Impact on costs and outcomes of multi-gene panel testing for advanced solid malignancies: a cost-consequence analysis using linked administrative data.

Background: To date, economic analyses of tissue-based next generation sequencing genomic profiling (NGS) for advanced solid tumors have typically required models with assumptions, with little real-world evidence on overall survival (OS), clinical trial enrollment or end-of-life quality of care. Methods: Cost consequence analysis of NGS testing (555 or 161-gene panels) for advanced solid tumors through the OCTANE clinical trial (NCT02906943). This is a longitudinal, propensity score-matched retrospective cohort study in Ontario, Canada using linked administrative data. Patients enrolled in OCTANE at Princess Margaret Cancer Centre from August 2016 until March 2019 were matched with contemporary patients without large gene panel testing from across Ontario not enrolled in OCTANE. Patients were matched according to 19 patient, disease and treatment variables. Full 2-year follow-up data was available. Sensitivity analyses considered alternative matched cohorts. Main Outcomes were mean per capita costs (2019 Canadian dollars) from a public payer's perspective, OS, clinical trial enrollment and end-of-life quality metrics. Findings: There were 782 OCTANE patients with 782 matched controls. Variables were balanced after matching (standardized difference <0.10). There were higher mean health-care costs with OCTANE ($79,702 vs. $59,550), mainly due to outpatient and specialist visits. Publicly funded drug costs were less with OCTANE ($20,015 vs. $24,465). OCTANE enrollment was not associated with improved OS (restricted mean survival time [standard error]: 1.50 (±0.03) vs. 1.44 (±0.03) years, log-rank p = 0.153), varying by tumor type. In five tumor types with ≥35 OCTANE patients, OS was similar in three (breast, colon, uterus, all p > 0.40), and greater in two (ovary, biliary, both p < 0.05). OCTANE was associated with greater clinical trial enrollment (25.4% vs. 9.5%, p < 0.001) and better end-of-life quality due to less death in hospital (10.2% vs. 16.4%, p = 0.003). Results were robust in sensitivity analysis. Interpretation: We found an increase in healthcare costs associated with multi-gene panel testing for advanced cancer treatment. The impact on OS was not significant, but varied across tumor types. OCTANE was associated with greater trial enrollment, lower publicly funded drug costs and fewer in-hospital deaths suggesting important considerations in determining the value of NGS panel testing for advanced cancers. Funding: T.P H holds a research grant provided by the Ontario Institute for Cancer Research through funding provided by the Government of Ontario (#IA-035 and P.HSR.158) and through funding of the Canadian Network for Learning Healthcare Systems and Cost-Effective 'Omics Innovation (CLEO) via Genome Canada (G05CHS).

Chronic Lymphocytic Leukemia IGHV Somatic Hypermutation Detection by Targeted Capture Next-Generation Sequencing.

BACKGROUND: Somatic hypermutation (SHM) status of the immunoglobulin heavy variable (IGHV) gene plays a crucial role in determining the prognosis and treatment of patients with chronic lymphocytic leukemia (CLL). A common approach for determining SHM status is multiplex polymerase chain reaction and Sanger sequencing of the immunoglobin heavy locus; however, this technique is low throughput, is vulnerable to failure, and does not allow multiplexing with other diagnostic assays. METHODS: Here we designed and validated a DNA targeted capture approach to detect immunoglobulin heavy variable somatic hypermutation (IGHV SHM) status as a submodule of a larger next-generation sequencing (NGS) panel that also includes probes for ATM, BIRC3, CHD2, KLHL6, MYD88, NOTCH1, NOTCH2, POT1, SF3B1, TP53, and XPO1. The assay takes as input FASTQ files and outputs a report containing IGHV SHM status and V allele usage following European Research Initiative on CLL guidelines. RESULTS: We validated the approach on 35 CLL patient samples, 34 of which were characterized using Sanger sequencing. The NGS panel identified the IGHV SHM status of 34 of 35 CLL patients. We showed 100% sensitivity and specificity among the 33 CLL samples with both NGS and Sanger sequencing calls. Furthermore, we demonstrated that this panel can be combined with additional targeted capture panels to detect prognostically important CLL single nucleotide variants, insertions/deletions, and copy number variants (TP53 copy number loss). CONCLUSIONS: A targeted capture approach to IGHV SHM detection can be integrated into broader sequencing panels, allowing broad CLL prognostication in a single molecular assay.

