1. Simoneaux, Richard

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The use of circulating tumor DNA (ctDNA) to perform liquid biopsies has been employed previously for a number of solid tumor-bearing malignancies, such as breast, colorectal, and lung cancer. In addition to malignancies with solid tumors, ctDNA analyses have also been performed for a number of hematologic malignancies, including multiple myeloma, diffuse large B-cell lymphoma, and most recently, classical Hodgkin lymphoma (cHL).

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In a recent publication, research was presented by Davide Rossi, MD, Deputy Head of the Division of Hematology, Oncology Institute of Southern Switzerland, Bellinzona, and colleagues concerning the use of ctDNA obtained from 112 cHL patients (Blood 2018;131:2413-2425).


"Longitudinal ctDNA profiling identified treatment-dependent clonal evolution patterns in patients that experienced relapse after chemotherapy and patients maintained in partial remission under immunotherapy. By measuring ctDNA changes during therapy, we propose ctDNA as a radiation-free means of tracking residual disease that may integrate positron emission tomography (PET) imaging for the early identification of patients with chemorefractory cHL," Rossi noted.


Advantages of ctDNA Liquid Biopsy

The attractiveness of liquid biopsy techniques is a result of their simple, expedient, non-invasive, and cost-effective means of monitoring the patient's disease status or therapeutic response. Liquid biopsy techniques offer several distinct advantages to more conventional tissue sampling.


First, liquid biopsy sampling is much less invasive and burdensome than a tissue biopsy, since bodily fluids such as blood, saliva, or urine are much more readily accessible. For many malignancies, taking a tissue biopsy is not clinically feasible, e.g., due to risks for hemorrhage, neurological injury, or further disease metastasis. In addition, tissue biopsies may not adequately reflect the complex genetic makeup of a primary or secondary tumor because of intra- and inter-tumoral heterogeneities. The only way to potentially address this issue with tissue biopsies is by sampling from multiple distinct tumor sites. In contrast, liquid biopsies, which contain DNA shed from tumor sites all over the body, may offer a more thorough cross-section of genetically heterogeneous diseases.


In addition, liquid biopsies may also provide genetic evidence of the underlying molecular mechanisms for different primary or secondary tumors, which may be significantly different in the same patient. Since sampling for liquid biopsies often only consists of a blood draw, which can often be combined with blood sampling for other routine analyses, this permits serial sampling of a patient at different stages throughout the treatment cycle. This longitudinal sampling permits surveillance of the disease's clonal evolution, an important benefit for the technique, as tumor cells have a genome that is often highly unstable and vulnerable to selective pressures such as therapy. In general, the liquid biopsy technique is quite complementary with the current approach of individualized treatment, providing a means for identifying ideal candidates for novel targeted therapies while permitting noninvasive disease assessment with important genomic information.


When asked to elaborate on why liquid biopsy techniques might be of special relevance to cHL patients, Rossi stated, "The rarity of Hodgkin and Reed-Sternberg tumor cells in cHL lymph nodes, which are usually present at less than 5 percent, and the routine formalin-fixed paraffin-embedded preservation of tissue biopsies imposed major technical limitations upon the identification of cHL genetic features by sequencing of tissue-derived DNA.


"Consistently, for other lymphomas as common as cHL, dozen to hundreds of genomes are available. However, for cHL, only 44 cases have been genomically sequenced after cumbersome single cell separation of Hodgkin and Reed-Sternberg tumor cells. Our study establishes ctDNA as a viable source of tumor DNA for cHL mutational profiling. By overcoming the major technical hurdles that have so far limited cHL genotyping, our technical approach, based on ctDNA, has allowed large scale assessment of mutations in different clinical phases of the disease ranging from newly diagnosed to refractory disease, and longitudinally during disease treatment," Rossi stated.


In noting the useful genetic information garnered in their study, Rossi commented, "First, STAT6 was identified as the most frequently mutated gene in cHL, present in approximately 40 percent of our patients, which is in keeping with the known importance of cytokine signaling in the biology of this tumor. It is of interest to note that this fact was not reported in previous exome sequencing studies of cHL cases."


"Second, a few major pathways emerged as recurrently mutated, including NF-[kappa]B, PI3K-AKT, cytokine and NOTCH signaling, as well as immune evasion," Rossi stated. "Of note, these pathways have been previously identified by gene expression profiling and functional genomic studies of cHL, indicating that mutations act as red flags highlighting cellular programs that are relevant for the biology of the disease, and thus potential therapeutic targets."


"The gold standard for lymphoma diagnosis is, and must be, the morphologic, immunophenotyping and molecular analysis performed on tissue samples," he said. "Though ctDNA cannot substitute tissue biopsy for diagnostic purposes, some advantages of the non-invasive liquid biopsy have been highlighted in recent years. Beyond decreasing procedure-related risks, including hemorrhage, infections, anesthetic risks for an open biopsy, ctDNA can serve an alternative source for tumor DNA for genotyping purposes when the presence of lymphoma cells is insufficient in the tissue sample, as is the case in the cHL subtype."


In the current ctDNA cHL study, a cohort of 24 advanced cHL patients were serially sampled during their treatment with ABVD (adriamycin, bleomycin, vinblastine, dacarbazine). "Those patients that attained a complete response and cure had a larger drop in ctDNA load after 2 courses of ABVD relative to those patients that underwent a relapse. Moreover, the magnitude of the drop was maintained until the end of the therapy, Rossi noted.


"A 2-log drop (i.e., 100-fold decrease) in ctDNA after 2 chemotherapy courses, a threshold value which was proposed and validated in diffuse large B-cell lymphoma (J Clin Oncol 2016;34:7511), was confirmed as the best cutoff to predict progression in our cohort and was associated with complete response and cure," he explained.


