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D. Rossi et al.
Lymphoma diagnosis by ctDNA
ctDNA cannot substitute tissue biopsy for lymphoma diagnosis. Only one single, rare, special scenario, name- ly the non-invasive diagnosis of PCNSL in those patients whose brain masses are surgically inaccessible, might one day be able to take advantage of the diagnostic potential of ctDNA. The diagnostic procedure of choice to establish the diagnosis of PCNSL is a stereotactic biopsy; if ocular or cerebrospinal fluid (CSF) involve- ment is evident, vitrectomy or CSF cytology may be suf- ficient. If a biopsy of the brain lesion is not possible, and CSF or ocular involvement is ruled out, histological diag- nosis can be difficult at both initial stages and at relapse. The MYD88 L265P mutation occurs in up to 85% of tis- sue biopsies from PCNSL patients but never in those from non-hematologic brain tumors, suggesting that this mutation is a fairly sensitive and highly specific bio- marker for differential PCSNL among central nervous system cancers.23-30 Droplet digital PCR assays probing the MYD88 L265P mutation in cfDNA samples from PCNSL patients known to harbor the MYD88 L265P have a 60% true positive rate.17 However, droplet digital PCR assays for detecting the MYD88 L265P mutation in cfDNA are far from being a validated non-invasive diag- nostic test of PCNSL. Indeed, apart from standardization of the technique to suppress the false positive rate orig- inating from the methodology, there are no data on the biological false positive rate of this assay. The MYD88 L265P mutation occurs in pre-malignant conditions such as monoclonal gammopathies of undetermined signifi- cance (MGUS) and monoclonal B-cell lymphocytosis (MBL). Both are relatively common in the older adult, and thus can co-occur by chance with a brain mass in the same subject, raising the issue of false positive results originating from a biological background (Figure 1).31,32 Plasma samples from large cohorts of patients diagnosed with a brain mass should be tested with stan- dardized droplet digital PCR assays for the MYD88 L265P mutation to precisely define its diagnostic accura- cy before bringing this test into diagnostic routine prac- tice for PCNSL.
Tumor genotyping by ctDNA
Tumor genotyping of lymphomas lacking a leukemic phase has so far relied on the analysis of the diagnostic tissue biopsy. However, multiregional sequencing showed that the diagnostic tissue biopsy might be sub- ject to a selection bias resulting from spatial heterogene- ity and, therefore, might not be representative of all the tumor genetics.33 Indeed, in follicular lymphoma, differ- ent areas of the same tumor may show different genetic profiles (i.e. intratumoral heterogeneity).34 A biopsy from one part of a tumor may miss mutations occurring in subclones residing in anatomically distant sites, including clinically relevant genetic biomarkers for treat- ment tailoring or anticipation of resistance.33 Furthermore, serial sampling of tumor material through repeat biopsies is not usually feasible in lymphomas lacking a leukemic phase, hampering efforts to under- stand patterns of genomic evolution during disease pro- gression and the development of treatment emergent resistant mutations. On the basis of this, lymphoma genotyping on ctDNA can complement, though not entirely substitute, the analysis of the diagnostic tissue biopsy in order to deal with the clinical need of a com-
prehensive and easily accessible tumor genotyping. ctDNA is representative of the entire lymphoma hetero- geneity, thus bypassing the bias imposed by tissue biop- sies in the reconstruction of the entire cancer clonal architecture, and identifying resistant clones that are dormant in non-accessible tumor sites. Accessing the blood stream has also a clear advantage for sampling in the serial monitoring of treatment emergent resistant mutations in real time.35
Independent studies have assessed the sensitivity and specificity of targeted gene mutation analysis in ctDNA versus tumor biopsy as gold standard from untreated DLBCL patients by using CAPP-Seq (Figure 1).15,36,37 The recovery rate of confirmed mutations (i.e. true positive rate) in the tumor biopsy ranges from 95% to 99%. The mutations confirmed by biopsy that were missed in ctDNA (i.e. false negative rate) range from 1% to 5% and are mostly of low allelic abundance in the tumor. After suppressing the biological background originating from clonal hematopoiesis by the sequencing of matched granulocyte DNA, such a false positive rate is represented by somatic variants recovered in cfDNA but absent in the tumor biopsy due to tumor mutations restricted to clones that are anatomically distant from the biopsy site.15,36,37 CAPP-seq of ctDNA thus stands as a robust and validated technology for accurate DLBCL genotyping. Genotyping of ctDNA by CAPP-seq allows recovery of 100% of tumor biopsy-confirmed action- able mutations of DLBCL, like EZH2, MYD88, CD79B, and longitudinal monitoring in the blood of the emer- gence of ibrutinib-resistant mutations.15,36-38 These data support the implementation in the clinic of this non- invasive technique in both settings. CAPP-seq standard- ization is, however, required before bringing this test into diagnostic routine practice for DLBCL (Figure 1).
ctDNA is an alternative source of tumor DNA when representation of lymphoma cells is insufficient in the tissue biopsy, as in classic Hodgkin lymphoma (cHL).16,39 The rarity of neoplastic Hodgkin and Reed-Sternberg cells in the biopsies is a limit to the genetic characteriza- tion of cHL, which can only be overcome by complex techniques for tumor cell enrichment that are beyond the budget of a diagnostic lab. By CAPP-seq, biopsy- confirmed tumor mutations are detectable in ctDNA samples with a true positive rate of 87% in cHL patients.16 Though clinical application is still a long way off, CAPP-seq of ctDNA opens up the opportunity of genotyping large cohorts of cHL patients for the identi- fication of genetic prognostic biomarkers and, within clinical trials, for the identification of biomarkers predic- tive of response to treatment.
Residual disease quantification by ctDNA
Due to the lack of a leukemic dissemination, MRD monitoring has so far been limited to tissue-born lym- phomas without bone marrow (BM) involvement, such as DLBCL and cHL. MRD monitoring in lymphomas is defined as any approach aimed at detecting, and possi- bly quantifying, residual tumor cells beyond the sensi- tivity level of routine imaging techniques. Whenever a patient achieves complete clinical remission, a number of different scenarios may actually be taking place, including full eradication of the neoplastic clone or per- sistence of residual tumor cells capable of giving rise to a full clinical relapse within months or years. According
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haematologica | 2019; 104(4)