Manuella S.S. Almeida

TGFβR2 aberrant methylation is a potential prognostic marker and therapeutic target in multiple myeloma

Multiple myeloma (MM) is an incurable hematological malignancy. Different studies demonstrated the occurrence of genetic and epigenetic alterations in MM. The aberrant methylation is one of the most frequent epigenetic alterations in human genome. This study evaluated the aberrant methylation status of 20 genes in 51 MM samples by quantitative methylation‐specific PCR (QMSP) and compared the methylation profile with clinicopathological characteristics of the patients. The QMSP analyses showed that PTGS2 (100.0%), SFN (100.0%), CDKN2B (90.2%), CDH1 (88.2%), ESR1 (72.5%), HIC1 (70.5%), CCND2 (62.7%), DCC (45.1%) and TGFβR2 (39.2%) are frequently hypermethylated in MM while aberrant methylation of RARβ (16.6%), MGMT (12.5%), AIM1 (12.5%), CDKN2A (8.3%), SOCS1 (8.3%), CCNA1 (8.3%) and THBS1 (4.1%) are rare events. There was no methylation of GSTP1, MINT31, p14ARF and RB1 in the samples tested. Hypermethylation of ESR1 was correlated positively with isotype IgA, while aberrant methylation of THBS1 correlated negatively with isotype IgG. Furthermore, hypermethylation of DCC and TGFβR2 were correlated with poor survival. The multivariate analysis showed ISS and TGFβR2 hypermethylation strongly correlated with poor outcome. This study represents the first quantitative evaluation of promoter methylation in MM and our data provide evidence that TGFβR2 hypermethylation, besides ISS, may be useful as prognostic indicator in this disease.

SAGE analysis highlights the importance of p53csv, ddx5, mapkapk2 and ranbp2 to multiple myeloma tumorigenesis

Serial analysis of gene expression (SAGE) allows a comprehensive profiling of gene expression within a given tissue and also an assessment of transcript abundance. We generated SAGE libraries from normal and neoplastic plasma cells to identify genes differentially expressed in multiple myeloma (MM). Normal plasma cells were obtained from palatine tonsils and MM SAGE library was generated from bone marrow plasma cells of MM patients. We obtained 29,918 SAGE tags from normal and 10,340 tags from tumor libraries. Computer-generated genomic analysis identified 46 upregulated genes in the MM library. Ten upregulated genes were selected for further investigation. Differential expression was validated by quantitative real-time PCR in purified plasma cells of 31 patients and three controls. P53CSV, DDX5, MAPKAPK2 and RANBP2 were found to be upregulated in at least 50% of the MM cases tested. All of them were also found upregulated in MM when compared to normal plasma cells in a meta-analysis using ONCOMINE microarray database. Antibodies specific to DDX5, RANBP2 and MAPKAPK2 were used in a TMA containing 57 MM cases and confirmed the expression of these proteins in 74%, 96%, and 21% of the MM samples, respectively. Analysis of differential expression using SAGE could identify genes important for myeloma tumorigenesis (P53CSV, DDX5, MAPKPK2 and RANBP2) and that could potentially be useful as therapeutic targets.

Analysis of polymorphism at site -174 G/C of interleukin-6 promoter region in multiple myeloma

It is well established that interleukin-6 (IL-6) is an essential growth factor for multiple myeloma (MM) and patients with increased IL-6 levels have a poor prognosis. In healthy subjects, the presence of the C allele at a polymorphic site (-174 G/C) of the IL-6 gene is related to low IL-6 levels. In view of the potential association of this particular polymorphism with IL-6 concentration, and the relevance of IL-6 in MM pathogenesis, the objective of the present study was to investigate the prevalence of IL-6 (-174 G/C) promoter polymorphism and its association with development of MM in Brazilian individuals. We investigated the prevalence of these alleles in 52 patients and 60 healthy subjects (matched by age, sex, and race) of a Brazilian population. Thirty patients were male (42.4%), 24 (46.2%) were white and the median age at diagnosis was 58.5 years (range: 28 to 84 years). To determine the IL-6 (-174 G/C) polymorphism, molecular analysis was performed by polymerase chain reaction followed by endonuclease restriction digestion. The genotype distributions observed in the group of patients were 4% CC, 42% GC and 54% GG. The C allele frequency was 0.25. These results were similar to the control group, suggesting no impact of this polymorphism on the susceptibility to MM.

