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miRNA in Richter syndrome
as a first link between non-coding RNA alterations and human diseases,4,5 the mechanistic involvement of miRNA in the CLL patients with the worst prognosis, those whose disease transforms to Richter syndrome (RS), has not been reported to date. RS occurs in up to 8% of untreated CLL patients6 and in 5-16% of patients treated with targeted therapies, such as ibrutinib or venetoclax for relapsed CLL.7,8 Abnormalities of regula- tors of tumor suppression (TP53), cell proliferation (NOTCH1, MYC) and cell cycle (CDKN2A), have been reported in RS,9 but biomarkers to predict the occur- rence of RS are lacking at present. RS is characterized by rapid progression and outcomes of patients treated with a variety of moderate or high-intensity chemoim- munotherapy regimens are uniformly dismal, with a median survival of less than 1 year,10-13 particularly for patients with clonally-related or TP53-mutated disease.14 Novel, molecularly targeted approaches are urgently required, but this is hampered by the limited under- standing of the molecular pathogenesis of RS. The paucity of molecular studies is mainly due to the scarce- ness of biopsy materials. Furthermore, the availability of non-invasive methods of diagnosis (such as the use of 2- deoxy-2-[(18)F] fluoroglucose/positron emission tomo- graphy,15 reduces the need for follow-up biopsies, which further limits the availability of material for research. Therefore, there is a strong need to develop RS biomark- ers and molecularly targeted therapies that could facili- tate early and accurate diagnosis, as well as assist cur- rent treatment strategies. In the present study, we inves- tigated the expression and potential roles of miRNA in the transformation from CLL to RS, as these miRNA could be therapeutically targeted.
Methods
Patients’ samples
The University of Texas MD Anderson Cancer Center (UTMDACC) cohort
The “paired” set: 14 bone marrow samples from seven patients with RS were collected at the UTMDACC. For each patient, samples from the time of CLL diagnosis (group 1a) and Richter transformation (group 1b) were available. In addition, we collected 14 bone marrow samples from seven age-, sex- and sample time-matched CLL control patients who did not develop RS over the course of follow-up at the UTMDACC. For each patient, a sample at the time of CLL diagnosis (group 2a) and at a time corresponding to the time of RS diagnosis of group 1 (group 2b) were available. Online Supplementary Table S1 shows that age at diagnosis, gender and time to transforma- tion were not significantly different between patients of this paired RS/CLL cohort.
The “extended” set : we also extended our initial paired RS/CLL cohort to include samples from 27 patients with RS [25 samples at CLL diagnosis (group 1a) and 9 samples at the time of Richter transformation (group 1b)] and 23 control CLL patients [17 samples at CLL diagnosis (group 2a) and 14 sam- ples at a time corresponding to the time of Richter transforma- tion in the RS group (group 2b)]. All samples used were forma- lin-fixed paraffin-embedded (FFPE) bone marrow cores, except for one lymph node sample in group 1b. A schematic represen- tation of the extended cohort is shown in Figure 1A,B, while the patients’ characteristics are presented in Table 1 and detailed in Online Supplementary Table S2.
Ulm University cohort
We used peripheral blood from 58 fludarabine-resistant patients. Samples were taken at enrollment before treatment. Eight of these 58 patients subsequently developed RS. These patients were described previously.16
The Bellinzona Institute of Oncology Research cohort
This cohort of patients comprised 737 cases of mature lym- phoid tumors including CLL, and were described previously.17
The study was approved by the institutional review boards and ethical committees of all the participating institutions.
Xenogeneic mouse transplantation
We used a previously described mouse model of CLL for in vivo experiments.18-20
Firefly microRNA profiling assay
We performed expression analysis of 40 human and viral miRNA known to be involved in the progression of CLL, asso- ciated with poor prognosis CLL, highly expressed in CLL as determined by a previously performed RNA-sequencing study,21 located in genomic regions reported to be deregulated in RS,17 or frequently reported in literature to be associated with CLL (Online Supplementary Table S3). We used a Firefly custom multiplex circulating miRNA assay (Abcam, Cambridge, MA, USA) for the extended cohort of CLL/RS sam- ples collected at the UTMDACC, as described in the Online Supplementary Methods.
Polymerase chain reaction analysis and microRNA gene expression profiling
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and miRNA gene expression profiling for miRNA profiling confirmation are described in detail in the Online Supplementary Methods.
Genome-wide DNA profiles
Genome-wide DNA profiles were obtained from high-mole- cular-weight genomic DNA using the Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA, USA), as previ- ously reported (GSE50252).17
Network analyses
The miRNA networks were generated as previously described;22 the method is detailed in the Online Supplementary Methods.
Results
A microRNA signature is involved in the process of Richter transformation
As illustrated in the workflow in Figure 1A, we first designed the UTMDACC cohort consisting of patients with RS and age-, sex- and sample time-matched CLL “controls”. For all of these cases FFPE bone marrow sam- ples, taken at the time of CLL diagnosis and at the time of RS diagnosis, or a time corresponding to the RS diag- nosis in the case of the matched CLL controls, were available and analyzed (Figure 1B and Online Supplementary Table S1). We decided to use the Firefly custom multiplex miRNA assay due to the best data generation/cost ratio, and well annotated and selected miRNA (see Methods). We identified nine miRNA potentially involved in the RS transformation process,
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