No Recurrent Mutation of SF3B1, U2AF1 and SRSF2 Spliceosomal Gene in Multiple Myeloma but Polymorphisms of These Genes Are Associated with the Prediction of Worse Prognosis

Abstract Abstract 3958 Background: Recently, a striking example of the effects on acquired somatic mutations in splicing factors such as SF3B1, U2AF1, and SRSF2 has been described. The sequencing of the DNA from abnormal blood cells from patients with several types of leukemia such as AML, CLL, CMML, pre-leukemic syndromes, and MDS, has shown that a high proportion of these cases are associated with somatic mutations in spliceosomal proteins. Also, evidence of cancer-specific alternative splicing and oncogenic somatic mutations in spliceosome subunits has been steadily growing. However, there is not much research regarding aberrant splicing pathways in Multiple myeloma (MM) patients. Therefore, we tried to investigate the presence and prognostic implication of mutation of the SF3B1 and U2AF1 protein in these patients in South Korea. Materials and Methods: We examined a cohort of 87 MM patients and 100 healthy controls for somatic mutations in SF3B1, U2AF1 and SRSF2 by using direct sequencing method. The medical records were reviewed for age, sex, plasma cell percentage, serum M protein, immunoglobulin level, free light chain ratio, calcium, creatinine, hemoglobin, bone lesion, albumin, beta 2 microglobulin, lactate dehydrogenase, treatment outcome, and so on. The collected data was analyzed by SPSS for Windows version 18.0. We performed Pearson's chi-square tests, one way ANOVA analysis, and Student t-test. Survival rates of myeloma patients according to the result of SF3B1, U2AF1 and SRSF2 sequencing were analyzed using Kaplan-Meier log-rank test. Results: Our 87 MM patients showed no mutation including known recurrent ones in SF3B1, U2AF1 and SRSF2 genes. However, the patients displayed 39198T>T/C polymorphism (70.1%) in exon 18 of SF3B1, 8345T>T/G polymorphism (13.8%) in exon 2 of U2AF1 and 5399C/T polymorphism (100%) in exon 1 of SRSF2. In the entire cohort, the number of patients with no polymorphism, one polymorphism, two polymorphisms and three polymorphisms was counted up to 0%, 24.1%, 67.8% and 8.0%, respectively. The T/C polymorphism at position 39198 of SF3B1 exon 18 and the T/G polymorphism at position 8345 of U2AF1 exon 2 were analyzed by allele-specific PCR using normal control. Results in 100 normal controls, polymorphism of SF3B1 exon 18 was taken into account of 82.0%, the remaining is non polymorphism while U2AF1 exon 2 showed 10.0% polymorphism and 90.0% non polymorphism. Sex (p=0.048) and free light chain ratio (p=0.002) showed significant results according to polymorphism status while other clinical characteristics were not associated. The patient with polymorphisms in both SF3B1 and U2AF1 had worse overall survival (P=0.042) and disease-free survival (P<0.01), compared to patients without polymorphism. Conclusion: Our results show no recurrent SF3B1, U2AF1 and SRSF2 mutations in MM patients rather polymorphisms in SF3B1 and U2AF1 gene were significantly implicated in the prediction of poor prognosis. Disclosures: No relevant conflicts of interest to declare.

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No Mutations of SF3B1, U2AF1, and SRSF2 spliceosomal Gene but Polymorphisms Associated with the Worse Prognosis in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.4190.4190 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4190-4190

Author(s):

Min-Gu Kang ◽

Hye-Ran Kim ◽

Jun Hyung Lee ◽

Thashma Pemmanda Ganapathy ◽

Seung-Hyeon Yang ◽

...

