Clean and unmarked supplemental data

BACKGROUND:Chromosome 1 abnormalities in multiple myeloma (MM) are increasingly recognized as high risk-defining features. The authors report the prognostic value of del(1p13.3) by fluorescence in situ hybridization (FISH) at enrollment in subjects treated on total therapy clinical trials 2-6.METHODS:FISH probes were generated from specific BAC DNA clones for the AHCYL1 gene locus (1p13.3) and the CKS1B locus (1q21).RESULTS:A total of 1133 patients were included in this analysis. Although del(1p13.3) was detected in 220 (19.4%) patients, 1q21gain or 1q21amp were observed in 300 (26.5%) and 150 (13.2%) patients, respectively. Concomitant del(1p13.3) with 1q21 gain or amp was observed in 65 (5.7%) and 29 (2.5%) patients, respectively. There was enrichment of high-risk features such as International Staging System (ISS) stage 3 disease and gene expression profiling (GEP)70 high risk (HR) in the group with del(1p13.3). Presence of del(1p13.3) confers inferior progression-free survival (PFS) and overall survival (OS). On multivariate analysis, the presence of ISS stage 3 disease, GEP70 HR, 1q21gain, and 1q21amp were independent predictors of PFS or OS.CONCLUSIONS:The PFS and OS of patients with combined abnormalities of del (1p13.3)/1q21gain or amp was significantly worse compared to del(1p13.3) alone and 1q21gain or 1q21 amp alone, which identifies a subset of patients with poor clinical outcomes.

采用数字全息方法对物体经毛玻璃成像复原机理进行研究.首先,在建立毛玻璃数学模型的基础上,提出了消除毛玻璃干扰的理论算法.然后,利用数字全息再现原理建立了数学模型,并搭建了实验光路,将经毛玻璃后的物体信息通过消除毛玻璃干扰算法后进行再现,并将消除毛玻璃干扰前后的全息图进行了对比分析.最后,通过仿真与实验验证了理论算法和实验方法的正确性,对于物体透过复杂介质成像研究有着极其重要的参考价值.

Introduction- Chromosome 1 abnormalities in multiple myeloma (MM) are increasingly recognized as high risk defined features. While there is robust data on 1q21 gain and amplification (amp), the clinical characteristics and outcome of patients with del 1p is less defined. Novel agents are incorporated into a backbone of multi-agent chemotherapy and tandem autologous stem cell transplantation (ASCT) in successive Total Therapy (TT) protocols for MM patients. We hereby report the prognostic value of del 1p by FISH at enrollment in subjects treated on TT protocols.

Introduction: Flow cytometric immunophenotyping is considered an indispensable tool for the diagnosis, classification, and monitoring of plasma cell disorders. Herein, we seek to study the clinical significance of expression of phenotype markers in monoclonal gammopathy of unknown significance (MGUS).

