Beta 2M Levels Research Articles

#4195 CAN A BLOOD TEST FOR MIDDLE MOLECULES BE USED TO MEASURE RESIDUAL KIDNEY FUNCTION TO PERFORM INCREMENTAL DIALYSIS?

Abstract Background and Aims Incremental dialysis involves combining residual kidney functions (RKF) with dialysis dose to individualise treatment, increasing the dialysis dose as RKF falls. Potential benefits include quality of life benefits, less treatment burden and health economic benefits. Kidney Disease Outcome Quality Initiative (KDOQI) recommends incremental haemodialysis may be performed when renal urea clearance (KRU) is > = 2ml/min. To avoid underdialysis, incremental approach requires frequent interdialytic urine collections to monitor RKF, which can be inconvenient. An easier assessment of RKF would likely increase the uptake of incremental dialysis. Blood levels of middle molecules (e.g Beta 2 Microglobulin (Beta2M) and Beta Trace Protein (BTP)) have been studied as predictors of RKF. However, their role in identifying patients for incremental dialysis have not been tested. There are a few published methods of predicting RKF using meddle molecules. A proposed simple method is identification of patients with KRU > = 2 ml/min, based on having a blood middle molecules level below a certain cut-off. Alternatively direct prediction of KRU from middle molecule levels can be performed with an algorithm/equation. We set out to establish if these methods may identify patients with significant RKF who can benefit from incremental dialysis. Method We conducted a retrospective analysis on the data from a multicentre feasibility randomised controlled trial to assess the impact of incremental versus conventional haemodialysis initiation. As a part of this trial 55 participants were followed up for up to 12 months with monthly RKF measurement using interdialytic urine collections. Monthly Beta2M and BTP measurements were also performed. We used (1) a published middle molecules-based KRU equation (2) published Beta2M cut-off levels, to predict RKF. We, then, compared the predicted RKF with conventionally measured RKF from urine collection. The focus was to establish reliability of these methods in identifying patients with KRU > 2 ml/min, which is the recommended cut-off for incremental dialysis, and to identify where underdialysis might occur if these methods were used in clinical practice. Results The middle molecules-based KRU equation had 62% sensitivity and 84% specificity to identify those with KRU >2, whereas a beta2M cut-off level of <19.15mg/L had 78% sensitivity and 82% specificity. 6/55 (10.9%) participants would have had underdialysis whilst performing incremental dialysis guided by a middle molecules-based KRU equation and 7/55 (12.7%) would under dialyse using the Beta2M cut-off level to predict KRU. The predicted mean underdialysis that would occur using these methods was 0.2 – 0.3 standard kt/v units. Combining these methods with urine volume improved specificity and sensitivity. Beta2M level of <19.15 mg/L and Urine volume of >0.5 litre/day combined predicted KRU >2 with 70% sensitivity and 98% specificity. In other words, only 1/55 (1.8%) patients would have had under dialysis if incremental dialysis were performed based on a combination of Beta2M cut-off and urine volume. Conclusion Blood Beta2M level, when combined with urine volume assessment reliably predicts adequate RKF to perform incremental dialysis safely. If dialysis patients can reliably estimate or measure their urine volume, this tool can potentially replace the need for cumbersome interdialytic urine collection to determine RKF and offer an easy way to perform incremental dialysis.