Keyword(s):

Multiple Myeloma ◽

Dna Sequences ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Disease Free Survival ◽

Cellular Growth ◽

Exon 2 ◽

Number Of Patients ◽

Significant Difference ◽

Worse Prognosis

Abstract Recently, a striking example of the effects of acquired somatic mutations in splicing factors such as SF3B1, U2AF1, and SRSF2 has been described. The DNA sequences from abnormal blood cells of patients with hematologic malignancies has shown that a high proportion of these diseases are associated with somatic mutations of spliceosomal proteins. However, little is known about the aberrant splicing pathways in multiple myeloma (MM) patients. Therefore, this study investigated the presence and prognostic implication of splicing gene aberration in MM patients. Also, we planned functional experiments in cultured normal and Hela cell line to determine the effects of the common spliceosome mutations on cellular growth rate, cell count, and apoptosis. In total, 87 MM patients and 100 healthy controls were examined for somatic mutations in SF3B1, U2AF1, and SRSF2 by direct sequencing. Also, the clinical and laboratory characteristics of MM patients were reviewed. Regarding to the initial findings in recent reports, this study expressed the wild-type and the mutant (Q157P) U2AF1 in Hela cells and examined their proliferation. MM patients did not show mutation in SF3B1, U2AF1,and SRSF2 splicing genes. However, the patients displayed 39198T>C or 39198T>T/C polymorphism (70.1 %) in exon 18 of SF3B1, 8345T>T/G polymorphism (13.8 %) in exon 2 of U2AF1, and 5399C>T or 5399C>C/T polymorphism (100.0 %) in exon 1 of SRSF2 (Fig. 1). In the entire cohort, the number of patients with one polymorphism, two polymorphisms, and three polymorphisms was counted up to 24.1 %, 67.8 %, and 8.0 %, respectively. Meanwhile, in 100 normal controls, polymorphism of SF3B1 exon 18 and U2AF1 exon 2 was counted up to 82.0 % and 10.0 %, respectively. Free light chain (FLC) kappa/lambda (K/L) ratio and chromosome analysis showed significant difference according to polymorphism status (p=0.002) while other clinical characteristics did not. The patient with polymorphisms in both SF3B1 and U2AF1 gene had unfavorable overall survival (p =0.067) and disease-free survival (p=0.001), compared to patients without polymorphism. Our results show no recurrent SF3B1 and U2AF1 mutations in MM patients. Instead, polymorphisms were found in SF3B1 or U2AF1, which were significantly implicated in worse prognosis. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Improving Outcomes in Patients with Renal Failure Secondary to Multiple Myeloma: Results From ChAR2M2.

Blood ◽

10.1182/blood.v114.22.1875.1875 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1875-1875

Author(s):

Colin Hutchison ◽

Parisa Airia ◽

Mark Cook ◽

Daniel Grima

Keyword(s):

Multiple Myeloma ◽

Renal Failure ◽

Side Effects ◽

Light Chain ◽

De Novo ◽

Conflicts Of Interest ◽

Treatment Patterns ◽

Free Light Chain ◽

High Rate ◽

Sustained Reduction

Abstract Abstract 1875 Poster Board I-900 Study purpose: To explore how free light chain (FLC) removal by high cut-off haemodialysis (HCO-HD) has been adopted into clinical practice for the management of renal failure secondary to multiple myeloma. Describing treatment patterns and the laboratory and clinical outcomes associated with its use. Methods: A chart audit of patients treated with FLC removal by HCO-HD, using the Gambro HCO 1100 dialyser, was performed in 16 dialysis centers across 9 countries. Patient demographics, treatment patterns and dialysis side-effects were recorded. In addition, the following outcomes were measured: dialysis independence and reductions in serum FLCs concentrations at 12 and 21 days. Results: Data for 66 patients was entered. Patients had an average age of 65.1 (SD×10.1); 42 of them (63.64%) were male and 24 (36.36%) were female. Sixteen (24%) presented with relapsing myeloma and 50 (76%) had de novo disease. On average, each patient received 13 HCO-HD sessions (SD×8). Forty-one patients became dialysis independent (62.12%), after an average of 12 sessions. Dialysis related side-effects were reported in 6% of all patients. Forty patients (60.61%) were reported to have a sustained reduction in serum FLC concentrations by day 12. By day 21 this had increased to forty-one (62.12%). Among the patients who achieved a sustained reduction in serum FLC concentrations, 28 (70%) had a decline in FLC levels of more than 50% by day 12 and 34 (82.93%) by day 21. Among patients who achieved sustained reduction of more than 50% in serum FLC concentrations by day 12, 75% became dialysis independent. In comparison only 53% of those with a reduction of less than 50% became dialysis independent (p×0.007). Furthermore, among patients who achieved sustained FLC reduction of greater than 75%, 81% became dialysis independent. The rate of dialysis independence was also significantly higher in patients with de novo disease compared with those with relapsing myeloma (64% versus 56%, p×0.04). Conclusion: Free light chain removal by HCO-HD was well tolerated and associated with a very high rate of dialysis independence in patients with renal failure secondary to multiple myeloma. Rates of renal recovery were greater in patients with de novo myeloma and those who achieved an early reduction in serum FLC concentrations. Disclosures: No relevant conflicts of interest to declare.

