The Liquid Biopsy Consortium: Challenges and opportunities for early cancer detection and monitoring.

The emerging field of liquid biopsy stands at the forefront of novel diagnostic strategies for cancer and other diseases. Liquid biopsy allows minimally invasive molecular characterization of cancers for diagnosis, patient stratification to therapy, and longitudinal monitoring. Liquid biopsy strategies include detection and monitoring of circulating tumor cells, cell-free DNA, and extracellular vesicles. In this review, we address the current understanding and the role of existing liquid-biopsy-based modalities in cancer diagnostics and monitoring. We specifically focus on the technical and clinical challenges associated with liquid biopsy and biomarker development being addressed by the Liquid Biopsy Consortium, established through the National Cancer Institute. The Liquid Biopsy Consortium has developed new methods/assays and validated existing methods/technologies to capture and characterize tumor-derived circulating cargo, as well as addressed existing challenges and provided recommendations for advancing biomarker assays.

Pemetrexed in Recurrent or Progressive Central Nervous System Lymphoma: A Phase I Multicenter Clinical Trial.

LESSONS LEARNED: The findings from this study using monotherapy with pemetrexed in a pretreated patient population are, overall, encouraging. Unlike high-dose methotrexate, which requires several days of inpatient hospitalization, pemetrexed is relatively easy to administer in the outpatient setting and remains a viable treatment option in this patient population. The maximum tolerated dose of pemetrexed administered (900 mg/m2 every 2 weeks) was generally well tolerated and showed activity in patients with relapsed or refractory CNSL. BACKGROUND: There is currently no standard salvage treatment for patients with relapsed/refractory central nervous system (CNS) lymphoma (CNSL). We report the results of a phase I study of pemetrexed, an antifolate drug with broader activity than methotrexate (MTX). We provide the safety, tolerability, and maximum tolerated dose (MTD) of pemetrexed in patients with recurrent CNSL. METHODS: Through October 2015, 17 patients with relapsed/refractory CNSL received pemetrexed every 2 weeks with the first cohort receiving 600 mg/m2 and dose escalation in increments of 300 mg/m2 to a maximum of 1,200 mg/m2 . Three patients were to enroll at each dose level with expansion to six patients in the event of dose-limiting toxicity. Patients with both primary CNS lymphoma (PCNSL) and secondary CNS lymphoma (SCNSL) could be enrolled. RESULTS: Seventeen patients were evaluable with a median age of 63.7 years. Main adverse events included fatigue (82.4%), anemia (82.4%), and neutropenia (70.6%). The MTD was established at 900 mg/m2 . Dose-limiting toxicities were recorded in one patient in the 600 mg/m2 cohort and in two patients in the 1,200 mg/m2 cohort. Fourteen patients were evaluable for response assessment; 21.4% achieved a complete response, 35.7% had a partial response, 14.3% had stable disease, and 28.6% had progressive disease. The median progression-free survival was 4.2 months. The median overall survival was 44.5 months. In the original study protocol, the plan was to add an expansion cohort of six patients at MTD level. However, the first phase of the study was characterized by slow recruitment. Therefore, after achieving the primary objective of the study and establishing the MTD, the investigators decided to amend the protocol and to close the study. CONCLUSION: Pemetrexed administered at 900 mg/m2 every 2 weeks exhibits single-agent activity in patients with recurrent CNSL; it is well tolerated, and side effects are manageable.

Characterization of plasma-derived protoporphyrin-IX-positive extracellular vesicles following 5-ALA use in patients with malignant glioma.

