p15基因在多发性骨髓瘤中的甲基化异常及其意义

目的：探讨p15基因甲基化与多发性骨髓瘤的发生、发展及预后的关系。方法：采用甲基化特异性聚合酶链反应（MSP）研究33例多发性骨髓瘤（MM）及20例对照组的p15基因甲基化情况。结果：33例MM患者中有24例p15基因发生甲基化，20例对照组中无p15基因甲基化存在，差异有统计学意义（P〈0.01）。MM患者组中8例Ⅰ期、Ⅰ例Ⅱ期骨髓象为分化成熟的浆细胞型MM，p15基因未发生甲基化；在Ⅰ、Ⅱ、Ⅲ期中为浆细胞型或混合型中的幼稚浆细胞病例易发生甲基化，两者差异有统计学意义（PdO．01）。在Ⅰ、Ⅱ、Ⅲ期中p15基因甲基化率分别为27．27%（3／11例）、92．31％（12／13例）、100%（9／9例），Ⅱ、Ⅲ期甲基化率明显高于Ⅰ期（均P〈0．01）。结论：p15基因甲基化可能是MM发病的原因之一，与MM的发展及预后有一定的关系。     

Tumor suppressor p16 methylation in multiple myeloma: biological and clinical implications

The biological and clinical implications of p16 gene methylation in multiple myeloma (MM) are still unclear despite previous studies. In this comprehensive study, using methylation-specific PCR (MS-PCR), we show that p16 methylation is relatively common and occurs in monoclonal gammopathy of undetermined significance (MGUS; n=17), smoldering multiple myeloma (SMM; n=40), and MM (n=522) at a prevalence of 24%, 28%, and 34%, respectively. However, p16 methylation does not appear to affect gene expression level. In a large cohort of patients with long-term follow-up information (n=439), there was no difference in overall survival between patients with or without p16 methylation. We also found no association between p16 methylation and the main cytogenetic categories, although it was more common among patients with 17p13.1 deletions (p53 locus), a genetic progression event in MM. In addition, p16 methylation has no apparent effect on the cycle because there was also no difference in the plasma cell labeling index (a direct measurement of proliferation) between patients with and without p16 methylation. Our results question a major role for p16 methylation in the oncogenesis of the PC neoplasm, and we now believe p16 methylation may be a marker for overall epigenetic changes associated with disease progression, with no obvious direct biological or clinical consequences.     

ABCG2 expression, function, and promoter methylation in human multiple myeloma

We investigated the role of the breast cancer resistance protein (BCRP/ABCG2) in drug resistance in multiple myeloma (MM). Human MM cell lines, and MM patient plasma cells isolated from bone marrow, were evaluated for ABCG2 mRNA expression by quantitative polymerase chain reaction (PCR) and ABCG2 protein, by Western blot analysis, immunofluorescence microscopy, and flow cytometry. ABCG2 function was determined by measuring topotecan and doxorubicin efflux using flow cytometry, in the presence and absence of the specific ABCG2 inhibitor, tryprostatin A. The methylation of the ABCG2 promoter was determined using bisulfite sequencing. We found that ABCG2 expression in myeloma cell lines increased after exposure to topotecan and doxorubicin, and was greater in logphase cells when compared with quiescent cells. Myeloma patients treated with topotecan had an increase in ABCG2 mRNA and protein expression after treatment with topotecan, and at relapse. Expression of ABCG2 is regulated, at least in part, by promoter methylation both in cell lines and in patient plasma cells. Demethylation of the promoter increased ABCG2 mRNA and protein expression. These findings suggest that ABCG2 is expressed and functional in human myeloma cells, regulated by promoter methylation, affected by cell density, up-regulated in response to chemotherapy, and may contribute to intrinsic drug resistance.     

Aberrant global methylation patterns affect the molecular pathogenesis and prognosis of multiple myeloma

We used genome-wide methylation microarrays to analyze differences in CpG methylation patterns in cells relevant to the pathogenesis of myeloma plasma cells (B cells, normal plasma cells, monoclonal gammopathy of undetermined significance [MGUS], presentation myeloma, and plasma cell leukemia). We show that methylation patterns in these cell types are capable of distinguishing nonmalignant from malignant cells and the main reason for this difference is hypomethylation of the genome at the transition from MGUS to presentation myeloma. In addition, gene-specific hypermethylation was evident at the myeloma stage. Differential methylation was also evident at the transition from myeloma to plasma cell leukemia with remethylation of the genome, particularly of genes involved in cell-cell signaling and cell adhesion, which may contribute to independence from the bone marrow microenvironment. There was a high degree of methylation variability within presentation myeloma samples, which was associated with cytogenetic differences between samples. More specifically, we found methylation subgroups were defined by translocations and hyperdiploidy, with t(4;14) myeloma having the greatest impact on DNA methylation. Two groups of hyperdiploid samples were identified, on the basis of unsupervised clustering, which had an impact on overall survival. Overall, DNA methylation changes significantly during disease progression and between cytogenetic subgroups.     

