NGS Analysis of Clonality and Minimal Residual Disease in a Patient with Concurrent Richter’s Transformation and CLL/SLL

B-cell lymphomas are neoplastic proliferations of clonal B lymphocytes. Clonality is generally determined by PCR amplification of VDJ rearrangements in the IgH heavy chain or VJ rearrangements in Igκ/Igλ light chain genes followed by capillary electrophoresis. More recently, next-generation sequencing (NGS) has been used to detect clonality in B-cell lymphomas because of the exponential amount of information that is obtained beyond just detecting a clonal population. The additional information obtained is useful for diagnostic confirmation, prognosis assessment, and response to therapy. In this study, we utilized NGS analysis to characterize two histologically distinct lymphomas (DLBCL and CLL/SLL) that were detected contemporaneously in an asymptomatic patient. NGS analysis showed that the same VDJ rearrangement was present in nodal (DLBCL) and marrow (CLL/SLL) biopsies confirming that the DLBCL resulted from Richter’s transformation of a subclinical CLL/SLL. The V region of the rearrangement remained unmutated without somatic hypermutation. In silico analysis showed that the HCDR3 sequence was heterogeneous and not stereotypic. Minimal residual disease analysis by NGS showed that the tumor clone decreased by 2.84 logs in the bone marrow after R-CHOP therapy. However, a small number of tumor cells were still detected in the peripheral blood after R-CHOP therapy. Subsequent allogeneic transplantation was successful in eradicating the tumor clone and achieving deep molecular remission. We show that NGS analysis facilitated clinical management in our patient by helping to characterize the VDJ rearrangement in detail and by tracking minimal residual disease with high sensitivity and specificity.

Liquid Biopsy and Lymphoma Monitoring in Clinical Practice

INTRODUCTION Lymphomas represent the fourth most frequent type of cancer, 90% of them arising from B cell lymphocytes. Despite their high prevalence, around 40% remain incurable because of refractoriness to current chemoimmunotherapy or disease relapse after obtaining response (Li et al., 2018; Meng et al., 2020). The cell of origin is a B lymphocyte with a unique B cell receptor (BCR). The BCR is an immunoglobulin composed of two heavy chains (IgH) and two light chains (IgL) whose genes have multiple coding segments that through rearrangement first and further somatic hypermutation in the germinal center, generate a unique sequence that could be used to monitor the treatment response (Seifert et al., 2019; Wang et al., 2020). This project studies the use of next-generation sequencing (NGS) to characterize and monitor such IgH clonality. The use of liquid biopsy samples would provide a minimally invasive method to identify refractory or relapse-risk patients since all of them will have residual tumor cells after treatment. METHODS The sample size of the study was 53 patients with several types of B-cell lymphomas. The IgH gene of the tumor clone was characterized from DNA of tumor samples at diagnosis by NGS and Sanger. The monitoring of this clone was studied by NGS from DNA and circulating tumor nucleic acids (ctNAs) during and after receiving treatment and at different times after clinical response was stablished (CR or PR). Relapse samples were analyzed by NGS and Sanger. RESULTS Characterization of the tumor clone IgH rearrangement is achieved in 45 of the 53 patients with B-cell lymphoma included in the study. As shown in Figure 1, after the two different amplification PCRs results are similar. In contrast, after sequencing the results obtained by Sanger and NGS are very different. In NGS, thanks to the massive amplification prior to sequencing, it is identify the tumor clone in approximately 80% of the samples. It is shown that the effectiveness in characterization is dependent on the origin of the DNA sample, with fresh material samples being the optimal (Figure 1). In monitoring, samples of different origin are used as shown in Figure 2. About 50% of these samples are plasma ctNAs whose average efficiency in the detection of IgH gene rearrangement is 73.3%, with a clear positive correlation between the sensitivity and the toal volume of plasma processed more starting plasma used (2 mL efficiency of 84.09%). Monitoring makes it possible to classify patients into three different groups (Figure 3): patients with complete remission, patients refractory to the different lines of treatment and patients with apparent complete response and subsequent relapse. In patients with complete response, the tumor clone decreases during treatment and at the end of the line it is no longer detectable, nor in subsequent follow-up samples. With respect to refractory patients, it is observed that the tumor clone remains present despite subsequent lines of treatment. Finally, in patients achieving CR with subsequent relapse, the clone can be detected in a small percentage at the end of the treatment schedule and remains present until relapse. A section of patients under treatment is also shown (Figure 3) to demonstrate the application of the study to clinical practice. Two patients with apparent complete response, one of them in complete remission and the other with a high risk of relapse, requiring a more exhaustive follow-up. The monitoring results obtained by flow cytometry are shown being these, in general, concordant. In some cases NGS shows greater sensitivity. CONCLUSION The use of NGS and liquid biopsy samples provides a minimally invasive method to monitor the IgH gene rearrangement of the tumor clone of patients with B-cell lymphomas. In our experience,, patients in remission can be clearly differentiated from those who are refractory or at risk of relapse. facilitating their treatment strategy and clinical decision making. Figure 1 Figure 1. Disclosures Ferrer Lores: Janssen: Membership on an entity's Board of Directors or advisory committees. Terol: BMS: Consultancy; Hospital Clinico Valencia: Current Employment; Roche: Consultancy; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel.