BACKGROUND: Malignant gliomas are rapidly progressive brain tumors with high mortality. Fluorescence guided surgery (FGS) with 5-aminolevulinic acid (5-ALA) provides fluorescent delineation of malignant tissue, which helps achieve maximum safe resection. 5-ALA-based fluorescence is due to preferential accumulation of the fluorophore protoporphyrin-IX (PpIX) in malignant glioma tissue. Additionally, gliomas cells release extracellular vesicles (EVs) which carry biomarkers of disease. Herein, we performed animal and human studies to investigate whether 5-ALA dosed glioma cells, in vitro and in vivo, release PpIX positive EVs in circulation which can be captured and analyzed. METHODS: We used imaging flow cytometry (IFC) to characterize PpIX-positive EVs released from 5-ALA-dosed glioma cells, glioma-bearing xenograft models, as well as patients with malignant glioma undergoing FGS. FINDINGS: We first show that glioma cells dosed with 5-ALA release 247-fold higher PpIX positive EVs compared to mock dosed glioma cells. Second, we demonstrate that the plasma of glioma-bearing mice (n = 2) dosed with 5-ALA contain significantly higher levels of circulating PpIX-positive EVs than their pre-dosing background (p = 0.004). Lastly, we also show that the plasma of patients with avidly fluorescent tumors (n = 4) undergoing FGS contain circulating PpIX-positive EVs at levels significantly higher than their pre-dosing background (p = 0.00009) and this rise in signal correlates with enhancing tumor volumes (r 2  = 0.888). INTERPRETATION: Our findings highlight the potential of plasma-derived PpIX-positive EV-based diagnostics for malignant gliomas, offering a novel liquid biopsy platform for confirming and monitoring tumor status.

Wide Range of Clinical Outcomes in Patients with Gliomatosis Cerebri Growth Pattern: A Clinical, Radiographic, and Histopathologic Study.

BACKGROUND: The 2016 World Health Organization Classification of Central Nervous System Tumors categorizes gliomatosis cerebri growth pattern (GC) as a subgroup of diffuse infiltrating gliomas, defined by extent of brain involvement on magnetic resonance imaging (MRI). Clinical and radiographic features in GC patients are highly heterogeneous; however, prognosis has historically been considered poor. SUBJECTS, MATERIALS, AND METHODS: We performed a retrospective search for patients at our institution meeting radiographic criteria of primary, type I GC (defined as diffuse tumor infiltration without associated tumor mass and contrast enhancement on MRI) and analyzed their clinical, imaging, and histopathologic features. RESULTS: A total of 34 patients met radiographic criteria of primary, type I GC, and 33 had a confirmed histologic diagnosis of an infiltrating glial neoplasm. Age >47 years at diagnosis was associated with worse overall survival (OS) compared with age ≤47 years (hazard ratio [HR] 1.04, 95% confidence interval [CI] 1.01-1.07, p = .003). Patients with grade 2 tumors demonstrated a trend for improved OS compared with those with grade 3 tumors (HR 2.65, 95% CI 0.99-7.08, p = .051). Except for brainstem involvement, extent or location of radiographic involvement did not detectably affect clinical outcome. IDH mutation status identified a subgroup of GC patients with particularly long survival up to 25 years and was associated with longer time to progression (HR 4.81, 95% CI 0.99-23.47, p = .052). CONCLUSION: Patients with primary, type I GC do not uniformly carry a poor prognosis, even in the presence of widespread radiographic involvement. Consistent with other reports, IDH mutation status may identify patients with improved clinical outcome. Molecular characterization, rather than MRI features, may be most valuable for prognostication and management of GC patients. IMPLICATIONS FOR PRACTICE: Patients with gliomatosis cerebri growth pattern (GC) constitute a challenge to clinicians, given their wide range of clinical, histologic, and radiographic presentation, heterogeneous outcome patterns, and the lack of consensus on a standardized treatment approach. This study highlights that radiographic extent of disease-albeit category-defining-does not detectably influence survival and that IDH mutations may impact clinical outcome. Practicing oncologists should be aware that select GC patients may demonstrate exceptionally favorable survival times and prognosticate patients based on molecular markers, rather than imaging features alone.

Extracellular vesicles: the growth as diagnostics and therapeutics; a survey.

