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ctDNA analysis in Lymphoma
to the Lugano criteria, positron emission tomography (PET)/computed tomography (CT) has become the rec- ommended imaging strategy for sensitive disease response assessment in DLBCL and cHL.40 The best clas- sification of patients with good versus poor prognosis is reached by the end-of-treatment PET/CT. However, this timepoint would be rather late to adapt treatment strategies according to the quality and depth of response. Interim PET/CT performed after two cycles of treatment has been tested for the early identification of chemorefractory patients, as they are candidates for treatment intensification to maximize the chances of cure, as well as to identify good-risk patients early, as they are candidates for treatment de-escalation to avoid both short- and long-term complications of chemo- radiotherapy.41 The accuracy of interim PET/CT has been considered adequate to inform early treatment intensification or de-escalation in both limited and advanced stage cHL.42 However, even in the ideal techni- cal and analytical setting, interim PET/CT results are inconsistent with the final outcome in approximately 20-30% of patients, who are thus still exposed to over- or under-treatment.41,42
In DLBCL, interim PET/CT does not correctly inform on the subsequent outcome in a larger number of patients than in cHL. Indeed, the positive predictive value of interim PET/CT in DLBCL is 50%.43 This means that half DLBCL patients are misclassified by interim PET/CT as being R-CHOP resistant, but ultimately are converted to a negative PET/CT at the end of treatment and cured by R-CHOP. The negative predictive value of interim PET/CT is 70%.43 This means that 30% of DLBCL patients are misclassified by interim PET/CT as R-CHOP sensitive, but ultimately relapse after R-CHOP. On the basis of this, interim PET/CT can not yet be adopted for clinical use to guide treatment decisions in individual DLBCL patients and remains a subject for research.
Minimal residual disease can be measured in tissue- born lymphomas without BM involvement and lacking a leukemic component by using ctDNA technologies. Compared to genomic DNA extracted from circulating mononuclear cells, plasma cfDNA harbors a 150-fold higher representation of tumor DNA, which makes cfDNA more reliable than genomic DNA from circulat- ing cells for MRD monitoring in DLBCL.12 By using the immunoglobulin gene rearrangements to quantify ctDNA in plasma, DLBCL patients with undetectable ctDNA after two chemotherapy courses show a superior progression-free survival compared with patients with positive ctDNA.37 Despite its value as a prognostic tool, using the immunoglobulin gene rearrangement to quan- tify ctDNA in DLBCL has several shortcomings. This includes limited sensitivity in low tumor burden settings and reduced applicability because of the somatic hyper- mutation process, leading to difficulties in identifying clonotypic sequences.12 In addition, as for PET/CT, also
for high throughput sequencing of the immunoglobulin genes the best informative timepoint is end of treat- ment.37
By covering a large spectrum of genetic lesions, ctDNA quantification by CAPP-seq is cross-validated by multiple tumor tags, and avoids false negative results caused by treatment-induced clonal shift. In both DLBCL and cHL, the change in ctDNA measured by CAPP-seq after two cycles of therapy associates with both event-free and overall survival.15,16,44 A drop of 100- fold (or 2-log drop) in ctDNA levels after two chemotherapy courses is associated with an eventual complete response and cure. Conversely, a drop of less than 2-log in ctDNA after two treatment courses is asso- ciated with an eventual progression.16,44 Quantification of ctDNA coupled with PET/CT improves the accuracy of residual disease assessment at the interim time com- pared to the sole PET/CT in both DLBCL and cHL. Indeed, patients inconsistently judged as interim PET/CT positive, but having a negative (i.e. >2-log drop in ctDNA) liquid biopsy, are actually cured, while patients inconsistently judged as interim PET/CT nega- tive, but having a positive (i.e. <2-log drop in ctDNA) liquid biopsy, are actually not cured.16,44 These results generate the hypothesis that ctDNA may complement interim PET/CT in informing on DLBCL and cHL patients’ outcome (Figure 1). Before translating this tech- nology into the management of DLBCL, the precise cumulative sensitivity and specificity of PET/CT and ctDNA monitoring in anticipating the clinical course of patients should be precisely defined in clinical trials.
Further investigations
At the clinical level, it is critical that well-designed tri- als validate current concepts and further explore applica- tions of ctDNA for interim monitoring, surveillance monitoring, and response assessment in lymphomas. The most immediate implementation of ctDNA technol- ogy in lymphoma clinical trials includes: i) non-invasive diagnostics of PCNSL; ii) baseline screening for the iden- tification of patients harboring actionable mutations; iii) early and accurate identification of non-responding patients; iii) monitoring the development of resistance mutations against targeted agents (Figure 1).
At the technological level, standardization and harmo- nization projects, like those performed before the imple- mentation of clinical MRD assessment in leukemias, should be designed and implemented also in lymphoma in order to meet clinical standards, and allow accurate, robust and reproducible results of ctDNA genotyping and quantification.
Acknowledgments
Work by the authors has been supported by Grant n. KFS- 3746-08-2015, Swiss Cancer League, Bern, Switzerland; AIRC 5 x 1000, "Metastatic disease: the key unmet need in oncology", project code 21198, Milan, Italy
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