Urinary cell-free DNA analysis had near-100% accuracy for detecting

mutations associated with histiocytic disease, according to a small clinical study

reported at the 2014 ASCO Annual Meeting in Chicago.

Analysis of cell-free DNA in urine and plasma specimens from 22 patients with Erdheim-Chester disease or Langerhans cell histiocytosis showed the DNA test correctly identified all 10 patients with biopsy-confirmed mutations and ruled out mutations in all six patients with biopsy-confirmed wild-type tumors. Additionally, four patients had failed biopsy tests, and two patients did not have biopsy results.

The high level of performance by the cell-free DNA analysis has persisted in ongoing testing that increased the total number of patients to 26, said Omar Abdel-Wahab, MD, a myeloid diseases specialist at Memorial Sloan Kettering Cancer Center in New York. The test also has wide-ranging potential for identifying other types of

Diamond is a neurologist at Memorial Sloan Kettering Cancer Center.

&ldquo;The same technology could be applied to any

-mutant disease,&rdquo; said Abdel-Wahab. &ldquo;In fact, this is ongoing currently in a study

at MD Anderson Cancer Center by Filip Janku, where he is measuring

-mutant allele burden in urine and plasma of many cancer patients of various types.&rdquo;

The technology can be adapted to identify other types of oncogenic mutations, Abdel-Wahab continued. Work has already begun on development of cell-free DNA tests for detection of

mutations. Urine-based cell-free DNA testing for

mutations is already commercially available.

&ldquo;The same technology can be used to detect virtually any point mutations,&rdquo; he said. &ldquo;Several groups are working on sequencing large numbers of genes in cell-free DNA as well.&rdquo;

Though a rare clinical entity, histiocytic disease provided a good candidate for evaluating the cell-free DNA assay because of the disease&rsquo;s high frequency of the

mutations and high response to treatment with RAF inhibition.

Abdel-Wahab and colleagues evaluated the DNA test in patients with known

mutation status, using droplet digital polymerase chain reaction (PCR) technology. In addition to patients with interpretable biopsy results, two patients without biopsy data were evaluated by urinary cell-free DNA assay, and both tested positive for the

mutation. Analysis of cell-free DNA from plasma of the patients with histiocytic disease did not identify additional patients with

-mutant disease that had not been identified by analysis of urinary cell-free DNA.

mutations by droplet digital PCR in cell-free DNA is feasible and was completely concordant with CLIA-certified tumor mutation testing,&rdquo; Abdel-Wahab and colleagues concluded in a poster presentation. &ldquo;Moreover, the ability to detect

mutations in the urine of patients with repeated tumor tissue testing failure is potentially practice changing and demonstrates the clinical utility of cell-free DNA technologies.&rdquo;

The study is notable as one of the few reported thus far to incorporate mutation analysis of tissue, urine, and blood, said invited discussant Ben Ho Park, MD, PhD, of Johns Hopkins Medicine in Baltimore, Maryland.

&ldquo;The results are pretty exciting because there were even some cases where tissue could not adequately be queried formeaning that the quality of the tissue wasn&rsquo;t there so that they could reliably do mutation analysis,&rdquo; said Park. &ldquo;You can now take urine and blood and use that almost as a surrogate, for a liquid biopsy purpose, to see whether or not a patient is harboring a

A published abstract from the ASCO meeting provided evidence that longitudinal analysis of urinary cell-free DNA might have utility in monitoring patients with

-mutant tumors for treatment response. Submitted by investigators headed by Janku, an assistant professor of investigational cancer therapeutics at MD Anderson Cancer Center, the study involved 17 patients with advanced, biopsy-proven

-mutant cancers, including melanoma, non-small cell lung cancer, and colorectal cancer.