Multisite clinical cross-validation and variant interpretation of a next generation sequencing panel for lymphoid cancer prognostication.

AIMS: Genomic sequencing of lymphomas is under-represented in routine clinical testing despite having prognostic and predictive value. Clinical implementation is challenging due to a lack of consensus on reportable targets and a paucity of reference samples. We organised a cross-validation study of a lymphoma-tailored next-generation sequencing panel between two College of American Pathologists (CAP)-accredited clinical laboratories to mitigate these challenges. METHODS: A consensus for the genomic targets was discussed between the two institutes based on recurrence in diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukaemia and T-cell lymphomas. Using the same genomic targets, each laboratory ordered libraries independently and a cross-validation study was designed to exchange samples (8 cell lines and 22 clinical samples) and their FASTQ files. RESULTS: The sensitivity of the panel when comparing different library preparation and bioinformatic workflows was between 97% and 99% and specificity was 100% when a 5% limit of detection cut-off was applied. To evaluate how the current standards for variant classification of tumours apply to lymphomas, the Association for Molecular Pathology/American Society of Clinical Oncology/CAP and OncoKB classification systems were applied to the panel. The majority of variants were assigned a possibly actionable class or likely pathogenic due to more limited evidence in the literature. CONCLUSIONS: The cross-validation study highlights the benefits of sample and data exchange for clinical validation and provided a framework for reporting the findings in lymphoid malignancies.

Case Report: Identification of a novel STAT3 mutation in EBV-positive inflammatory follicular dendritic cell sarcoma.

EBV-positive inflammatory follicular dendritic cell sarcoma (EBV+ IFDCS) is an uncommon disease primarily observed in Asia. It is characterized by the development of tumors believed to originate from follicular dendritic cells (FDC). The consistent association between this condition and clonal EBV infection suggests EBV's involvement as an etiological factor. However, diagnosing EBV+ IFDCS can be challenging due to its morphological variability and diverse immunohistochemical staining patterns. The genetic characteristics of EBV+ IFDCS remain insufficiently understood. To address this knowledge gap, we present a case study of a 47-year-old male patient diagnosed with EBV+ IFDCS. We utilized a Next-generation sequencing (NGS) platform to investigate the genetic profile of the tumor cells. We identified a single pathogenic mutation (G618R) in the STAT3 gene. This finding provides valuable insights into the genetic alterations associated with EBV+ IFDCS and potentially contributes to our understanding of the disease's pathogenesis.

Development of a comprehensive approach to adult hereditary cancer testing in Ontario.

BACKGROUND: Genetic testing for hereditary cancer susceptibility has advanced over time due to the discovery of new risk genes, improved technology and decreased cost. In the province of Ontario, testing eligibility criteria were initially developed to include hereditary breast, ovarian and colorectal cancer syndromes. The rapid evolution of genetic technologies has facilitated the ability to interrogate a large number of genes concurrently. This, coupled with new knowledge about risk genes, necessitated a coordinated approach to expanding the scope of genes and indications tested and synchronisation of access and test utilisation across the province as required in a publicly funded universal healthcare system. METHODS: Ontario Health-Cancer Care Ontario convened expert working groups to develop a standardised and comprehensive cancer gene list for adults and accompanying hereditary cancer testing (HCT) criteria using an evidence-based framework and broad laboratory and clinical genetics engagement. RESULTS: A standardised 76-cancer-gene panel, organised into 13 larger disease site panels and 25 single/small gene panels, was developed and endorsed by the working groups. Provincial genetic testing eligibility criteria were updated to align with the new panels and to guide clinical decision-making. In the first year following the implementation of these changes, 10 564 HCT panels were performed with an overall mutation detection rate of 12.2%. CONCLUSION: Using an evidence framework and broad clinical engagement to develop and endorse an updated guidance document, cancer genetic testing for adults in Ontario is now standardised and coordinated across the province.

Myeloma immunoglobulin rearrangement and translocation detection through targeted capture sequencing.

