Survival Based on Metaphase Versus Interphase Cells for 154 Patients with Newly Diagnosed Multiple Myeloma.

Abstract Background: Multiple myeloma (MM) has been studied by conventional cytogenetics and fluorescence in situ hybridization using DNA probes (FISH) on interphase nuclei and metaphase cells, but the relative clinical significance of proliferating (metaphase) versus non-proliferating (interphase) cells at diagnosis is poorly understood. We investigated a consecutive series of 154 patients (pts) with newly diagnosed untreated MM, and compared results for metaphase and interphase cells with survival. Methods: Bone marrow within 30 days of diagnosis was analyzed by standard cytogenetics. Leftover cells were stored at −70°C. Interphase and metaphase FISH was done using probes for IGH, FGFR3, CCND1, c-MAF, D13S319, LAMP1, p53 and D17Z1 to detect t(4;14), t(11;14), t(14;16) and chromosome 13 or 17 anomalies, respectively. We analyzed <10 metaphases and 200 unselected interphase nuclei for each FISH probe set for each pt. Survival rates were estimated using Kaplan-Meier and differences in survival curves compared using log-rank test. Results: 61 (39.6%) pts had abnormal metaphases by cytogenetics (43 pts) and/or FISH (46 pts): 12 (19.7%) had t(11;14) by cytogenetics (3 pts) and/or FISH (11 pts), 11 (18.0%) had t(4;14) by FISH but not cytogenetics, and 3 (4.9%) had t(14;16) by FISH but not cytogenetics. Metaphases with 13q- were seen in 30 pts (49.2%). Interphase FISH results were abnormal for 133 (86.3%) pts: mean percent neoplastic nuclei was 26.9 ± 21.6 (3.9 to 99.0). Mean percent plasma cells was 54.7 ± 24.6 (3.0 to 99.0). Each of the 61 pts with abnormal metaphases was also abnormal in interphase nuclei. Median survival for pts with ≥50% (20 pts) abnormal nuclei was 202 days vs. 1351 days for pts with <50% (134 pts) (p<0.001). Median survival for pts with abnormal metaphases (61 pts) was 587 days vs. 1404 days for pts with normal metaphases (93 pts) (p=0.002). Median survival was 381 days for 30 pts with 13q- metaphases compared with 1404 days for 38 pts with normal metaphases and 13q- interphase nuclei (p<0.001). Metaphase and interphase analysis along with other potential risk factors (hemoglobin <10, serum calcium ≥11, serum creatinine ≥2, plasma cell labeling index and percent bone marrow plasma cells) were evaluated by multivariate analysis using a stepwise selection in a Cox proportional hazards model. Only the observation of 13q- in metaphases or ≥50% abnormal interphase nuclei were significant predictors of survival. Conclusions: Analysis of metaphases at diagnosis is important to detect proliferating disease which is a strong indicator of poor prognosis. Metaphase FISH is a necessary adjunct to conventional cytogenetics to detect cryptic t(4;14) and t(14;16), and accurately detect prognostic chromosome anomalies such as 13q-. Observing a 13q- in metaphase cells is a strong predictor of survival compared with observing 13q- in only interphase nuclei. At diagnosis, analysis of interphase nuclei is efficient, identifies an abnormal clone in 86% of pts and defines a subset of patients with ≥50% nuclei that have a poor prognosis. Since analysis of metaphases gives better prognostic information, interphase FISH is not a substitution in the cytogenetic work up of patients with MM.

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Increased Expression of Cyclin-D1 on Trephine Bone Marrow Biopsies Independently Predicts for Shorter Overall Survival In Patients with Multiple Myeloma Treated with Novel Agents

Blood ◽

10.1182/blood.v116.21.2965.2965 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2965-2965

Author(s):

Evangelos Terpos ◽

Maria Roussou ◽

Anna Tasidou ◽

Magdalini Migkou ◽

Maria Gavriatopoulou ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Overall Survival ◽