The patients underwent serial analyses of urinary cell-free DNA, which demonstrated statistically significant correlation in

mutation load and imaging-confirmed treatment response to targeted therapy. Specifically, patients with a decrease in

load had a significantly longer median time to treatment failure (259 days vs 61 days,

mutation analysis for the 7 most common mutations from urine is currently available through Trovagene&rsquo;s Clinical Laboratory Improvement Amendments (CLIA) lab, which uses next-generation sequencing technology. The company is currently working on an

mutation analysis for lung cancer, including the

mutation, which is a marker of resistance to erlotinib.

mutations associated with Erdheim-Chester disease or Langerhans cell histiocytosis.

-mutant allele burden in urine and plasma of many cancer patients of various types.

Work has begun on development of cell-free DNA tests for

Analysis of cell-free DNA from plasma of the patients with histiocytic disease did not identify additional patients with

The study is one of the few reported to incorporate mutation analysis of tissue, urine, and blood and provided evidence that longitudinal analysis of urinary cell-free DNA might have use in monitoring patients with

mutation analysis for the 7 most common mutations from urine is currently available through Trovagene&rsquo;s CLIA lab. NRAS and PIK3CA are among the future markers also being developed. Studies in KRAS are already open and accruing.

NRAS and PIK3CA are among the future markers also being developed. Studies in KRAS are already open and accruing. The University of Southern California is the study site for the colorectal study, and US Oncology/Scottsdale Healthcare/University of California, San Francisco are the sites for the pancreatic cancer study. A melanoma BRAF study collaboration was just announced with the Dana-Farber Cancer Institute as well, with Jason Luke as the principal investigator.

Abdel-Wahab OI, Diamond MP, Holley VR, et al. Detection of BRAF mutations in urine and plasma cell-free DNA: application to the diagnosis and management of histiocytic disorder patients. Poster presented at: 50th Annual Meeting of the American Society of Clinical Oncologists; May 30-June 2, 2014, Chicago, IL. Abstract 11012.

Janku F, Vibat CR, Falchook GS, et al. Longitudinal monitoring of BRAF V600E mutation in urinary cell-free DNA of patients with metastatic cancers.

. 2014;32(suppl). Abstract e22175. http://meetinglibrary.asco.org/content/130132-144. Accessed June 18, 2014.

Articles

Clinical Articles

Urinary cell-free DNA analysis had near-100% accuracy for detecting BRAF mutations associated with histiocytic disease, according to a small clinical study reported at the 2014 ASCO Annual Meeting in Chicago.

DNA in Urine Shows Promise as a Liquid Biopsy for Cancers and Rare Diseases

Eli L. Diamond, MD, neurologist, Memorial Sloan Kettering Cancer Center, discusses an analysis that looked at urine and plasma cell-free DNA

testing compared with tissue biopsy mutation testing. This analysis was presented at the 2014 ASCO Annual Meeting.

Read about DNA in urine as a liquid biopsy for cancers and rare diseases > >

Eli L. Diamond, MD, neurologist, Memorial Sloan Kettering Cancer Center, discusses an analysis that looked at urine and plasma cell-free DNA BRAFV600E testing compared with tissue biopsy mutation testing.

Comparing Urine and Plasma Cell-Free DNA <em>BRAFV600E</em> Testing to Tissue Biopsy Mutation Testing

Filip Janku, MD, PhD, assistant professor, Department of Investigational Cancer Therapeutics, University of Texas, MD Anderson Cancer Center, discusses the importance of having a urine test to detect for cancer mutations.

Learn more about mutational analysis using urinary cell-free DNA > >

Filip Janku, MD, PhD, assistant professor, Department of Investigational Cancer Therapeutics, University of Texas, MD Anderson Cancer Center, discusses the importance of having a urine test to detect for cancer mutations.  

The Importance of Urine Tests for Mutation Detection

Mutational analysis is emerging as a powerful tool in oncology; from the identification and validation of a tumor-specific genetic lesion, to the development of therapeutic agents designed to target that lesion, to the subsequent, longitudinal assessment of acquired mutations that may be associated with therapeutic resistance.