scONE-seq: A one-tube single-cell multi-omics method enables simultaneous dissection of molecular phenotype and genotype heterogeneity from frozen tumors

Genomic and transcriptomic heterogeneity both play important roles in normal cellular function as well as in disease development. To be able to characterize these different forms of cellular heterogeneity in diverse sample types, we developed scONE-seq, which enables simultaneous transcriptome and genome profiling in a one-tube reaction. Previous single-cell-whole-genome-RNA-sequencing (scWGS-RNA-seq) methods require physical separation of DNA and RNA, often by physical separation of the nucleus from the cytoplasm. Most of these methods are labor-intensive and technically demanding, time-consuming, or require special devices, and they are not applicable to frozen samples that cannot generate intact single-cell suspensions. scONE-seq is a one-tube reaction which eliminates loss due to transfer steps, and thus is highly scalable and compatible with frozen biobanked tissue, generating data that is superior in quality compared to other applicable methods. We benchmarked scONE-seq against existing methods using cell lines and lymphocytes from a healthy donor, and we applied it to a 2-year-frozen astrocytoma sample profiling over 1,200 nuclei, subsequently identifying a unique transcriptionally normal-like tumor clone. scONE-seq makes it possible to perform large-scale single-cell multi-omics interrogation with ease on the vast quantities of biobanked tissue, which could transform the scale of future multi-omics single-cell cancer profiling studies.

scONE-seq: A one-tube single-cell multi-omics method enables simultaneous dissection of molecular phenotype and genotype heterogeneity from frozen tumors

Genomic and transcriptomic heterogeneity both play important roles in normal cellular function as well as in disease development. To be able to characterize these different forms of cellular heterogeneity in diverse sample types, we developed scONE-seq, which enables simultaneous transcriptome and genome profiling in a one-tube reaction. Previous single-cell-whole-genome-RNA-sequencing (scWGS-RNA-seq) methods require physical separation of DNA and RNA, often by physical separation of the nucleus from the cytoplasm. These methods are labor-intensive and technically demanding, time-consuming, or require special devices, and they are not applicable to frozen samples that cannot generate intact single-cell suspensions. scONE-seq is a one-tube reaction, thus is highly scalable and is the first scWGS-RNA-seq method compatible with frozen biobanked tissue. We benchmarked scONE-seq against existing methods using cell lines and lymphocytes from a healthy donor, and we applied it to a 2-year-frozen astrocytoma sample profiling over 1,200 nuclei, subsequently identifying a unique transcriptionally normal-like tumor clone. scONE-seq makes it possible to perform large-scale single-cell multi-omics interrogation with ease on the vast quantities of biobanked tissue, which could transform the scale of future multi-omics single-cell cancer profiling studies.

The attack of the “seeding” clones

Tumor clone tracking in breast cancer xenografts identifies a small subset of circulating tumor cells as “seeders” associated with metastasis.

Genetic subclone architecture of tumor clone-initiating cells in colorectal cancer

A hierarchically organized cell compartment drives colorectal cancer (CRC) progression. Genetic barcoding allows monitoring of the clonal output of tumorigenic cells without prospective isolation. In this study, we asked whether tumor clone-initiating cells (TcICs) were genetically heterogeneous and whether differences in self-renewal and activation reflected differential kinetics among individual subclones or functional hierarchies within subclones. Monitoring genomic subclone kinetics in three patient tumors and corresponding serial xenografts and spheroids by high-coverage whole-genome sequencing, clustering of genetic aberrations, subclone combinatorics, and mutational signature analysis revealed at least two to four genetic subclones per sample. Long-term growth in serial xenografts and spheroids was driven by multiple genomic subclones with profoundly differing growth dynamics and hence different quantitative contributions over time. Strikingly, genetic barcoding demonstrated stable functional heterogeneity of CRC TcICs during serial xenografting despite near-complete changes in genomic subclone contribution. This demonstrates that functional heterogeneity is, at least frequently, present within genomic subclones and independent of mutational subclone differences.