Frequency and prognostic relevance of cancer testis antigen 45 expression in multiple myeloma.

OBJECTIVE This study aims to analyze the expression of cancer testis antigen 45 (CT45) in normal tissues and in plasma cell disorders and to identify possible associations with clinical data and prognosis in multiple myeloma (MM) patients. MATERIALS AND METHODS Expression of CT45 was studied in 20 normal tissues (testis, placenta, skeletal muscle, bladder, lung, spleen, heart, brain and fetal brain, thymus, uterus, stomach, mammary gland, pancreas, prostate, small intestine, kidney, adrenal gland, spinal cord, colon, and one pool of 10 normal bone marrow samples) and bone marrow aspirates from 3 monoclonal gammopathies of undetermined significance, 5 solitary plasmacytomas, 61 newly diagnosed MM patients and MM cell line U266 by reverse transcriptase polymerase chain reaction. RESULTS CT45 was positive in 3 of 20 (15%) normal tissues tested: lung, brain (both fetal and adult), and spinal cord. Among monoclonal gammopathies, CT45 was positive in 2 of 5 (40%) solitary plasmacytomas bone marrow aspirates, 10 of 61 (16%) MM bone marrow aspirates, and in the U266 MM cell line. CONCLUSIONS We did not find associations between bone marrow histology and CT45 expression. However, we demonstrated for the first time that positive expression of CT45 was associated with poor prognostic (International Staging System) and poor outcomes in MM patients, meaning that CT45-positive cases presented seven times more chance of worse evolution than the negative ones.

Bilateral Central Retinal Vein Occlusion Associated with Multiple Myeloma

Purpose: To report a case of simultaneous bilateral central retinal vein occlusion (CRVO) associated with multiple myeloma. Methods: A 65-year-old woman had sudden, painless loss of vision in both eyes for 20 days. Ophthalmologic examination revealed bilateral CRVO. Appropriate medical workup was conducted, and multiple myeloma was diagnosed as the underlying cause. Results: Clinical support and chemotherapy effectively controlled paraprotein production, leading to improvement of both systemic and ocular alterations. Conclusions: Many conditions have been noted to be associated with CRVO. Based on a Medline search, this is the first report of simultaneous bilateral CRVO as the first manifestation of multiple myeloma, illustrating the need for a primary care ophthalmologist to be involved in the basic assessment for associated underlying diseases in retinal disorders.

Outcomes of autologous transplantation for multiple myeloma according to different induction regimens.

Background Induction therapy followed by high-dose chemotherapy and autologous transplantation is the standard treatment for suitable patients with multiple myeloma. Objective The aim of this study was to assess whether induction therapy with thalidomide-containing regimens was associated with improved results compared to vincristine, doxorubicin, and dexamethasone, and whether cyclophosphamide, thalidomide, and dexamethasone were associated with better results than thalidomide and dexamethasone. Methods The records of 152 patients who underwent autologous transplantation at this institution from August of 2004 to January of 2012 were reviewed, selecting those with at least partial response to a maximum of eight cycles of induction therapy and sufficient follow-up information for analysis. Results This study included 89 patients; 44 were female, with a mean age of 55 years (there was a significant trend for increasing age over the years of the study). The median number of induction therapy cycles was four, again with a trend of increase over the years. At least a very good partial response to induction therapy was achieved more often in the cyclophosphamide, thalidomide, and dexamethasone group (61.1%) and in the thalidomide and dexamethasone group (59.2%) than in the vincristine, doxorubicin, and dexamethasone group (16.2%). The overall median progression-free survival was 34 months, with no statistically significant difference between the three groups. The overall median survival was not reached, and there was no significant difference between the three groups; the estimated five-year overall survival was 55%. Conclusion Although the quality of responses appeared to be better with thalidomide-containing regimens, these improvements did not translate into improved long-term outcomes. Given its track record, cyclophosphamide, thalidomide, and dexamethasone is currently considered the preferred regimen for first-line induction therapy in the Brazilian public health system.

Response of plasmacytomas to low-dose thalidomide in a patient with refractory multiple myeloma.

To the Editor: The ideal dose of thalidomide (Thal) for multiple myeloma (MM) remains unknown and its actual effect on plasmacytomas is questionable. We report on the case of a patient with refractory MM with several bone and extramedullary plasmacytomas who showed a clinical response with a low-dose Thal schedule. The patient, a 60-year-old male, was diagnosed in January/2002 with Salmon-Durie stage IIIA, IgG kappa MM and simultaneous plasmacytoma in the right jaw and left humerus. The bone marrow (BM) had massive plasmablastic infiltration and high microvessel density (Fig. 1A and 1B). The lytic lesions and the plasmacytomas progressed rapidly during the three cycles of VAD (IV vincristine 0.4 mg D1 /4; IV doxorubicin 9 mg/m D1-4; PO dexamethasone 40 mg D1 /4, 9 /12, 17 /20). The jaw plasmacytoma became very prominent requiring local radiotherapy, given only at a dose of 26 Gy because of severe mucositis and disease progression. Following radiotherapy, systemic treatment was restarted with melphalan and prednisone (PO melphalan 8 mg/m D1 /4; PO prednisone 100 mg D1 /4), but after three cycles the patient’s clinical condition continued to deteriorate (severe bone pain, pathologic fractures, volume increase of the pre-existing plasmacytomas and appearance of another one) (Table 1). The patient was then treated with one cycle of Thal plus dexamethasone (Thal 200 mg/day; PO dexa-

Autologous stem cell transplantation improves quality of life in economically challenged, Brazilian multiple myeloma patients

OBJECTIVES: 1) To characterize the impact of multiple myeloma on the quality of life of patients treated in two public institutions in São Paulo State, Brazil, using a generic Short Form 36 Health Survey and a questionnaire specific for oncologic patients (QLQ-C30) upon diagnosis, after the clinical treatment, and at day +100 after autologous stem cell transplantation; 2) to evaluate whether autologous stem cell transplantation can improve the quality of life of our economically challenged population aside from providing a clinical benefit and disease control. METHODS: We evaluated 49 patients with multiple myeloma (a total of 70 interviews) using the two questionnaires. The scores upon diagnosis, post-treatment/pre-autologous stem cell transplantation, and at D+100 were compared using ANOVA (a comparison of the three groups), post hoc tests (two-by-two comparisons of the three groups), and paired t-tests (the same case at two different times). RESULTS : Of the included patients, 87.8% had a family budget under US

Number of expressed cancer/testis antigens identifies focal adhesion pathway genes as possible targets for multiple myeloma therapy

600 (economic class C, D, or E) per month. The generic Short Form 36 Health Survey questionnaire demonstrated that physical function, role-physical, and bodily pain indices were statistically different across all three groups, favoring the D+100 autologous stem cell transplantation group (ANOVA). The questionnaire specific for oncologic patients, the QLQ-C30 questionnaire, confirmed what had been demonstrated by the Short Form 36 Health Survey with respect to physical function and bodily pain, with improvements in role functioning, fatigue, and lack of appetite and constipation, favoring the D+100 autologous stem cell transplant group (ANOVA). The post hoc tests and paired t-tests confirmed a better outcome after autologous stem cell transplantation. CONCLUSION: The questionnaire specific for cancer patients seems to be more informative than the generic Short Form 36 Health Survey questionnaire and reflects the real benefit of autologous stem cell transplantation in the quality of life of multiple myeloma patients in two public Brazilian institutions that provide assistance for economically challenged patients.

Plasmablastic multiple myeloma is associated with increased vascular endothelial growth factor immunoexpression

Considering that the importance of cancer/testis (CT) antigens in multiple myeloma (MM) biology is still under investigation, the present study aimed to: (1) identify genes differentially expressed in MM using microarray analysis of plasma cell samples, separated according to the number of expressed CTs; (2) examine possible pathways related to MM pathogenesis; (3) validate the expression of candidate genes by quantitative real-time PCR (RQ-PCR). Three samples predominantly positive (>6 expressed), including the U266 cell line, and three samples predominantly negative (0 or 1 expressed CT for the 13 analyzed CT antigens), were submitted for microarray analysis. Validation by RQ-PCR from 24 MM samples showed that the ITGA5 gene was downregulated in predominantly positive (>6 expressed CTs, p = 0.0030) and in tumor versus normal plasma cells (p = 0.0182). The RhoD gene was overexpressed in tumor plasma cells when compared to normal plasma cells (p = 0.0339). Results of the microarray analysis corroborate the hypothesis that MM could be separated into predominantly positive and predominantly negative expression. The differential expression of ITGA5 and RhoD suggests disruption of the focal adhesion pathway in MM and offers a new target field to be explored in this disease.