This article aims to document the growth in extracellular vesicle (EV) research. Here, we report the growth in EV-related studies, patents, and grants as well as emerging companies with major intent on exosomes. Four different databases were utilized for electronic searches of published literature: two general databases - Scopus/Elsevier and Web of Science (WoS), as well as two specialized US government databases - the USA Patent and Trademark Office and National Institutes of Health (NIH) of the Department of Health and Human Services. The applied combination of key words was carefully chosen to cover the most commonly used terms in titles of publications, patents and grants dealing with conceptual areas of EVs. Within the time frame from 1 January 2000 to 31 December 2016, limited to articles published in English, we identified output using search strategies based upon Scopus/Elsevier and WoS, patent filings and NIH Federal Reports of funded grants. Consistently, USA and UK universities are the most frequent among the top 15 affiliations/organizations of the authors of the identified records. There is clear evidence of upward streaming of EV-related publications. By documenting the growth of the EV field, we hope to encourage a roster of independent authorities skilled to provide peer review of manuscripts, evaluation of grant applications, support of foundation initiatives and corporate long-term planning. It is important to encourage EV research to further identify biomarkers in diseases and allow for the development of adequate diagnostic tools that could distinguish disease subpopulations and enable personalized treatment of patients.

Engineered nanointerfaces for microfluidic isolation and molecular profiling of tumor-specific extracellular vesicles.

Extracellular vesicles (EVs) carry RNA, DNA, proteins, and lipids. Specifically, tumor-derived EVs have the potential to be utilized as disease-specific biomarkers. However, a lack of methods to isolate tumor-specific EVs has limited their use in clinical settings. Here we report a sensitive analytical microfluidic platform (EVHB-Chip) that enables tumor-specific EV-RNA isolation within 3 h. Using the EVHB-Chip, we achieve 94% tumor-EV specificity, a limit of detection of 100 EVs per µL, and a 10-fold increase in tumor RNA enrichment in comparison to other methods. Our approach allows for the subsequent release of captured tumor EVs, enabling downstream characterization and functional studies. Processing serum and plasma samples from glioblastoma multiforme (GBM) patients, we can detect the mutant EGFRvIII mRNA. Moreover, using next-generation RNA sequencing, we identify genes specific to GBM as well as transcripts that are hallmarks for the four genetic subtypes of the disease.

Coding and noncoding landscape of extracellular RNA released by human glioma stem cells.

Tumor-released RNA may mediate intercellular communication and serve as biomarkers. Here we develop a protocol enabling quantitative, minimally biased analysis of extracellular RNAs (exRNAs) associated with microvesicles, exosomes (collectively called EVs), and ribonucleoproteins (RNPs). The exRNA complexes isolated from patient-derived glioma stem-like cultures exhibit distinct compositions, with microvesicles most closely reflecting cellular transcriptome. exRNA is enriched in small ncRNAs, such as miRNAs in exosomes, and precisely processed tRNA and Y RNA fragments in EVs and exRNPs. EV-enclosed mRNAs are mostly fragmented, and UTRs enriched; nevertheless, some full-length mRNAs are present. Overall, there is less than one copy of non-rRNA per EV. Our results suggest that massive EV/exRNA uptake would be required to ensure functional impact of transferred RNA on brain recipient cells and predict the most impactful miRNAs in such conditions. This study also provides a catalog of diverse exRNAs useful for biomarker discovery and validates its feasibility on cerebrospinal fluid.

A cerebrospinal fluid microRNA signature as biomarker for glioblastoma.

PURPOSE: To develop a cerebrospinal fluid (CSF) miRNA diagnostic biomarker for glioblastoma. EXPERIMENTAL DESIGN: Glioblastoma tissue and matched CSF from the same patient (obtained prior to tumor manipulation) were profiled by TaqMan OpenArray® Human MicroRNA Panel. CSF miRNA profiles from glioblastoma patients and controls were created from three discovery cohorts and confirmed in two validation cohorts. RESULTS: miRNA profiles from clinical CSF correlated with those found in glioblastoma tissues. Comparison of CSF miRNA profiles between glioblastoma patients and non-brain tumor patients yielded a tumor "signature" consisting of nine miRNAs. The "signature" correlated with glioblastoma tumor volume (p=0.008). When prospectively applied to cisternal CSF, the sensitivity and specificity of the 'signature' for glioblastoma detection were 67% and 80%, respectively. For lumbar CSF, the sensitivity and specificity of the signature were 28% and 95%, respectively. Comparable results were obtained from analyses of CSF extracellular vesicles (EVs) and crude CSF. CONCLUSION: We report a CSF miRNA signature as a "liquid biopsy" diagnostic platform for glioblastoma.