While sustained minimal residual disease (MRD) negativity is an ideal therapeutic goal,1, 2 there is a subset of patients in whom MRD negativity is never achieved even with most intensive treatment approaches. In the current study, we describe the clinical characteristics and outcome of newly diagnosed multiple myeloma (NDMM) patients with continuous persistent MRD disease. NDMM patients treated in our institution between 2013 and 2019, with more than one serial BM MRD assessments regardless of disease response were included in this analysis. The frequency of MRD assessments ranged from every 3–6 months during the first 2 years of treatment to 6–12 months thereafter determined mainly by treatment phase (on or off maintenance therapy) and disease response.2, 3 All patients were treated with induction chemotherapy that included a combination of immunomodulatory agents (IMiD) and proteasome inhibitor (PI) added to a backbone of multiagent chemotherapy followed by high dose chemotherapy and autologous stem cell transplantation (ASCT) and maintenance therapy consisting of a combination of an IMiD,PI and dexamethasone.4 BM aspirate MRD assessment was performed using eight-color next generation flow cytometric (NGF) analysis with a minimal sensitivity of 10−5 cells.5, 6 Persistent MRD was defined as never attaining MRD negativity throughout the disease course. Patients who had a transient MRD negativity that lasted for ≤3 months were considered to have persistent MRD disease for this analysis. Sustained MRD negativity was defined as combined BM and imaging MRD negativity confirmed on two assessment ≥12 months apart. Patients with MRD conversion from negative to positive were excluded from the current analysis. Risk status was also assessed according to the 70 gene model (GEP70) as previously described.7 Of the 207 patients identified, 129 achieved sustained MRD negativity and 78 had persistent BM MRD positivity at all points of disease assessment. Overall, an average of 15 (range, 3–17) serial BM MRD assessments per patient were performed between 2013 and 2019. The median percentage of atypical myeloma plasma cells as determined by flow cytometry in the group with persistent MRD positivity was 0.04% (range 0.001–0.91). The median follow-up of the entire cohort from the first transplant was 2.70 (0.01–6.31) years. Baseline characteristics are shown in Table S1. In the group with persistent MRD positivity, the median age was 64 (range 56–69) years with 62% (n = 48) of patients >60 years of age; 44% (n = 34) of the patients were female and 12% (n = 9) African American. All patients received at least one autologous stem cell transplant (ASCT) and 73 of the 78 (94%) of patients received a second ASCT either planned (n = 33) or as salvage therapy (n = 40). Overall, there were no significant differences in the baseline demographics between the two groups. The distribution of GEP 70 subtypes was significantly different between the two groups. Patients with persistent MRD positivity expressed more frequently a HR GEP70 pattern (22% vs. 7.8%, p = 0.007). The distribution of CD2 subtype was 16% and 22% in the sustained MRD negative and persistent MRD positivity group, respectively. High-risk FISH markers such as (del)17p, t (14;16) and t (4;14) were observed 12% versus 17% (p = 0.5), 11% versus 12% (p = 0.9) and 6.2% versus 2% (p = 0.4), respectively and not statistically different between the two groups. On multivariate analysis, the effect of age >70 years (OR 1.18, 95% CI 0.98–1.44, p = 0.095), ISS stage III (OR 1.34, 95% CI 0.57–3.18, p = 0.5) or GEP70 HR signature (OR 2.88 95% CI 0.91–9.46, p = 0.075) were not statistically significant between the two groups, partly due to the relatively low numbers in the subgroups (Table S2). Patients with persistent residual MRD had an overall disease response (ORR) of 84% (n = 43/51); 49% (25/51) achieved ≥VGPR at 1-year post-ASCT2 (Figure S2). A second ASCT was performed in 94% (n = 73/78) of the patients with 51% (n = 40) of those as planned tandem ASCT and 42% (n = 33) as salvage procedure at disease progression. Next, we investigated clinical outcomes including relapsed free survival (RFS) and overall survival (OS) between the two groups. The RFS at 1, 2 and 5 years in the group with sustained MRD negativity (n = 129) was 96% (93–100), 91% (86–96),78% (71–86) compared to only 66% (57–78), 48% (38–60) and 24% (16–37) at 1, 2 and 5 years respectively in the group with persistent MRD positivity (n = 78). Similarly, the OS at 1, 2 and 5 years were 99% (98–100), 96% (93–100) and 85% (79–91) respectively in patients with sustained MRD negativity compared to 88% (82–96), 75% (66–85) and 40% (31–53) in patients with persistent MRD positivity. The median RFS and OS in the group with persistent MRD positivity was 1.6 (1.0–3.2) and 3.8 (3.2–6.3) years, respectively compared to median not reached in sustained MRD negative group. (Figure 1(A), (B)). Looking specifically at the distribution of GEP 70 persistent MRD positivity, HR disease was associated with dismal RFS of 1.6 years and OS of 1 year. Patients with LR disease and persistent MRD positivity had a median RFS and OS of 2.6(1.6–4.8) and 5.2(3.9-NR), respectively (Figure 1(C), (D)). Patients with baseline GEP 70 HR who showed sustained MRD negativity had a median RFS of 6.2 years (1.8-NR) and the median OS was not reached (5.5-NR) (Figure 1(C), (D)). The median RFS and OS were not reached in GEP 70 LR with sustained MRD negativity (Figure 1(C), (D)). In the context of persistent MRD disease, tandem ASCT (HR 0.57, 95% CI 0.25–1.28, p = 0.2) and age failed to meet statistically significance, while ISS stage III disease (HR 2.69, 95% CI 1.13 6.39, p = 0.025), HR GEP70 signature (HR 4.30, 95% CI 1.73–10.7, p = 0.002), GEP subtype MF (HR 5.87, 95%CI 1.61–21.4, p = 0.007) and MS (HR 5.37 95% CI 1.67–17.3, p = 0.005) were independently associated with inferior RFS and OS on multivariate analysis (Tables S3 and S4). We also analyzed 71 patients with persistent MRD positivity and paired BM GEP studies obtained at diagnosis and at the time of progression or relapse. At presentation, the majority (n = 61; 78.2%) of patients had GEP 70 defined LR disease. Overall, among the patients with LR disease at diagnosis, who had serial GEP assessment (n = 61), 10 (16.3%) patients changed from LR to HR GEP70 