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T. Mährle et al.
acterized by refractory anemia, often thrombocythemia with atypical megakaryocytes in the bone marrow (BM) and a relatively low progression rate to acute myeloid leukemia compared to the rate of transformation of other types of MDS.2 The pathogenesis seems to be partially driven by haploinsufficiency of genes located in the deleted area on chromosome 5.3-8 Moreover, there is evi- dence of adaptive and innate immune dysregulation with altered cytokine networks, diminished NK cell activity and an anergic T-cell environment failing to suppress the defective del(5q) clone.9
In 2005, lenalidomide was approved in the USA for the treatment of lower-risk, transfusion-dependent del(5q) MDS patients as the first immunomodulatory drug. In the placebo-controlled phase III trial MDS-004, 56% of patients who received 10 mg/day lenalidomide achieved red blood cell transfusion independency for more than 26 weeks.10 Response to lenalidomide was shown to be asso- ciated with significant improvement in survival with a median overall survival of 4 years.11 Cereblon (CRBN), a substrate receptor of the CRL4 ubiquitin ligase complex, is the primary molecular target through which lenalidomide mediates its anti-tumor effects. Krönke et al. demonstrated that lenalidomide induces the ubiquitination and degrada- tion of IKZF1, IKZF3 and CK1a by binding to CRBN.12,13 CK1a, a negative regulator of p53, may be of particular pathophysiological relevance in the context of MDS del(5q), since it is encoded by a gene within the deleted chromosomal region and haploinsufficient expression sen- sitizes cells to lenalidomide therapy.12 Furthermore, the ubiquitin-independent physiological chaperone-like func- tion of CRBN is targeted by lenalidomide. Eichner et al. found that lenalidomide outcompetes CRBN for binding to and stabilizing the CD147-MCT1 complex.14 This com- plex promotes various biological functions, including angiogenesis, proliferation and invasion. Since recent evi- dence suggests a negative regulatory role of CD147 in T- cell-mediated immune responses,15-17 lenalidomide- induced CD147 destabilization has been speculated to explain not only direct anti-tumor effects, but also immune cell activation.14 Functionally, the immunomodu- lation induced by lenalidomide was described as a reversal of pathological T-cell tolerance by increasing T-cell effec- tor function and secretion of interleukin-2, interferon-γ and tumor necrosis factor-a by CD4+ and CD8+ T cells.18 Expanding on this, a recent RNA sequencing study on non-del(5q) MDS patients suggested that the immunomodulatory signal delivered by lenalidomide may also affect the composition rather than only the functional state of the local BM immune cells.19 However, the immunomodulatory effects of lenalidomide on the microenvironment have not yet been fully defined.
In this study, we analyzed local BM and peripheral blood (PB) environments to understand the clonal immune architecture of MDS del(5q) and its modulation by lenalidomide. To do this, we used state-of-the-art next-generation sequencing (NGS) of immunoglobulin heavy chain (IGH) and T-cell receptor beta (TRB) rearrangements as an advanced technology that enables simultaneous identification of tens of thousands of unique B-cell and T-cell receptor rearrangements from a single tissue sample as well as tracking over time. These analyses demonstrated that lenalidomide shapes T-cell adaptive immune responses in the BM niche of patients with MDS del(5q).
Methods
Patients’ characteristics
Patients’ BM samples were collected between 2013 and 2016 at baseline and after a median of 12 months of lenalidomide treat- ment in the course of the LE-MON 5 and Bioregister trials. The cohort comprised 15 patients with del(5q) MDS. Matching pre- and post-treatment PB samples were available for eight of these patients. All del(5q) patients were treated in the LE-MON 5 trial,20 a German multicenter, single-arm, open-label, phase II study [Safety of Lenalidomide Monotherapy and Markers for Disease Progression in Patients With IPSS Low- or Intermediate-1 Risk Myelodysplastic Syndromes (MDS) Associated With an Isolated Deletion 5q Cytogenetic Abnormality] according to the standard lenalidomide protocol.10 As a reference, seven BM and 22 PB sam- ples from age-matched healthy controls without hematologic abnormalities were included. Five BM samples were collected during hip replacement surgery and two were obtained from iliac crest aspiration of healthy individuals, without apparent differ- ences. Hemograms of all the healthy donors were normal as were their C-reactive protein levels. The BM of all MDS patients was obtained by standardized aspiration and biopsy of the iliac crest. All MDS del(5q) samples analyzed in this study were previously subjected to NGS of TP53,21 and were negative for TP53 muta- tions. The patients’ characteristics are shown in Table 1.
Study approval
Informed consent was obtained from all patients and healthy controls for the use of their diagnostic material for scientific pur- poses approved by the institutional review board (Ethikkommission der Medizinischen Fakultät, Henrich Heine University Düsseldorf, Germany, project number MC-LKP-392).
Isolation of genomic DNA
Genomic DNA was isolated from >106 frozen BM or PB mononuclear cells using a Gen Elute mammalian genomic DNA miniprep kit (Sigma-Aldrich, Taufkirchen, Germany) according to the supplier’s suggestions.
Multiplex polymerase chain reaction analysis for IGH and TRB repertoire amplification for Illumina targeted next-generation sequencing
Both the IGH and TRB genes containing the entire rearranged V, D and J segments were amplified with BIOMED2-FR1 (IGH), -TRB-A and -B primer pools starting from 250 ng of input genomic DNA.22 Amplicons were tagged with Illumina adapters and indices in two consecutive polymerase chain reactions (PCR), as previously described.23 Briefly, previously published primers22-24 for the first PCR annealed within either the IGH or TRB loci and contained Illumina-compatible adapters for NGS primer annealing. A second PCR was performed using outer primers to extend the Illumina adapter for later hybridization of amplicons to the Illumina flow cell and to add a seven nucleotide barcode, allowing each sequence to be matched during data analysis to a certain patient and time point. All primers were purchased from Metabion (Martinsried, Germany). All PCR were performed using Phusion HS II polymerase (Thermo Fisher Scientific Inc., Darmstadt, Germany). Amplicons with the expected size were purified after agarose gel electrophoresis using a NucleoSpin® Gel and PCR Clean-up kit (Macherey- Nagel, Düren, Germany). The concentration of the amplicons was determined on Qubit (QIAGEN, Hilden, Germany) and the quality of the amplicon pools was controlled on an Agilent 2100 Bioanalyzer (Agilent technologies, Böblingen, Germany) before being subjected to NGS.
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haematologica | 2019; 104(7)