Targeted locus amplification to detect molecular markers in mantle cell and follicular lymphoma

Abstract Minimal residual disease (MRD) monitoring by PCR methods is a strong and standardized predictor of clinical outcome in mantle cell lymphoma (MCL) and follicular lymphoma (FL). However, about 20% of MCL and 40% of FL patients lack a reliable molecular marker, being thus not eligible for MRD studies. Recently, targeted locus amplification (TLA), a next‐generation sequencing (NGS) method based on the physical proximity of DNA sequences for target selection, identified novel gene rearrangements in leukemia. The aim of this study was to test TLA in MCL and FL diagnostic samples lacking a classical, PCR‐detectable, t(11; 14) MTC (BCL1/IGH), or t(14; 18) major breakpoint region and minor cluster region (BCL2/IGH) rearrangements. Overall, TLA was performed on 20 MCL bone marrow (BM) or peripheral blood (PB) primary samples and on 20 FL BM, identifying a novel BCL1 or BCL2/IGH breakpoint in 16 MCL and 8 FL patients (80% and 40%, respectively). These new breakpoints (named BCL1‐TLA and BCL2‐TLA) were validated by ASO primers design and compared as MRD markers to classical IGH rearrangements in eight MCL: overall, MRD results by BCL1‐TLA were superimposable (R Pearson = 0.76) to the standardized IGH‐based approach. Moreover, MRD by BCL2‐TLA reached good sensitivity levels also in FL and was predictive of a primary refractory case. In conclusion, this study offers the proof of principle that TLA is a promising and reliable NGS‐based technology for the identification of novel molecular markers, suitable for further MRD analysis in previously not traceable MCL and FL patients.

Fondazione Da Rosa, Torino, Italy; Fondi di Ricerca Locale, Università degli Studi di Torino, Italy; Fondazione Neoplasie Del Sangue (Fo.Ne.Sa), Torino, Italy rearrangements in eight MCL: overall, MRD results by BCL1-TLA were superimposable (R Pearson = 0.76) to the standardized IGH-based approach. Moreover, MRD by BCL2-TLA reached good sensitivity levels also in FL and was predictive of a primary refractory case. In conclusion, this study offers the proof of principle that TLA is a promising and reliable NGS-based technology for the identification of novel molecular markers, suitable for further MRD analysis in previously not traceable MCL and FL patients. 18), translocation causing BCL1 or BCL2/IGH rearrangements. 1 Despite considerable therapeutic progress, eventually both cancers tend to relapse, even after long periods of clinical remission. 2,3 To predict disease relapse, it is necessary to early assess the presence of residual clonal cells after a successful treatment. Therefore, minimal residual disease (MRD) analysis, based on the highly sensitive PCR, for BCL1 and BCL2/IGH [4][5][6] and IGH clonal rearrangements detection 7,8 has become a widely used and standardized approach to monitor the disease persistence. 9 Overall, current standard PCR techniques allow the identification of a molecular marker in 75%-80% of MCL patients, namely 65%-70% carrying an IGH clonal rearrangement and up to 30%-35% an amplifiable BCL1/IGH rearrangement (with a breakpoint located in the major translocation cluster [MTC] region ). 10,11 In FL patients, a molecular marker, either BCL2/IGH major breakpoint region (MBR) or minor cluster region (MCR), is available in 55%-60% of affected cases. [12][13][14] Thus, about 20% of MCL and 40% of FL cases lacking a molecular marker are currently still not eligible for MRD assessment with the classical PCR techniques (i.e., "no marker patients").
Actually, the introduction of next-generation sequencing (NGS) opened new perspectives in this context. 15,16 The recently developed targeted locus amplification (TLA) technology is able to sequence structural variants, usually not detected by conventional PCR approaches. 17 TLA, by selective amplification and sequencing of entire genes on the basis of the crosslinking of physically proximal DNA loci, was able to identify novel candidate oncogenes and not yet described gene fusion partners in B-and T-cell acute lymphoblastic leukemia (ALL), increasing the knowledge of genomic diversity and driving oncogenic lesions. 18 The aim of this study was to apply TLA on samples collected from MCL and FL patients enrolled in prospective clinical trials of the Fondazione Italiana Linfomi (FIL) and lacking a PCR-detectable BCL1 (MTC) or BCL2 (MBR and MCR) rearrangement, in order to assess the feasibility of TLA as marker screening approach. In both sample populations, new BCL1 and BCL2/IGH breakpoints (namely "BCL1-TLA and BCL2-TLA") were addressed and their performance as MRD markers was evaluated. Additionally, MRD levels were compared by ASO-qPCR in those MCL cases with an already available IGH clonal rearrangement. Finally, MRD trends were monitored in MCL and FL "no marker" patients, when follow-up samples were available. The clinical trials, as well as the MRD study, were approved by the ethical committees of all the enrolling centers. All patients provided written informed consent for the use of their biological samples for research purposes, in accordance with Institutional Review Boards requirements and the Helsinki's declaration.