Analysis of extracellular RNA in cerebrospinal fluid.

We examined the extracellular vesicle (EV) and RNA composition of pooled normal cerebrospinal fluid (CSF) samples and CSF from five major neurological disorders: Alzheimer's disease (AD), Parkinson's disease (PD), low-grade glioma (LGG), glioblastoma multiforme (GBM), and subarachnoid haemorrhage (SAH), representing neurodegenerative disease, cancer, and severe acute brain injury. We evaluated: (I) size and quantity of EVs by nanoparticle tracking analysis (NTA) and vesicle flow cytometry (VFC), (II) RNA yield and purity using four RNA isolation kits, (III) replication of RNA yields within and between laboratories, and (IV) composition of total and EV RNAs by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing (RNASeq). The CSF contained ~106 EVs/µL by NTA and VFC. Brain tumour and SAH CSF contained more EVs and RNA relative to normal, AD, and PD. RT-qPCR and RNASeq identified disease-related populations of microRNAs and messenger RNAs (mRNAs) relative to normal CSF, in both total and EV fractions. This work presents relevant measures selected to inform the design of subsequent replicative CSF studies. The range of neurological diseases highlights variations in total and EV RNA content due to disease or collection site, revealing critical considerations guiding the selection of appropriate approaches and controls for CSF studies.

Detection of wild-type EGFR amplification and EGFRvIII mutation in CSF-derived extracellular vesicles of glioblastoma patients.

BACKGROUND: RNAs within extracellular vesicles (EVs) have potential as diagnostic biomarkers for patients with cancer and are identified in a variety of biofluids. Glioblastomas (GBMs) release EVs containing RNA into cerebrospinal fluid (CSF). Here we describe a multi-institutional study of RNA extracted from CSF-derived EVs of GBM patients to detect the presence of tumor-associated amplifications and mutations in epidermal growth factor receptor (EGFR). METHODS: CSF and matching tumor tissue were obtained from patients undergoing resection of GBMs. We determined wild-type (wt)EGFR DNA copy number amplification, as well as wtEGFR and EGFR variant (v)III RNA expression in tumor samples. We also characterized wtEGFR and EGFRvIII RNA expression in CSF-derived EVs. RESULTS: EGFRvIII-positive tumors had significantly greater wtEGFR DNA amplification (P = 0.02) and RNA expression (P = 0.03), and EGFRvIII-positive CSF-derived EVs had significantly more wtEGFR RNA expression (P = 0.004). EGFRvIII was detected in CSF-derived EVs for 14 of the 23 EGFRvIII tissue-positive GBM patients. Conversely, only one of the 48 EGFRvIII tissue-negative patients had the EGFRvIII mutation detected in their CSF-derived EVs. These results yield a sensitivity of 61% and a specificity of 98% for the utility of CSF-derived EVs to detect an EGFRvIII-positive GBM. CONCLUSION: Our results demonstrate CSF-derived EVs contain RNA signatures reflective of the underlying molecular genetic status of GBMs in terms of wtEGFR expression and EGFRvIII status. The high specificity of the CSF-derived EV diagnostic test gives us an accurate determination of positive EGFRvIII tumor status and is essentially a less invasive "liquid biopsy" that might direct mutation-specific therapies for GBMs.

Extracellular vesicles derived from human vestibular schwannomas associated with poor hearing damage cochlear cells.

