Humanized Mouse Model of Myeloma Reveals Clinically Occult Genomic Changes in Primary Tumor Cells
Authors: Verma Rakesh , Strowig Till , Das Rituparna , Koduru Srinivas , Hafemann Anja , Hopf Stephanie , Kocoglu Mehmet H. , Borsotti Chiara , Zhang Lin , Branagan Andrew R , Eynon Elizabeth , Manz Markus , Flavell Richard , Dhodapkar Madhav V 
Published: October 2019

Abstract
Next generation sequencing of purified plasma cells from myeloma patients has revealed the genomic complexity of tumors and is being utilized for developing personalized approaches to therapy. Evaluation of functional properties of these genetic changes and the growth potential of human tumors / their subclones has been limited by difficulties in reliable engraftment of primary human tumor cells in mice. The growth of human cells in mice is limited in part due to the presence of species non-cross-reactive cytokines/growth factors and innate immune rejection mechanisms. In order to overcome these barriers, we designed MISKI TRG6 mice, wherein human IL6 was additionally knocked in on the backbone of MISKI TRG mice. Both MISTRG and MISKI TRG support enhanced engraftment of human hematoipoietic cells (Rongvaux et al. Nat Biotechnol. 2014) and the addition of IL6 provides an essential MM growth factor. Injection of primary tumor cells from MM patients led to reliable engraftment of primary tumor cells in these mice. In this study, we utilized whole exome sequencing (WES) to compare the genomic changes in paired tumors isolated from (n=3) patients versus those growing in xenografts. In some patients, tumor cells from more than one xenografted mouse were analyzed separately to allow for evaluation of tumors growing in each individual mouse. Tumor cells were sorted by flow cytometry and subjected to DNA isolation followed by whole exome capture and sequencing. Germline and tumor DNA were captured on a Roche NimbleGen Sequence Capture V2.0 human exome array following the manufacturer's protocol, with protocol modifications at the Yale Center for Genome Analysis. Captured libraries were sequenced on the HiSeq 2500 sequencing system. Analysis of sequencing data was performed with the Yale exome-sequencing pipeline, as described (Choi et al. Nat. Genet. 2015). Mean depth of coverage for primary and xenografted samples was comparable. Comparison of loss of heterozygosity (LOH) patterns revealed that the majority of LOH changes in baseline tumors were also observed in xenografts, but the latter also contained additional changes. Profile of somatic copy number alterations (CNA) also revealed that while the xenografts captured the majority of CNA detected in freshly isolated tumor cells, they also exhibited additional genomic changes not detected in the initial tumor. Importantly, this included genomic changes in chromosome 1 typically associated with high-risk MM. Interestingly, the pattern of LOH and CNA were identical in individual mice transplanted with the same tumor cells, indicating that the new patterns of genomic changes observed in xenografts were likely already present at baseline, but clinically occult as in a minor subclone. Analysis of somatic non-synonymous variants (SNVs) revealed that the great majority of SNVs detected in the parent tumor were also identified in the xenografts, which also contained additional SNVs not detected at baseline. This included some targetable SNVs with known oncogenic potential. Together, these data demonstrate the capacity of MISKI TRG6 mice to recapitulate the genomic complexity of primary MM tumor cells and reveal their growth potential. These data provide a novel approach to investigate the genetics of human plasma cell neoplasia in vivo and reveal subclinical genetic lesions that may contribute to future relapse. Humanized models may be essential to fully understand genomic complexity of human tumors at a functional level and to optimize personalized approaches to therapy of human MM.

Disclosures
No relevant conflicts of interest to declare.