MM-301: Plasma Cell Leukemia, PCL

Context PCL is a rare entity and a very aggressive disease. The diagnosis is based on the presence of more than 20% of PCL in peripheral blood or an absolute plasma cell count of more than 20×109. It is classified as primary PCL when it occurs in a patient with no previous history of MM and as a secondary PCL when it is observed as the leukemic transformation of relapsed or refractory recognized MM. Objective Diagnostic challenges and treatment of PCL. Case Report A 34-year-old female was accepted in clinic in 2019. She has spleen: 161 mm; liver: 148 mm. High levels of beta-2M and LDH. More than 30% of plasma cells in peripheral blood. Severe anemia, leukocytosis, and thrombocytopenia. Flow cytometry showed clonality for plasma cells. Results We treated our patient with VTD-PACE regimen (a combination of bortezomib 1 mg/m2, s.c, D1, D5, D8, and D11; dexamethasone 40 mg D1–D4 i.v.; thalidomide 200 mg/d, D7–D28, p.o.; cisplatin 10 mg/m2 D1–D4, i.v.; adriamycin 10 mg/m2 D1–D4,i.v.; cyclophosphamide 400 mg/m2 D1–D4, and etoposide 40 mg/m2 D1–D4, i.v.). We recommended HSCT, and she went abroad because this procedure cannot be performed in our country yet. Conclusions PCL is a rare entity, very aggressive, with a poor prognosis. Early diagnosis and early treatment are very important prognostic factors. Treatment with immunomodulatory drugs such as bortezomib-based regimens, followed by stem cell transplant, is recommended. References Harousseau JL, Atal M, Avet-Loiseau H, et al. Bortezomib plus dexamethasone is superior to vincristine plus doxorubicin plus dexamethasone as induction treatment prior to autologous stem-cell transplantation in newly diagnosed multiple myeloma: results of the IFM 2005-01 phase III trial. J Clin Oncol. 2010;28:4621-4629. Reeder CB, Reece DE, Kukreti V, et al. Cyclophosphamide, bortezomib and dexamethasone induction for newly diagnosed multiple myeloma: high response rates in phase II clinical trial. Leukemia. 2009;23:1337-1341. Kumar SK, Dispenzieri A, Lacy MQ, et al. Continued improvement in survival in multiple myeloma; changes in early mortality and outcomes in older patients. Leukemia. 2013;28(5):1122-1128.

The relationship between concentrations of magnesium and oxidized low-density lipoprotein and Beta2-microglobulin in the serum of patients on the end-stage of renal disease.

The end-stage of renal disease is associated with increased oxidative stress and oxidative modification of low-density lipoproteins (LDLs). Beta2 microglobulin (beta2M) is accumulated in the serum of dialysis patients. Magnesium (Mg) plays a protective role in the development of oxidative stress in healthy subjects. We studied the relationship between concentrations of magnesium and oxidized LDL (ox-LDL) and beta2M in the serum of patients on the end stage of renal disease. In 96 patients on on-line- predilution hemodiafiltration, beta2M and intact parathormone were measured by radioimmunoassays. High-sensitivity C-reactive protein (hsCRP) and ox-LDL were measured using ΕLISA. Serum bicarbonate levels were measured in the blood gas analyser gas machine. We performed logistic regression analysis models to investigate Mg as an important independent predictor of elevated ox-LDL and high beta2M serum concentrations, after adjustment to traditional and specific for dialysis patients' factors. We observed a positive correlation of Mg with ox-LDL (r = 0.383, P = 0.001), but the association of Mg with beta2M, hsCRP, and serum bicarbonate levels was significantly inverse (r = -0.252, P = 0.01, r = -0.292, P = 0.004, and r = -0.282, P = 0.04 respectively). The built logistic-regression analysis showed that Mg act as a significant independent factor for the elevated ox-LDL and beta2M serum concentrations adjusting to traditional and specific factors for these patients. We observed a positive relationship between magnesium and acidosis status- related ox-LDL concentrations, but the inverse association between magnesium and beta2M serum concentrations in hemodialysis patients.

LBP17: A SCIENTIFIC MYTH: THERE IS MORE HLA CLASS I ON B CELLS THAN ON T CELLS

In 1978, before class II Ags were defined, Pellegrino et al. reported that there is more HLA on B cells than T cells with equivalent beta2m levels on each cell type. Since then, the report has been widely misinterpreted to have shown there is more class I on B cells than on T cells and has led to a long standing interpretation of a T−/B+ FXM result as presence of either class II Ab or low level class I. A T+/B− FXM result has generally been interpreted as non-HLA. We studied the relative expression of CI antigen on T and B cells in 60 pts to determine validity of this dogma.” The aim of our study was to provide direct experimental evidence for HLA-class I Ag expression on T and B cells by probing class I with pan class I MoAb and class I-DSA positive serum. Class I Probing: Biotinylated W6/32 MoAb in various concentrations (conc) was incubated with 0.1X106 PBMCs for 30 mins/RT. After washes, PerCP-CD3, PE-Cy7-CD19 and SA-PE were added and incubated for another 30 mins/RT. The final cells were acquired on a Canto-II and the PE intensities on T and B cells were measured. FXM: Standard T/B FXM procedure using (1) class I-DSA serum: doubling dilutions vs corresponding cells and (2) humanized W6/32 MoAb (hW6/32; InvivoGen): same conc as class I probing expt above. LMX-IgG SAB: One Lambda LabScreen. class I expression was evaluated on T and B cells with the W6/32 probing assay and FXM using hW6/32 MoAb and no significant difference was found (Fig. 1). FXM with class I DSA showed no significant differences between MCS on T cells and B cells (Fig. 2). In three examples (Fig. 3), T+/B− FXM results were observed in patients where only class I DSA was detected by the LMX-SAB. Our study shows that class I antigen is evenly distributed on T and B cells. A T+/B− FXM cannot exclude the existence of class I DSA and the presence of class I Ab alone can result in T+/B−; T−/B+ or both T+/B+ FXM. Thus, a T+/B− FXM can reflect class I DSA, be potentially clinically relevant, and warrants further investigation before attributing to non-HLA antibody or no clinical risk.

Long-Term Exposure to Pomalidomide-Dexamethasone in Pts with Refractory Myeloma

Background. The IFM2009-02 study was launched in 2009, and randomized 84 patients (pts) with pomalidomide (oral 4 mg daily) and dexamethasone (oral 40 mg weekly) given either 21 days out of 28 or continuous. Whilst the overall median PFS was 4.6 months - this end stage very advanced RRMM population, we observed that 40% of the patients had a prolonged PFS and subsequently OS in the trial.We sought to analyze the characteristics of 58 pts that had more than 3 months of pomalidomide to study the effect of long exposure to Pomalidomide.Method. IFM 2009-02 was a multicentre phase 2 study of pts with RRMM who had at best a stable disease with the last course of bortezomib and of lenalidomide, or who were refractory to bortezomib and lenalidomide (IMWG). This analysis was performed on the ITT population combining data from the 2 study arms. We have analyzed the characteristics of pts according to duration of treatment with pomalidomide and dexamethasone 3 months to one year (<1 year) or more than one year (≥1 year).Results. 60% and 40% of pts were exposed to pomalidomide for <1 year and ≥1 year, respectively. The ORR for the <1 year group was 43%, the median PFS 4.6 months (CI95% 4;6) with only 6% at 12 months, and the median OS 15 months (4;6) and 65% at 12 months, 40% at 18 months. For the ≥1 year group, the response rate and survival were strikingly different, ORR at 83%, PFS 20.7 months, OS not reached (CI95% 40;-) and 100% at 12 months, 91% at 18 months. Of the pts in the <1 year, 87% have died versus 35% in the ≥1 year group. Of note, death of most pts occurred during the follow up period post pomalidomide therapy , however in a far greater extent for the <1 year group, 96% versus 67%, that could suggest it was more difficult to salvage these pts post pomalidomide.We next sought to identify the characteristics of the 2 groups. Interestingly, the median number of prior lines was similar across groups, 5 (range 1-10), with 89% and 79% of the pts exposed to more than 3 lines and 17% and 22% exposed to more than 6 lines, for the <1 year and ≥1 year groups, respectively. 40% and 48% of pts had Bortezomib as last line, 29% and 43% Lenalidomide, 43% and 26% and alkylating agent. Similarly, 80% and 74% of pts were refractory to Bortezomib, 89% versus 87% to Lenalidomide, 65% versus 75% to alkylating agents, 74% and 87% to the last line of therapy, respectively. 74% and 70% were double refractory (Bortezomib and Lenalidomide) and 60% and 70% were triple refractory (double +last line), respectively. The time from diagnosis to IFM 2009-02 study entry was also similar between cohorts, 5.8 and 6.5 years for <1 year and ≥1 year groups; however with 6% versus 26% of pts, entering the study in less than 3 years since diagnosis. There was no difference in terms of patients characteristics between groups, either patients-based such as gender, age, weight, or myeloma-based characteristics.However, serum beta 2m level was higher at diagnosis in the <1 year compared to the ≥1 year, 54% versus 35%, with a slightly more adverse cytogenetic profile 35% versus 12%, with the limitation that this was not available for all the patients. Presence of plasmacytoma/EMD was also greater in the former group, 20% versus 4%, respectively. It seems that the <1 year had more intrinsic adverse features of the tumor cells compared to the ≥1 year group.There was no clear difference in terms of safety management of pomalidomide and/or dexamethasone, with respect to the daily dose intensity of Pomalidomide (median, 3.0 and 2.9 mg/day), and the relative dose intensity of pomalidomide that was 89% and 84%, respectively; similar to the rate of dose reduction and dose interruption.Conclusion. Pomalidomide and dexamethasone is effective and well tolerated in these heavily pre-treated MM pts refractory to Bortezomib and Lenalidomide, with approximately 40% of the patients having a prolonged exposure to treatment, which translated into a significantly prolonged OS. Our study suggests that patients with more intrinsic adverse features of Myeloma tumor cells could not have prolonged exposure to pomalidomide as they progressed within a year from start of pomalidomide. Future studies should examine optimizing pomalidomide therapy in those patients, such as using multidrug pomalidomide-based combined regimens to prolong exposure to pomalidomide and improve the survival of these patients. DisclosuresKarlin:Janssen: Honoraria; celgene: Consultancy, Honoraria; Sandoz: Consultancy. Stoppa:Celgene Jansen: Honoraria.