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Immunoglobulin Heavy/Light Chain Immunoassays for Response Evaluation in Multiple Myeloma: Comparison with Immunofixation, Serum Free Light Chain, and Multicolor Flow- Cytometry

Blood ◽

10.1182/blood.v124.21.3366.3366 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3366-3366 ◽

Cited By ~ 1

Author(s):

Yasuhito Suehara ◽

Keisuke Seike ◽

Kouta Fukumoto ◽

Manabu Fujisawa ◽

Masafumi Fukaya ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Light Chain ◽

Conflicts Of Interest ◽

Free Light Chain ◽

Multicolor Flow Cytometry ◽

Serum Free ◽

Serum Free Light Chain ◽

Normal Ranges ◽

Best Response

Abstract BACKGROUND: Monitoring of multiple myeloma (MM) patients is required to assess and determine the treatment response. The serum immunoglobulin (Ig) heavy/light chain immunoassays are a new method that enables separate quantification of the different light chain types of each Ig class, i.e., IgGk, IgGl, IgAk, and IgAl. Although the contribution of these tests to disease evaluation has been assessed, available data are still limited. Here, we compared the serum Ig heavy/light chain immunoassays with the conventional methods for disease evaluation. PATIENTS AND METHODS: Three hundred and three samples were obtained from a total of 101 patients with IgG and IgA type MM recruited at Kameda Medical Center and Kanazawa University Hospital. The median age of the patients was 68 years (range: 44 – 89). The median follow-up was 36 months (range: 2.5 – 189.6). The proportions of patients in International Scoring System (ISS) stages I, II, and III were 25%, 35%, and 40%, respectively. Paraprotein type was IgG in 64 patients (44 Gk, 20 Gl) and IgA in 37 (20 Ak, 17 Al). Samples were taken at various times after treatment and analyzed retrospectively with serum Ig heavy/light chain immunoassays (Hevylite®; Binding Site, Birmingham, UK) on a SPAPLUS® turbidimeter. The heavy/light chain ratio (HLCR) was calculated with the Ig' k (either G or A) as the numerator and compared to normal ranges (IgGk:3.84-12.07g/L; IgGl:1.91-6.74g/L; IgGk/IgGl: 1.12-3.21; IgAk:0.57-2.08g/L; IgAl:0.44-2.04g/L; IgAk/IgAl:0.78-1.94). Results were compared to serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE), and serum free light chain immunoassays (FLC). HLC-pair suppression was defined as the uninvolved immunoglobulin of the same isotype 50% below the lower limit of the normal range (IgGk, IgGl, IgAk, IgAl). Residual neoplastic plasma cells (MM-PCs) in bone marrow samples were assessed by multicolor flow cytometry (MFC) simultaneously in 140 samples from 64 patients at very good partial response (VGPR), complete response (CR), and stringent CR (sCR). The MFC negativity was defined at a level of < 10-4 MM-PCs. Overall survival (OS) and progression-free survival (PFS) were estimated by the Kaplan–Meier method, and survival was compared using the log rank test. RESULTS: Myeloma responses were assessed serially after induction therapy and were assigned according to international response criteria. Patients' samples at various responses were: sCR, n = 86 (28%); CR, n = 31 (10%); VGPR, n = 116 (38%); and PR, n = 70 (23%). Normal HLCR was obtained at sCR, CR, and VGPR in 73 (85%), 28 (90%), and 69 (59%) cases, respectively. Discordance in the depth of response between standard electrophoretic methods and HLCR was more commonly seen in IgG compared to IgA MM; possibly reflecting the dose dependent half life of IgG immunoglobulins. In the sera from patients who achieved a CR or better, abnormal HLCR and FLC ratio were seen at rates of 12.8% and 26.5%, respectively. Discordance between normalization of HLCR and FLC ratio was seen in 42% of patients with a CR or better; however, a good correlation between normalization of HLCR and FLC ratio was still observed (Fisher's exact test, P = 0.004). Among the 71 sera from patients with a CR or better in which simultaneous MFC data were available, 16 (22.5%) sera were MFC-negative. Among the patients who achieved a VGPR or better, patients with a normal HLCR had fewer MM-PCs than those with an abnormal HLCR (median 9.8x10-4vs. 46.0x10-4, respectively, P=0.062), although the difference was not statistically significant. Abnormal HLCR in CR samples was seen more frequently in patients with IgA type (19%) than IgG type (7.7%). Longer PFS and OS were observed in patients who achieved HLCR normalization at best response than in those who did not (PFS; 83.8 months vs. 41.8 month, respectively, P = 0.016 and OS; not reached vs. not reached, P = 0.018). When patients at best response were divided into with or without uninvolved HLC pair suppression, the latter group showed significantly better OS compared to the former group (not reached vs. not reached, P=0.019). CONCLUSIONS: The findings presented here suggest the potential usefulness of heavy/light chain immunoassays for monitoring of myeloma response in patients with IgA myeloma and residual MM-PC assessment at VGPR or better. Presence of abnormal HLCR at best response and HLC pair suppression were also associated with poorer survival. Disclosures No relevant conflicts of interest to declare.

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Role of Free Light Chain Assay Ratio to Distinguish Between CR According EBMT Criteria or Stringent Complete Remission (sCR) According IMWG in Multiple Myeloma Patients.

Blood ◽

10.1182/blood.v114.22.1787.1787 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1787-1787

Author(s):

Svetlana Asenova ◽

Ulrike Bacher ◽

Andreas Gerritzen ◽

Axel R. Zander ◽

Nicolaus Kröger

Keyword(s):

