Liquid Biopsies Reveal Complex Landscape of Lymphoma

Alexander M. Castellino, PhD

November 18, 2016

The sequencing of circulating tumor DNA (ctDNA) in blood samples – so-called liquid biopsies ― has emerged as a promising and noninvasive approach to detect the clonal evolution of lymphoma. Previously, the only way to follow the disease as it evolved was by tissue biopsy.

The new findings come from a study conducted at Stanford University, where researchers carried out cancer personalized profiling by deep sequencing (CAPP-Seq) on lymphoma tissue samples and ctDNA from blood samples. They showed that molecular profiling seen in blood samples matched that seen in lymphoma tissue.

In addition, they were able to identify specific genetic variations associated with adverse outcomes, likelihood of response to current therapy, and emergence of resistance, and they could predict the likelihood of relapse before it occurred.

"We can now measure several unique features of B-cell lymphomas in the blood that were once only possible to do with tissue samples," Ash A. Alizadeh, MD, PhD, assistant professor of medicine at Stanford University, told Medscape Medical News.

"The blood biopsy could also measure risk-associated features of the disease, differentiate between subtypes, and monitor changes from low-grade to high-grade lymphoma," he added.

Watching how the lymphoma changes over time and seeing this much earlier in the blood provides us a window of opportunity to intervene sooner. Dr Maximilian Diehn

"Watching how the lymphoma changes over time and seeing this much earlier in the blood provides us a window of opportunity to intervene sooner before disease burden sets in," Maximilian Diehn, MD, PhD, assistant professor of radiation oncology at Stanford University, told Medscape Medical News.

Dr Alizadeh and Dr Diehn share senior authorship of the article, which was published online November 9 in Science Translational Medicine.

"This is a landmark paper and represents a significant technological advance in managing patients with lymphoma," said lymphoma expert Mark Roschewski, MD, from the Center for Cancer Research at the National Cancer Institute. He was not involved in the study and was approached for comment by Medscape Medical News.

Diffuse large B-cell Lymphoma (DLBCL) evolves differently in different patients. Dr Roschewski explained that the heterogeneity of DLBCL is at the molecular level and cannot be seen under the microscope. Once it begins, treatment adds selection pressure, and "DLBCL has the ability to further evolve and change its molecular biology," he added.

"Now for the first time in lymphoma, we have seen a broad panel noninvasively interrogate genes and other molecular aberrations that are specific to DLBCL and other lymphoma subtypes. The ability to pick up individual mutations that confer drug resistance could prove to be very valuable in the clinical management of these patients," Dr Roschewski commented.

Unmasking the Landscape of DLBCL

For their study, which included 92 patients, the Stanford team profiled DLBCL at "various disease milestones" to evaluate whether CAPP-Seq could identify DLBCL genotypes.

By profiling 76 diagnostic DLBCL tumor biopsy specimens and 144 longitudinal blood samples (45 obtained before treatment), the researchers were able to show somatic changes in 100% of the tumors (134 variants, which included the V(D)J rearrangement) and 89% of translocations typically identified through fluorescence in situ hybridization (FISH). Pretreatment blood samples could detect ctDNA in 100% of patients with 99.8% specificity.

Several specific driver mutations and FISH-confirmed translocations previously identified from tissue samples could then be identified from ctDNA extracted from blood samples.

The researchers also showed that these mutations seen in tumor biopsy specimens and pretreatment blood samples could also be captured over time in serial blood samples of patients.

"These data suggest that, in most DLBCL patients, ctDNA is a robust surrogate for direct assessment of primary tumor genotypes," the researchers note.

CAPP-Seq genotyping of blood samples applied to patients whose disease progressed on treatment with ibrutinib (Imbruvica, Janssen Biotech) — a Bruton kinase inhibitor under investigation in the treatment of DLBCL — showed that the technique could capture mutations in BTK previously seen only in ibrutinib-refractory chronic lymphocytic leukemia and mantle cell lymphoma. The clonal dynamics of the tumors was different in different patients and suggested that the technique used on blood samples could follow clonal evolution of the tumors.

CAPP-Seq was also able to detect clinical risk equivalents. ctDNA concentrations in pretreatment blood samples were higher in patients with higher blood lactate dehydrogenase levels or higher metabolic tumor volume and correlated with Ann Arbor stage — all risk equivalents in DLBCL.

"Pretreatment ctDNA in DLBCL can complement traditional clinical indices and serve as an independent prognostic biomarker," the authors write.

