCs, at the single cell level, has shown that CTCs are heterogenous, disseminate early and could represent only a minor subpopulation of the primary tumor responsible for disease relapse. While the impact of molecular profiling of CTCs has not yet been translated to the clinic, CTC enumeration has been widely used as a prognostic biomarker to monitor treatment response and to predict disease relapse. However, previous studies have revealed a major challenge: the low abundance of CTCs in the bloodstream of patients with cancer, especially in early stage disease where the identification and characterization of subsequently "lethal" cells has potentially the greatest clinical relevance. The CTC field is rapidly evolving with development of new technologies to improve the sensitivity of CTC detection, enumeration, isolation, and molecular profiling. Here we examine the technical and analytical validity of CTC technologies, we summarize current data on the biology of CTCs that disseminate early and review CTC-based clinical applications.

Technical Evaluation of Commercial Mutation Analysis Platforms and Reference Materials for Liquid Biopsy Profiling.

Molecular profiling from liquid biopsy, in particular cell-free DNA (cfDNA), represents an attractive alternative to tissue biopsies for the detection of actionable targets and tumor monitoring. In addition to PCR-based assays, Next Generation Sequencing (NGS)-based cfDNA assays are now commercially available and are being increasingly adopted in clinical practice. However, the validity of these products as well as the clinical utility of cfDNA in the management of patients with cancer has yet to be proven. Within framework of the Innovative Medicines Initiative (IMI) program CANCER-ID we evaluated the use of commercially available reference materials designed for ctDNA testing and cfDNA derived from Diagnostic Leukaphereses (DLA) for inter- and intra-assay as well as intra- and inter-laboratory comparisons. In three experimental setups, a broad range of assays including ddPCR, MassARRAY and various NGS-based assays were tested. We demonstrate that both reference materials with predetermined VAFs and DLA samples are extremely useful for the performance assessment of mutation analysis platforms. Moreover, our data indicate a substantial variability of NGS assays with respect to sensitivity and specificity highlighting the importance of extensive validation of the test performance before offering these tests in clinical routine practice.

Cell-free DNA analysis reveals POLR1D-mediated resistance to bevacizumab in colorectal cancer.

BACKGROUND: Bevacizumab, a monoclonal antibody against soluble VEGFA, is an approved and commonly administered anti-angiogenic drug in patients with metastasized colorectal cancer (mCRC). The survival benefit of anti-VEGF therapy in mCRC patients is limited to a few months, and acquired resistance mechanisms are largely unknown. Here, we employed whole-genome sequencing of plasma DNA to evaluate the tumor genome of patients undergoing treatment with bevacizumab to determine novel aberrations associated with resistance. METHODS: Using longitudinal plasma analyses, we studied the evolution of tumor genomes in a mCRC cohort (n = 150) and conducted analyses of CRC cases from The Cancer Genome Atlas (TCGA) database (n = 619) to identify associations between genomic aberrations and clinical features. We employed whole-genome sequencing to identify the most frequently occurring focal somatic copy number alterations (SCNAs). Using the TCGA data as a comparative and supporting dataset, we defined the minimally amplified overlapping region and studied the mechanistic consequences of copy number gain of the involved genes in this segment. In addition, we established an in vitro cell model and conducted downstream gene expression and cell viability assays to confirm our findings from the patient dataset. RESULTS: We observed a recurrent focal amplification (8.7% of cases) on chromosome 13q12.2. Analysis of CRC cases from the TCGA database suggested that this amplicon is associated with more advanced stages. We confirmed that this 13q12.2 amplicon frequently emerges later during the clinical course of disease. After defining the minimally amplified region, we observed that the amplification and expression of one gene, POLR1D, impacted cell proliferation and resulted in upregulation of VEGFA, an important regulator of angiogenesis which has been implicated in the resistance to bevacizumab treatment. In fact, in several patients, we observed the emergence of this 13q12.2 amplicon under bevacizumab treatment, which was invariably associated with therapy resistance. CONCLUSIONS: Non-invasive analyses of cell-free DNA from patients undergoing treatment with bevacizumab enabled the tracking of evolving tumor genomes and helped identify a recurrent focal SCNA of clinical relevance. Here, we describe a novel resistance mechanism against a widely applied treatment in patients with mCRC which will impact the clinical management of patients.