Multiple myeloma is a plasma cell neoplasm characterized by clonal immunoglobulin V(D)J signatures and oncogenic immunoglobulin gene translocations. Additional subclonal genomic changes are acquired with myeloma progression and therapeutic selection. PCR-based methods to detect V(D)J rearrangements can have biases introduced by highly multiplexed reactions and primers undermined by somatic hypermutation, and are not readily extended to include mutation detection. Here, we report a hybrid-capture approach (CapIG-seq) targeting the 3' and 5' ends of the V and J segments of all immunoglobulin loci that enable the efficient detection of V(D)J rearrangements. We also included baits for oncogenic translocations and mutation detection. We demonstrate complete concordance with matched whole-genome sequencing and/or PCR clonotyping of 24 cell lines and report the clonal sequences for 41 uncharacterized cell lines. We also demonstrate the application to patient specimens, including 29 bone marrow and 39 cell-free DNA samples. CapIG-seq shows concordance between bone marrow and cfDNA blood samples (both contemporaneous and follow-up) with regard to the somatic variant, V(D)J, and translocation detection. CapIG-seq is a novel, efficient approach to examining genomic alterations in myeloma.

Integrating comprehensive genomic sequencing of non-small cell lung cancer into a public healthcare system.

OBJECTIVES: Standard molecular testing for patients with stage IV non-small cell lung cancer (NSCLC) in the Canadian publicly funded health system includes single gene testing for EGFR, ALK, and ROS-1. Comprehensive genomic profiling (CGP) may broaden treatment options for patients. This study examined the impact of CGP in a publicly funded health system. METHODS: Consenting patients with stage IV NSCLC without known targetable alterations underwent CGP on diagnostic samples. Patients that had progressed on targeted therapy were also eligible. The CGP assay was a hybrid capture next generation sequencing (NGS) panel (Oncomine Comprehensive Assay Version 3, ThermoFisher). The number of actionable alterations, changes in treatment, clinical trial eligibility and costs as a result of CGP were evaluated and patient willingness-to-pay. RESULTS: Of 182 screened patients,134 (74%) had successful CGP testing. Twenty percent had received prior targeted therapy. Incremental actionable alterations were identified in 31% of patients. The most common novel targets identified were mutations in ERBB2 (exon 20 insertions), MET (exon 14 skipping) and KRAS (G12C). At data cut off (31/12/2020), 16% of patients had a change in treatment as a result of CGP. Additional clinical trial options were identified for 75% of patients. The incremental direct laboratory cost for CGP beyond public reimbursement for single gene tests was $747 CAD/case. CONCLUSION: CGP identifies additional actionable targets beyond single gene tests with a direct impact on patient treatment and increased clinical trial eligibility. These benefits highlight the value of CGP in patients with NSCLC in public health systems.

DNA Methylation-Based Classification of Small B-Cell Lymphomas: A Proof-of-Principle Study.

Although most small B-cell lymphomas (SBCLs) can be diagnosed using routine methods, challenges exist. For example, marginal zone lymphomas (MZLs) can be difficult to rule-in, in large part because no widely-used, sensitive, and specific biomarker is available for the marginal zone cell of origin. In this study, it was hypothesized that DNA methylation array profiling can assist with the classification of SBCLs, including MZLs. Extramedullary SBCLs, including challenging cases, were reviewed internally for pathology consensus and profiled. By combining the resulting array data set with data sets from other groups, a set of 26 informative probes was selected and used to train machine learning models to classify 4 common SBCLs: chronic lymphocytic leukemia/small lymphocytic lymphoma, follicular lymphoma, mantle cell lymphoma, and MZL. Prediction probability cutoff was used to separate classifiable from unclassifiable cases, and show that the trained model was able to classify 95% of independent test cases (n = 264/279). The concordance between model predictions and pathology diagnoses was 99.6% (n = 262/263) among classifiable test cases. One validation reference test case was reclassified based on model prediction. The model was also used to predict the diagnoses of two challenging SBCLs. Although the differential examined and data on difficult cases are limited, these results support accurate methylation-based classification of SBCLs. Furthermore, high specificities of predictions suggest that methylation signatures can be used to rule-in MZLs.

Consensus Recommendations for MRD Testing in Adult B-Cell Acute Lymphoblastic Leukemia in Ontario.