The researchers showed that CAPP-Seq could detect occult disease long before it could be seen in the clinic using current radiographic techniques. They molecularly profiled blood samples taken from patients who had shown a complete radiographic response, from those whose disease recurred, and from those who ultimately experienced disease progression. Although ctDNA was not able to detect mutations in patients who had a complete response, the molecular profile at relapse compared well with the currently used assay to detect V(D)J rearrangements.

Indeed, patients with detectable ctDNA showed inferior progression-free survival. What was most significant was the observation that in those patients who eventually relapsed, CAPP-Seq was able to capture minimal residual disease with a mean lead time of longer than 2 months. The mean time between the first positive ctDNA time point and clinical relapse was 188 days (>6 months).

"CAPP-Seq can identify patients who will relapse, often months before recurrence can be clinically identified." Dr Diehn told Medscape Medical News.

That is because it requires a billion cells for a 1-cm lesion to be detected on imaging, but modern assays that utilize next-generation sequencing have limits of detection that are approximately 1 molecule in a million cellular equivalents, Dr Roschewski explained.

Important Patterns in DLBCL

A significant prognostic factor in DLBCL is the ability to identify the cell from which it originates — germinal center B-cell-like, or activated B-cell-like, each of which is enriched in specific mutations. CAPP-Seq was able to identify these in ctDNA with accuracy similar to that from tissue samples, the researchers showed.

The researchers were able to use CAPP-Seq on ctDNA to follow the transformation of indolent lymphoma to aggressive lymphoma. The data suggest that the imprint of the transformation is seen in the blood long before it is clinically manifested.

"Once validated, it should be possible to detect and monitor for transformation using the same tool," Dr Alizadeh said.

"Each year, patients with indolent lymphoma roughly have a 2% risk of transformation into aggressive disease," Dr Alizadeh told Medscape Medical News. "In a lifetime, this risk can gradually accumulate to nearly 25%," he added. It is difficult to monitor this routinely, he explained. Which lymph node to choose for biopsy, where to look for the transformation, and how to image are some of the issues facing clinicians following patients with indolent disease, he explained.

Until now, clinical management has often taken a watch-and-wait approach for patients with indolent lymphoma. Only after changes are seen on CT scans or symptoms ensue is intervention indicated. CAPP-Seq and other molecular monitoring assays are major advancements that may inform clinicians as to the best time to start or stop therapy, Dr Roschewski pointed out. "Ultimately, these tools set the framework for research studies that attempt to cure patients of indolent lymphomas," he told Medscape Medical News.

Technology Available, but Not Yet Ready For the Clinic

Getting serial tissue biopsy specimens from patients is difficult, and currently available technology requires fresh tissue, which poses a challenge in the clinic, Dr Roschewski explained. "Accessing molecular data without a tissue biopsy is a major technological advancement," he told Medscape Medical News.

He was enthusiastic about the potential implications from this study – that such information could be obtained from blood samples. However, he pointed out that this test is not ready for ordering off the shelf. "This is the most robust assay to be published. There is reason to be excited. This is one of the early papers to provide proof of principle and demonstrate feasibility," Dr Roschewski said. Questions remain as to how to take this forward and make it clinically applicable, he added.

The two senior authors of the study agreed. "This is the first study that generated these data, and we need to validate these findings," Dr Diehn said. He explained that the next steps are validation studies in independent cohorts. Two validation studies will be presented at the annual meeting of the American Hematologic Society, which will be held next month in San Diego, Dr Alizadeh told Medscape Medical News.

In addition, Dr Diehn indicated it is yet to be seen whether CAPP-Seq can pass the ultimate test by improving patient outcomes in the context of a prospective clinical trial.

"Processing questions also need to be answered," Dr Roschewski said. There are also other tests, he explained. It remains to be determined what the best assay is, what the best timing is, and what the different advantages are, he pointed out.

"Ultimately, before the test is offered in the clinic, it will need to be offered in a CLIA-certified lab," Dr Diehn said. "The infrastructure is in place, the tools are in place; now it is a matter of time before it can be rolled out in the clinic," Dr Roschewski told Medscape Medical News.

Dr Alizadeh and Dr Diehn are among the coinventors with patent applications related to CAPP-Seq and are consultants for Roche Molecular Systems. Dr Rochewski has disclosed no relevant financial relationships.

Sci Transl Med. Published online November 9, 2016. Abstract


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