Circulating Tumour Cells in Lung Cancer.

Circulating tumour cells (CTCs) constitute a potential tumour surrogate that could serve as "liquid biopsy" with the advantage to be a minimally invasive approach compared to traditional tissue biopsies. As CTCs are thought to be the source of metastatic lesions, their analysis represents a potential means of tracking cancer cells from the primary tumour en route to distant sites, thus providing valuable insights into the metastatic process. However, several problems, such as their rarity in the peripheral blood, the technical limitations of single-cell downstream analysis and their phenotypic variability, make CTC detection and molecular characterisation very challenging. Nevertheless, in the last decade, there has been an exponential increase of interest in the development of powerful cellular and molecular methodologies applied to CTCs. In this chapter, we focus on the recent advances of functional studies and molecular profiling of CTCs. We will also highlight the clinical relevance of CTC detection and enumeration, and discuss their potential as tumour biomarkers with special focus on lung cancer.

Profiling of Circulating Free DNA Using Targeted and Genome-wide Sequencing in Patients with SCLC.

INTRODUCTION: SCLC accounts for approximately 250,000 deaths worldwide each year. Acquisition of adequate tumor biopsy samples is challenging, and liquid biopsies present an alternative option for patient stratification and response monitoring. METHODS: We applied whole genome next-generation sequencing to circulating free DNA (cfDNA) from 39 patients with limited-stage (LS) SCLC and 30 patients with extensive-stage SCLC to establish genome-wide copy number aberrations and also performed targeted mutation analysis of 110 SCLC associated genes. Quantitative metrics were calculated for copy number aberrations, including percent genome amplified (PGA [the percentage of genomic regions amplified]), Z-score (a measure of standard deviation), and Moran's I (a measure of spatial autocorrelation). In addition CellSearch, an epitope-dependent enrichment platform, was used to enumerate circulating tumor cells (CTCs) from a parallel blood sample. RESULTS: Genome-wide and targeted cfDNA sequencing data identified tumor-related changes in 94% of patients with LS SCLC and 100% of patients with extensive-stage SCLC. Parallel analysis of CTCs based on at least 1 CTC/7.5 mL of blood increased tumor detection frequencies to 95% for LS SCLC. Both CTC counts and cfDNA readouts correlated with disease stage and overall survival. CONCLUSIONS: We demonstrate that a simple cfDNA genome-wide copy number approach provides an effective means of monitoring patients through treatment and show that targeted cfDNA sequencing identifies potential therapeutic targets in more than 50% of patients. We are now incorporating this approach into additional studies and trials of targeted therapies.

Analysis of circulating cell-free DNA identifies KRAS copy number gain and mutation as a novel prognostic marker in Pancreatic cancer.

Serial biopsy of pancreatic ductal adenocarcinoma (PDAC), to chart tumour evolution presents a significant challenge. We examined the utility of circulating free DNA (cfDNA) as a minimally invasive approach across a cohort of 55 treatment-naïve patients with PDAC; 31 with metastatic and 24 with locally advanced disease. Somatic mutations in cfDNA were detected using next generation sequencing in 15/24 (62.5%) and 27/31 (87%) of patients with locally advanced and metastatic disease, respectively. Copy number changes were detected in cfDNA of 10 patients of whom 7 exhibited gain of chromosome 12p harbouring KRAS as well as a canonical KRAS codon 12 mutation. In multivariable Cox Regression analysis, we show for the first time that patients with KRAS copy number gain and KRAS mutation have significantly worse outcomes, suggesting that this may be linked to PDAC progression. The simple cfDNA assay we describe will enable determination of the presence of KRAS copy number gain and KRAS mutations in larger studies and clinical trials.

Utility of ctDNA to support patient selection for early phase clinical trials: the TARGET study.

Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) supports blood-based genomic profiling but is not yet routinely implemented in the setting of a phase I trials clinic. TARGET is a molecular profiling program with the primary aim to match patients with a broad range of advanced cancers to early phase clinical trials on the basis of analysis of both somatic mutations and copy number alterations (CNA) across a 641 cancer-associated-gene panel in a single ctDNA assay. For the first 100 TARGET patients, ctDNA data showed good concordance with matched tumor and results were turned round within a clinically acceptable timeframe for Molecular Tumor Board (MTB) review. When a 2.5% variant allele frequency (VAF) threshold was applied, actionable mutations were identified in 41 of 100 patients, and 11 of these patients received a matched therapy. These data support the application of ctDNA in this early phase trial setting where broad genomic profiling of contemporaneous tumor material enhances patient stratification to novel therapies and provides a practical template for bringing routinely applied blood-based analyses to the clinic.

In silico error correction improves cfDNA mutation calling.

MOTIVATION: Circulating-free DNA (cfDNA) profiling by sequencing is an important minimally invasive protocol for monitoring the mutation profile of solid tumours in cancer patients. Since the concentration of available cfDNA is limited, sample library generation relies on multiple rounds of PCR amplification, during which the accumulation of errors results in reduced sensitivity and lower accuracy. RESULTS: We present PCR Error Correction (PEC), an algorithm to identify and correct errors in short read sequencing data. It exploits the redundancy that arises from multiple rounds of PCR amplification. PEC is particularly well suited to applications such as single-cell sequencing and circulating tumour DNA (ctDNA) analysis, in which many cycles of PCR are used to generate sufficient DNA for sequencing from small amounts of starting material. When applied to ctDNA analysis, PEC significantly improves mutation calling accuracy, achieving similar levels of performance to more complex strategies that require additional protocol steps and access to calibration DNA datasets. AVAILABILITY AND IMPLEMENTATION: PEC is available under the GPL-v3 Open Source licence, and is freely available from: https://github.com/CRUKMI-ComputationalBiology/PCR_Error_Correction.git. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Challenges and unanswered questions for the next decade of circulating tumour cell research in lung cancer.

Since blood borne circulating tumour cells (CTCs) initially shed from the primary tumour can seed and initiate metastasis at distant sites a better understanding of the biology of CTCs and their dissemination could provide valuable information that could guide therapeutic intervention and real time monitoring of disease progression. Although CTC enumeration has provided a reliable prognostic readout for a number of cancers, including lung cancer, the precise clinical utility of CTCs remains to be established. The rarity of CTCs together with the vanishingly small amounts of nucleic acids present in a single cell as well as cell to cell heterogeneity has stimulated the development of a wide range of powerful cellular and molecular methodologies applied to CTCs. These technical developments are now enabling researchers to focus on understanding the biology of CTCs and their clinical utility as a predictive and pharmacodynamics markers. This review summarises recent advances in the field of CTC research with focus on technical and biological challenges as well the progress made towards clinical utility of characterisation of CTCs with emphasis on studies in lung cancer.

Vasculogenic mimicry in small cell lung cancer.

Small cell lung cancer (SCLC) is characterized by prevalent circulating tumour cells (CTCs), early metastasis and poor prognosis. We show that SCLC patients (37/38) have rare CTC subpopulations co-expressing vascular endothelial-cadherin (VE-cadherin) and cytokeratins consistent with vasculogenic mimicry (VM), a process whereby tumour cells form 'endothelial-like' vessels. Single-cell genomic analysis reveals characteristic SCLC genomic changes in both VE-cadherin-positive and -negative CTCs. Higher levels of VM are associated with worse overall survival in 41 limited-stage patients' biopsies (P<0.025). VM vessels are also observed in 9/10 CTC patient-derived explants (CDX), where molecular analysis of fractionated VE-cadherin-positive cells uncovered copy-number alterations and mutated TP53, confirming human tumour origin. VE-cadherin is required for VM in NCI-H446 SCLC xenografts, where VM decreases tumour latency and, despite increased cisplatin intra-tumour delivery, decreases cisplatin efficacy. The functional significance of VM in SCLC suggests VM regulation may provide new targets for therapeutic intervention.

mFast-SeqS as a Monitoring and Pre-screening Tool for Tumor-Specific Aneuploidy in Plasma DNA.