Cyclin D1 ◽

Plasma Cells ◽

Univariate Analysis ◽

Novel Agents ◽

Positive Staining ◽

Newly Diagnosed ◽

Immunohistochemical Expression

Abstract Abstract 2965 The cyclin-D1 proto-oncogene is an important cell regulator of G1 to S phase progression. The overexpression of cyclin-D1 has been linked to the development and progression of several malignancies. The aim of our study was to evaluate the impact of the immunohistochemical expression of cyclin-D1on the plasma cells of trephine biopsies on survival of newly-diagnosed patients with multiple myeloma (MM) who were treated with novel agents. We evaluated formalin-fixed, paraffin-embedded, bone marrow sections of 130 consecutive patients with newly-diagnosed MM (67M/63F; median age 68 years) before any kind of therapy administration. One hundred and fifteen patients had symptomatic disease that required therapy: 29 (25%) received bortezomib-based regimens and 31 (26%) received thalidomide-based regimens as first line therapy, while all patients received regimens containing bortezomib or an IMiD at some point during the course of their disease. Immunohistochemistry was performed in all trephine biopsies using monoclonal antibodies against cyclin-D1 (Cell Marque Corp., Rocklin, CA, USA), but also against CD56 (Cell Marque Corp., Rocklin, CA, USA), CD27 (Novocastra, Newcastle upon Tyne, UK), CD117 and MUM-1 (DAKO A/S, Glostrup, Denmark), as recommended by the manufacturers. A case was considered positive if there was unequivocal positive staining of at least 20% of the plasma cells for cyclin-D1, CD56 and MUM-1 and a positive staining of at least 10% of the plasma cells for CD117 and CD27. Among patients with symptomatic myeloma (N=115), positive staining for cyclin-D1 was found in 35 (30%) patients, for CD56 in 45 (39%), for CD117 in 94 (81%) and for CD27 in 72 (62%) patients. In patients with asymptomatic myeloma, positive staining for Cyclin-D1 was found only in 1 (7%) patient, for CD56 in 9 (64%), and for CD117 in 6 (43%) (p<0.01 for all comparisons compared to symptomatic patients). There were significant positive correlations between positivity for CD27 and CD56 (p<0.001), between positivity for cyclin-D1 and CD117 (p=0.045) and a negative correlation between positivity for CD117 and CD56 (p=0.001). We also observed significant correlations between CD56 positivity and ISS-1 or ISS-2 (p=0.01) and between CD117 positivity and ISS-3 disease (p=0.002). The median overall survival (OS) for patients with symptomatic MM was 57 months (range 22–120 months). In the univariate analysis, positivity for cyclin-D1 (41 vs. 62 months, p=0.03) and for CD117 (50 vs. 75 months p=0.018) were associated with inferior survival, while positivity for CD56 (47 vs. 62 months, p=0.286), MUM-1 (52.7 vs. 63.8 months, p=0.528) and CD27 (57 vs. 50 months, p=0.445) were not. Other factors associated with inferior OS, in the univariate analysis, included ISS-3 (median OS 37 months, vs. 57 months for ISS-2 and 73 months for ISS-1, p=0.005), Hb <10 g/dl (56 vs. 73 months, p=0.044), corrected serum calcium >11.5 g/dl (29 vs. 62 months, p=0.02), serum LDH above upper normal limit (31 vs. 61 months, p=0.05), serum creatinine >2 mg/dl (26 vs. 64 months, p=0.007), low platelet counts (<100,000/ml) (22 vs. 62 months, p=0.031) and age >65 years (45 months vs. not reached for younger patients, p=0.002). In the multivariate analysis, positivity for cyclin-D1 (HR: 2.6; p=0.001), ISS stage (HR: 1.8; p=0.001) and age >65 (HR 2.7, p=0.003) were independently associated with inferior survival. Immunohistochemistry for cyclin-D1 identified subgroups of patients in ISS-2 and in ISS-3 who had extremely poor outcome. Patients with cyclin-D1 positivity had a median survival of 22 months in ISS-2 (vs. 64 months for the rest of ISS-2 patients, p=0.01) and of 13 months in ISS-3 (vs. 47 months for the rest of ISS-3, p=0.012). Our findings underline that the immunohistochemical expression of cyclin-D1 in the bone marrow trephine biopsies has independent prognostic value in MM patients, even in the era of novel agents. This marker can easily be assessed in patients who undergo a trephine biopsy as part of their initial evaluation and offers significant prognostic information. Furthermore, novel agents targeting cyclin-D1 may be of therapeutic value in MM. Disclosures: No relevant conflicts of interest to declare.

