Genetic and Molecular Characterization of Novel Richter Syndrome Patient-Derived Xenograft Models

Background. Richter syndrome (RS) is the transformation of a pre-existing chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) into an aggressive lymphoma, most commonly a diffuse large B-cell lymphoma (DLBCL), an event which occurs in approximately 2% to 10% of CLL patients. RS is highly aggressive, commonly refractory to treatment and associated with poor survival. A combination of germline and somatic genetic characteristics (del17p13, trisomy 12, CD38 and LRP4 polymorphisms), clinical and biological features (stereotyped B-cell receptor, CD38 and CD49 expression, advanced Rai stage) of CLL B cells and likely some CLL therapies are associated with higher risk of RS development. CDKN2A, TP53, MYC and NOTCH1 have been recently recognized as recurrently mutated in RS patients. In spite of significant advancement in treatment options for CLL patients, RS is still a drug-orphan disease.

Aim of the work. The aim of this work is to genetically and molecularly characterize two novel RS patient-derived xenograft (RS PDX) models, recently established in the laboratory, to gain insight into the molecular mechanisms critical for RS biology and potentially uncover signaling pathways of therapeutic interest.

Results. Neoplastic cells from peripheral blood or lymph node of 2 clinically diagnosed RS patients were sub-cutaneously (s.c.) injected in severe immunocompromised mice (NOD/SCID/gamma chain^-/-, NSG). Primary cells engrafted with different kinetics (3 vs 24 weeks). Lymphoma cells were purified from tumor masses and re-implanted for at least 7 passages to obtain stable PDX models. Flow cytometry analyses indicated that PDX-derived RS cells maintained the same phenotype (e.g., CD19, CD5, CD20, CD21) of primary cells through the passages. These models were then genetically characterized, both by targeted and whole exome sequencing (WES). Comparison of primary RS cells with tumor cells of xenografted mice at different passages (1, 3, 5 and 7) revealed that PDX-derived cells retain their original genomic architecture with minor changes. The main molecular pathways altered in these models were related to the BCR signaling (e.g., BTK and MYC), transcription factors (e.g., IRF4, TP53 and MED12), epigenetic remodeling (e.g., SETD2), RAS (e.g., KRAS) and Notch (e.g., NOTCH1 and NOTCH2) signaling pathways. Copy-number estimation obtained from WES confirmed a stable landscape of chromosomal aberrations: both primary samples were characterized by a complex karyotype, with several gains and losses in different chromosomes, all maintained in the xenografts.

RNA-sequencing analysis performed on both primary and PDX-derived cells, corresponding to sequential in vivo passages, highlighted that most of the differentially modulated genes were involved in intracellular signaling cascades, apoptosis, cell cycle, DNA repair and remodeling.

The PDX s.c. models were then implemented by intra-venous (i.v.) injection of RS cells in NSG mice. Preliminary data indicated that neoplastic cells engrafted predominantly in the spleen and in the bone marrow, with disease also detectable in the peripheral blood. Phenotypic and genetic characterization of lymphoma cells purified from these target organs indicated that no changes at the molecular or the genetic levels occurred.

Conclusions. Taken together, these results indicate that two PDX models of RS have been successfully established from primary samples. They maintain the original tumor characteristics including biomolecular signature, malignant phenotypes and genomic architecture. Based on these results, these RS-PDX models offer a unique opportunity to gain relevant insights into the molecular and genetic drivers of RS biology and to explore the efficacy of novel therapies.