Recent progress in the analysis of cell-free DNA fragments (cell-free circulating tumor DNA, ctDNA) now allows monitoring of tumor genomes by non-invasive means. However, previous studies with plasma DNA from patients with cancer demonstrated highly variable allele frequencies of ctDNA. Comprehensive genome-wide analysis of tumor genomes is greatly facilitated when plasma DNA has increased amounts of ctDNA. In order to develop a fast and cost-effective pre-screening method for the identification of plasma samples suitable for further extensive qualitative analysis, we adapted the recently described FAST-SeqS method. We show that our modified FAST-SeqS method (mFAST-SeqS) can be used as a pre-screening tool for an estimation of the ctDNA percentage. Moreover, since the genome-wide mFAST-SeqS z-scores correlate with the actual tumor content in plasma samples, changes in ctDNA levels associated with response to treatment can be easily monitored without prior knowledge of the genetic composition of tumor samples.

Homozygosity mapping identified a novel protein truncating mutation (p.Ser100Leufs*24) of the BBS9 gene in a consanguineous Pakistani family with Bardet Biedl syndrome.

BACKGROUND: Bardet Biedl Syndrome (BBS) is a rare condition of multi-organ dysfunction with characteristic clinical features of retinal degeneration, truncal obesity, postaxial polydactyly, genital anomaly, intellectual disability and renal dysfunction. It is a hetero-genetic disorder and nineteen BBS genes have been discovered so far. METHODS: Whole genome SNP genotyping was performed by using CytoScan® 750 K array (Affymetrix). Subsequently, the segregation of the disease locus in the whole family was carried out by genotyping STS markers within the homozygous interval. Finally, the mutation analysis was performed by Sanger DNA sequencing. RESULTS: In the present molecular study a consanguineous Pakistani family, with autosomal recessive BBS, was analyzed. The clinical analysis of affected individuals presented with synpolydactyly, obesity, intellectual disability, renal abnormality and retinitis pigmentosa. The presented phenotype was consistent with the major features of BBS syndrome. Homozygosity mapping identified a common homozygous interval within the known BBS9 locus. Sequence analysis of BBS9/PTHB1 gene revealed a single base deletion of c.299delC (p.Ser100Leufs*24) in exon 4. This frame-shift mutation presumably leads to a 122 amino acid truncated protein with complete loss of its C-terminal PTHB1 domain in combination with a partial loss of the N-terminal PTHB1 domain as well. BBS9/PTHB1 gene mutations have been shown to be associated with BBS syndrome and to the best of our knowledge this study reports the first Pakistani family linked to the BBS9 gene. CONCLUSION: Our molecular findings expand the mutational spectrum of BBS9 gene and also explain the genetic heterogeneity of Pakistan families with BBS syndrome. The growing number of mutations in BBS genes in combination with a detailed phenotypical description of patients will be helpful for genotype-phenotype correlation, targeted genetic diagnosis, prenatal screening and carrier testing of familial and non-familial BBS patients.

Clinical evaluation of a novel microfluidic device for epitope-independent enrichment of circulating tumour cells in patients with small cell lung cancer.