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High Trip13 and Low P31 Comet Cause Drug Resistance and Poor Prognosis In Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.3820.3820 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3820-3820

Author(s):

Yi Tao ◽

Zhimin Gu ◽

Ye Yang ◽

Hongwei Xu ◽

Xiaojing Hu ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Cell Lines ◽

Poor Prognosis ◽

Caspase 3 ◽

Plasma Cells ◽

Myeloma Cell ◽

Newly Diagnosed ◽

Caspase 9 ◽

Over Expression

Abstract Background We have recently established that increased chromosomal instability (CIN) signature is linked to drug resistance and poor outcome in multiple myeloma (MM) and other cancers. Thyroid Hormone Receptor Interactor 13 (Trip13), one of the 56 drug-resistant genes, plays a key role in chromosomal recombination and structure development during meiosis and has been reported to be increased in some malignancies including lung cancer, prostate cancer and breast cancer. In this study, we investigated how important Trip13 is in myelomagenesis and progression. Materials and Methods Gene expression profiling (GEP) was analyzed on plasma cells from 22 healthy donors, 44 patients with monoclonal gammopathy of undetermined significance (MGUS), 351 patients with newly diagnosed multiple myeloma, and 9 human myeloma cell lines, as well as on 36 sequential samples at diagnosis, pre-1st, pre-2nd and post-2nd autologous stem cell transplantation (ASCT). Over-expression and knock-down experiments of Trip13 were performed on myeloma cell lines by lentivirus transfection. Cell viability was assessed by trypan exclusion assay. Western blots were used to detect the expression of Trip13, P31 comet, caspase-8, caspase-9, caspase-3 and PARP, and checkpoint related proteins MAD2 and CDC20 in Trip13 overexpressed or Trip13 shRNA-transfected myeloma cells. Results Sequential GEP samples showed that Trip13 expression increased in 8 of 9 patients after chemotherapy and ASCT compared to the samples at diagnosis strongly suggesting that increased Trip13 is associated with drug resistance. Trip13 was already significantly increased in MGUS patients, newly diagnosed MM patients and MM cell lines compared with normal plasma cells. Furthermore, Trip13 was significantly higher in high-risk MMs than in low-risk MMs and increased Trip13 was linked to an inferior event-free survival (EFS) (p<0.01) and overall survival (OS) (p<0.01) in 351 newly diagnosed MMs. In contrast, the Trip13-interacting gene P31 comet was down-regulated in high-risk MMs and high expression of P31 was associated with good outcome. Interestingly, patients with high Trip13 and low P31 comet have the worst outcome compared to patients with only one of these, suggesting the interaction of Trip 13 and p31 has a synergistic effect on MM progression. Transfection of Trip13 into ARP1 and OCI-My5 cells significantly increased cell proliferation, while knock-down Trip13 in OCI-My5, H929, RPMI8226 cells inhibited cell growth and induced MM cell apoptosis with increases of cleaved caspase-8, caspase-9, caspase-3 and PARP. Mechanistic studies showed that Trip13 over-expression decreased P31comet and MAD2 expression by western blotting, but increased CDC20. Conclusions The association of increased Trip13 and decreased p31 is a good biomarker for MM drug resistance and poor prognosis. Our results also show Trip13 and P31 comet could be potential targets to overcome drug resistance in MM. Disclosures: No relevant conflicts of interest to declare.

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The Frequency of Genetic Anomalies According to Clonal Plasma Cells' Phenotype in Patients with Newly Diagnosed (ND) Multiple Myeloma (MM)

Blood ◽

10.1182/blood.v126.23.5316.5316 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5316-5316

Author(s):

Andrei Garifullin ◽

Irina Martynkevich ◽

Sergei Voloshin ◽

Alexei Kuvshinov ◽

Ludmila Martynenko ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Risk Group ◽