Multiple Myeloma ◽

Complete Remission ◽

Light Chain ◽

Partial Remission ◽

Conflicts Of Interest ◽

Free Light Chain ◽

Normal Range ◽

Light Chain Immunoglobulin ◽

Definition Of ◽

Additional Value

Abstract Abstract 1787 Poster Board I-813 Introduction The qualitative assay for free light chain has been reported to be sensitive and specific for detecting and monitoring diseases caused by monoclonal gammopathies such as multiple myeloma. More recently the International Myeloma Working Group proposed uniform response criteria including a new definition of stringent complete remission (sCR). The definition of stringent complete remission requires beside absence of clonal cells in bone marrow by immunohistochemistry or immunofluorescence also normalization of free light chain ratio in serum. Patients and Methods We evaluate the value of free light chain assay to determine stringent CR by monitoring 87 patients with multiple myeloma who achieved complete remission (n=52) or very good partial remission (n=35) between January 2003 and December 2008. Free light chain measurements were performed with the commercially available Free liteTM Kit (Binding Site, Heidelberg, Germany). Because of the shorter half-life of free light chain assay ratio, only patients were included, if the complete or very good partial remission remains stable for at least 3 months. The comparison between immunofixation and free light chain ratio was performed at least 6 weeks after immunofixation becomes negative for the first time. 87 patients (50 mal and 37 female) were included, 67 had intact immunoglobulin and 11 had light chain immunoglobulin at time of diagnosis. The remission status was determined either after allogeneic (n = 73) or autologous (n = 7) stem cell transplantation or after conventional bortezomib or lenalidomide containing chemotherapy (n = 7). Results 35 out of 87 patients achieved a very good partial or near complete remission with still positive immunofixation. In 22 out of those 35 patients the free light chain kappa ratio was within the normal range of 0.26 – 1.65 mg/l (63 %). Only in 13 patients with persistent immunofixation positivity the free light chain ratio was outside the normal range (37%). 52 patients achieved complete remission according to the EBMT criteria with negative immunofixation for at least 3 months. In those patients all (100 %) had a normal free light chain kappa/lambda ratio. In a subgroup of patients (n = 10) who relapsed during follow-up from complete remission sequential monitoring of immunofixation and free light assay was performed as recently described [4]. In 9 out of 10 patients a free light chain ratio became abnormal at a median 90 days before immunofixation became positive. Conclusions The free light chain assay ratio is a useful marker for a faster detection of remission or progression in myeloma patients. However, these results do not support additional value of free light chain ratio to determine the depth of remission in immunofixation negative patients. More sensitive methods such as imunophenotyping analysis by FACS or molecular primer should be used to determine depth of complete remission since these methods have shown relevant clinical impact. Disclosures No relevant conflicts of interest to declare.

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Low Mutation Rate and No Significant Prognostic Implication of SF3B1, U2AF1 and SRSF2 Genes in Myelodysplastic Syndrome without Harboring Ring Sideroblast

Blood ◽

10.1182/blood.v120.21.4950.4950 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4950-4950