多发性骨髓瘤实验数据分析

目的探讨多发性骨髓瘤（multiple myeloma,MM）细胞的主要形态以及其他实验室相关指标特征,了解MM发病的显著特点,从而降低误诊率。方法对30例MM患者的实验室检查资料进行回顾性分析。结果30例MM患者中,22例（73．3％）血红蛋白平均为88g／L,出现有不同程度的贫血特征;24例（80．O％）血沉增加,平均为117．6mm／h;蛋白尿18例（60．0％）,平均为（＋＋）;16例（53．3％）肾功能发生病理性改变,尿素氮平均为19．26mmol／L。30例均做尿本周氏,阳性13例（43．3％）;26例（86．7％）检出M蛋白;28例（93．3％）骨髓片中发现原、幼浆细胞及浆细胞形态异常。30例免疫分型中,IgG型16例,占53．3％;IgA型8例,占26．7％;IgM型1例,占3．3％;游离轻链型5例,占16．7％。其中ire和IgA型以K型为主。结论由于MM临床表现较复杂,误诊率较高,因此对MM患者诊断时,应进行实验室相关指标检查和MM细胞形态学诊断。     

Concurrent p16 methylation pattern as an adverse prognostic factor in multiple myeloma: a methylation-specific polymerase chain reaction study using two different primer sets

Disruption of cell cycle control genes, including p16, is known to contribute to the cancerogenesis of multiple myeloma (MM). We investigated the methylation status of p16 and its association with common cytogenetic changes, clinicolaboratory findings, and survival in MM. Methylation-specific polymerase chain reaction was performed in 99 newly diagnosed MM patients using two different sets of primers (p16M1 and p16M2). Four patterns of p16 promoter methylation were observed: (1) concurrent methylation of p16M1 and p16M2 (P1P2), 27.3%; (2) methylation of p16M1 alone (P1N2), 7.1%; (3) methylation of p16M2 alone (N1P2), 26.3%; and (4) no methylation (N1N2), 39.4%. Patients with p16P1P1 showed shorter survivals than those with the other methylation patterns (P1N2, N1P2, or N1N2; median survival, 12 vs. 43?months; P?<?0.001), regardless of the treatment protocol. In a multivariate analysis, p16P1P2 was an independent prognostic factor of adverse outcome in MM. According to International Staging System (ISS), the study population could be divided into 21.2% (20/94) for stage I, 22.3% (21/94) for stage II, and 56.4% (53/94) for stage III (P?=?0.003). ISS can divide patients into prognostic groups. Of note, in patients older than 60?years, ISS was not reflective of disease stage (P?=?0.114). If p16P1P2 sets up as stage 4 of ISS, modified ISS could be a more reliable staging system irrespective of age in Korean MM patients (P?=?0.003 and P?=?0.004 in patients younger than 60?years and in patients older than 60?years, respectively). Our study suggests the potential use of p16 methylation status in predicting the outcome of MM patients and the applicability of demethylating agents in MM.     

Analysis of methylation in the c-MYC gene in five human myeloma cell lines

Genomic alterations of the human c-MYC gene were analysed in five human myeloma cell lines established in Kawasaki Medical School and compared with those of normal lymphocytes, Raji cells from Burkitt's lymphoma, and an Epstein-Barr virus positive lymphoblastoid cell line (LCL). Although no structural chromosome aberrations at 8q24, the c-MYC locus, were distinct, the mRNA level of c-MYC in these myeloma cell lines was 30-50-fold that in normal peripheral blood lymphocytes. Regarding the methylation of c-MYC, DNAs of the myeloma cell lines were digested with MspI plus EcoRI or HpaII plus EcoRI, and hybridized with three genomic 32P-labelled probes; the first, second and third exons of the human c-MYC gene, respectively. The extent of methylation in cytosine at a single CCGG site in the third exon substantially decreased in these myeloma cell lines as compared with that in normal tonsillar B, LCL and Raji cells. No significant differences in hypomethylation between these myeloma, normal B, LCL and Raji cells was detected in the first and second exon of c-MYC. These results suggest that the hypomethylation in the third exon of c-MYC might be related to the enhanced expression of c-MYC in these human myeloma cell lines.     

