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L. Raman et al.
(Figure 5A). These zones are represented by irregularities in the scatter plot trend between the profiles (Figure 5B).
Overall, observations were consistent for refractory and relapsed patients: when detectable, copy number patterns were very similar across different longitudinal staging moments (Figure 5C). One exception was found in patient 73 (rightmost patient in Figure 5C), who appeared to relapse with a divergent copy number profile (Online Supplementary File S1: Longitudinal samples). This case con- cerned an aggressive EBV-positive plasmablastic lymphoma that is known to present complex karyotypic abnormalities and, presumably, an instable genome.36
The most recent follow-up of all 17 patients in CR showed that none had remaining copy number alterations; in contrast, this was the case in only 5/14 patients in whom CR was thought not to be reached (Figure 5D). In total, five EBV-positive patients, according to sWGS of cfDNA, were detected at diagnosis of the 31 patients with follow-up. Of these, three went into CR and, consequently, no longer had abnormally elevated EBV levels; one refractory patient (patient 6) was noted to have recurrently high EBV levels and one relapsed patient (patient 73) was detected with an unusually high EBV read fraction, following an earlier drop to 0 ppm at first response evaluation (Figure 5E). It should be noted that, for this patient, no tumor was detected under PET/CT at the fourth time point (Online Supplementary File 2: Table S4), however, the steadily increasing EBV fraction anticipated actual relapse at the fifth time point, after which the MTV increased rapidly along with the plasma concen- tration of viral fragments (Online Supplementary File S1: Longitudinal samples).
A similar analysis, concerning the CPA score, was per- formed for all patients with HL (Figure 5F) and nHL (Figure 5G) with follow-up. Similar conclusions were made accord- ing to the trend lines, which largely summarize the longitu- dinal analyses: a prompt drop for patients in CR; varying values for refractory patients; and a drop, followed by a newly-triggered rise, for relapsed patients. It is significant how the CPA decreased rapidly when treatment was initi- ated: interim samples had normal CPA scores unless they represented a refractory patient. Finally, 6/31 patients never transcended the CPA abnormality cutoff, meaning that these, probably, have insufficient ctDNA. In two cases (patients 66 and 71), however, the MTV reached extreme levels (>1000 cm3) while the CPA score stayed low (Online Supplementary File S1: Longitudinal samples). For these patients, it is more likely that copy number alterations real- ly were absent.
Of the above cases, additional clinical information from four interesting patients further illustrates the potential of shallow-depth sequencing for disease monitoring - e.g., when dealing with ambiguous PET/CT images or tumor and viral entities beyond initial suspicion. These are dis- cussed as short case-reports in Online Supplementary File S1: Case reports.
Copy number anomalies detected in liquid biopsies correlate with clinical parameters
Five lymphoma-related clinical variables were included in an extensive concordance analysis of the baseline and lon- gitudinal liquid biopsies: Ann Arbor stage; International Prognostic Index/Score; lactate dehydrogenase concentra- tion; b2 microglobulin concentration; and MTV (Online Supplementary File S1: Figure S2). The estimated tumor frac- tion, according to copy number profiles, positively and sig-
nificantly correlates with all of these clinical variables, ren- dering the former estimate relevant. The CPA score is intrinsically correlated to the tumor fraction, so similar associations as for the tumor fraction arise. Since all these variables are naturally cross-related, the derived relations are obviously not claimed to be independently significant.
Discussion
To date, molecular profiling of lymphoma has largely been focused on the characterization of actionable targets, such as point mutations and translocations. This profiling requires panel sequencing, currently using tissue biopsies as the DNA source and targeting a substantial number of genes. However, until now, the availability of this precise information has contributed little to actual progress in patient care, despite its tempting rationale.37 In addition, applying these concepts in a minimally-invasive manner requires ultra-deep sequencing (~2000x),38 of which the implementation in routine practice remains challenging.
In this study, we evaluated sWGS of cfDNA for the diag- nosis, differential diagnosis, and disease monitoring of HL and DLBCL. In contrast to targeted sequencing, this approach provides genomic insight by copy number profil- ing in an accessible and standardized manner. Indeed, non- invasive prenatal testing, which involves the same laborato- ry and computational steps, has evolved into an application performed daily in molecular diagnostic laboratories. The short turnaround time (approximately, 4 days) and low- price tag (around $200, including processing costs) renders sWGS a fortiori feasible.
Copy number profiling is not a surrogate for targeted sequencing, nor does it enable the characterization of phe- notypic traits, such as CD20 expression. However, to a lim- ited extent, it does enable the prediction of targetable genes, such as JAK2, PD-L1 and REL. Further, regarding tumor fraction and specificity, it has the advantage of easier and more stable interpretation: once a copy number alteration is visible, the presence of sufficient ctDNA is most likely ascertained given that large somatic copy number alter- ations are rarely detected in unaffected subjects.14
For DLBCL, the sensitivity of copy number profiling is a shortcoming: only 74.1% of baseline liquid biopsies were found to have detectable aberrations, which is lower than that reported from targeted ultra-deep duplex sequencing studies (e.g., 98% in Kurtz et al.39). On the other hand, the sensitivity was surprisingly high for HL (84.2% for HL over- all and 88.6% for cHL in this study; 88.9% in a smaller proof-of-concept study16) and compares to that of SNV- based studies (e.g., 81.2% in Spina et al.11). It ought to be noted that the lower limit of detection for CNA is often considered to be 3% tumor fraction.21 It is worth mention- ing that this limit could be lowered as paired-end sequenc- ing at greater depths (1x-1.5x) is expected to become avail- able for routine diagnosis at a similar cost. This would empower computational tumor-read enrichment by insert- size filtering, as shorter cfDNA fragments are more likely be tumor-derived, thus increasing sensitivity.20,40,41
The signs and symptoms of aggressive lymphoma are often non-specific, having much in common with a broad range of different disorders, including autoimmune, immunodeficiency and systemic diseases, infections and other malignancies. Therefore, currently, accurate lym- phoma diagnosis is essentially only possible through inva-
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haematologica | 2022; 107(1)