signature. Altogether, at relapse 32.8% (20/61) were HR GEP 70 (20/61) (Figure S3). The current study indicates for the first time to our knowledge that the outcome of NDMM patients with persistent BM MRD positivity at all assessments resembles that of primary refractory disease; in both circumstances, sequential intensive therapies failed to eradicate residual MRD clone(s).8, 9 Notably, at diagnosis the distribution of FISH-defined HR disease or ISS stage were not different between persistent MRD negative and MRD positive patients, suggesting that persistent MRD positivity confers a poor outcome regardless of these traditional risk factors routinely used in clinical practice. As previously shown, MRD status supersedes IMWG response with no statistically significant difference in RFS and OS in patients who attained CR/VGPR versus PR/SD.10 However, the prognosis of MRD positive patients is worse in the setting of higher stage of the disease (ISS stage) and high-risk disease (GEP 70) consistent with previous studies.6 Recent reports indicate that phenotypic and genomic features of MRD tumor cells show the presence of therapy-induced clonal selection with overexpression of adhesion molecules, integrins and chemokine receptors conferring a survival advantage in the BM microenvironment.11 Recent studies also show that residual MRD especially in the setting of HR disease is associated with acquisition of new mutations conferring increased genomic instability in residual tumor cells.12 Not surprisingly, the HR GEP70 signature drives the poor outcome in this group of patients with RFS and OS of 1 (0.7–1.5) and 1.5(0.91–2.6) years, respectively (Figure 1(C), (D)). In agreement with previous reports, we observed a higher percentage of patients with HR GEP 70 score and a shift to PR with disease progression in patients with LR disease at presentation.13 These interpretations should be viewed cautiously in the context of intra and inter-tumoral and spatial heterogeneity especially in advanced disease.13, 14 On the other hand, patients with GEP 70 LR disease and assigned to CD2 subtype on GEP 70 less frequently changed to a PR subtype and/or HR GEP 70 signature compared to HY and CD-1 subtypes (Figure S3).6, 15, 16 Using serial BM MRD as the only discriminating tool, we were able to identify a group of NDMM patients with dismal clinical outcomes despite intensive transplant-based approach.17, 18 Thus, in the current study persistence MRD positivity clearly trumps traditional risk stratification tools and clinical response assessment and identifies patients who behave like HR GEP70 disease in need for alternative treatment approaches. We acknowledge that this study has several limitations inherent to its retrospective nature including small sample size and heterogenous treatment regimens. The data reported here is reflective of clinical outcome with PI, IMiD, and monoclonal antibody daratumumab-based therapies at relapse, which are FDA approved medications in the relapsed MM setting. Other immunotherapy approaches like T cell directed therapies and antibody drug conjugates should be tested in this setting.19 The authors thank the patients and staff of the Myeloma Center, University of Arkansas for Medical Sciences. The authors declare no potential conflict of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Figure S1. Kaplan Meier curves showing overall survival of patients with persistent MRD positivity when stratified based on application of tandem transplants. Figure S2. Kaplan Meier curves showing shows the relapse free and overall survival of patients with persistent MRD positivity stratified by best response at D100 post ASCT. Figure S3. Figure showing the shift in the GEP 70 signature and subtype at relapse in patients with low risk GEP70 at presentation. Figure compares the distribution of GEP 70 subtypes at diagnosis to those at relapse. Table S1. Table comparing the baseline characteristics of a cohort of persistent MRD positivity to sustained MRD negativity. Table S2. Table showing the results of multivariate analysis comparing distribution of the clinical variable between the group with sustained MRD negativity and persistent MRD disease. Table S3. Table showing multivariate analysis of predictors of Progression free survival in group of patients with persistent MRD positivity. Table S4. Table showing multivariate analysis of predictors of overall survival in patients with persistent MRD positivity. 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Multiple myeloma (MM) patients frequently present with extensive osteolytic bone lesions. However, the impact of myeloma treatment on focal lytic lesion remineralization has not been extensively studied. In this study, the effect of anti-myeloma treatment on the extent of bone remineralization was examined and potential mediators identified. Newly diagnosed MM patients enrolled in the Total Therapy 4 and 5 (TT4; n = 231, TT5; n = 64) protocols were longitudinally evaluated for changes in radiological parameters for a median of 6.1 years. Bone remineralization was defined as a sclerotic CT change within the lytic lesion and quantified as a percentage of remineralization, using the initial lesion size as a reference. Such changes were correlated to clinical and biochemical parameters, and the gene expression profile of bone marrow biopsy. Overall, remineralization occurred in 72% of patients (213/295). Of those patients that experienced remineralization, 36% (107/295) achieved at least 25% of bone remineralization. Patients with high-risk disease defined by gene expression profile signature (GEP70 ≥ 0.66) experienced significant remineralization compared to low-risk MM. Female patients were also more likely to experience bone remineralization and in a shorter median time (2.0 vs. 3.3 y). Factors such as serum alkaline phosphatase along with high levels of RUNX2 and SOX4 gene expression correlated with increasing extent of bone remineralization. This analysis demonstrated significant remineralization of lytic lesions in MM patients treated on TT clinical trials. While the underlying mechanism remains elusive these findings support the hypothesis that patient baseline bone-related factors play a fundamental role in the skeletal repair of bone lesions in MM that provide new opportunities for improving patient outcomes.