Author(s):

Hye-Ran Kim ◽

Trang Nguyen Thi Dai ◽

Min-Gu Kang ◽

Stephanie J. Won ◽

Hwan-Young Kim ◽

...

Keyword(s):

Myelodysplastic Syndrome ◽

Gene Mutation ◽

Mutation Rate ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Study Cohort ◽

Prognostic Implication ◽

Hematopoietic Stem ◽

Exon 2 ◽

Number Of Patients

Abstract Abstract 4950 Background: Myelodysplastic syndrome (MDS) is an hematopoietic stem cells (HSCs) disorder, leading to malignant cells that ultimately grow uncontrollably. Somatic mutations of spliceosomal gene such as SF3B1, U2AF1 and SRSF2 has been widely described in MDS and other hematological malignancies. Many reports state that hematopoietic malignancies mostly result from somatic mutations in HSCs in the bone marrow. Some functional studies have been performed and have shown that a high proportion of these cases are associated with somatic mutations in spliceosomal proteins. Also, such kinds of mutation are hallmarks of dominantly acting and growing specific cancer. Many efforts have been made to unravel the mutation considered disease background of MDS. Genotype–phenotype associations have been demonstrated for somatic spliceosomal gene mutation in MDS with ring sideroblasts but there is not much research regarding aberrant splicing pathways in MDS without harboring ring sideroblast. Therefore, this study investigated the prevalence and prognostic implication of the SF3B1, U2AF1 and SRSF2 splice gene mutation in these patients in Korea. Materials and Methods: The study cohort of 92 MDS patients was examined for somatic mutations in SF3B1, U2AF1 and SRSF2 splicing gene using direct sequencing method. The clinical and hematologic data were also recorded. The collected data was analyzed by SPSS for Windows version 18. 0. Pearson's chi-square tests, one way ANOVA analysis, and Student t-test were performed. Survival rates of multiple myeloma patients according to the mutation result of SF3B1, U2AF1 and SRSF2 splicing gene were analyzed using Kaplan-Meier log-rank test. Results: Our 92 MDS patients showed recurrent mutation and polymorphisms. Mutations in K666N, H662Q and K700E of SF3B1; S34T, S34P and Q157P of U2AF1; P95H, P95R and P95L of SRSF2 were found in 8 (8. 7%), 6 (6. 5%), and 11 (11. 9%) patients, respectively. The patients displayed 39198T>T/C polymorphism (88. 0%) in exon 18 of SF3B1, 8345T>T/G polymorphism (7. 6%) in exon 2 of U2AF1 and 5399C/T polymorphism (100%) in exon 1 of SRSF2. In the entire cohort, the number of patients with no polymorphism, one polymorphism, two polymorphisms and three polymorphisms was counted up to 0%, 12%, 80. 4% and 7. 6%, respectively. The T/C polymorphism at position 39198 of SF3B1 exon 18 and the T/G polymorphism at position 8345 of U2AF1 exon 2 were analyzed by allele-specific PCR using normal control. Results in 100 normal controls, polymorphism of SF3B1 exon 18 was taken into account of 82. 0%, the remaining is non polymorphism while U2AF1 exon 2 showed 10. 0% polymorphism and 90. 0% non polymorphism. The patient with either polymorphisms or mutations in both SF3B1, U2AF1 and SRSF2 had no effect on overall survival and disease-free survival. Conclusion: Our results show that mutation rate of SF3B1, U2AF1 and SRSF2 splice gene in Korean MDS patients without harboring ring sideroblast displayed relatively rare and infrequent molecular event. Moreover, alteration of SF3B1, U2AF1 and SRSF2 splice gene was not significantly implicated in the clinical outcomes and prognosis. Disclosures: No relevant conflicts of interest to declare.

