High applicability of ASO-RQPCR for detection of minimal residual disease in multiple myeloma by entirely patient-specific primers/probes.

Allele-specific oligonucleotide real-time quantitative PCR (ASO-RQPCR) is a standardized technique for detection and monitoring of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) but not multiple myeloma (MM) due to a low applicability inherent with presence of somatic hypermutation. Herein, by a staged PCR approach and sequencing, clonality and tumor-specific complementarity-determining region 3 (CDR3) sequence were identified in 13/13 MM by sequential PCR of IgH VDJ (n = 10), IgH DJ (n = 2), or IgK VJ (n = 1). Using consensus primers/probes conventionally employed in ALL, ASO-RQPCR worked in three (23.1 %) cases only. Conversely, using entirely patient-specific primers/probes, ASO-RQPCR was applicable in eight (61.5 %) cases with a sensitivity of 5 × 10-4-10-5. Moreover, using standard curves constructed by serial dilution of plasmids cloned with patient-specific CDR3, ASO-RQPCR was successful in 12 (92.3 %) cases with a sensitivity of 10-4-10-5, but not in a case lacking an N region, in which design of a tumor-specific ASO primer was precluded. Finally, in a patient in complete response (CR), further reduction of MRD after autologous stem cell transplantation (ASCT) was demonstrated. In summary, using entirely patient-specific primers/probes, ASO-RQPCR was applicable in >90 % MM patients and enabled detection of dynamic changes of MRD before and after ASCT despite conventional CR.

Methylation profiling of SOCS1, SOCS2, SOCS3, CISH and SHP1 in Philadelphia-negative myeloproliferative neoplasm.

Janus kinase-signal transducer and activator of transcription (JAK/STAT) signalling, pivotal in Philadelphia-negative (Ph-ve) myeloproliferative neoplasm (MPN), is negatively regulated by molecules including SOCSs, CISH and SHP1. SOCS1, SOCS2 and SOCS3 methylation have been studied in MPN with discordant results. Herein, we studied the methylation status of SOCS1, SOCS2 and SOCS3, CISH and SHP1 by methylation-specific polymerase chain reaction (MSP) in cell lines and 45 diagnostic marrow samples of Ph-ve MPN. Moreover, we attempted to explain the discordance of methylation frequency by mapping the studied MSP primers to the respective genes. Methylation was detected in normal controls using SOCS2 MSP primers in the 3'translated exonic sequence, but not primers around the transcription start site in the 5' untranslated regions (5'UTR). SOCS1, SOCS2, SOCS3 and CISH were completely unmethylated in primary MPN samples and cell lines. In contrast, methylation of SHP1 was detected in 8.9% primary marrow samples. Moreover, SHP1 was completely methylated in K562 cell line, leading to reversible SHP1 silencing. A review of methylation studies of SOCS1 and SOCS3 showed that spuriously high rates of SOCS methylation had been reported using MSP primers targeting CpG sites in the 3'translated exonic sequence, which is also methylated in normal controls. However, using MSP primers localized to the 5'UTR, methylation of SOCS1, SOCS2 and SOCS3 is infrequent across all studies. In summary, methylation of SOCS1, SOCS2, SOCS3 and CISH is infrequent in Ph-ve MPN. Appropriate MSP primers are important for accurate estimation of the methylation frequency. The role of SHP1 methylation in the pathogenesis of MPN warrants further investigation.

Methylation of miR-34a, miR-34b/c, miR-124-1 and miR-203 in Ph-negative myeloproliferative neoplasms.

BACKGROUND: MicroRNA (miR) miR-34a, -34b/c, -124-1 and -203 are tumor suppressor miRs implicated in carcinogenesis. METHODS: We studied DNA methylation of these miRs in Philadelphia-negative (Ph-ve) myeloproliferative neoplasms (MPNs). Methylation-specific PCR (MSP), verified by direct sequencing of the methylated MSP products, was performed in cell lines, normal controls and diagnostic marrow samples of patients with MPNs. RESULTS: Methylation of these miRs was absent in the normal controls. miR-34b/c were homozygously methylated in HEL cells but heterozygously in MEG-01. In HEL cells, homozygous miR-34b/c methylation was associated with miR silencing, and 5-aza-2'-deoxycytidine treatment led to re-expression of both miR-34b and miR-34c, consistent with that both miRs are under the regulation of the same promoter CpG island. miR-34a was heterozygously methylated in MEG-01 and K-562. miR-203 was completely unmethylated in K-562 and SET-2 but no MSP amplification was found in both HEL and MEG-01, suggestive of miR deletion. In primary samples, four each had miR-34b/c and -203 methylation, in which two had concomitant methylation of miR-34b/c and -203. miR-34a was methylated in one patient and none had methylation of miR-124-1. Seven patients (15.6%) had methylation of at least one of the four miRs. miR methylation did not correlate with clinical parameters, disease complications or JAK2 V617F mutation. CONCLUSION: This is the first report of miR hypermethylation in MPNs. miR-203 hypermethylation is not specific to Ph+ve leukemias but also present in Ph-ve MPNs. miR-34b/c methylation was associated with reversible miR silencing. There was no correlation of miR methylation with clinical demographic data or outcome.

