In multiple myeloma malignant plasma cells expand within the bone marrow. Since this site is well-perfused, a rapid dissemination of “fitter” clones may be anticipated. However, an imbalanced distribution of multiple myeloma is frequently observed in medical imaging. Here, we perform multi-region sequencing, including iliac crest and radiology-guided focal lesion specimens from 51 patients to gain insight into the spatial clonal architecture. We demonstrate spatial genomic heterogeneity in more than 75% of patients, including inactivation of CDKN2C and TP53, and mutations affecting mitogen-activated protein kinase genes. We show that the extent of spatial heterogeneity is positively associated with the size of biopsied focal lesions consistent with regional outgrowth of advanced clones. The results support a model for multiple myeloma progression with clonal sweeps in the early phase and regional evolution in advanced disease. We suggest that multi-region investigations are critical to understanding intra-patient heterogeneity and the evolutionary processes in multiple myeloma.

• Hits in driver genes and bi-allelic events affecting tumor suppressors increase apoptosis resistance and proliferation rate-driving relapse.• Excessive biallelic inactivation of tumor suppressors in highrisk cases highlights the need for TP53-independent therapeutic approaches.To elucidate the mechanisms underlying relapse from chemotherapy in multiple myeloma, we performed a longitudinal study of 33 patients entered into Total Therapy protocols investigating them using gene expression profiling, high-resolution copy number arrays, and whole-exome sequencing. The study illustrates the mechanistic importance of acquired mutations in known myeloma driver genes and the critical nature of biallelic inactivation events affecting tumor suppressor genes, especially TP53, the end result being resistance to apoptosis and increased proliferation rates, which drive relapse by Darwinian-type clonal evolution. The number of copy number aberration changes and biallelic inactivation of tumor suppressor genes was increased in GEP70 high risk, consistent with genomic instability being a key feature of high risk. In conclusion, the study highlights the impact of acquired genetic events, which enhance the evolutionary fitness level of myeloma-propagating cells to survive multiagent chemotherapy and to result in relapse. (Blood. 2016;128(13):1735-1744

Nephrotoxicity from the chemotherapeutic drug cisplatin is associated with DNA fragmentation and cell death. We have recently demonstrated that DNase I knockout mice are significantly protected against cisplatin nephrotoxicity, but it is unknown whether the DNA fragmentation that occurs is produced by DNase I or another endonuclease. In this study we assessed the expression of several endonucleases involved in cell death after injection of cisplatin and found that the expression of endonuclease G (EndoG) increased whereas the expression of DNase I decreased almost to zero. Immunostaining showed that some nuclei contained both fragmented DNA and EndoG, suggesting that EndoG may cause DNA fragmentation induced by cisplatin. The increase in expression of EndoG was greater in wild-type mice than in DNase I knockout mice, indicating a potential link between the two endonucleases. In support of such a link, overexpression of DNase I in cultured mouse tubular epithelial cells also induced EndoG. Furthermore, gene silencing of EndoG in vitro provided significant protection against cell death. Taken together, our data suggest that both DNase I and EndoG mediate cisplatin injury to tubular epithelial cells.

Degradation of DNA during gene delivery is an obstacle for gene transfer and for gene therapy. DNases play a major role in degrading foreign DNA. However, which of the DNases are involved and whether their inactivation can improve gene delivery have not been studied. We have recently identified deoxyribonuclease I (DNase I) and endonuclease G (EndoG) as the major degradative enzymes in the mouse kidney proximal tubule epithelial (TKPTS) cells. In this study, we used immortalized mouse TKPTS cells and primary tubular epithelial cells isolated from DNase I or EndoG knockout (KO) mice and examined the degradation of plasmid DNA during its uptake. DNase I and EndoG KO cells showed a higher rate of transfection by pECFP-N1 plasmid than wild-type cells. In addition, EndoG KO cells prevented the uptake of fluorescent-labeled RNA. Complete inhibition of secreted DNase I by G-actin did not improve plasmid transfection, indicating that only intracellular DNase I affects DNA stability. Data demonstrate the importance of DNase I and EndoG in host cell defense against gene and RNA delivery to renal tubular epithelial cells in vitro.