AbstractMultiple myeloma (MM) patients frequently attain a bone marrow (BM) minimal residual disease (MRD) negativity status in response to treatment. We identified 568 patients who achieved BM MRD negativity following autologous stem cell transplantation (ASCT) and maintenance combination therapy with an immunomodulatory agent and a proteasome inhibitor. BM MRD was evaluated by next-generation flow cytometry (sensitivity of 10−5 cells) at 3- to 6-month intervals. With a median follow-up of 9.9 years from diagnosis (range, 0.4-30.9), 61% of patients maintained MRD negativity, whereas 39% experienced MRD conversion at a median of 6.3 years (range, 1.4-25). The highest risk of MRD conversion occurred within the first 5 years after treatment and was observed more often in patients with abnormal metaphase cytogenetic abnormalities (95% vs 84%; P = .001). MRD conversion was associated with a high risk of relapse and preceded it by a median of 1.0 years (range, 0-4.9). However, 27% of MRD conversion-positive patients had not yet experienced a clinical relapse, with a median follow-up of 9.3 years (range, 2.2-21.2). Landmark analyses using time from ASCT revealed patients with MRD conversion during the first 3 years had an inferior overall and progression-free survival compared with patients with sustained MRD negativity. MRD conversion correctly predicted relapse in 70%, demonstrating the utility of serial BM MRD assessment to complement standard laboratory and imaging to make informed salvage therapy decisions.