Epigenetic dysregulation of the death-associated protein kinase/p14/HDM2/p53/Apaf-1 apoptosis pathway in multiple myeloma.

AIM: To study the role of gene promoter hypermethylation of the putative tumour suppressor genes involved in the death-associated protein (DAP) kinase/p14/HDM2/p53/Apaf-1 apoptosis pathway in multiple myeloma (MM). METHOD: DNAs from 55 primary MM marrow samples and myeloma cell lines were analysed for aberrant promoter methylation of DAP kinase, p14 and Apaf-1 genes by methylation-specific polymerase chain reaction (MSP). RESULT: In the methylated positive control, the sensitivity of M-MSP for DAP kinase was 1 x 10(-3). Aberrant hypermethylation of DAP kinase was found in 29/55 (52.7%) primary MM samples, whereas hypermethylation of p14 or Apaf-1 was undetectable in any of the samples tested. 5-Azacytidine treatment of two myeloma cell lines, WL2 and HS-Sultan, led to de-methylation and re-expression of DAP kinase, thereby confirming gene silencing associated with promoter hypermethylation. Hypermethylation of DAP kinase did not correlate with age, sex, paraprotein subtype or Durie-Salmon stage, but negatively affected the overall survival. CONCLUSION: Of the putative tumour suppressor genes in the DAP kinase/p14/HDM2/p53/Apaf-1 apoptosis pathway, only DAP kinase is frequently methylated in MM, which is associated with gene silencing and might be of prognostic significance. p14 and Apaf-1 were not methylated in MM.

Therapy-related lymphomas in patients with autoimmune diseases after treatment with disease-modifying anti-rheumatic drugs.

Ten patients developing lymphomas after disease modifying anti-rheumatic drugs (DMARD) (methotrexate, n = 3, mean cumulative dose = 3.4 g; cyclophosphamide, n = 2, mean dose = 70 g; azathioprine, n = 6, mean dose = 243 g) were investigated. Methotrexate-related lymphomas were Epstein-Barr virus (EBV)-positive, had infrequent aberrant methylation of p15 and p16, and responded well to methotrexate withdrawal or anti-CD20 antibody (rituximab) alone without concomitant chemotherapy, implying that defective immunosurveillance was important in lymphomagenesis. However, 75% of cyclophosphamide/azathioprine-related lymphomas were EBV-negative, had frequent p15 and p16 methylation, and responded poorly to drug withdrawal and chemotherapy, implying that direct drug-induced mutagenesis might be involved in lymphomagenesis.

SOCS1 and SHP1 hypermethylation in multiple myeloma: implications for epigenetic activation of the Jak/STAT pathway.

SOCS1 and SHP1 negatively regulate the Janus kinase/signal transducer and activator of transcription (Jak/STAT) signaling pathway. The role of promoter hypermethylation leading to epigenetic inactivation of SOCS1 and SHP1 in myeloma was investigated. The methylation-specific polymerase chain reaction (MSP) was used to define SOCS1 and SHP1 methylation in 34 diagnostic myeloma samples. For SOCS1, MSP primers 3' to the translation start site were unreliable and gave positive results in normal controls. However, primers in the 5' promoter region were specific, although no myeloma samples showed methylation. For SHP1, 27 of 34 (79.4%) myeloma samples showed SHP1 hypermethylation. The biologic significance of SHP1 methylation was investigated in the U266 human myeloma line. U266 contained completely methylated SHP1. Furthermore, there was constitutive STAT3 phosphorylation. Treatment with 5-azacytidine led to progressive demethylation of SHP1 on days 2 to 5, with consequent increasing reexpression of SHP1 as shown by reverse transcription-polymerase chain reaction (RT-PCR). Concomitant with increasing SHP1, a parallel down-regulation of phosphorylated STAT3 occurred, so that by day 5 phosphorylated STAT3 was barely detectable. The overall survivals of patients with and without SHP1 methylation were similar. SHP1 methylation leading to epigenetic activation of the Jak/STAT pathway might have a tentative role in the pathogenesis of myeloma, which should be further confirmed by functional studies in primary myeloma samples.