Measurable (minimal) residual disease (MRD) is an established, key prognostic factor in adult B-cell acute lymphoblastic leukemia (B-ALL), and testing for MRD is known to be an important tool to help guide treatment decisions. The clinical value of MRD testing depends on the accuracy and reliability of results. Currently, there are no Canadian provincial or national guidelines for MRD testing in adult B-ALL, and consistent with the absence of such guidelines, there is no uniform Ontario MRD testing consensus. Moreover, there is great variability in Ontario in MRD testing with respect to where, when, and by which technique, MRD testing is performed, as well as in how the results are interpreted. To address these deficiencies, an expert multidisciplinary working group was convened to define consensus recommendations for improving the provision of such testing. The expert panel recommends that MRD testing should be implemented in a centralized manner to ensure expertise and accuracy in testing for this low volume indication, thereby to provide accurate, reliable results to clinicians and patients. All adult patients with B-ALL should receive MRD testing after induction chemotherapy. Philadelphia chromosome (Ph)-positive patients should have ongoing monitoring of MRD during treatment and thereafter, while samples from Ph-negative B-ALL patients should be tested at least once later during treatment, ideally at 12 to 16 weeks after treatment initiation. In Ph-negative adult B-ALL patients, standardized, ideally centralized, protocols must be used for MRD testing, including both flow cytometry and immunoglobulin (Ig) heavy chain and T-cell receptor (TCR) gene rearrangement analysis. For Ph-positive B-ALL patients, MRD testing using a standardized protocol for reverse transcription real-time quantitative PCR (RT-qPCR) for the BCR-ABL1 gene fusion transcript is recommended, with Ig/TCR gene rearrangement analysis done in parallel likely providing additional clinical information.

Validation of the RHL30 digital gene expression assay as a prognostic biomarker for relapsed Hodgkin lymphoma.

Despite continuing improvements in the management of classical Hodgkin lymphoma (cHL), relapse remains associated with a risk of lymphoma-related mortality. The biological composition of relapse tumour biopsies shows interpatient variability, which can be leveraged to design prognostic biomarkers. Here, we validated the RHL30 assay, a previously reported gene expression model in an independent cohort of 41 patients with relapsed cHL. Patients classified as high-risk by the RHL30 assay had inferior failure-free survival (FFS) after autologous stem cell transplantation (2-year FFS 41% vs. 92%, P = 0·035). The RHL30 model is a robust biomarker that risk-stratifies patients considered for autologous stem cell transplantation.

Molecular profiling for precision cancer therapies.

The number of druggable tumor-specific molecular aberrations has grown substantially in the past decade, with a significant survival benefit obtained from biomarker matching therapies in several cancer types. Molecular pathology has therefore become fundamental not only to inform on tumor diagnosis and prognosis but also to drive therapeutic decisions in daily practice. The introduction of next-generation sequencing technologies and the rising number of large-scale tumor molecular profiling programs across institutions worldwide have revolutionized the field of precision oncology. As comprehensive genomic analyses become increasingly available in both clinical and research settings, healthcare professionals are faced with the complex tasks of result interpretation and translation. This review summarizes the current and upcoming approaches to implement precision cancer medicine, highlighting the challenges and potential solutions to facilitate the interpretation and to maximize the clinical utility of molecular profiling results. We describe novel molecular characterization strategies beyond tumor DNA sequencing, such as transcriptomics, immunophenotyping, epigenetic profiling, and single-cell analyses. We also review current and potential applications of liquid biopsies to evaluate blood-based biomarkers, such as circulating tumor cells and circulating nucleic acids. Last, lessons learned from the existing limitations of genotype-derived therapies provide insights into ways to expand precision medicine beyond genomics.

Unique MLH1 mutations in colonic adenomas in an obligate germline Lynch syndrome carrier.

BACKGROUND: An obligate germline Lynch syndrome carrier had four colonic adenomas removed. MATERIALS AND METHODS: The adenomas were evaluated for grade of dysplasia, MLH1, PMS2, MSH2 and MSH6 protein expression, microsatellite instability (MSI), BRAF, methylation status and a next-generation sequencing (NGS) panel of 52 cancer genes. RESULTS: There were four tubular or tubulovillous adenomas from the hepatic flexure, rectosigmoid and rectum; one with low-grade and high-grade dysplasia, one with high-grade dysplasia only and two with low-grade dysplasia. All four adenomas showed retention of MLH1, MHS2 and MSH6 but complete loss of PMS2 in both low-grade and high-grade dysplasia areas.Two of the four adenomas were MSI-high, BRAF V600E wild type and were not MLH1 methylated. NGS identified an MLH1 germline variant: NM_000249.3: c.1558+1 G>A, p.(?) in all tissue (adenomas and normal), which likely explains the pathophysiology of Lynch syndrome in this patient. Other variants were also detected in MLH1 and MSH6 in all four adenomas tested; these being reported previously in somatic colorectal cancers. CONCLUSION: We highlight an MLH1 variant in the colonic adenomas in an obligate Lynch syndrome carrier that resulted in PMS2 protein loss in the absence of mutations of the PMS2 gene.