BACKGROUND: Vestibular schwannoma (VS) is a tumor of the vestibular nerve that transmits balance information from the inner ear to the brain. Sensorineural hearing loss occurs in 95% of patients with these tumors, but the cause of this loss is not well understood. We posit a role of VS-secreted extracellular vesicles (EVs) as a major contributing factor in cochlear nerve damage. METHODS: Using differential centrifugation, we isolated EVs from VS cell line HEI-193 and primary cultured human VS cells from patients with good hearing or poor hearing. The EVs were characterized using a Nanosight device and transmission electron microscopy and by extracting their RNA content. The EVs' effects on cultured murine spiral ganglion cells and organotypic cochlear cultures were studied using a transwell dual-culture system and by direct labeling of EVs with PKH-67 dye. EV-induced changes in cochlear cells were quantified using confocal immunohistochemistry. Transfection of VS cells with a green fluorescent protein-containing plasmid was confirmed with reverse transcription PCR. RESULTS: Human VS cells, from patients with poor hearing, produced EVs that could damage both cultured murine cochlear sensory cells and neurons. In contrast, EVs derived from VS cells from patients with good hearing did not damage the cultured cochlear cells. CONCLUSIONS: This is the first report on EVs derived from VSs and on the capacity of EVs from VSs from patients with hearing loss to selectively damage cochlear cells, thereby identifying a potential novel mechanism of VS-associated sensorineural hearing loss.

ExRNA in Biofluids as Biomarkers for Brain Tumors.

Patients with high-grade gliomas and glioblastomas (GBMs) have poor survival despite optimal surgical and drug therapy. Minimally invasive diagnostic biomarkers would enable early diagnosis and tumor-specific treatments for 'personalized targeted' therapy, and would create the basis for response tracking in patients with GBM. Extracellular vesicles (EVs) isolated from cerebrospinal fluid and blood contain glioma-specific molecules, including tumor-derived EV RNAs that are detectable in small copy numbers in these biofluids. EV RNA mutations or expression changes are also detectable, the analysis of which gives rise to 'liquid biopsy' tumor profiling.

Comparative Analysis of Technologies for Quantifying Extracellular Vesicles (EVs) in Clinical Cerebrospinal Fluids (CSF).

Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patients. However, the optimal method for quantitative assessment of EVs in clinical bio-fluid remains a point of contention. Multiple high-resolution platforms for quantitative EV analysis have emerged, including methods grounded in diffraction measurement of Brownian motion (NTA), tunable resistive pulse sensing (TRPS), vesicle flow cytometry (VFC), and transmission electron microscopy (TEM). Here we compared quantitative EV assessment using cerebrospinal fluids derived from glioblastoma patients using these methods. For EVs <150 nm in diameter, NTA detected more EVs than TRPS in three of the four samples tested. VFC particle counts are consistently 2-3 fold lower than NTA and TRPS, suggesting contribution of protein aggregates or other non-lipid particles to particle count by these platforms. While TEM yield meaningful data in terms of the morphology, its particle count are consistently two orders of magnitude lower relative to counts generated by NTA and TRPS. For larger particles (>150 nm in diameter), NTA consistently detected lower number of EVs relative to TRPS. These results unveil the strength and pitfalls of each quantitative method alone for assessing EVs derived from clinical cerebrospinal fluids and suggest that thoughtful synthesis of multi-platform quantitation will be required to guide meaningful clinical investigations.

Extracellular RNAs: development as biomarkers of human disease.

Ten ongoing studies designed to test the possibility that extracellular RNAs may serve as biomarkers in human disease are described. These studies, funded by the NIH Common Fund Extracellular RNA Communication Program, examine diverse extracellular body fluids, including plasma, serum, urine and cerebrospinal fluid. The disorders studied include hepatic and gastric cancer, cardiovascular disease, chronic kidney disease, neurodegenerative disease, brain tumours, intracranial haemorrhage, multiple sclerosis and placental disorders. Progress to date and the plans for future studies are outlined.

Depot delivery of dexamethasone and cediranib for the treatment of brain tumor associated edema in an intracranial rat glioma model.