| Targeted locus amplification
TLA was carried out starting from 3-5 � 10 6 cells or 5 μg of gDNA. 23 Circular TLA template (of on average 2 kb in size) was created and amplified with IGH enhancer primers (AGCAATTAAGACCAGTTCCC and CTCCACAACCTCTGAATGG; Cergentis), then indexed by Nextera XT transposon-based technology and sequenced on MiSeq platform (Illumina).
Sequence reads were mapped against the human genome version hg19 using BWA-SW, which is a Smith-Waterman alignment tool.
This allows partial mapping, which is optimally suited for identifying

| TLA molecular markers sequences validation and MRD monitoring
ASO primers were designed on the BCL1 and BCL2-TLA juxtaposed chromosomic breakpoints. 25 MRD quantification was performed on FU samples, according to the Euro MRD criteria. 9 The correlation between the classical IGH and the BCL1-TLA molecular markers in MCL samples was performed using bivariate Pearson's regression analysis: quantitative discordances between the two molecular markers were defined as "minor" if one target was positive and the other positive not quantifiable (PNQ) 9 and "major" if a positive versus negative result occurred.
Moreover, 23 BM samples of FL patients were tested by TLA, too. In this group, 20 out of 23 (87%) patients resulted in BCL-2/IGH (both MBR and MCR) negative by PCR, thus considered "no marker FL" (Table 1). Otherwise, 3 out of 23 (13%) patients had a wellcharacterized MBR translocation by classic quantitative PCR approach, with very low tumor burden levels (1E−05 and 1E−06 or positive but not quantifiable, according to EURO-MRD qPCR guidelines).
Additionally, DOHH2 cellular line, harboring MBR breakpoint, and four healthy donors were tested as positive and negative controls, respectively. with a quantifiable disease (1E-05), and no rearrangements were found in those two cases defined as positive but not quantifiable (1E −06 or PNQ). 9 Finally, as expected, in all the four healthy donors, no rearrangement was found.

| Chromosomal breakpoints detection
To define the exact chromosomal breakpoints regions, binary align-

| TLA sequences validation as molecular markers
The new BCL1-TLA and BCL2-TLA markers were validated by ASO-qPCR strategy. 25 With ASO primer design, a sequence validation was possible in 14 out of 16 (86%) MCL patients. In two cases no validation was possible due to an uncommon sequence structure, leading to no target and polyclonal background amplification, respectively (data not shown).
In FL cases, five out of six (83%) BCL2-TLA sequences were validated using the same ASO strategy. The BCL2-TLA sequence validation failed in one sample (TLA 23), probably due to the close proximity but incomplete involvement of IGHJ6 region that prevented the optimal annealing of the IGHJ consensus primer (reads mapped 48bp at 3'side of IGHJ6; data not shown).

| Tumor burden quantification and MRD monitoring by TLA molecular markers
Out of the 12 patients of the "double positive marker" MCL group, 10 could be evaluated for comparative MRD analysis by ASO q-PCR, using both BCL1-TLA and IGH as molecular markers. As mentioned before, two patients (TLA 9 and TLA 11) were excluded because the BCL1-TLA sequences were not validated.   Figure 5, panels I and L, respectively) and was nicely able to describe the MRD persistence in a primary refractory patient (i.e., TLA 26, see Figure 5, panel I).