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Clinical Significance of Serum Free Light Chain Test in the First Onset of Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.4999.4999 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4999-4999

Author(s):

Pan Hong ◽

Xiaojian Meng ◽

Jingsong He ◽

Wenjun Wu ◽

Yi Li ◽

...

Keyword(s):

Multiple Myeloma ◽

Survival Analysis ◽

Clinical Significance ◽

Light Chain ◽

Conflicts Of Interest ◽

Free Light Chain ◽

Serum Free ◽

Serum Free Light Chain ◽

Sensitivity Assessment ◽

First Onset

Abstract Abstract 4999 Objective: Clinical significance of serum free light chain (sFLC) test in the diagnosis, efficacy evaluation, prognosis judgment of first onset of multiple myeloma (MM). Method: Medical records of 39 cases of first-onset MM were gathered in our hospital from 1/2010 to 2/2012, including 25 male and 14 female patients with the median age being 63 years old. sFLC-kappa and sFLC-lambda were determined by immune turbidimetric assay, sFLC-kappa/lambda were calculated and the results were compared with that of immunofixation electrophoresis (IFE) as well as serum protein electrophoresis (SPE) for sensitivity assessment. All patients were treated with chemotherapy followed by sFLC detection and efficacy assessment. Patients with a sFLC results higher than median were marked as high-sFLC, the rest were marked as low-sFLC, and survival analysis were made comparing the two groups. Results: Of the 39 first-onset MM patients, 38 were detected as sFLC- kappa/lambda positive, that's a sensitivity of 97. 4%, higher than IFE (79. 5%), SPE (53. 8%), and uPE (38. 5%). As condition improves after treatment, sFLC gradually decline and sFLC- kappa/lambda level falls back to normal. Evaluation of efficacy by sFLC results has an accuracy of 77. 27%, significantly higher than that of corresponding M protein test£̈ P=0. 009£©. Follow-up survival analysis showed that the OS time and TTP time of low-sFLC group are significantly higher than those of high-sFLC group (P=0. 037, 0. 009 respectively). Conclusion: sFLC detection is a highly sensitive quantitative method, can help the diagnosis of MM, gives a quicker assessment of treatment efficacy, and can predict disease recurrence. When it's the first onset, sFLC level can also roughly forecast the prognosis. Disclosures: No relevant conflicts of interest to declare.

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