Risk Groups ◽

Fish Analysis ◽

Newly Diagnosed ◽

Definition Of ◽

Standard Risk

Abstract Background. Genetic anomalies (GA) are primary link of pathogenesis in MM. GA lead to formation of clonal plasma cells, which has different phenotype. Aim. To estimate the incidence of GA and their correlation with clonal plasma cells' phenotype in patients with ND MM. Methods. We analysed 22 patients with ND MM (median age 57 years, range 38-80; male/female - 1:1.75). Cytogenetic analysis was performed on bone marrow samples using standard GTG-method. Metaphase FISH analysis was performed according to the manufacturer's protocol using DNA probes: LSI 13(RB1)13q14, IGH/CCND1, IGH/FGFR3, LSI TP53 (17q13.1). 8-color immunophenotypic by flow cytometry using antibody to CD45, CD38, CD138, CD56, CD19, CD20, CD27 and CD117 antigenes. Results. Translocation t(11;14) was detected in 3/14 (21.4%) patients, del(13q) - 2/14 (14.3%), t(11;14) - 3/14 (21.4%), hypodyploidy - 1/20 (5%), del(17р) - 0% patients. Clonal plasma cells' phenotype CD38+CD138+CD45- was detected in 100%. Expression CD56+ was revealed in 11/22 (50%) patients, CD19+ in 9/22 (40.9%), CD117+ in 5/22 (22.7%), CD20+ in 1/22 (4.5%), CD27+ in 1/22 (4.5%). The frequency of GA didn't depend on clonal plasma cells' phenotype and was 27.3%(3/11) in CD56+ phenotype, 23.8%(5/21) - CD20-, 23.8%(5/21) - CD27-, 23.5%(4/17) - CD117-, 23%(3/13) - CD19-, 22.2%(2/9) - CD19+, 20%(1/5) - CD117+, 18.2%(2/11) - CD56-, 0%(0/1) - CD20+, 0%(0/1) - in CD27+ phenotype. Patients of standard risk group according to mSMART 2.0 with GA had CD19-negative plasma cells' phenotype vs. CD19-positive phenotype in patients of intermediate and high-risk groups (p<0.05). 3-years overall survival in standard risk group with CD19- phenotype was 92,3%, CD19+ - 77,7% (p>0.05). Conclusion . Identification of GA, which has adverse forecast, correlates with CD19+ plasma cells phenotype. The combined definition of plasma cells phenotype and GA can improve the system of risk stratification in MM. Disclosures No relevant conflicts of interest to declare.

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Targeting EZH2 in Multiple Myeloma Could be Promising for a Subgroup of MM Patients in Combination with IMiDs

Blood ◽

10.1182/blood.v128.22.311.311 ◽

2016 ◽

Vol 128 (22) ◽

pp. 311-311 ◽

Cited By ~ 4

Author(s):

Laurie Herviou ◽

Alboukadel Kassambara ◽

Stephanie Boireau ◽

Nicolas Robert ◽

Guilhem Requirand ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Newly Diagnosed ◽

Advisory Committees ◽

Myeloma Cells ◽

Ezh2 Expression ◽

Bone Marrow Plasma

Abstract Multiple Myeloma is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow.Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes. We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio > 2 and FDR (false discovery rate) < 5%) were identified, including the EZH2 histone methyltransferase. EZH2, the enzymatic subunit of Polycomb Repressive Complex 2, is a histone methyltransferases able to repress gene expression by catalyzing H3K27me3 histone mark. EZH2 overexpression has been associated with numerous hematological malignancies, including MM. We thus studied EZH2 role in MM physiopathology and drug resistance. EZH2 expression was analyzed in normal bone marrow plasma cells (BMPCs; N=5), primary myeloma cells from newly diagnosed patients (MMCs; N=206) and human myeloma cell lines (HMCLs; N=40) using Affymetrix microarrays. EZH2 gene is significantly overexpressed in MMCs of patients (median 574, range 105 - 4562) compared to normal BMPCs (median = 432; range: 314 - 563) (P < 0.01). The expression is even higher in HMCLs (median 4481, range 581 - 8455) compared to primary MMCs or BMPCs (P < 0.001). High EZH2 expression is associated with a poor prognosis in 3 independent cohorts of newly diagnosed patients (Heidelberg-Montpellier cohort - N=206, UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N =158). Furthermore, GSEA analysis of patients with high EZH2 expression highlighted a significant enrichment of genes involved in cell cycle, downregulated in mature plasma cells vs plasmablasts, and EZH2 targets. Specific EZH2 inhibition by EPZ-6438 EZH2 inhibitor induced a significant decrease of global H3K27me3 in all the HMCLs tested (P < 0.01) and inhibited MM cell growth in 5 out of the 6 HMCLs tested. The inhibitory effect of EZH2 inhibitor on MM cell growth appeared at day 6 suggesting that it is mediated by epigenetic reprogramming. To confirm that EZH2 is also required for the survival of primary MMCs from patients, primary MM cells (n = 17 patients) co-cultured with their bone marrow microenvironment and recombinant IL-6 were treated with EPZ-6438. As identified in HMCLs, EZH2 inhibition significantly reduced the median number of viable myeloma cells by 35% (P = 0.004) from a subset of patients (n=9) while the other group (n=8) was resistant. Of interest, EPZ-6438 induced a significant global H3K27me3 decrease in both groups of patient. RNA sequencing of 6 HMCLs treated with EPZ-6438 combined with H3K27me3 ChIP analyses allowed us to create an EZ GEP-based score able to predict HMCLs and primary MM cells sensitivity to EZH2 inhibitors. We also observed a synergy between EPZ-6438 and Lenalidomide, a conventional drug used for MM treatment. More interestingly, pretreatment of myeloma cells with EPZ-6438 significantly re-sensitize drug-resistant MM cells to Lenalidomide. Investigating the effect of EPZ-6438/Lenalidomide combination in MMC, we identified that IKZF1, IRF4 and MYC protein levels were significantly more inhibited by the combination treatment (65.5%, 63.9% and 14.8% respectively) compared with Lenalidomide (51.5%, 43% and 2.2%) or EPZ-6438 (45.2%, 38.7% and 6.2%) alone. Clinical trials are ongoing with EZH2 inhibitors in lymphoma and could be promising for a subgroup of MM patients in combination with IMiDs. Furthermore, the EZ score enables identification of MM patients with an adverse prognosis and who could benefit from treatment with EZH2 inhibitors. Disclosures Goldschmidt: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