Treatments of brain tumor associated edema with systemically delivered dexamethasone, the standard of care, and cediranib, a novel anti-edema agent, are associated with systemic toxicities in brain tumor patients. A tunable, reservoir-based drug delivery device was developed to investigate the effects of delivering dexamethasone and cediranib locally in the brain in an intracranial 9L gliosarcoma rat model. Reproducible, sustained releases of both dexamethasone and solid dispersion of cediranib in polyvinylpyrrolidone (AZD/PVP) from these devices were achieved. The water-soluble AZD/PVP, which exhibited similar bioactivity as cediranib, was developed to enhance the release of cediranib from the device. Local and systemic administration of both dexamethasone and cediranib was equally efficacious in alleviating edema but had no effect on tumor growth. Edema reduction led to modest but significant improvement in survival. Local delivery of dexamethasone prevented dexamethasone-induced weight loss, an adverse effect seen in animals treated with systemic dexamethasone. Local deliveries of dexamethasone and cediranib via these devices used only 2.36% and 0.21% of the systemic doses respectively, but achieved similar efficacy as systemic drug deliveries without the side effects associated with systemic administration. Other therapeutic agents targeting brain tumor can be delivered locally in the brain to provide similar improved treatment outcomes.

Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma.

Real-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical problem as serial re-biopsy of primary tumours is often not a clinical option. MGMT (O(6)-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy. Yet, serial clinical analysis remains difficult, and, when done, primarily relies on promoter methylation studies of tumour biopsy material at the time of initial surgery. Here we present a microfluidic chip to analyse mRNA levels of MGMT and APNG in enriched tumour exosomes obtained from blood. We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients. We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.

Extracellular vesicles, tissue factor, cancer and thrombosis - discussion themes of the ISEV 2014 Educational Day.

Although the association between cancer and venous thromboembolism (VTE) has long been known, the mechanisms are poorly understood. Circulating tissue factor-bearing extracellular vesicles have been proposed as a possible explanation for the increased risk of VTE observed in some types of cancer. The International Society for Extracellular Vesicles (ISEV) and International Society on Thrombosis and Haemostasis (ISTH) held a joint Educational Day in April 2014 to discuss the latest developments in this field. This review discusses the themes of that event and the ISEV 2014 meeting that followed.

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

Phase 2 trial of high-dose rituximab with high-dose cytarabine mobilization therapy and high-dose thiotepa, busulfan, and cyclophosphamide autologous stem cell transplantation in patients with central nervous system involvement by non-Hodgkin lymphoma.

BACKGROUND: High-dose thiotepa, busulfan, and cyclophosphamide (TBC) with autologous stem cell transplantation (ASCT) has been used in patients with central nervous system (CNS) involvement by non-Hodgkin lymphoma (NHL). Despite limited penetration into the CNS, rituximab is active in primary CNS NHL. Therefore, high-dose rituximab was combined with TBC for ASCT in patients with CNS NHL. METHODS: A single-arm phase 2 trial using high-dose rituximab with cytarabine for stem cell mobilization followed by high-dose rituximab combined with thiotepa, busulfan, and cyclophosphamide (R-TBC) for ASCT was conducted. Doses of rituximab at 1000 mg/m(2) were given on days 1 and 8 of mobilization and on days -9 and -2 of TBC. The primary endpoint was efficacy. RESULTS: Thirty patients were enrolled. Eighteen patients had primary CNS NHL (12 with complete remission (CR)/first partial remission (PR1) and 6 with CR/PR2), and 12 patients had secondary CNS lymphoma (5 with CR/PR1 and 7 with CR/PR2 or beyond). All patients were in partial or complete remission. Twenty-nine patients proceeded to R-TBC ASCT. Two patients developed significant neurotoxicity. The 100-day nonrelapse mortality rate was 0%, and 1 patient died because of nonrelapse causes 5 months after ASCT. For all patients, at a median follow-up of 24 months (range, 12-40 months), the estimated 2-year progression-free survival rate was 81% (95% confidence interval, 59%-92%), and the 2-year overall survival rate was 93% (95% confidence interval, 76%-98%). There were no relapses or deaths among the 18 patients with primary CNS lymphoma. CONCLUSIONS: For patients with CNS involvement by B-cell NHL and especially for patients with primary CNS NHL, R-TBC ASCT shows encouraging activity and merits further study.