Bendamustine in multiple myeloma

The advent of high‐dose melphalan with autologous stem‐cell transplantation (ASCT), the availability of novel agents such as thalidomide, lenalidomide (immunomodulatory drugs or IMiDs) and bortezomib (proteasome inhibitor) and improvements in supportive care have allowed to increase overall survival in multiple myeloma (MM) patients; nevertheless, MM remains an incurable pathology. For this reason, newer agents are required for continued disease control. Bendamustine is an old drug rediscovered in the last decade. In fact, its unique mechanism of action with structural similarities to both alkylating agents and antimetabolities, but which is not cross‐resistant to alkylating agents, has reawakened interest in the use of this drug in the treatment of MM. Studies have proven the safety and efficacy of bendamustine administered alone or in combination with new drugs in both upfront and relapse/refractory settings of MM patients, including those with renal impairment. Moreover, bendamustine has been successfully used as conditioning for autologous stem‐cell transplantation. Finally, the use of bendamustine does not compromise peripheral blood stem‐cell collection. This drug is generally well tolerated, with the majority of adverse events being due to myelosuppression. Non‐haematological adverse events are infrequent and usually mild.     

Hyperphosphatemia in multiple myeloma

Summary We report three cases of IgG kappa multiple myeloma with pseudohyperphosphatemia. The patients' serum calcium levels were normal, and the hyperphosphatemia was not related to impaired renal function. No hypoparathyroidism was found, and no exogenous phosphate preparation had been given. Since the hyperphosphatemia was of no obvious clinical or physiological significance, as evidenced by normal serum levels of 1,25 dihydroxy vitamin D₃, it was diagnosed as spurious and was connected to interference of the paraprotein with the chromogenic assay. In two of the patients major fluctuations in serum phosphate levels were seen, induced by the changes in globulin and paraprotein levels that occurred during therapy and relapse.     

Bortezomib in multiple myeloma

Multiple myeloma (MM) remains an incurable disease, and novel agents are therefore needed to improve outcome. Bortezomib is the first proteasome inhibitor to be approved by the US Food and Drug Administration and the European Agency for the Evaluation of Medicinal Products for the treatment of refractory/relapsed MM. Bortezomib has demonstrated significant anti-myeloma activity as a single agent in refractory/relapsed MM. When used in combination with other agents, responses have suggested the possibility of chemosensitization and synergy. All these facts have been the rationale for the use of bortezomib-based regimens as upfront treatment in young and elderly newly diagnosed MM patients. Furthermore, bortezomib does not appear to have an adverse effect on subsequent stem-cell collection. Bortezomib is well tolerated; most side-effects are only mild to moderate and manageable. Practical management of these side-effects is given so that they can be recognized and minimized by dose modification or concomitant therapy.     

Lenalidomide in multiple myeloma

For many years the treatment of multiple myeloma was limited to such regimens as melphalan-prednisone, high-dose dexamethasone, and vincristine-doxorubicin-dexamethasone (VAD). These combinations provided response rates of 45-55%, with complete remission rates of up to 10%. With the advent of thalidomide- and bortezomib-based combinations, response rates to induction therapy have risen to 85-95% in previously untreated patients and are associated with complete remission rates up to 25%. However, these agents are associated with such side-effects as somnolence, constipation and neuropathy. Lenalidomide, a thalidomide analog, was developed with the hope of improving both the efficacy and toxicity profile of thalidomide, and has subsequently shown significant clinical activity in patients with multiple myeloma. We describe the role of lenalidomide in patients with symptomatic multiple myeloma that is newly diagnosed, relapsed and/or refractory to other therapies, or concurrent with primary amyloidosis.     

Chemokines in multiple myeloma

OBJECTIVE: In this article we focus on the role that chemokines and chemokine receptors play in the pathogenesis of multiple myeloma and the associated bone destructive process, and consider their utility as novel therapeutic targets for treating this devastating disease. METHODS: Current research on the role that chemokine and chemokine receptors play in the pathogenesis of myeloma is reviewed. RESULTS: The chemokines, MIP-1alpha, MCP-1, IL-8, and SDF-1, and their receptors play important roles in homing of MM cells, tumor growth, and bone destruction in myeloma. They are attractive therapeutic targets for treating myeloma patients. CONCLUSION: Addition of chemokine antagonists to current treatment regimens for myeloma should result in better therapeutic responses because of the loss of both the protective effect of the marrow microenvironment on the MM cells and the induction of osteoclast activity.     

Hyperammonemia in multiple myeloma

Two patients with multiple myeloma who appeared to be producing ammonia are reported. Both patients showed hyperammonemia and amino acid disturbances, such as a low Fischer ratio. One patient had Bence Jones protein (lambda) type myeloma and became comatose, but the hyperammonemia and disturbance of consciousness were improved by chemotherapy for the myeloma. The other patient had IgA kappa type myeloma and somnolence and died of malignant pleurisy despite intensive chemotherapy. Autopsy showed widespread multiple myeloma and an almost normal liver. Ammonia levels in the supernatant of cultured myeloma cells from the patient's pleural effusion increased almost linearly from the time of cell seeding. These observations showed that ammonia was produced at a high level by these human myeloma cells. We also found that one of the common myeloma cell lines, RPMI 8226, could produce ammonia as well.