However, a less than 2-log drop in ctDNA after 2 courses of ABVD had an association with progression and inferior survival. The quantification of ctDNA was complementary to interim PET/CT in assessing residual disease.


"Indeed, cured patients who were inconsistently judged as interim PET/CT-positive had a more than 2-log drop in ctDNA, whereas those patients who were inconsistently judged as interim PET/CT-negative had a less than 2-log drop in ctDNA," Rossi stated. "The lack of correlation between log fold change of maximum standardized uptake value in PET/CT and log fold change of ctDNA between baseline and interim evaluations is consistent with the notion that the maximum standardized uptake value largely reflects the metabolic activity of the inflammatory component of the mass in cHL, whereas ctDNA reflects the tumor load levels."


Implications for Treatment Options

"PET/CT imaging is the most sensitive tool for residual disease identification in cHL at the clinical grade," Rossi stated. "Interim PET/CT, which is performed after 2 cycles of treatment, has been tested to preliminarily identify residual disease in chemorefractory patients, as they are prime candidates for treatment intensification in order to maximize the chances of their cure.


"In addition, this technique can also identify at an early stage those patients who are already cured, as they are candidates for treatment de-escalation to avoid both short- and long-term complications of chemo-radiotherapy. The ideal analytical tool to be used for such response-adapted therapy needs to be reliable and provide results that carry a strong correlation with the final treatment outcome. Unfortunately, interim PET/CT results are inconsistent with the final patient outcomes, primarily because of false-positive findings which occur in roughly 20-30 percent of patients, who are thus overexposed to their therapy."


"By substituting an invasive tissue biopsy, which is the gold standard for identifying false-positive PET/CT results, the liquid biopsy proved to be a novel precision biomarker for confirming or excluding the presence of residual disease in the case of positive PET/CT findings in various lymphoma types, including cHL.


"Quantification of ctDNA, when coupled with PET/CT, improves the accuracy of residual disease assessment at the interim time (i.e., after 2 treatment cycles) compared to PET/CT alone in cHL patients," Rossi further elaborated.


When asked about future directions for research of ctDNA techniques in cHL, Rossi answered, "In order to translate ctDNA monitoring as a routine response assessment tool for cHL, this methodology should be incorporated into cHL-based clinical trials. Doing so would first allow us to precisely assess the accuracy with which ctDNA can anticipate disease course; second, this would also serve as a test to verify if ctDNA can provide predictive and prognostic information for guiding treatment decisions."


Richard Simoneaux is a contributing writer.


The Value & Future of ctDNA in Non-Hodgkin Lymphoma

In a recent interview with Oncology Times, Kieron Dunleavy, MD, Professor of Medicine and Director of the Lymphoma Program at George Washington (GW) University and GW Cancer Center, discussed the use of ctDNA in non-Hodgkin lymphoma (NHL).


What are examples of NHLs where ctDNA liquid biopsy has been applied for patient testing?


These techniques have been applied for patients with a number of different B-cell lymphomas. In particular, a number of studies have evaluated the use of ctDNA in diffuse large B-cell lymphoma (DLBCL) patients; some studies have also been performed in patients with other lymphomas such as mantle cell lymphoma (MCL) and follicular lymphoma (FL). However, these liquid biopsy methods have not been validated in prospective studies.


In a NCI study including 126 DLBCL patients, among the 107 that attained complete remission, those that subsequently showed the presence of ctDNA were more than 200-fold more likely to undergo disease progression than those who did not (Lancet Oncol 2015;16:541-549).


Another smaller study utilized ctDNA for 26 patients with MCL, a rare B-cell lymphoma. In that study, lower pre-treatment ctDNA levels were shown to correlate with normalized granulocyte-macrophage colony-stimulating factor response to idiotype vaccine and may be a surrogate marker of ongoing host anti-tumor immunity. Moreover, ctDNA levels did not correlate with traditional tumor proliferation markers. There was also a trend towards improved OS in those achieving minimal residual disease-zero (Blood 2016;128:2943).


In another study, ctDNA analyses were utilized to identify genetic predictors of response in DLBCL and FL patients being treated with the selective EZH2 inhibitor tazometostat (Blood 2017;130:4013).


What is the benefit for using this technology?


One clear area of immediate impact would be the monitoring of patients therapy during treatment to assess how they are responding. Currently, the major means of determining treatment success are PET/CT studies using fludeoxyglucose (18F) FDG as an imager. While the negative predictive value for FDG-PET is good, there are a fair percentage of false positives that can arise; that is, patients who are cancer-free may actually appear to be tumor-bearing. The use of ctDNA might help sort these instances out. In addition, this modality exposes the patient to radiation for monitoring.


What are some advantages to applying ctDNA methods to patients with NHLs?


The fact that sampling is non-invasive permits easy longitudinal sampling, which could enable following the genomic evolution of a patient's disease over time. Additionally, this access could yield new mutations (e.g., BCl-2, BCL6) for which targeted therapies can be applied. A research group at Stanford University is using CAPP-Seq (CAncer Personalized Profiling by deep Sequencing) methodologies to do just that. Also, as was previously mentioned, genetic predictors of response were identified in DLBCL and FL patients taking tazometostat.


What do you see as future research for ctDNA in NHLs?


I think we will see prospective studies utilizing ctDNA in interim assessments. Also, another logical topic would be the assessment of ctDNA levels for prognostic and predictive value at the end of therapy and at follow-up or following induction therapy for MCL. All of these studies will take years to complete.