Heterogenous loss of mismatch repair (MMR) protein expression: a challenge for immunohistochemical interpretation and microsatellite instability (MSI) evaluation.

Immunohistochemistry (IHC) for mismatch repair (MMR) proteins is used to identify MMR status: being diffusely positive (intact/retained nuclear staining) or showing loss of nuclear tumour staining (MMR protein deficient). Four colonic adenocarcinomas and a gastric adenocarcinoma with associated dysplasia that displayed heterogenous IHC staining patterns in at least one of the four MMR proteins were characterised by next-generation sequencing (NGS). In order to examine a potential molecular mechanism for these staining patterns, the respective areas were macrodissected, analysed for microsatellite instability (MSI) and investigated by NGS and multiplex ligation-dependent probe amplification (MLPA) analysis of MLH1, MSH2, MSH6 and PMS2 genes, including MLH1 methylation analysis. One colonic adenocarcinoma showed heterogenous MSH6 IHC staining and molecular analysis demonstrated increasing allelic burden of two MSH6 frameshift variants (c.3261delC and c.3261dupC) in areas with MSH6 protein loss compared to areas where MSH6 was retained. Two colonic adenocarcinomas with heterogenous MLH1 staining showed no differences in sequence variants. In one of these cases, however, MLH1 was hypermethylated in the area of MLH1 loss. Another colon carcinoma with heterogenous PMS2 staining (but with retained MSH6) showed both MSH6 c.3261dupC and 3260_3261dupCC where PMS2 protein was lost and only c.3261dupC where PMS2 was retained. The gastric carcinoma showed complete loss of MSH6 in dysplastic foci, while the underlying invasive carcinoma showed retention of MSH6. Both these areas, however, were MSI-high and showed the same MSH6 variant: c.3261delC. The gastric dysplasia additionally showed MSH6 c.3261dupC. In four of the five cases where MMR protein was lost, these areas were MSI-high. Heterogenous MMR IHC (focal and/or zonal within the same tumour or between invasive and dysplastic preinvasive areas) is not always due to artefact and is invariably related to MSI-high status in the areas of loss. An interesting aspect to this study is the presence of MSH6 somatic mutations irrespective of whether MSH6 IHC staining was intact or lost.

Stable transmission of an unbalanced chromosome 21 derived from chromoanasynthesis in a patient with a SYNGAP1 likely pathogenic variant.

BACKGROUND: Complex genomic structural variations, involving chromoanagenesis, have been implicated in multiple congenital anomalies and abnormal neurodevelopment. Familial inheritance of complex chromosomal structural alteration resulting from germline chromoanagenesis-type mechanisms are limited. CASE PRESENTATION: We report a two-year eleven-month old male presenting with epilepsy, ataxia and dysmorphic features of unknown etiology. Chromosomal microarray identified a complex unbalanced rearrangement involving chromosome 21. G-banding and FISH for targeted regions of chromosome 21 revealed that the copy number imbalances were limited to gains dispersed throughout the long arm of chromosome 21, characteristic of a chromosome derived from chromoanagenesis. Family studies showed that the unbalanced chromosome had been stably inherited, as it was present in both his healthy mother and maternal grandfather. Further molecular testing for non-syndromic intellectual disability genes found a likely pathogenic mutation in SYNGAP1 (NM_006772.2:c.3722_3723del). CONCLUSIONS: This study indicates that complex rearrangements involving an unbalanced chromosome derived from chromoanasynthesis can be familial and should be not be presumed pathogenic.

Heterozygous mutations in ERF cause syndromic craniosynostosis with multiple suture involvement.

Craniosynostosis is a clinically and genetically heterogeneous condition. Knowledge of the specific genetic diagnosis in patients presenting with this condition is important for surgical and medical management. The most common single gene causes of syndromic craniosynostosis are mutations in FGFR1, FGFR2, FGFR3, TWIST1, and EFNB1. Recently, a new single gene cause of craniosynostosis was published, together with phenotype data that highlight the clinical importance of making this specific molecular diagnosis. Phenotypic features of "ERF-related craniosynostosis" include sagittal or multiple-suture synostosis, Chiari malformation, and language delay. In order to determine the contribution of ERF mutations to genetically undiagnosed patients with craniosynostosis, we sequenced the coding regions of ERF in 40 patients with multi-suture or sagittal suture synostosis. We identified heterozygous ERF mutations in two individuals (5%). One mutation positive individual had pansynostosis, while the second had bilateral coronal and metopic synostosis. Both presented in infancy or childhood (age 3 months, and 6 years 9 months, respectively). One had CNS abnormalities including Chiari I malformation. Dysmorphic features included hypertelorism, proptosis, depressed nasal bridge, and retrognathia, in keeping with previously reported cases. The individuals did not require repeated cranial surgeries. ERF-related craniosynostosis should be suspected in patients presenting with multiple suture or sagittal synostosis.