To realize the full potential of this technology, however, rapid, minimally invasive, and cost-effective approaches for mutational analysis of tumors are needed not only at surgery and diagnosis, but also throughout an extended course of treatment. While polymerase chain reaction (PCR)-based technologies allow for the amplification and identification of mutant DNA from small quantities of biopsy material, material for analysis cannot always be readily obtained, or obtained in sufficient quantity or quality, for the desired analysis. Furthermore, the invasive and expensive nature of tumor biopsies precludes their routine use to evaluate tumor response or resistance to a given targeted therapy.

Filip Janku, MD, PhD, on the Importance of Urine Tests for Mutation Detection

Janku is an assistant professor at MD Anderson Cancer Center.

In a publication for this year&rsquo;s American Society of Clinical Oncology (ASCO) Annual Meeting, Filip Janku, MD, PhD, and coworkers at the M.D. Anderson Cancer Center in Houston, Texas, described a technique for longitudinal assessment of a specific mutation,

, in patients with metastatic tumors using urinary cell-free DNA (cfDNA).1 Janku noted that mutational analysis is quickly moving toward becoming the standard of care. He emphasized that not only is this important from the standpoint of identifying patients with a specific mutation who are candidates for targeted therapy, but also for excluding those who are not appropriate candidates for a given targeted therapy, and indeed, who may do worse on that therapy. He noted, for example, that while patients with tumors containing

inhibitors, have good responses, those without mutations given the same therapies in fact do worse on the therapy; for this reason, the Food and Drug Administration (FDA) now mandates that those patients initiating

David Hyman, MD, a medical oncologist at Memorial Sloan Kettering Cancer Center in New York City, noted that tumor genetic profiling can be challenging because of the limited amount of tumor material present in a biopsy, and that the benefit of urine-based cfDNA genetic profiling is that it allows for testing even when adequate tumor material is unavailable. &ldquo;This avoids the need for additional, and possibly invasive, biopsies,&rdquo; Hyman said.

In their study, Janku&rsquo;s group used urine samples collected sequentially at 4 or more week intervals from patients with advanced cancers known to harbor the

mutation, predetermined by tumor tissue assessment from a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory.

Mutation-containing fragments were enriched within the samples by preamplification of the

mutation in urinary cfDNA was quantitatively evaluated using droplet digital PCR methodology.

Janku emphasized that there are several benefits to the urine-based approach for obtaining cfDNA; first, urine is easier to obtain, allowing for multiple time points, which (in the case of plasma) would require multiple blood draws. He also noted that, in the clinical trial setting, patients are required to have multiple blood draws for other purposes, and there is a defined limit, according to the Institutional Review Board (IRB)-approved protocol, to the volume of blood that can be drawn. Another benefit is stability; while blood samples need to be carefully and rapidly processed, urine samples, by comparison, are relatively stable and nonperishable. The higher sensitivity of the PCR assay used for the urine-based approach also shows a clear benefit over other methods; Janku noted that normal testing of tumor biopsy material, even with the most sensitive methods, &ldquo;is capable of detecting

-mutant DNA in the tissue sample, as long as you have at least 5%, maybe even 10%, of mutant alleles in the sample.&rdquo; With the urine-based cfDNA system, he noted that a much higher sensitivity is needed for advanced cancers.

&ldquo;You need to find 1 mutant allele in 1000 wild types, at the least,&rdquo; Janku said. Likewise, Hyman emphasized that the digital droplet PCR technology used here is &ldquo;very sensitive and capable of detecting even a very low level of mutations.&rdquo;

The results of Janku&rsquo;s studies demonstrated that, among patients with advanced cancers harboring the

mutation in the tumor (n = 17), the same mutation was detected using urinary cfDNA in 15 patients (88%). In previous studies applying the same methodology, agreement between

mutation as detected by tumor tissue versus urinary cfDNA was 100% (3 of 3 samples) among patients with Erdheim-Chester Disease (ECD), 95% (19/20) among patients with various metastatic cancers, and 88% among 33 patients with advanced cancers or ECD.

G12 mutations showed agreement between the CLIA laboratory assessed- and urinary cfDNA-assessed KRAS G12 mutation in 7 of 7 patients (100%).