Circulating tumour cells (CTCs) have potential utility as minimally-invasive biomarkers to aid cancer treatment decision making. However, many current CTC technologies enrich CTCs using specific surface epitopes that do not necessarily reflect CTC heterogeneity. Here we evaluated the epitope-independent Parsortix system which enriches CTCs based on size and rigidity using both healthy normal volunteer blood samples spiked with tumour cells and blood samples from patients with small cell lung cancer (SCLC). Blood samples were maintained unfractionated at room temperature for up to 4 days followed by plasma removal for circulating free DNA (cfDNA) isolation and direct application of the remaining cell component to the Parsortix system. For tumour cells expressing the EpCAM cell surface marker the numbers of spiked cells retained using the Parsortix system and by EpCAM-positive selection using CellSearch® were not significantly different, whereas only the Parsortix system showed strong enrichment of cells with undetectable EpCAM expression. In a pilot clinical study we banked both enriched CTCs as well as plasma from SCLC patient blood samples. Upon retrieval of the banked Parsortix cellular samples we could detect cytokeratin positive CTCs in all 12 SCLC patients tested. Interestingly, processing parallel samples from the same patients by EpCAM enrichment using CellSearch® revealed only 83% (10/12) with cytokeratin positive CTCs indicating the Parsortix system is enriching for EpCAM negative SCLC CTCs. Our combined results indicate the Parsortix system is a valuable tool for combined cfDNA isolation and CTC enrichment that enables CTC analysis to be extended beyond dependence on surface epitopes.

D2HGDH regulates alpha-ketoglutarate levels and dioxygenase function by modulating IDH2.

Isocitrate dehydrogenases (IDH) convert isocitrate to alpha-ketoglutarate (α-KG). In cancer, mutant IDH1/2 reduces α-KG to D2-hydroxyglutarate (D2-HG) disrupting α-KG-dependent dioxygenases. However, the physiological relevance of controlling the interconversion of D2-HG into α-KG, mediated by D2-hydroxyglutarate dehydrogenase (D2HGDH), remains obscure. Here we show that wild-type D2HGDH elevates α-KG levels, influencing histone and DNA methylation, and HIF1α hydroxylation. Conversely, the D2HGDH mutants that we find in diffuse large B-cell lymphoma are enzymatically inert. D2-HG is a low-abundance metabolite, but we show that it can meaningfully elevate α-KG levels by positively modulating mitochondrial IDH activity and inducing IDH2 expression. Accordingly, genetic depletion of IDH2 abrogates D2HGDH effects, whereas ectopic IDH2 rescues D2HGDH-deficient cells. Our data link D2HGDH to cancer and describe an additional role for the enzyme: the regulation of IDH2 activity and α-KG-mediated epigenetic remodelling. These data further expose the intricacies of mitochondrial metabolism and inform on the pathogenesis of D2HGDH-deficient diseases.

Rapid Identification of Plasma DNA Samples with Increased ctDNA Levels by a Modified FAST-SeqS Approach.

BACKGROUND: Recent progress in the analysis of cell-free DNA fragments [cell-free circulating tumor DNA (ctDNA)] now allows monitoring of tumor genomes by noninvasive means. However, previous studies with plasma DNA from patients with cancer demonstrated highly variable allele frequencies of ctDNA. The comprehensive analysis of tumor genomes is greatly facilitated when plasma DNA has increased amounts of ctDNA. Therefore, a fast and cost-effective prescreening method to identify such plasma samples without previous knowledge about alterations in the respective tumor genome could assist in the selection of samples suitable for further extensive qualitative analysis. METHODS: We adapted the recently described Fast Aneuploidy Screening Test-Sequencing System (FAST-SeqS) method, which was originally established as a simple, effective, noninvasive screening method for fetal aneuploidy from maternal blood. RESULTS: We show that our modified FAST-SeqS method (mFAST-SeqS) can be used as a prescreening tool for an estimation of ctDNA percentage. With a combined evaluation of genome-wide and chromosome arm-specific z-scores from dilution series with cell line DNA and by comparisons of plasma-Seq profiles with data from mFAST-SeqS, we established a detection limit of ≥10% mutant alleles. Plasma samples with an mFAST-SeqS z-score >5 showed results that were highly concordant with those of copy number profiles obtained from our previously described plasma-Seq approach. CONCLUSIONS: Advantages of this approach include the speed and cost-effectiveness of the assay and that no prior knowledge about the genetic composition of tumor samples is necessary to identify plasma DNA samples with >10% ctDNA content.

Changes in colorectal carcinoma genomes under anti-EGFR therapy identified by whole-genome plasma DNA sequencing.

Monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR), such as cetuximab and panitumumab, have evolved to important therapeutic options in metastatic colorectal cancer (CRC). However, almost all patients with clinical response to anti-EGFR therapies show disease progression within a few months and little is known about mechanism and timing of resistance evolution. Here we analyzed plasma DNA from ten patients treated with anti-EGFR therapy by whole genome sequencing (plasma-Seq) and ultra-sensitive deep sequencing of genes associated with resistance to anti-EGFR treatment such as KRAS, BRAF, PIK3CA, and EGFR. Surprisingly, we observed that the development of resistance to anti-EGFR therapies was associated with acquired gains of KRAS in four patients (40%), which occurred either as novel focal amplifications (n = 3) or as high level polysomy of 12p (n = 1). In addition, we observed focal amplifications of other genes recently shown to be involved in acquired resistance to anti-EGFR therapies, such as MET (n = 2) and ERBB2 (n = 1). Overrepresentation of the EGFR gene was associated with a good initial anti-EGFR efficacy. Overall, we identified predictive biomarkers associated with anti-EGFR efficacy in seven patients (70%), which correlated well with treatment response. In contrast, ultra-sensitive deep sequencing of KRAS, BRAF, PIK3CA, and EGFR did not reveal the occurrence of novel, acquired mutations. Thus, plasma-Seq enables the identification of novel mutant clones and may therefore facilitate early adjustments of therapies that may delay or prevent disease progression.

Establishment of tumor-specific copy number alterations from plasma DNA of patients with cancer.

With the increasing number of available predictive biomarkers, clinical management of cancer is becoming increasingly reliant on the accurate serial monitoring of tumor genotypes. We tested whether tumor-specific copy number changes can be inferred from the peripheral blood of patients with cancer. To this end, we determined the plasma DNA size distribution and the fraction of mutated plasma DNA fragments with deep sequencing and an ultrasensitive mutation-detection method, i.e., the Beads, Emulsion, Amplification, and Magnetics (BEAMing) assay. When analyzing the plasma DNA of 32 patients with Stage IV colorectal carcinoma, we found that a subset of the patients (34.4%) had a biphasic size distribution of plasma DNA fragments that was associated with increased circulating tumor cell numbers and elevated concentration of mutated plasma DNA fragments. In these cases, we were able to establish genome-wide tumor-specific copy number alterations directly from plasma DNA. Thus, we could analyze the current copy number status of the tumor genome, which was in some cases many years after diagnosis of the primary tumor. An unexpected finding was that not all patients with progressive metastatic disease appear to release tumor DNA into the circulation in measurable quantities. When we analyzed plasma DNA from 35 patients with metastatic breast cancer, we made similar observations suggesting that our approach may be applicable to a variety of tumor entities. This is the first description of such a biphasic distribution in a surprisingly high proportion of cancer patients which may have important implications for tumor diagnosis and monitoring.

Factor XIIa regulates the structure of the fibrin clot independently of thrombin generation through direct interaction with fibrin.

Recent data indicate an important contribution of coagulation factor (F)XII to in vivo thrombus formation. Because fibrin structure plays a key role in clot stability and thrombosis, we hypothesized that FXII(a) interacts with fibrin(ogen) and thereby regulates clot structure and function. In plasma and purified system, we observed a dose-dependent increase in fibrin fiber density and decrease in turbidity, reflecting a denser structure, and a nonlinear increase in clot stiffness with FXIIa. In plasma, this increase was partly independent of thrombin generation, as shown in clots made in prothrombin-deficient plasma initiated with snake venom enzyme and in clots made from plasma deficient in FXII and prothrombin. Purified FXII and α-FXIIa, but not ß-FXIIa, bound to purified fibrinogen and fibrin with nanomolar affinity. Immunostaining of human carotid artery thrombi showed that FXII colocalized with areas of dense fibrin deposition, providing evidence for the in vivo modulation of fibrin structure by FXIIa. These data demonstrate that FXIIa modulates fibrin clot structure independently of thrombin generation through direct binding of the N-terminus of FXIIa to fibrin(ogen). Modification of fibrin structure by FXIIa represents a novel physiologic role for the contact pathway that may contribute to the pathophysiology of thrombosis.