Danon Disease Due to a Novel LAMP2 Microduplication.

Danon disease is a rare X-linked disorder comprising hypertrophic cardiomyopathy, skeletal myopathy, intellectual disability, and retinopathy; mutations of the lysosome-associated membrane protein gene LAMP2 are responsible. Most affected persons exhibit "private" point mutations; small locus rearrangements have recently been reported in four cases. Here, we describe the clinical, pathologic, and molecular features of a male proband and his affected mother with Danon disease and a small LAMP2 microduplication. The proband presented at age 12 years with exercise intolerance, hypertrophic cardiomyopathy, and increased creatine kinase. Endomyocardial biopsy findings were nonspecific, showing myocyte hypertrophy and reactive mitochondrial changes. Quadriceps muscle biopsy demonstrated the characteristic autophagic vacuoles with sarcolemma-like features. LAMP2 tissue immunostaining was absent; however, LAMP2 sequencing was normal. Deletion/duplication testing by multiplex ligation-dependent probe amplification (MLPA) assay revealed a 1.5kb microduplication containing LAMP2 exons 4 and 5. RT-PCR studies were consistent with the inclusion of these two duplicated exons in the final spliced transcript, resulting in a frameshift. The proband's mother, who had died following cardiac transplantation due to suspected myocarditis at age 35, was reviewed and was shown to be affected upon immunostaining of banked myocardial tissue. This case constitutes the second report of a pathogenic microduplication in Danon disease, and illustrates a number of potential diagnostic pitfalls. Firstly, given the imperfect sensitivity of LAMP2 sequencing, tissue immunostaining and/or MLPA should be considered as a diagnostic adjunct in the workup for this disorder. Secondly, the pathological findings in myocardium may be falsely indicative of relatively common conditions such as myocarditis.

Type VIII collagen signals via ß1 integrin and RhoA to regulate MMP-2 expression and smooth muscle cell migration.

The extracellular matrix signals and regulates the behavior of vascular cells during the pathogenesis of atherosclerosis. Type VIII collagen, a short chain collagen, is scarcely present in normal arteries, but is dramatically upregulated in atherosclerosis and after other types of vascular injury. Cell culture studies have revealed that this protein supports smooth muscle cell (SMC) adhesion and stimulates migration, however little is known about the signaling or the mechanisms by which this occurs. SMCs isolated from wild-type C57BL/6 and type VIII collagen deficient mice were studied using assays to measure chemotactic and haptotactic migration, and remodeling and contraction of 3-dimensional type I collagen gels. Col8(-/-) SMCs exhibited impairments in migration, and a strongly adhesive phenotype with prominent stress fibers, stable microtubules and pronounced central basal focal adhesions. The addition of exogenous type VIII collagen to the Col8(-/-) SMCs rescued the impairments in migration, and restored cytoskeletal architecture so that it was similar to Col8(+/+) cells. We measured elevated levels of active GTP-RhoA in the Col8(-/-) cells, and this too was reversed by treatment with exogenous type VIII collagen. We showed that type VIII collagen normally suppresses RhoA activation through a beta-1 integrin dependent mechanism. MMP-2 levels were reduced in the Col8(-/-) SMCs, and knockdown of MMP-2 in Col8(+/+) SMCs partially recapitulated the decreases in migration and 3D gel contraction seen in Col8(-/-) cells, showing that type VIII collagen-stimulated migration was dependent on MMP-2. Inhibition of Rho restored MMP-2 activity in the Col8(-/-) cells, and partially rescued migration, demonstrating that the elevations in RhoA activity were responsible for the suppression of migration of these cells. In conclusion, we have shown that type VIII collagen signals through beta-1 integrin receptors to suppress RhoA, allowing optimal configuration of the cytoskeleton, and the stimulation of MMP-2-dependent cell migration.