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CD43 Expression Is an Adverse Prognostic Factor in Newly Diagnosed Multiple Myeloma

Blood ◽

10.1182/blood-2021-146754 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4753-4753

Author(s):

Xueqin Ning ◽

Yongqiang Wei ◽

Xiaolei Wei ◽

Ru Feng ◽

Bingyuan Chen ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Prognostic Factor ◽

Plasma Cells ◽

Newly Diagnosed ◽

Negative Group ◽

Hematopoietic Stem ◽

Female Ratio ◽

Positive Group ◽

Adverse Prognostic Factor

Abstract CD43 expression is an adverse prognostic factor in newly diagnosed multiple myeloma Introduction CD43 is an abundant, heavily-glycosylated, cell surface protein expressed on bone marrow hematopoietic stem cells and most white blood cells. Previous studies have found that CD43 also expressed in some types of lymphoma cells and associated with adverse outcomes. However, the prognosis value of CD43 expression in multiple myeloma (MM) remain unknown. Patients and Methods A total of 109 MM patients, newly diagnosed in Nanfang Hospital of Southern Medical University from January 2017 to December 2019, were included in the study. CD43 was detected by flow cytometre as follows: 2 ml of heparin anticoagulated bone marrow was collected from the patients at the time of diagnosis，1×10 6 cells were detected, and a gate was set for identifying abnormal plasma cells characterized by CD138 expression and CD38. CD43 positive was defined as more than 20% of the plasma cells expressed CD43. Results A total of 109 patients with newly diagnosed MM were enrolled in the study, including 77 patients (70.6%) for CD43 positive and 32 patients (29.4%) for CD43 negative . The median age was 58 years, and the male-female ratio was 1.7:1. Patients in the CD43 positive group were more likely to have international staging system (ISS) stage III (67.5% VS 46.9%, P= 0.044), hemoglobin < 85g/L (64.9% VS 37.5%, P= 0.008), 13q deletion (31.4% VS 10.4%), and higher percentage of bone marrow monoclonal plasma cells detected by flow cytometry (5.5% VS 1.4%, P=0.003). Most patients enrolled in the study received bortezomib-based treatment. The very good partial response (VGPR) or better rate after 4 induction cycles was significantly lower in the CD43 positive group than CD43 negative group (35.1% VS 56.3%, P=0.041), and the overall response rate (ORR) in the CD43 positive group was lower than that in CD43 negative group (75.3% VS 84.4%, P=0.299), but no significantly difference. The median follow-up time was 22 months. Patients with CD43 positive had significantly lower PFS (median PFS 24 months VS not reached, P =0.012), and OS (median OS not reached, P = 0.023) than those with CD43 negative. Multivariate analysis indicated that CD43 positive expression was an independent poor risk factor for PFS (HR 2.517 95%CI 1.178-5.376, P = 0.017) and OS (HR 3.664 95%CI 1.100-12.075, P = 0.034). Conclusion Our study showed that CD43 expression was an adverse prognosis for multiple myeloma. Disclosures No relevant conflicts of interest to declare.