Abstract Purpose: Copy-number changes and translocations have been studied extensively in many datasets with long-term follow-up. The impact of mutations remains debated given the short time to follow-up of most datasets. Experimental Design: We performed targeted panel sequencing covering 125 myeloma-specific genes and the loci involved in translocations in 223 newly diagnosed myeloma samples recruited into one of the total therapy trials. Results: As expected, the most commonly mutated genes were NRAS, KRAS, and BRAF, making up 44% of patients. Double-Hit and BRAF and DIS3 mutations had an impact on outcome alongside classical risk factors in the context of an intensive treatment approach. We were able to identify both V600E and non-V600E BRAF mutations, 58% of which were predicted to be hypoactive or kinase dead. Interestingly, 44% of the hypoactive/kinase dead BRAF-mutated patients showed co-occurring alterations in KRAS, NRAS, or activating BRAF mutations, suggesting that they play a role in the oncogenesis of multiple myeloma by facilitating MAPK activation and may lead to chemoresistance. Conclusions: Overall, these data highlight the importance of mutational screening to better understand newly diagnosed multiple myeloma and may lead to patient-specific mutation-driven treatment approaches.

In vertebrates, biomineralization is a feature considered unique to mature osteoblasts and odontoblasts by which they synthesize hydroxyapatite (HAP), which is deposited in the collagen matrix to construct endoskeleton. For many decades, the mechanisms that modulate differentiation and maturation of these specialized cells have been sought as a key to understanding bone-remodeling defects. Here, we report that biomineralization is an innate ability of all mammalian cells, irrespective of cell type or maturation stage. This innate biomineralization is triggered by the concomitant exposure of living cells to three indispensable elements: calcium ion, phosphoester salt, and alkaline phosphatase. Any given somatic cell, including undifferentiated mononuclear cells, can undergo a biomineralization process to produce calcium-phosphate agglomerates. The biologically generated minerals under such conditions are composed of genuine HAP crystallites of Ca10(PO4)6(OH)2 and 5–10 nanometer (nm) in size. This discovery will profoundly improve our understanding of bone metabolism and ectopic calcifications.

Gain of chromosome 1q21 and the gene expression-based GEP70 risk score are established prognostic markers for newly diagnosed Multiple Myeloma (MM) patients. Here we addressed the prognostic impact of these two markers in 81 relapsed/refractory (RR) MM patients treated with the CD38-antibody daratumumab. Fluorescence in situ hybridization for 1q21 was performed at initial presentation, while the GEP70 score was determined at initial presentation and prior to daratumumab treatment. While the GEP70 at initial presentation showed a trend for inferior survival, the GEP70 collected prior to daratumumab treatment was significantly associated with poor outcome (P < 0·05). The worst outcome was seen for patients who were positive for gain(1q) and classified as GEP70 high risk prior to daratumumab [progression-free (PFS) and overall survival (OS) of 0·3 years (95% CI: 0·15-1·4 years) and 0·8 years (95% CI: 0·5-1·9 years) respectively], while the median PFS and OS were not reached by patients without gain(1q) and GEP70 low-risk status. In conclusion, gain(1q) and the GEP70 are powerful prognostic markers for RR MM patients treated with daratumumab, and patients classified as high risk according to these markers experience shorter treatment response.

8023 Background: The amplification of the proximal 1q21 region has been reported by interphase FISH (iFISH) in about 40% of newly diagnosed patients, and in 70% of MM patients at relapse. We hereby report the prognostic value of 1q21 amplification/gain by iFISH at enrollment in subjects treated on total therapy (TT) 4, 5 and 6 protocols. Methods: TT4 protocol enrolled newly diagnosed patients with LR disease as defined GEP 70 model ≤ 0.66. TT5 was designed for newly diagnosed patients with HR MM.TT6 enrolled previously treated patients with both HR and LR disease. iFISH was performed on bone marrow samples obtained at the time of study enrollment. FISH probes were generated from specific BAC DNA clone for CKS1B gene locus (1q21) gene locus. MM cells were identified post hybridization using isotype specific antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) to stain Ig-Kappa or Ig-Lambda light chain in cytoplasm (cIg) of MM cells. The iFISH signals in 100 of MM cells were recorded. A 20% cutoff point was used for detection of significant abnormalities i.e. gain of 1q21 (≥ 3 copies). Results: 607 patients were included in this analysis. With a median age of 61 years, 39% had high risk and 88% low risk disease. 1q21 abnormalities were present in 55% of patients at enrollment. 591 patients (97%) received at least one HDT ASCT. The analysis included 382 TT4 patients with a median PFS of 7.2 years and OS not yet reached. 75 patients enrolled on TT5 protocol experienced median PFS of 2.2 years and median OS of 5.6 years.150 TT6 enrolled patients experienced median PFS of 4.1 years and median OS of 7.5 years. Statistically significant difference in PFS (p < 0.0001) and OS (p < 0.0001) was observed between 1q21 defined groups (figure 1). No differences in survival were observed based on the 1q21 copy number > 3. Conclusions: This retrospective analysis clearly showed that 1q21 amplification/gain detected by interphase iFISH in different stages of disease can identify patients with significant shorter progressive free and overall survival even if exposed to total therapy regimens. Clinical trial information: NCT00734877, NCT00869232.

MYC is a widely acting transcription factor and its deregulation is a crucial event in many human cancers. MYC is important biologically and clinically in multiple myeloma, but the mechanisms underlying its dysregulation are poorly understood. We show that MYC rearrangements are present in 36.0% of newly diagnosed myeloma patients, as detected in the largest set of next generation sequencing data to date (n=1,267). Rearrangements were complex and associated with increased expression of MYC and PVT1, but not other genes at 8q24. The highest effect on gene expression was detected in cases where the MYC locus is juxtaposed next to super-enhancers associated with genes such as IGH, IGK, IGL, TXNDC5/BMP6, FAM46C and FOXO3. We identified three hotspots of recombination at 8q24, one of which is enriched for IGH-MYC translocations. Breakpoint analysis indicates primary myeloma rearrangements involving the IGH locus occur through non-homologous end joining, whereas secondary MYC rearrangements occur through microhomology-mediated end joining. This mechanism is different to lymphomas, where non-homologous end joining generates MYC rearrangements. Rearrangements resulted in overexpression of key genes and chromatin immunoprecipitation-sequencing identified that HK2, a member of the glucose metabolism pathway, is directly over-expressed through binding of MYC at its promoter.

The human kidney embryonic 293 cell line (293 cells) is extensively used in biomedical and pharmaceutical research. These cells exhibit a number of numerical and structural chromosomal anomalies. However, the breakpoints responsible for these structural chromosomal rearrangements have not been comprehensively characterized. In addition, it is not known whether chromosomes with structural rearrangement are more sensitive to external toxic agents, such as ionizing radiation. We used G-banding, spectral karyotyping (SKY), and locus- and region-specific fluorescence in situ hybridization (FISH) probes designed in our lab or obtained from commercial vendor to address this gap. Our G-banding analysis revealed that the chromosome number varies from 66 to 71, with multiple rearrangements and partial additions and deletions. SKY analysis confirmed 3 consistent rearrangements, two simple and one complex in nature. Multicolor FISH analysis identified an array of breakpoints responsible for locus- and region-specific translocations. Finally, SKY analysis revealed that radio-sensitivity of structurally rearranged chromosomes is dependent on radiation dose. These findings will advance our knowledge in 293 cell biology and will enrich the understanding of radiation biology studies.

Our systematic investigation on MYC (C-Myc) - a crucial pro-regeneration transcription factor - has determined that genetic aberrations (chromosomal rearrangements) and overexpression of the gene are highly prevalent in multiple myeloma (MM). At diagnosis, the rearrangements of MYC locus (8q24) were detected in 51% of MM patients using karyotyping and interphase FISH with DNA probe sets to MYC and the flank genomic sequences. The abnormalities of 8q24 in MM include monosomy (5%), amplification (≥3 copies; 13%), break-apart (14%), jumping (11%), and others (8%). Such aberrations were present in 49% of low-risk MM (183/376) and in 62% of high-risk MM (51/82) defined by the gene expression profiling cancer-risk model (GEP-70). The GEP also indicated that 74% of MM patients had excessively high levels of MYC overexpression. Nonetheless, there was no significant correlation between the MYC cytogenetic abnormalities and the levels of MYC expression in myeloma tumor cells - unlike the primary IGH translocations that drive transcriptional spikes of its partner genes. MYC expression by GEP was unable to precisely predict the presence of MYC chromosomal rearrangements in MM. In fact, the clinical outcomes of MM patients with MYC aberrations vs. the patients without detectable 8q24 events showed that neither the numerical variations nor the structural rearrangements of 8q24 had significant impact on the overall survival of patients who were treated on Total Therapy protocols. Further, we have shown that chromosomal rearrangements and the levels of MYC overexpression were not intrinsically relevant to the translation of MYC protein in myeloma. Importantly, the fate of myeloma cells was associated with the presence of two endogenous polypeptide-isoforms of MYC - the canonical MYC protein alias (P01106; 48.8kD) and an alternative MYC protein alias (alt-MYC; > 50kD). In newly diagnosed MM, P01106 was absent in most of the patients; whereas, it was a constitutive alias well maintained across the myeloma tumor cells from the patients with relapsed and progressive diseases and in all of the self-renewable myeloma cell lines. In contrast, alt-MYC alias was present as a stand-alone alias or co-existing with P01106 alias in newly diagnosed myeloma. Most importantly, both MYC aliases were absent in some cell lines and primary tumor cells suggesting cancer cell growth may not be dependent on any of MYC aliases despite of high levels of MYC transcription. Since the MYC gene originated from a viral integration, MYC mRNA has dual-translation features due to an internal ribosome entry segment (IRES) at 5'UTR. Previous studies have determined that the translations can be alternatively initiated from an in-frame CUG (Leucine) codon to generate NP_002458 alias or at a downstream canonical cap-dependent AUG (Methionine) codon to generate P01106 alias. Although these two aliases share identical C-termini, the translational switch can extend the N-terminal in NP_002458 alias. In vitro, the switch of P01106 to alt-MYC has resulted in cell growth arrest. Such alternation was induced by a transient gene transfection of P53 tumor suppressor (TP53); seemingly, the alternative MYC alias acts as an antagonist to P01106 in cell proliferation. Overall, it was generally unpredictable which of the MYC aliases were the dominant driver; therefore, MYC cytogenetic rearrangements and overexpression may have unknown indirect impacts on the outcomes. We are currently examining the precise amino acid sequences on both N-terminal and C-terminal ends of MYC aliases to study the regulatory mechanisms of the translational switching. This may indicate a novel anti-cancer strategy to cease cancer proliferation by deliberately inducing alternative translation of the antagonistic alias of an oncogene, and eventually overcome cooperation of the chromosomal rearrangements and dysregulated oncogene transcriptions in progressive cancer cells. Disclosures van Rhee: Adicet Bio: Consultancy; Karyopharm Therapeutics: Consultancy; EUSA: Consultancy; Castleman Disease Collaborative Network: Consultancy; Takeda: Consultancy; Sanofi Genzyme: Consultancy; Kite Pharma: Consultancy.

Patients with multiple myeloma (MM) accumulate adverse copy number aberrations (CNAs), gains of 1q21, and 17p deletions during disease progression. A subset of these patients develops heightened 1q12 pericentromeric instability and jumping translocations of 1q12 (JT1q12), evidenced by increased copy CNAs of 1q21 and losses in receptor chromosomes (RC). To understand the progression of these aberrations we analyzed metaphase cells of 50 patients with ≥4 CNAs of 1q21 by G-banding, locus specific FISH, and spectral karyotyping. In eight patients with ≥5 CNAs of 1q21 we identified a chromosome instability phenotype similar to that found in ICF syndrome (immunodeficiency, centromeric instability, and facial anomalies). Strikingly, the acquired instability phenotype identified in these patients demonstrates the same transient structural aberrations of 1q12 as those found in ICF syndrome, suggesting similar underlying pathological mechanisms. Four types of clonal aberrations characterize this phenotype including JT1q12s, RC deletions, 1q12-21 breakage-fusion-bridge cycle amplifications, and RC insertions. In addition, recurring transient aberrations include 1q12 decondensation and breakage, triradials, and 1q micronuclei. The acquired self-propagating mobile property of 1q12 satellite DNA drives the continuous regeneration of 1q12 duplication/deletion events. For patients demonstrating this instability phenotype, we propose the term “Jumping 1q Syndrome.”

The application of fluorescence in situ hybridization (FISH) technology in diagnosis and molecular classification of cancer-risk has become an essential tool in the proceeding of personalized therapy. In multiple myeloma, the precise FISH detection of numerical and structural genetic aberrations can be carried out on metaphase chromosome spreads, interphase nuclei, and formalin fixed paraffin-embedded (FFPE) tissues. To dissect highly complex cancer genomes, a broad variety of novel DNA probes, which outpace supplies from commercial resources on the market, are also crucial to the advanced translational researches. Here, we provide the protocols for the creation of custom-made DNA probes and for conducting hybridizations on various targeting cells and tissues.

Introduction: The proto-oncogene MYC (locus 8q24.21) is a key transcription factor in multiple myeloma (MM) resulting in significant gene deregulation and impacting on many biological functions, including cell growth, proliferation, apoptosis, differentiation, and transformation. Chromosomal rearrangement and copy number change at the MYC locus are secondary events involved in MM progression, which are thought to lead to aggressive disease. Current analyses of the MYC locus have not been large and have reported rearrangements in 15% of new-diagnosed MM. However, more recent studies using advanced genomic techniques suggest that the frequency of MYC rearrangements may be much higher, and that a full reassessment of the role of MYC in MM pathogenesis may be critical. In this study, we analyzed 1280 MM patients to provide a better understanding of the role of this important genomic driver in MM pathogenesis.

Introduction Despite significant improvement in overall survival, most multiple myeloma (MM) patients inevitably, yet unpredictably, develop refractory disease. The purpose of this study was to evaluate the effect of daratumumab as single agent or in combination in high risk relapse refractory (RR) myeloma population. Methods The UAMS Myeloma data base was interrogated to identify patients with relapsed refractory Multiple Myeloma who received daratumumab either a monotherapy or in combination with other agents. Patient were risk stratified to high and low risk MM using gene expression profile (GEP) studies on bone marrow specimens collected within one year from the time of relapse. Patients were further risk stratified based on interphase FISH used for CKS1B (1q21), AHCYL1 (1p13), TP53 (17p13), and ERBB2 (17q11). A 20% cutoff point was used for detection of significant abnormalities. Gene expression profiling was performed on the U133 plus 2.0 microarrays. High risk was defined by ≥ 0.66 GEP-70 score. The patients were followed with serum Immunoglobin levels, Serum and urine M component, serum free light chains, PET CT and bone marrow examinations as indicated. Response rates were determined as per the IMWG criteria. Patients were treated using a dose of daratumumab at 16 mg/kg administered weekly for weeks 1 to 8; every two weeks for weeks 9 to 24; and then every four weeks until progression. Daratumumab and immunomodulatory drug combination (mostly pomalidomide) was used in 80% of treated patients, in the remaining 20% daratumumab was used in combination with proteasome inhibitors. Fourteen percent of patients received daratumumab in combination with both IMID and PI drugs. Results Eighty MM patients were identified with a median age of 58 years, 56% were male. The median time from diagnosis to daratumumab treatment was 5 years. All patients were previously exposed and failed proteasome inhibitors and IMiD drugs. Ninety-five percent progressed after autologous transplant. In 35 patients, interphase FISH at baseline identified 1q21 gain and 32 subjects carried high risk disease according to the GEP 70 score. Seven patients had deletion of 17p13. Seven patients received daratumumab as single agent 73 in combination therapy. The median event free survival for the entire study group was eight months, median for OS was not reached, however, patients with high risk GEP 70 and 1q21 gain experienced a significantly inferior outcome presenting with median EFS and OS of 4 and 12 months respectively (P Conclusions Patients with high risk relapse/refractory myeloma defined by interphase FISH 1q21 gain and or GEP 70 high risk disease do not have a satisfactory response to daratumumab even in combination with IMID or PI drugs. This data suggests that anti MM activity of daratumumab may be impacted by genomic risk factors. Prospective studies are warranted to assess efficacy of daratumumab containing regimens at earlier stage of disease in this higher risk population. Disclosures Davies: Seattle Genetics: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; Bristol-Myers: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees. Morgan: Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria.

Multiple myeloma (MM) is a malignant disorder of plasma cells with a heterogeneous clinical outcome that is affected by both numerical and structural chromosomal abnormalities, baseline characteristics (age, lactate dehydrogenase concentration, International Staging System score) and treatment