Commenting on the overall concordance between tissue-assessed mutational status and the urine-based cfDNA method, Janku said that &ldquo;when you collect cell-free DNA either in plasma or in urine, at the same time as you acquire the tissue [biopsy sample]&hellip; analyzing both samples at the same time, your concordance is really close to 100%, if not 100%.&rdquo; He also noted that, as was the case in the present study, if the tissue is collected at a different time as part of the standard patient care, the concordance rate goes down a little, but typically, &ldquo;concordance is roughly 85% plus&hellip; which I think is actually acceptable.&rdquo;

Further findings from the study showed a significant correlation (r = 0.69,

as detected by urinary cfDNA and the response to therapy targeting

, evaluated by percentage change as determined by the Response Evaluation Criteria In Solid Tumors (RECIST 1.1). There was also a longer median time to treatment failure (259 days vs 61 days,

= .002) for patients who experienced a decrease in

Commenting on the implications of the longitudinal data, Janku noted that, to date, all of the data have been evaluated in a retrospective manner, that is, patients are treated according to standard of care, sampled, and then evaluated for their response. He suggested that the next step in this research would be to have a situation where one would actually adjust treatment based on evaluation of cfDNA mutational status, that is, intervention based on the cell-free DNA test. &emsp;

Using this method of urinary cfDNA monitoring for mutations in patients with metastatic cancers, one can envision the next step toward individualized cancer care: a strategy whereby tumor burden, response to targeted therapies, and molecular mechanisms underlying the development of resistance to these can be readily evaluated in a simple and completely noninvasive manner. As a final thought, Janku also alluded to some emerging data indicating that the overall mutational load, as assessed by urinary cfDNA, might be additionally useful as a prognostic indicator for patient survival. Hyman also added, &ldquo;The ability to longitudinally measure and quantify mutations in cfDNA may help revolutionize the way we follow response to targeted therapies in the clinic&rdquo;, and he further emphasized that it may soon be possible to predict responses before observed computed tomography (CT) changes, and perhaps detect emergent resistance before the patient develops symptoms or the CT worsens.

2014 ASCO: Janku F, Vibat CRT, Falchook GS, et al. Longitudinal monitoring of

mutation in urinary cell-free DNA of patients with metastatic cancers.

Janku F, Vibat CRT, Kosco K, et al. BRAF V600E mutations in urine and plasma cell-free DNA from patients with Erdheim-Chester disease.

. 2014 [Epub ahead of print] www.impactjournals.com/oncotarget

Janku F, Falchook GS, Piha-Paul SA, et al. Detection and Monitoring of BRAF and KRAS Mutations in Cell-Free Urinary DNA of Metastatic Cancer Patients by Droplet Digital PCR. American Association for Cancer Research (AACR) 2013 Annual Meeting. Available at: http://www.trovagene.com/trovagene/media/Corporate-Media/TROVAGENE-POSTER-AACR-2.pdf. Accessed June 7, 2014.

Janku F, Falchook GS, Piha-Paul SA, et al. Droplet Digital PCR Mutant and Longitudinal Monitoring of BRAF Mutations in Cell-Free Urinary DNA of Patients Metastatic Cancers or Erdheim-Chester Disease. American Association for Cancer Research (AACR) 2014 Annual Meeting. Abstract LB170. Available at: http://www.trovagene.com/trovagene/media/Trovagene/140408-Trovagene_2014-AACR_Annual_Monitoring.pdf. Accessed June 7, 2014.

Mutational analysis is emerging as a powerful tool in oncology; from the identification and validation of a tumor-specific genetic lesion, to the development of therapeutic agents, to the subsequent, longitudinal assessment of acquired mutations.

Precision Diagnostics | Special Reports

DNA in Urine Shows Promise as a Liquid Biopsy for Cancers and Rare Diseases

Comparing Urine and Plasma Cell-Free DNA <em>BRAFV600E</em> Testing to Tissue Biopsy Mutation Testing

The Importance of Urine Tests for Mutation Detection

Tumor Mutational Analysis Using Urinary Cell-Free DNA