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The expression of p53 protein in patients with multiple myeloma

Srpski arhiv za celokupno lekarstvo ◽

10.2298/sarh0702043m ◽

2007 ◽

Vol 135 (1-2) ◽

pp. 43-47 ◽

Cited By ~ 2

Author(s):

Olivera Markovic ◽

Dragomir Marisavljevic ◽

Vesna Cemerikic ◽

Maja Perunicic ◽

Milica Colovic

Keyword(s):

Multiple Myeloma ◽

Clinical Significance ◽

Median Survival ◽

Plasma Cells ◽

Histological Grade ◽

Stage I ◽

Newly Diagnosed ◽

P53 Expression ◽

P53 Immunoreactivity ◽

Histological Stage

Introduction: Although mutations of p53 are one of the most often acquired genetic changes in malignant tumors, these mutations are rare events in patients with newly diagnosed multiple myeloma (MM). Moreover, there are a few literature data about clinical significance of p53 overexpression in multiple myeloma. Objective The aim of our study was to evaluate the clinical significance of p53 immunoexpression in multiple myeloma. Method A total of 58 patients with newly diagnosed MM (26 females and 32 males, mean age 62 years) were enrolled in the study. The diagnosis of MM was made according to criteria of Chronic Leukemia-Myeloma Task Force. Clinical staging was done according to Durie and Salmon classification (4 patients had disease stage I, 15 patients stage II and 39 patients stage III). The histological grade and histological stage were determined according to predominant plasma cell morphology and volume of myeloma infiltration, respectively. Standard immunohistochemical analysis with p53 antibody in B5-fixed and paraffin- embedded bone marrow specimens was used to evaluate the expression of p53 in myeloma cells. The specimens were considered positive when ?5% of plasma cells exhibited clear nuclear positivity. Results Out of 58 patients, p53 expression was detected in 9 (15.52%). No significant correlation was found between p53 expression and clinical stage (I+II vs. III), ?2-microglobulin level (?6 mg/L vs. >6mg/L), histological grade (I vs. II+III), histological stage (<20% vs. 21-50% vs. >50%) and the extent of osteolytic lesions (?3 vs. >3 lesions). Median survival of patients with p53 immunoreactivity in =>5% of plasma cells was 10 months, whilst median survival of patients with p53 immunoreactivity in <5% of plasma cells was 36 months. However, such difference was not significant (p=0.2). Conclusion The frequency of p53 immunoexpression in our group of newly diagnosed MM was relatively low. Although p53 immunoexpression was not associated with clinical and histological features of more aggressive disease, or with shorter survival, further investigations of larger group of patients will lead to final conclusions.

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The usefulness of interphase fluorescence in situ hybridization at diagnosis of myeloma in addition to metaphase cytogenetics

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.e19558 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. e19558-e19558

Author(s):

S. Park ◽

C. Kim ◽

H. Kim ◽

D. Hong ◽

S. Lee ◽

...

Keyword(s):

Multiple Myeloma ◽

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Plasma Cells ◽

Chromosomal Abnormalities ◽

Chromosome Abnormalities ◽

Interphase Fish ◽

Conventional Cytogenetics ◽

Igh Rearrangement

e19558 Background: Multiple myeloma is characterized by the accumulation of malignant plasma cells within the bone marrow and regarded as incurable, but remissions may be induced with steroids, chemotherapy, thalidomide and stem cell transplants. The clinical heterogeneity of myeloma is dictated by the cytogenetic aberrations present in the clonal plasma cells. Fluorescence in situ hybridization (FISH) overcomes the limitations of standard cytogenetics and allows for the detection of numerical and structural chromosomal abnormalities in both metaphase spreads and interphase nuclei. Methods: We evaluated the chromosome abnormalities in 34 MM patients using conventional cytogenetics and interphase FISH with 6 probes such as IGH/CCND1, IGH/FGFR3, IGH/MAF, DS13S319/LAMP1, IGH/BAP, and p53/CEP17. Results: Cytogenetic abnormalities were found in 24 (70.6%) of the 28 MM patients. 10 (35.7%) patients had abnormal metaphases by conventional cytogenetics. Interphase FISH results were abnormal in 21 (61.8%) patients and 11 (52.3%) patients had abnormal interphase FISH but normal metaphases. The evidence of the loss of D13S319 with or without loss of LAMP1 was found in 6 (21.4%) patients, and loss of p53±CEP17 for 2 patients, IGH-BAP for 9 (26.5%) patients, IGH/FGFR3 for 2 patients, and IGH/CCND1 for 7 (20.6%) patients, respectively. However, there were none positive for IGH/MAF. Chromosome 13 abnormalities and IGH rearrangement is correlated with poor clinical outcome. Conclusions: Interphase FISH can provide useful information to evaluate the presence of prognostic chromosome abnormalities in addition to metaphase cytogenetics. And it should be used in the routine evaluation of multiple myeloma. No significant financial relationships to disclose.