INTRODUCTION In multiple myeloma (MM) samples for diagnostics, prognostication and response evaluation are most commonly obtained from the patients' posterior iliac crest due to its accessibility and safety, assuming a homogenous spread throughout the bone marrow. However, imaging studies revealed a highly imbalanced distribution of the disease in the majority of the patients, presenting with accumulations of malignant plasma cells (PC) in restricted areas in the bone marrow (BM), so called focal lesions (FL). In line with this pattern, our recently reported preliminary results of paired FL and random BM (RBM) samples strongly indicate an unequal distribution of sub-clones in the BM. Spatial genomic heterogeneity has not been systematically analyzed in MM thus far, although its existence would have a high impact on interpretation of drug resistance studies, risk stratification and personalized treatment based on genomic markers. Here we report on an extended genomic analysis of regional heterogeneity in paired FL and RBM samples including 42 newly diagnosed and 11 extensively treated MM patients with 10 of these patients also being studied longitudinally. MATERIAL & METHODS MM PCs were CD138-enriched. Leukapheresis products were used as controls. For whole exome sequencing (WES) we applied the qXT kit and the SureSelect Clinical Research Exome bait design (Agilent). Paired-End sequencing was performed on an Illumina HiSeq 2500. Sequencing data were aligned to the GRCh37/hg19 reference using BWA. Somatic single nucleotide variants (SNV) were identified using MuTect. Copy number aberrations (CNA) were derived from Illumina HumanOmni 2.5 bead chip data using ASCAT. Subclonal reconstruction was performed using SciClone. Gene expression profiles (GEP) were generated using Affymetrix U133plus2 microarrays. Statistical analyses were carried out using the R software package 3.1.1. RESULTS In 42 newly diagnosed patients we detected a median number of 86 (34 to 807) mutations per patient with up to 42% (median 5%) of them being unique to a specific site (non-ubiquitous). Among known MM driver genes, BRAF (n=2) and KRAS (n=4) were the genes that most often showed non-ubiquitous mutations at baseline. In treated patients mutations in KRAS, NRAS and RB1 contributed to regional heterogeneity in one patient each. Furthermore, we found temporal heterogeneity in mutations affecting ATRin two patients, aberrations recently associated with poor outcome. Analyzing chromosomal aberrations with known prognostic value we observed three newly diagnosed patients with a site-specific del(1p) affecting CDKN2C and/or FAM46Cwith two of these patients also showing regional heterogeneity in del(17p13). Non-ubiquitous gain(1q) or amp(1q) was seen in two patients at baseline. Of note, in all of these cases the unique event was detected in a FL and one case with a unique gain(1q) at baseline presented with this aberration in subsequent samples. These observations strongly support the concept of FLs being sites of resistant clones able to cause relapse. In four patients a MYC translocation was seen at only one site. In the longitudinal analysis we found one patient in whom a MYC translocation clone was replaced by a clone with a different MYC translocation, indicating that events at the MYC locus are secondary and can be sub-clonal. In contrast, primary IgH translocations were always shared, confirming that they are initiating events. Paired samples from RBM and FLs derived from three newly diagnosed patients showed discordant GEP risk profiles, further supporting the existence of site-specific high risk (HR) clones. To investigate the clinical relevance of this finding we analyzed outcome data of 263 newly diagnosed patients with paired GEP data. The 34 cases with discordant GEP based risk scores showed no significant differences in outcome compared to cases with HR at both sites, suggesting that HR sub-clones drive prognosis even if they are not ubiquitously present. CONCLUSIONS We show that spatial genomic heterogeneity is common in MM. The existence of site-specific sub-clones highlights the importance of heterogeneity analyses for accurate risk prediction, detection of suitable targets for precision medicine and identification of aberrations contributing to treatment resistance. As a result we strongly recommend to include FL examinations into routine diagnostics and follow-up analyses in MM. Disclosures Ashby: University of Arkansas for Medical Sciences: Employment. Barlogie:Signal Genetics: Patents & Royalties. Davies:Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Morgan:Univ of AR for Medical Sciences: Employment; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria.