Abnormalities In Multiple Myeloma Research Articles

The role of 1q abnormalities in multiple myeloma: Genomic insights, clinical implications, and therapeutic challenges

Chromosome 1q copy number variations, collectively termed +1q, are 1 of the most common cytogenetic abnormalities in multiple myeloma. 1q abnormalities are associated with overexpression of a high-risk gene signature promoting cell proliferation, apoptosis resistance, genomic instability, and treatment resistance, and acquisition or expansion of +1q subclones mediate disease development and relapse. While there remains significant controversy as to whether the presence of +1q is itself an independent driver of poor prognosis or is simply a marker of other high-risk features, +1q has recently been incorporated into multiple prognostic scoring models as a new high-risk cytogenetic abnormality. In this review, we present possible underlying genetic mechanisms of high-risk disease in +1q myeloma, implications for subclonal development, its role in modifying the tumor microenvironment, current evidence for clinical significance in newly-diagnosed and relapsed patients, and current controversies in +1q classification and prognostication.

Cytoplasmic light-chain immunofluorescence combined with FISH in bone marrow smears to detect cytogenetic abnormalities in multiple myeloma

Objective: To analyze the sensitivity of cytoplasmic light-chain immunofluorescence with fluorescence in situ hybridization in bone marrow smears (new FISH) for detecting cytogenetic abnormalities in multiple myeloma (MM) . Methods: 42 MM patients admitted to the First Affiliated Hospital of Nanjing Medical University from April 2022 to October 2023 were enrolled. The patients with MM were detected by new FISH and CD138 immunomagnetic bead sorting technology combined with FISH (MACS-FISH) or cytoplasmic immunoglobulin FISH (cIg-FISH) to analyze cytogenetic detection results using combination probes which included 1q21/1p32, p53, IgH, IgH/FGFR3 [t (4;14) ], and IgH/MAF [t (14;16) ]. Results: In 23 patients with MM, the abnormality detection rates of cIg-FISH and new FISH were 95.7% and 100.0%, respectively (P>0.05). The detection rates of 1q21+, 1p32-, p53 deletion, and IgH abnormalities by cIg-FISH and new FISH were consistent, which were 52.2%, 8.7%, 17.4%, and 65.2%, respectively. The results of the two methods further performed with t (4;14) and t (14;16) in patients with IgH abnormalities were identical. The positive rate of t (4;14) was 26.7%, whereas t (14;16) was not detected. In 19 patients with MM, the abnormality detection rates of MACS-FISH and new FISH were 73.7% and 63.2%, respectively (P>0.05). The positivity rate of 1q21+, 1p32- and IgH abnormalities detected by MACS-FISH were slightly higher than those detected by new FISH; however, the differences were not statistically significant (all P values >0.05) . Conclusion: The new FISH method has a higher detection rate of cytogenetic abnormalities in patients with MM and has good consistency with MACS-FISH and cIg-FISH.

1p and 1q: Partners in crime in multiple myeloma.

7571 Background: Cytogenetic abnormalities in multiple myeloma (MM) highly influence the disease course, response to treatment and survival. Trisomies and immunoglobulin H chain translocations are primary CA while del(17p), gain(1q), del(1p) among others are secondary CA. Gain (3 copies) and amplification (>3 copies) 1q have been recognized as adverse prognostic markers and incorporated into the second revision of the International Staging System (R2-ISS). However, the role of del(1p) is less well defined, especially in the era of novel therapies. We aimed to analyze the outcomes of newly diagnosed MM (NDMM) patients with chromosome 1 abnormalities, mainly del 1p, treated with autologous stem cell transplant (ASCT) consolidation at our institution. Methods: We conducted a retrospective study of all NDMM patients who were treated with ASCT from 1/1/2015-2/13/2019 (n=511). High-risk cytogenetics (HRC) were defined by the presence of del(17p), t(4;14), or t(14;16) similar to R-ISS; standard-risk cytogenetics (SRC) were defined as the absence of HRC. Modified HR cytogenetics (mHRC) included gain/amp 1q and/or t(14;20) in addition to HRC, while ultra high-risk (uHRC) included 2 or more mHRC CA. Results: Of 511 pts transplanted, 453 had cytogenetic data at the time of diagnosis. SRC were seen in 353 pts (77.9%), while 100 (22.1%) had HRC, 156 (34.4%) had mHRC, and 43 (9.5%) had uHRC. Thirty-two (7.1%) pts had del(1p) while 105 (23.2%) had gain 1q and 30 (6.6%) had amplification 1q. As expected, compared to SRC pts, pts with HRC, mHRC and uHRC had higher risk of relapse or death. Patients with gain and amp 1q had inferior outcomes in terms of progression-free survival (PFS) (HR 1.35; 95% CI 1.06-1.73, p=0.016), time to next treatment (TTNT) (HR 1.84; 95% CI 1.40-2.42, p<0.001) and overall survival (OS) (HR 1.47; 95% CI 1.06-2.02, p=0.02) compared to those without, consistent with published literature. The median PFS, TTNT and OS from ASCT in pts with gain/amp 1q were 3.17 years (y), 3.95y and 7.13y, respectively, compared to 4.01y, 7.60y and 8.21y in pts without gain/amp 1q. Pts with del(1p) had inferior PFS (median 2.43y versus 3.98y; HR 1.75; 95% CI 1.16-2.64, p=0.008), TTNT (median 2.72y versus 6.17y; HR 1.96; 95% CI 1.22-3.14, p=0.005) and OS (median 4.11y versus 8.38y; HR 2.19; 95% CI 1.34-3.58, p=0.002) from the time of ASCT compared to those without del(1p). Conclusions: In our study of NDMM patients that underwent AHCT, del(1p) at diagnosis was an independent predictor of shorter PFS, TTNT and OS. Despite induction therapy involving novel drugs and ASCT consolidation, patients with del(1p) at diagnosis continue to have inferior outcomes. Larger analyses are needed to validate the prognostic value of del(1p) and investigate its role in predicting outcomes in MM.

Detection of cytogenetic abnormalities in multiple myeloma by using optical genome mapping

Multiple myeloma (MM) is a plasma cell neoplasm characterized by numerous chromosomal number and structural abnormalities, which are of great significance for risk stratification and response evaluation of MM patients. Optical genome mapping (OGM) is a novel technology that has the potential to resolve many of the issues and limitations associated with traditional cytogenetic methods. To date, the clinical utility of OGM has been validated in the fields of cancer, reproduction, and embryonic dysplasia, et al. In this study, we compared OGM to traditional techniques for the first time in five newly diagnosed MM patients, and evaluated the potential of OGM for detecting cytogenetic aberrations and its clinical application value in MM.

Gaps in CIBMTR Reporting of High Risk Abnormalities in Multiple Myeloma

Gaps in CIBMTR Reporting of High Risk Abnormalities in Multiple Myeloma

1q21+ is associated with poor prognosis in newly diagnosed multiple myeloma patients with extramedullary disease: a retrospective study.

1q21+ is a common cytogenetic abnormality in multiple myeloma (MM) and is considered an independent predictor of poor prognosis; however, its impact on extramedullary disease (EMD) remains unknown. Our study reviewed the clinical relevance and prognostic value of 1q21+ status in 92 patients with NDMM and EMD. 1q21+ was detected in 23.9% (22/92) of patients. Patients with 1q21+ presented with advanced International Staging System stages (P = 0.006), lower level of hemoglobin (P = 0.004), higher percentage of plasma cells in the bone marrow (P < 0.001), higher level of serum β2-microglobulin (7.24g/L vs. 3.85g/L, P = 0.003), and higher levels of lactic dehydrogenase (LDH) (206.5 U/L vs. 177 U/L, P = 0.019). The prevalence of soft tissue-related EMD (EMD-S) (54.5% vs. 18.6%, P < 0.001), renal dysfunction (50.5% vs. 17.7%, P = 0.002), and hypercalcemia (27.3% vs. 7.1%, P = 0.011) was also higher. 1q21+ was strongly associated with other high-risk cytogenetic abnormalities, including IgH/FGFR3 (22.7% vs. 4.3%, P = 0.007) and IgH/MAF translocations (22.7% vs. 1.4%, P < 0.001). 1q21+ patients had significantly shorter overall survival (OS) and progression-free survival (PFS) (OS: 24months vs. 47months, P = 0.002; PFS: 14months vs. 38months, P < 0.001); the poor survival outcomes could not be reversed by autologous hematopoietic stem cell transplantation. Multivariate analysis suggested that 1q21+ , EMD-S, elevated lactate dehydrogenase (LDH) levels, and P53 deletion were independent risk factors for poor prognosis in patients with EMD. In patients with 1q21+ EMD, hypercalcemia, elevated LDH levels, and P53 deletion were independent adverse risk prognostic factors.

MRI-based bone marrow radiomics for predicting cytogenetic abnormalities in multiple myeloma

To develop a radiomics signature applied to magnetic resonance imaging (MRI)-images to predict cytogenetic abnormalities in multiple myeloma (MM). Patients with newly diagnosed MM were enrolled retrospectively from March 2019 to September 2022. They were categorised into the high-risk cytogenetics (HRC) group and standard-risk cytogenetics (SRC) group. The patients were allocated randomly at a ratio of 7:3 into training and validation cohorts. Volumes of interest (VOI) was drawn manually on fat suppression T2-weighted imaging (FS-T2WI) and copied to the same location of the T1-weighted imaging (T1WI) sequence. Radiomics features were extracted from two sequences and selected by reproducibility and redundant analysis. The least absolute shrinkage selection operation (LASSO) algorithm was applied to build the radiomics signatures. The performance of the radiomics signatures to distinguish HRC with SRC was evaluated by ROC curves. The area under the curve (AUC), specificity, and sensitivity were also calculated. A total of 105 MM patients were enrolled in this study. The four and 11 most significant and relevant features were selected separately from T1WI and FS-T2WI sequences to build the radiomics signatures based on the training cohort. Compared to the T1WI sequence, the radiomics signature based on the FS-T2WI sequence achieved better performance with AUCs of 0.896 and 0.729 in the training and validation cohorts respectively. A sensitivity of 0.833, specificity of 0.667, and Youden index of 0.500 were achieved for the FS-T2WI radiomics signature in the validation cohort. The radiomics signature based on MRI provides a non-invasive and convenient tool to predict cytogenetic abnormalities in MM patients.

Whole-Genome Sequencing for Copy Number Abnormalities in Multiple Myeloma Supersedes Karyotype Analysis and Fluorescent in Situ Hybridization

Whole-Genome Sequencing for Copy Number Abnormalities in Multiple Myeloma Supersedes Karyotype Analysis and Fluorescent in Situ Hybridization Jiali Li 1, Shaobing Gao 2, Zhenling Li 3, Yinyin Chang 4, Xiangxiang He 4, Yuexin Cheng 5, Manqian Li 4, Chunxiao Yao 4, Shiyong Li 6, Dandan Zhu 4, Shichun Tu 4, Mao Mao 6,7, #, Xi Zhang 1, #, Li Gao 1, # 1 The Xinqiao Hospital of Third Military Medical University, Chongqing 214426, China 2The Affiliated Cancer Hospital of Zhengzhou University and Henan Cancer Hospital, Zhengzhou 450003, China 3China-Japan Friendship Hospital, Beijin100029,China 4Clinical Laboratories, Shenyou Bio, Zhengzhou 450000, China 5Yancheng No. 1 People's Hospital, The Yancheng Clinical College of Xuzhou Medical University, Yancheng 224006, China 6Research &amp; Development, SeekIn Inc., Shenzhen 518000, China 7Yonsei Song-Dang Institute for Cancer Research, Yonsei University, Seoul 03722, Korea Corresponding authors. Tel.: +86-15601973722(Maomao); 13808310064(Xi Zhang) 13228686076(Li Gao) Corresponding authors Email: mao_m@yahoo.com (Maomao); zhangxxi@sina.com (Xi Zhang); gaotiantiantiger@163.com (Li Gao) Introduction Prognosis and management of multiple myeloma (MM) relies on risk stratification based on copy number abnormalities (CNA) detection results. CNA is conventionally evaluated by cytogenetic methods including karyotyping and flurescence in situ hybridization (FISH), both limited by the invasiveness, low proliferative activity of plasma cell and the low plasma cell count. LeukoPrint is a novel shallow whole-genome sequencing (sWGS) based approach to profile CNA in bone marrow cells and/or circulating cell-free DNA (cfDNA). We compared the CNA detection results by LeukoPrint, karyotyping and FISH using bone marrow or plasma samples from multiple myeloma patients. Methods A total of 128 patients were enrolled in this study. Karyotyping and FISH were performed by conventional approaches. CNA was detected by sWGS in genomic DNA from bone marrow aspiration samples and CD138+ enriched plasma cells as well as in plasma cfDNA. CNA degree was measured by log R ratio. Significant mutational peak region was identified by GISTIC2. Risk stratification was performed following the Mayo Stratification of Myeloma and Risk-adapted Therapy (mSMART) criteria. Results In this study, 908 CNAs were identified by sWGS in 96 (75.0%, 96/128) patients, while the positive rate of CNA detected by karyotyping and FISH was 7.9% and 56.7% , respectively. A total of 368 whole chromosome aberration events were identified by sWGS, 66.0% (243/ 368) of which were trisomy that defined 39.6% (38/96) of patients as hyperdiploidy and the remaining 60.4% (58/96) as non-hyperdiploidy. The most frequently mutational CNA peak region were amp(1q21), del(1p), del(6q), del(8p), del(13q14) and del(14q). The concordance rate between sWGS and FISH in detecting the four conventional loci (1q21 gain, 13q14 del, 13q14.3 del, and 17p13 del) was 81.8%. Comparing to the conventional approaches, sWGS provided new CNA information for 75.0% of the patients, and redefined the risk stratification for 17.2% of patients according to mSMART criteria. CNA detection results using paired bone marrow aspiration samples and enriched plasma cell samples from 8 patients were compared. We found that more CNAs (81 vs 58) and higher Log R ratio of CNAs were detected using enriched plasma cells than that using bone marrow aspiration samples. The concordance rate of the CNA detection results by sWGS between paired bone marrow genomic DNA and plasma cfDNA from another 9 patients was 83.3%. Conclusions LeukoPrint is an automated, convenient and cost-effective approach to depict CNA profile in genomic DNA or cfDNA. This method is superior to conventional approaches when used for CNA testing, and the practice of this method could improve prognostic stratification of MM patients.

1q21+ Is Associated with Poor Prognosis in Newly Diagnosed Multiple Myeloma Patients with Extramedullary Disease: A Retrospective Study

Abstract: Background: 1q21+ is a common cytogenetic abnormality in multiple myeloma (MM) and is considered as an independent factor for poor prognosis, but its impact on extramedullary disease (EMD) remains unknown. Method: In our retrospective analysis, patients who met the diagnostic criteria of IMWG and first diagnosed with EMD from January 2011 to May 2022 were enrolled in the study. EMD patients were divided into 2 subgroups: 1q21+ and 1q21- according to the FISH analysis. 1q21+ abnormality was regarded as gain of 1q21 (gain [1q21], 3 copies) and amplification of 1q21 (amp [1q21], ≥4 copies). Continuous variables were analyzed by independent-sample t test or Mann-Whitney U test and categorical variables were compared using chi-square test or Fisher's exact test. Kaplan-Meier method was applied for PFS and OS curve made and differences were evaluated by Log-rank test. Cox proportional hazard models were constructed for prognostic factors analyze. Result: Patients with 1q21+ presented with advanced International Staging System stages (stage III percentage: 68.2% vs. 30.0%, P=0.006), lower level of hemoglobin (91g/dL vs. 113.5g/dL, P=0.004), higher tumor burden (BMPC infiltration≥50% : 45.0% vs. 11.9%, P＜0.001), higher level of serum β2-microglobulin (7.24 g/L vs. 3.85 g/L, P=0.003) and lactic dehydrogenase (LDH) (206.5 U/L vs. 177 U/L, P=0.019). Higher incidence of soft tissue related EMD (54.5% vs. 18.6%, P＜0.001), renal dysfunction (50.5% vs. 17.7%, P=0.002) and hypercalcemia (27.3% vs. 7.1%, P=0.011) was also observed in 1q21+ subgroup. 1q21+ was found to be strongly associated with other high risk cytogenetic abnormalities including IgH/FGFR3 translocation (22.7% vs. 4.3%, P=0.007) and IgH/MAF translocation (22.7% vs. 1.4%, P＜0.001). Patients with 1q21+ had a significantly shorter overall survival (OS) and progression-free survival (PFS) (OS: 24 months vs. 47 months, P=0.002; PFS: 14months vs. 38months, P＜0.001), and the poor survival outcome couldn't be reversed by autologous hematopoietic stem cell transplantation (auto-HSCT). Further investigation estimated the impact of cooccurrence of 1q21+ and del17p on survival outcome. For median OS, 1q21- patients without del17p was 47 months, 1q21+ patients without del17p were 28 months, 1q21- patients with del17p was 22 months, and patients presented with both 1q21+ and del17p was 18 months (p=0.02). The median PFS for 1q21- without del17p, 1q21+ without del17p, 1q21+ with del17p was 38 months, 20 months and 6.5 months respectively, while not reached for 1q21- patients (P＜0.001). Multivariate analysis suggested 1q21+ along with EMD-S, elevated LDH level and P53 deletion were independent risk factors for poor prognosis in EMD patients. For 1q21+ EMD patients, hypercalcemia, elevated LDH level and P53 deletion were defined as independent adverse risk prognostic factors. Conclusion: Taken together, our findings emphasized that 1q21+ increased the possibility of disease progression and predicted poor survival in EMD patients. 1q21+ EMD tends to be associated with other high risk disease factors. ASCT may not overcome the adverse effect of 1q21+ in EMD patients. We suggested 1q21+ should be considered as a high-risk factor and added into routine assessment of risk stratification in EMD patients.

Gain or Amplification of Chromosome 1q Mediate Multiple Myeloma Resistant to Xpo-1 Inhibitor Selinexor Via Transcription Factor ETV3

Background: Gain or amplification of chromosome 1q, which characterized as a poor prognosis indicater, is one of the most frequent cytogenetic abnormalities in multiple myeloma (MM), occurring in nearly 40% of newly diagnosed patients. Though several genes, such as IL6R and CKS1B, have been reported to related to its severity, there still remains a mist for us to explore. So, we further explore the potential mechanism that may illustrate the malignant phenotype of gain or amplification of chromosome 1q in multiple myeloma, and the ETS transcription factors ETV3 may make sense. Objective：Explore the functions ETV3 played in the malignant phenotype of gain or amplification of chromosome 1q in multiple myeloma. Methods：Dataset GSE2658 from GEO database was used to analyze the correlation of ETV3 expression and survival of newly diagnosed multiple myeloma (NDMM). Dataset GSE4581 from GEO database was used to investigate the relationship between the number of 1q copies and expression of ETV3. Next, ETV3 expression on different myeloma cell lines was detected by RT-PCR,WB and high ETV3 expression cell lines were selected for further study. Knocking down of ETV3 on these cell lines were performed and CCK-8 was used to detected the proliferation ability. XPO-1 inhibitor selinexor was used with different concentrations (0-300nM) to treat these cells for 24h and 48h, and apoptosis was detected by flow cytometry. Further, RNAseq was performed to explore the related changed genes that might be regulated by ETV3. Result s : Survival analysis showed MM patients with higher ETV3 expression exhibit worse prognosis according to Dataset GSE2658 (P mini=0.046). As for Dataset GSE4581, 414 patients were included, among which 186 patients carried more than 2 copies of 1q21. We found that along with the increasing of 1q21+ copies, expression of ETV3 increased (p&amp;lt;0.01). The results of RT-PCR and West-blots showed that all the MM cell lines expressed different level of ETV3, among which H929 and AMO1 were extremely high. Hence, we knocked down ETV3 on these two cell lines (H929 Sh-ETV3 and AMO1 Sh-ETV3) in subsequent experiments. Results showed that after knocking down of ETV3, the proliferation rate of myeloma cells was reduced (p&amp;lt;0.05). Besides, myeloma cells knocked down of ETV3 were more sensitive to selinexor (p&amp;lt;0.01). Based on the above results, we analyzed the genes changed after knocking down of ETV3 by RNAseq, results indicated several genes such as PKN1 might play a part in this process. Conclusion s : MM patients carrying with higher 1q21+ copies expressed higher level of ETV3 genes, which indicated a worse prognosis. Knocking down ETV3 on myeloma cells reduced their proliferation ability and enhanced their sensitivity to selinexor. PKN1 might involved in this process. Key words: ETV3, multiple myeloma, Gain or amplification of chromosome 1q, selinexor

Beyond the Primary: Unveiling the Prognostic Value of Secondary Cytogenetic Abnormalities in Multiple Myeloma

Introduction Fluorescent In-Situ Hybridization (FISH) has been an indispensable tool to characterize the cytogenetic landscape of multiple myeloma (MM), mainly for identifying primary abnormalities such as heavy chain gene (IgH) translocations and odd-number chromosome trisomies. These aberrations play a crucial role in risk stratifying the disease given their correlation with disease aggressiveness, treatment response and overall prognosis. High-risk (HR) cytogenetics are identified through the presence of t(14;16), t(14;20) or loss of the p53 gene locus. Less is known about the prognostic implication of secondary abnormalities, in particular gain of 1q21. We aimed to evaluate the association between primary and secondary cytogenetic abnormalities and their impact on overall survival (OS) in MM patients. Methods We performed a retrospective analysis on a FISH records database from three Mayo Clinic campuses. Cohort included patients &amp;gt;18 years of age, diagnosed with multiple myeloma between 2005 and 2022, with FISH performed within two years of diagnosis. Demographics including age, date of diagnoses and OS data were retrieved from electronic medical records. Statistical analyses were done using BlueSky Statistics software. Study was conducted in accordance with the principles of the Declaration of Helsinki; informed consent was obtained from all participants. Results We analyzed a total of 2221 patients, among which 59.8% were male, with a median age of diagnosis 65 years. Hyperdiploidy was the most prevalent primary cytogenetic abnormality observed in 44.3% of patients, followed by t(11;14) as the most common translocation (23%), and t(4;14) (8.2%). Gain(1q21) was assessed in 925 patients, among which 64%, 30.3% and 5.7% had 2, 3 and ≥4 copies respectively. Furthermore, 11.4% (n=253) exhibited either mutated TP53 or del17p, and 37.8% (n=839) presented with 13q loss or monosomy 13. The median follow-up for the overall population was 102 months (8.5 years), with a median OS of 75.5 months. Patients with high-risk translocations had significantly shorter median OS of 40.01 months compared to 80.65 months for those with standard risk translocations (p &amp;lt;0.001). The presence of ≥4 1q copies was associated with significantly reduced overall survival (2 copies: not reached, 3 copies: 69.4 months, ≥4 copies: 40.1774; p &amp;lt;0.001). This was consistent even when considering the presence of high-risk translocations (Figure 1). To confirm the independent significance of these findings, we performed multivariate Cox regression models, incorporating age at diagnosis, year of diagnosis, gender, presence of hyperdiploid disease, and presence of high-risk translocations. Given that overall survival outcomes were analyzed irrespective of treatment received, we incorporated year of diagnosis as a variable to control for potential impact of evolving treatment regimens over the years. The presence of ≥4 1q copies was associated with reduced OS, with a hazard ratio (HR) of 2.12 (CI: 1.38-3.26; p &amp;lt;0.001). The presence of 3 copies also showed prognostic significance (HR: 1.43; p: 0.012). Similarly, both the presence of TP53mut/mon17 (HR: 1.96, CI: 1.66-2.31; p &amp;lt;0.001) and 13qdel/mon13 (HR: 1.21, CI: 1.08-1.38) were associated with decreased OS. On the other hand, 1p deletion did not have any significant prognostic implications (HR: 1.84, CI: 0.38-1.59; p 0.113). When incorporating all cytogenetic events into a multivariate model, variables associated with reduced OS included age at diagnosis, the presence of both of 3 and ≥4 1q copies, TP53mut/mon17, t(4;14), t(14;16) and t(14;20). However, 13qDel/mon13 lost significance when incorporated with other abnormalities (Table 1). Conclusion Our investigation sheds light on the less explored realm of secondary cytogenetic abnormalities and confirm a strong association between increasing number of 1q copies and reduced overall survival, independent of other cytogenetic abnormalities and treatment regimens. These results underscore the utility of FISH-based comprehensive cytogenetic profiling at baseline, which enables reliable identification of high-risk patients. Future prospective trials should further elucidate the pathogenic mechanism for these findings. Our analysis used OS as primary endpoint and included over 2000 MM patients, reinforcing the clinical relevance of our findings.

Prospective Molecular Characterization of Multiple Myeloma Patient Samples Identifies High-Risk Patients and Informs Treatment Sequences through Resistance Mechanisms to Immunotherapies

Introduction. Multiple myeloma (MM) genetic diagnostics has traditionally been performed using fluorescence in situ hybridization (FISH), giving prognostic information based on immunoglobulin (Ig) translocations and key copy number abnormalities such as 1p, 1q, and 17p. However, in recent years it has become clear that more mutations, biallelic events, and focal deletions are also prognostically important and can be captured better through molecular techniques such as DNA sequencing. In addition, as targeted and immunotherapies become standard, monitoring the molecular targets will be important in determining the optimal sequence of therapies. Methods. Bone marrow aspirates from patients with MGUS/amyloid (n=3), smoldering MM (n=3), newly diagnosed MM (n=15), or progression/relapse MM (n=33) were collected. All patients were enrolled in the Indiana Myeloma Registry and consented for use of data for research purposes. CD138+ MACS cell separation was performed in a clinical pathology laboratory and DNA was extracted from CD138+ cells and patient-matched buccal swabs. 100 ng DNA from both the tumor and control sample were processed using the HyperPlus kit (KAPA Biosystems) and hybridized to the Myeloma Genome Project (MGP) sequencing panel in a CLIA-certified laboratory. The panel consisted of 228 genes for mutation and copy number analysis, as well as the immunoglobulin and MYC loci for translocation analysis. Samples were sequenced using paired-end reads to a median depth of 1063x. Data were analyzed using a clinical workflow and genomic abnormalities reported by a pathologist. Results. Of the 54 patients, Ig translocations were detected in 30 and included the known t(4;14), t(6;14), t(11;14), t(14;16) and t(12;14) as well as rare Ig translocations to partners such as GADD45B, LYN, PPCDC, and RCBTB2, of which over-expression of GADD45B and LYN are potentially targetable. A complex t(1;8;11;14) was also detected involving CCND1, MYC and the IGH loci which would not have been detectable by FISH. High-risk markers, such as gain/amp1q (22.2%) and deletion/mutation TP53 (16.6%) were detected including biallelic TP53 alterations in 5 patients (9.3%), which were mostly deletion plus mutation (4/5) and would not have been detected by traditional techniques. Of the MM patients who had been previously treated (n=33), 27 had received an IMiD and 30 had received a proteasome inhibitor. One patient showed mutation of CRBN (p.N236D) in conjunction with del3p and another patient with mutation of CUL4B (p.E277Ter) with delX indicating complete inactivation of the genes as a resistance mechanism to IMiDs. In addition, 17 patients had received an immunotherapeutic regimen including anti-CD38 or anti-BCMA. Of those that had received an anti-CD38 treatment (n=17), three had deletion of 4p (the location of CD38) including one patient, who had received three previous anti-CD38 regimens, with focal deletion of CD38. Six patients had also received anti-BCMA therapy, of which one had a mutation in the gene encoding BCMA, TNFRSF17, resulting in premature termination within the extracellular domain (p.S48Ter) in 42% of cells. This sample came from a patient who relapsed after an initial response to a BCMA CAR-T, followed by a progression occurring quickly while on a BCMA bispecific antibody Teclistamab, suggesting the loss of BCMA expression as a mechanism of resistance in both treatments. Interestingly, two patients harbored somatic mutations in the transmembrane domains of either CD38 (p.S29I) or TNFRSF17 (p.L60S) prior to the start of the respective treatments indicating that not all mutations seen in these genes are a result of treatment resistance. Conclusions. DNA sequencing approaches are cost-effective alternatives to FISH, the current standard diagnostic technology, to detect genomic abnormalities in multiple myeloma and they can identify clinically meaningful markers related to prognosis and treatment, particularly in the emerging era of immunotherapeutics where antigen evasion is a key mechanism of molecular resistance.

Optical Genome Mapping Reveals the Complex Genetic Landscape of Myeloma.

Fluorescence in situ hybridization (FISH) on enriched CD138 plasma cells is the standard method for identification of clinically relevant genetic abnormalities in multiple myeloma. However, FISH is a targeted analysis that can be challenging due to the genetic complexity of myeloma. The aim of this study was to evaluate the potential of optical genome mapping (OGM) to detect clinically significant cytogenetic abnormalities in myeloma and to provide larger pangenomic information. OGM and FISH analyses were performed on CD138-purified cells of 20 myeloma patients. OGM successfully detected structural variants (SVs) (IGH and MYC rearrangements), copy number variants (CNVs) (17p/TP53 deletion, 1p deletion and 1q gain/amplification) and aneuploidy (gains of odd-numbered chromosomes, monosomy 13) classically expected with myeloma and led to a 30% increase in prognosis yield at our institution when compared to FISH. Despite challenges in the interpretation of OGM calls for CNV and aneuploidy losses in non-diploid genomes, OGM has the potential to replace FISH as the standard of care analysis in clinical settings and to efficiently change how we identify prognostic and predictive markers for therapies in the future. To our knowledge, this is the first study highlighting the feasibility and clinical utility of OGM in myeloma.

Evaluation of the Sensitivity of Conventional Cytogenetic and Fluorescence in Situ Hybridization Methods for the Detection of Cytogenetic Abnormalities in Multiple Myeloma Patients: A Retrospective Study

Background: Identification of genetic abnormalities in multiple myeloma (MM) patients is of particular importance in order to design their treatment and management. Therefore, it is necessary to use the diagnostic methods with high sensitivity to detect abnormalities. In this study, we investigated the sensitivity of conventional cytogenetic and FISH methods in the diagnosis of genetic abnormalities in MM patients. Methods: This retrospective study included 246 patients who referred to the Kariminejhad Center for the Diagnosis of Genetic Abnormalities between 2009-2019. All patients were diagnosed based on diagnostic tests, as well as the approval of the relevant physician. The diagnosis of cytogenetic abnormality was made based on the two methods of conventional cytogenetic and FISH. Result: The results showed that out of 246 patients examined by conventional cytogenetic, only 17.8% had abnormal karyotypes. While out of 67 patients examined by FISH, 64.1% had abnormal results. The results also showed that 31 out of 50 patients with normal karyotype had abnormal FISH result. In the present study, the results showed that 25% of the patients had hyperdiploidy (57-47), which was diagnosed by conventional cytogenetic. Also, 40.90% had diploid abnormalities (pseudodiploid or structural abnormalities). FISH detected del 13q in 27.9% and t(11;14) IGH-CCND1 in 18.6% of patients, which were the most frequent compared to other abnormalities. Conclusion: Considering that the variety of mutations and translocations is high in different parts of the world and every day new mutations are detected, using both methods together can help to identify genetic disorders.

P-367 High-risk cytogenetic abnormalities in multiple myeloma: PETHEMA-GEM experience

P-367 High-risk cytogenetic abnormalities in multiple myeloma: PETHEMA-GEM experience

Concomitant deletion of the short arm (del(1p13.3)) and amplification or gain (1q21) of chromosome 1 by fluorescence in situ hybridization are associated with a poor clinical outcome in multiple myeloma.

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. FISH probes were generated from specific BAC DNA clones for the AHCYL1 gene locus (1p13.3) and the CKS1B locus (1q21). 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. 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.

Presentation and outcomes of patients with multiple myeloma harboring gain or amplification of 1q21 and receiving novel agent therapies: results from a single-center study

ABSTRACTObjectiveGain or amplification 1q21 (1q21+) is one of the most common recurrent cytogenetic abnormalities in multiple myeloma (MM). Our aim was to explore the presentation and outcomes of patients with MM harboring 1q21 + .MethodsWe retrospectively analyzed the clinical features and survival outcomes in 474 consecutive patients with MM receiving immunomodulatory drugs or proteasome inhibitor-based regimens as first-line therapies.Results1q21 + was detected in 249 (52.5%) patients. Patients with 1q21 + had a higher proportion of subtypes of IgA, IgD, and λ-light chain than non-1q21 + . 1q21 + was associated with more advanced ISS stage and was more frequently accompanied by del(13q), elevated lactate dehydrogenase and lower levels of hemoglobin and platelets. Patients with 1q21 + had shorter PFS (21 months vs. 31 months, P = 0.001) and OS (43 months vs. 72 months, P < 0.001) than those without 1q21 + . Multivariate Cox regression analysis confirmed that 1q21 + was an independent prognostic factor for both PFS (HR 1.277, P = 0.031) and OS (HR 1.547, P = 0.003). Patients with 1q21 + del(13q) double-abnormality had shorter PFS (P < 0.001) and OS (P = 0.001) than those with no FISH abnormalities, and they also had shorter PFS (P = 0.018) and OS (P = 0.026) than those with del(13q) single abnormality. No significant difference in PFS (P = 0.525) or OS (P = 0.245) was found between patients with 1q21 + del(13q) double-abnormality and 1q21 + del(13q) multiple-abnormality.ConclusionsPatients with 1q21 + were more likely to have coexisting negative clinical features and del(13q). 1q21 + was an independent prognostic factor associated with poor outcomes. Concurrence with such unfavorable features may account for poor outcomes given 1q21 + .

Cytogenetic Abnormalities in Multiple Myeloma: Incidence, Prognostic Significance, and Geographic Heterogeneity in Indian and Western Populations

Multiple myeloma (MM) is a genetically complex and heterogeneous neoplasm in which cytogenetics is a major factor playing an important role in the risk stratification of disease. High-risk MM based upon cytogenetic classification includes primary IGH translocations t(4;14), t(14;16), t(14;20), and secondary progressive aberrations such as gain/amp(1q), 1p deletion, del(17p), and hypodiploidy. Several studies have proved that interphase FISH can detect primary as well as secondary cryptic aberrations very efficiently in lowest 5–10% abnormal plasma cell population. The present large-scale study was undertaken to evaluate the incidence of cytogenetic abnormalities, to analyse the correlation of conventional karyotyping with FISH, and to seek the geographic heterogeneity in the incidence of primary as well as secondary aberrations in our Indian versus Western populations. We conducted prospective studies of 1,104 patients consecutively referred from the primary, secondary, and tertiary oncology centres from all over India. Interphase FISH was performed on isolated plasma cells. Karyotype analysis was done as per ISCN 2016 and 2020. FISH could detect cytogenetic abnormalities in 67.6% of the cases with an incidence of 59% non-hyperdiploidy. The incidence of IGH translocation was 26% versus literature frequency of 40–50% which was mainly due to a low incidence (6%) of t(11;14) in contrast to 15–20% in other series. Additionally, the association of secondary progressive aberrations in the hyperdiploid group rather than the non-hyperdiploid group in our patients is not a common finding. A biallelic inactivation of TP53 as an ultra-high risk factor was detected in old-aged patients. These observations disclose the novel findings and strongly indicate the racial disparity which leads to geographic heterogeneity. In contrast to FISH, conventional karyotyping could detect MM-related aberrations in 50% of cases, of which 44% revealed highly complex karyotypes with common aberrations of chromosome 1q. Overall, FISH was found to be a novel, easy approach with high success rate and capability of detection of all cytogenetic abnormalities that add valid information for the risk stratification of disease. This, in future, in combination with mutation profile and gene expression profile will help in further refinement of disease and identification of actionable targets.

A novel human multiple myeloma cell line with a 1q21 gain genetic abnormality and CKS1B overexpression.

Multiple myeloma (MM) is an incurable hematologic malignancy mainly due to its cytogenetic abnormalities. Therefore, it is important to establish permanent malignant MM cell lines as tools to develop more effective therapies. Pleural effusion cells of a 70-year-old patient was collected to establish the CZ2 cell line. Characterization of CZ2 was determined with nephelometry, flow cytometry, fluorescence in situ hybridization (FISH), and chromosomal microarray analysis (CMA). Western blotting analysis was adopted to determine protein expression. Cell viability was measured by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. We established and characterized a new MM cell line, CZ2. Using nephelometry and flow cytometry, cells with typical plasma cell morphology but not classical plasma cell phenotype were found to be non-immunoglobulin-secretary cells. FISH analysis of cells revealed a unique characteristic, namely, that there was only gain of the 1q21 region (1q21+). No other common cytogenetic abnormalities in MM, such as deletion of 17p (17p-), deletion of 13q (13q-), or translocation of immunoglobulin heavy chain (IgH), were observed. In addition, the original cell line maintains its single cytogenetic abnormality. Meanwhile, we observed through western blotting that CDC28 protein kinase regulatory subunit 1B (CKS1B), an adverse prognostic gene located in the 1q21 region, was highly expressed in CZ2. Knockdown of CKS1B reduced cell viability and also increased the levels of cleaved-poly(ADP-ribose) polymerase (cleaved-PARP) and cleaved-caspase3. CZ2 provides a suitable material for cellular and molecular studies of MM with only a 1q21 abnormality. This cell line is characterized by a gain of 1q21, and the high expression of CKS1B is an important model for studies of myeloma cell growth and drug resistance during therapy.

CD20 expression: A risk stratification factor for newly diagnosed multiple myeloma with t(11;14).

Translocation (11;14) is one of the most frequent recurrent cytogenetic abnormalities in multiple myeloma (MM), while its clinical prognostic value remains controversial. CD20 expression is uncommon in MM while strongly associated with t(11;14). This study aimed to investigate whether CD20 could provide further prognostic value in MM patients harboring t(11;14). CD20 expression detected by flow cytometry was retrospectively analyzed in 211 newly diagnosed MM patients with t(11;14). The clinical characteristics and outcomes were analyzed between CD20 positive and negative patients. CD20 expression was found in 34.6% (73/211) newly diagnosed MM (NDMM) patients with t(11;14), associated with lower serum creatine levels and lower incidence of plasmacytoma. Based on similar treatment regimens, CD20 positive patients had a comparable overall response rate to CD20 negative patients, whereas had a lower CR/sCR (complete response/stringent complete response) rate than the latter (31.4% vs. 46.4%, P =0.045). Nevertheless, CD20 positive patients had a longer tendency of progression-free survival (PFS) (59.0 vs. 29.0 months, P =0.163) and significantly longer overall survival (OS) (99.0 vs. 56.0 months, P=0.003) than CD20 negative patients. Further investigation among CD20 expression proportion showed that strong expression of CD20 (>80% of bone marrow plasma cells) exhibited the longest OS (median not reached, P =0.011). However, the favorable impact of CD20 expression on survival was eliminated with the contaminant presence of cytogenetic abnormalities besides t(11;14). Autologous stem cell transplantation (ASCT) could improve the prognosis of CD20 negative t(11;14) patients. Multivariate analysis confirmed that CD20 expression was an independent favorable indicator for longer OS in t(11;14) MM patients. CD20 expression is a favorable prognostic factor in NDMM with t(11;14) and could provide further risk-stratification value in this heterogeneous disease subgroup.