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Comparison of Newly Diagnosed and Relapsed Refractory Multiple Myeloma Using Transcriptional Profiling of Myeloma Cells.

Blood ◽

10.1182/blood.v106.11.1555.1555 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1555-1555

Author(s):

Shaji Kumar ◽

Philip R. Greipp ◽

Jessica L. Haug ◽

Michael Kline ◽

Wee Joo Chng ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Differential Expression ◽

Median Survival ◽

Plasma Cell ◽

Plasma Cells ◽

Expression Profiles ◽

Significant Proportion ◽

Newly Diagnosed ◽

Myeloma Cells

Abstract Background: Multiple myeloma (MM) is a plasma cell malignancy that is incurable with current approaches. The median survival for patients with MM is around four years and a significant proportion of patients experience a course characterized by multiple relapses treated with different therapies. The median survival for patients relapsing after the initial therapy is nearly 18 months and successive treatment strategies result in decreasing response durations, likely reflecting acquired drug resistance. In order to better understand the biological changes associated with advanced, relapsed, refractory MM, we compared gene expression profiles (GEP) of malignant plasma cells isolated from patients with relapsed refractory MM and compared them to plasma cells from patients with newly diagnosed MM. Methods: In order to obtain two relatively homogenous group of patients, we compared samples from 44 patient with newly diagnosed MM enrolled in the ECOG E1A00 clinical trial (comparing thalidomide and dexamethasone to dexamethasone alone) to 44 patients with relapsed refractory MM enrolled in a phase II trial of Velcade (SUMMIT), where most patients had four or more previous relapses. Plasma cells from bone marrow aspirates were separated by magnetic bead selection of CD138 positive cells and studied using Affymetrix HG-U133A chips using standard methodology. The arrays were analyzed using Genespring 7.2 software following GCRMA normalization and genes with differential expression between the two datasets were examined. Differentially expressed genes were further analyzed using Ingenuity Pathways Analysis program. Results: A total of 864 genes were identified which were at least two fold and significantly different between the newly diagnosed and relapsed patients. Using Ingenuity software, 437 of these genes were mapped to different biological networks. Examination of the canonical pathways demonstrated several important cellular pathways differentially regulated between the two groups. Several important mediators of the cytokines, receptors and respective signaling pathways appear to be down regulated in the relapsed group and included IGF-1, HGF, SDF-1 alpha, gp130 and importantly the MEK/ERK pathway. Additionally expression of adhesion molecules such as VCAM1 and PECAM was decreased in the relapsed group compared to newly diagnosed pts. There appear to increased tissue hypoxia in the relapsed marrow as indicated by up regulation of HIF-1 alpha as well increased levels of Placental growth factor. Myeloma cells from relapsed disease were characterized by decreased expression of mcl1, FLIP1, and bcl-xL and increased caspase 8 relative to newly diagnosed group. Also seen was decreased expression of the glucocorticoid and interferon receptors in the relapsed setting. Conclusion: Comparison of the GEP between MM cells from newly diagnosed and relapsed pts demonstrates important differences that have potential biological relevance. The plasma cell in the relapsed setting appears to be more independent of the tumor microenvironment. Additionally, differential expression of some of the genes provides clues to mechanisms of drug resistance commonly observed in the relapsed pts. We are in the process of validating some of the key findings from these analyses.

