Scientists from the National Cancer Institute’s (NCI) Center for Cancer Research, and the Washington University School of Medicine in St. Louis have developed a blood test that, they believe, could one day offer a highly sensitive and inexpensive approach to detect cancer early in people with neurofibromatosis type 1 NF1. The blood test could also help doctors monitor how well patients are responding to treatment for their cancer.

The team’s findings. “Cell-free DNA ultra-low-pass whole genome sequencing to distinguish malignant peripheral nerve sheath tumor (MPNST) from its benign precursor lesion: A cross-sectional study” are published in PLOS Medicine.

“The leading cause of mortality for patients with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome is the development of malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma. In the setting of NF1, this cancer type frequently arises from within its common and benign precursor, plexiform neurofibroma (PN). Transformation from PN to MPNST is challenging to diagnose due to difficulties in distinguishing cross-sectional imaging results and intralesional heterogeneity resulting in biopsy sampling errors,” write the investigators.

“This multi-institutional study from the National Cancer Institute and Washington University in St. Louis used fragment size analysis and ultra-low-pass whole genome sequencing (ULP-WGS) of plasma cell-free DNA (cfDNA) to distinguish between MPNST and PN in patients with NF1. Following in silico enrichment for short cfDNA fragments and copy number analysis to estimate the fraction of plasma cfDNA originating from tumor (tumor fraction), we developed a noninvasive classifier that differentiates MPNST from PN with 86% pretreatment accuracy (91% specificity, 75% sensitivity) and 89% accuracy on serial analysis (91% specificity, 83% sensitivity).

“Tumor fraction levels derived from cfDNA fragment size and copy number alteration analysis of plasma cfDNA using ULP-WGS significantly correlated with MPNST tumor burden, accurately distinguished MPNST from its benign PN precursor, and dynamically correlated with treatment response. In the future, our findings could form the basis for improved early cancer detection and monitoring in high-risk cancer-predisposed populations.”

NF1 is the most common cancer predisposition syndrome, affecting 1 in 3,000 people worldwide. The condition, caused by a mutation in a gene called NF1, is almost always diagnosed in childhood. Roughly half of people with NF1 will develop large but benign tumors on nerves, called plexiform neurofibromas.

In up to 15% of people with plexiform neurofibromas, these benign tumors turn into an aggressive form of cancer known as malignant peripheral nerve sheath tumor (MPNST). Patients with MPNST have a poor prognosis because the cancer can quickly spread and often becomes resistant to both chemotherapy and radiation. Among people diagnosed with MPNST, 80% die within five years.

“Imagine going through life with a cancer predisposition syndrome like NF1. It’s kind of like a ticking bomb,” said study co-author Jack F. Shern, MD, a Lasker Clinical Research Scholar in NCI’s Pediatric Oncology Branch. “The doctors are going to be watching for cancerous tumors, and you’re going to be watching for them, but you really want to discover that transformation to cancer as early as possible.”

Imaging scans or biopsy findings are currently used in diagnosis

Doctors currently use either imaging scans (MRI or PET scan) or biopsies to determine if plexiform neurofibromas have transformed into MPNST. However, biopsy findings aren’t always accurate, and the procedure can be extremely painful for patients because the tumors grow along nerves. Imaging tests, meanwhile, are expensive and can also be inaccurate.

“What we don’t have right now is a tool to help us determine if within that big, bulky benign plexiform neurofibroma, something bad is cooking and it’s turning into an MPNST,” Shern said. “So we thought, ‘What if we developed a simple blood test where instead of a full-body MRI or a fancy PET scan, we could just draw a tube of blood and say whether or not the patient has an MPNST somewhere?’”

In pursuit of this goal, Shern and study co-leads Aadel A. Chaudhuri, MD, PhD, and Angela C. Hirbe, MD, PhD, of Washington University School of Medicine, and their collaborators collected blood samples from 23 people with plexiform neurofibromas. Fourteen patients with MPNST that had not yet been treated, and 16 healthy people without NF1. Most of the study participants were adolescents and young adults, the age group in which MPNST most often develops.

The researchers isolated cell-free DNA from the blood samples and used whole-genome sequencing technology to look for differences in the genetic material among the three groups.

The cell-free DNA in patients with MPNST had several features that distinguished it from the DNA in the other two groups. For example, patients with MPNST had pieces of cell-free DNA that were shorter than those in people with plexiform neurofibromas or without NF1. In addition, the proportion of cell-free DNA that comes from tumors (plasma tumor fraction) in the blood samples was much higher in people with MPNST than in those with plexiform neurofibromas.

Together, these differences allowed the researchers to differentiate, with 86% accuracy, between patients with plexiform neurofibromas and those with MPNST.

In the study participants with MPNST, the plasma tumor fraction also aligned with how well they responded to treatment. In other words, if their plasma tumor fraction decreased following treatment, the size and number of their tumors (as measured by imaging scans) also decreased. An increase in plasma tumor fraction was associated with metastatic recurrence.

“You can imagine treating a patient with a chemotherapy regimen. This blood test could easily and rapidly allow us to determine whether the disease is going down or maybe even going away entirely,” Shern said. “And if you had done surgery and taken out an MPNST, and the blood test was negative, you could use that to monitor the patient going forward to see if the tumor returns.”

Shern noted that one limitation of the current study is its small size, even though it included people with NF1 from two large hospitals. The researchers are planning to conduct a larger trial with more patients. Shern said the team’s goal is to increase the accuracy of the blood test from 86% to closer to 100%. One approach would be to refine the genetic analysis to focus on genes known to be involved in MPNST.

A simple and inexpensive blood test to detect MPNST early in NF1 patients would be especially useful in developing countries and other resource-poor areas, where access to the equipment and expertise needed to perform imaging is limited, according to Shern.

Blood tests of this type also have applications in the early detection and monitoring of patients with other cancer-predisposing genetic disorders, such as multiple endocrine neoplasia, in which benign tumors can turn cancerous, or Li-Fraumeni syndrome, which increases one’s risk for developing several types of cancer.

“This is the perfect opportunity to apply these technologies where we can use a simple blood test to screen an at-risk population,” noted Shern. “If the test shows something abnormal, that’s when we know to act and go looking for a tumor.”

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