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MAGE-A3 or NY-ESO1 Expression and Spontaneous Antibody Responses to NY-ESO1 in Newly Diagnosed Multiple Myeloma Patients Are Associated with Worse Overall Survival

Blood ◽

10.1182/blood.v112.11.5110.5110 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5110-5110

Author(s):

Adam D Cohen ◽

Ping Lu ◽

Sacha Gnjatic ◽

James Hoffman ◽

Erika Ritter ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Overall Survival ◽

Soft Tissue ◽

Induction Therapy ◽

Plasma Cells ◽

Prognostic Significance ◽

Antibody Responses ◽

Newly Diagnosed ◽

Bone Marrow Plasma

Abstract The cancer-testis antigens (CTA) are highly immunogenic antigens expressed in various tumors but not in normal tissues (except during gametogenesis), making them an attractive target for cancer immunotherapy. Expression of CTAs such as MAGE-A3, MAGE-C1 (CT7), MAGE-C2 (CT10), NY-ESO1 and the SSX antigens has been previously reported in multiple myeloma (MM). To date, however, these reports have included a heterogeneous group of newly diagnosed and relapsed/refractory patients, all in different stages of treatment. Therefore, the extent and prognostic significance of CTA expression, and of de novo immune responses against CTA in newly-diagnosed MM patients are not known. We now report on both CTA expression and antibody responses in MM patients at diagnosis and on their prognostic significance. From 8/00-11/04, we treated 67 newly-diagnosed, symptomatic patients with a thalidomide, doxorubicin, and dexamethasone-based induction regimen. (Brit J Haematol2006;132:155). Median age was 58; 54% were ISS stage I, 28% ISS II, and 18% ISS III. Nine of 63 tested (14%) had deletion 13q by FISH, while 24% had soft tissue involvement by MM. Responses to induction therapy included 10 (15%) CR, 16 (24%) VGPR, 26 (39%) PR, 6 (8%) stable or progressive disease, and 9 (13%) inevaluable. Post-induction 54 underwent autoSCT and 9 also underwent alloSCT.. Median overall survival (OS) has not been reached with 61% alive at median follow up of 65 months. Cryopreserved pre-treatment bone marrow plasma cells were used to assess CTA expression by RT-PCR. Pre- and post-treatment sera were used to assess antibody (Ab) responses against CTA proteins by ELISA. Fifty-two patients had sufficient RNA for PCR, and 46 had baseline serum for ELISA. OS of these groups did not differ significantly from the entire cohort. At least 1 CTA was expressed in 77% of cases, including MAGE-A3 (52%), SSX1 (40%), CT7 (29%), CT10 (25%), NY-ESO1 (21%), and SSX5 (17%). Three or more CTA were expressed in 29% of cases. Individually MAGE-A3 or NY-ESO1 expression at diagnosis conferred a poorer prognosis (MAGE-A3: median OS 66 mos. vs. not reached, p=0.02 by log-rank; NY-ESO1: median OS 65 mos. vs. not reached, p=0.09). These poorer outcomes were independent of ISS stage, presence of del 13q, or response to induction therapy. No other CTA was associated with an OS difference, nor was the total number of CTA expressed prognostically significant. Baseline Ab responses, all at titers > 1:1600, were noted to NY-ESO1 in 6/46 (13%) patients, 5 of whom also had Ab to the NY-ESO1 homologue LAGE-1. Ab responses were also noted to CT7 (n=2), CT10 (n=1) and SSX4 (n=1). No Ab responses were noted to MAGE-A3. The effect of induction therapy on antibody titers was inconsistent, with increases, decreases, and no changes seen. Interestingly, 2 of the 6 NY-ESO1 Ab+ patients had no NY-ESO1 expression in bone marrow plasma cells. Both, however, had extensive soft tissue (ST) plasmacytomas, suggesting another source of NY-ESO1 antigen. Presence of NY-ESO1 Ab correlated significantly with baseline ST involvement, with 67% of Ab+ patients having ST disease compared with 20% of Ab− patients (p=0.05). NY-ESO1 Ab+ patients also had significantly poorer OS (med 21 mos. vs. not reached, p=0.009), independent of other prognostic factors. In sum, CTA expression is frequent in newly diagnosed MM patients, and expression of MAGE-A3 or NY-ESO1 is associated with worse long-term survival. Spontaneous antibody responses against NY-ESO1 are seen in untreated patients, and are associated with ST involvement and poorer survival. Further exploration of biologic differences between CTA+ and CTA-MM, as well as immunotherapeutic strategies which target these antigens, are warranted.

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