Combination Therapy with Ofatumumab and Lenalidomide in Patients with Relapsed Chronic Lymphocytic Leukemia (CLL): Results of a Phase II Trial

Abstract 1788Patients with relapsed CLL have limited effective treatment options, and new therapies are needed. We report the results of a phase II study which investigated the efficacy and tolerability of a combination regimen with ofatumumab and lenalidomide in patients (pts) with relapsed CLL. These agents were evaluated as a combination regimen for pts with relapsed CLL based on their documented single-agent efficacy, distinct and potentially complimentary mechanisms of action and non-overlapping toxicities. Furthermore, the combination of rituximab and lenalidomide was active in relapsed CLL (Badoux et al. 2010). Pts with symptomatic disease who met treatment indications were eligible. Other inclusion criteria required prior treatment with purine analogs, an ECOG PS score of 0–2, and adequate organ function (creatinine clearance > 30ml/min, total bilirubin < to 2 mg/dl, ALT < 2 × ULN). Pts with any neutrophil count were eligible. Pts were excluded for platelets < 30,000 mm3, positivity for HIV, active hepatitis B or C or recent history of tuberculosis. Pts received ofatumumab IV weekly for four weeks (300mg week 1; 1,000 mg weeks 2 to 4), monthly during months 2–6, and every other month during months 7–24. Lenalidomide 10 mg PO daily was started on day 9 and continued daily for a maximum of 24 months. Allopurinol 300mg PO daily was given during the first two weeks of cycle 1. No pts received antibiotic or DVT prophylaxis. The use of growth factors was according to standard guidelines. Responses were assessed after 3, 6, 12, 18 and 24 months. Between January 2010 and January 2011, 36 pts were enrolled and 34 pts are evaluable (one pt withdrew consent prior to treatment and one was excluded because of concomitant MDS). The median age of the pts was 62 yrs (34–82). Twenty pts (59%) had Rai III-IV disease. The median beta-2M level at start of therapy was 4.4 mg/dL (1.7-16.5). The median number of prior treatments was 2 (1–8). All pts had received prior chemoimmunotherapy with fludarabine, cyclophosphamide and rituximab (FCR). Twelve pts (35%) were fludarabine-refractory. Twenty-two pts (65%) had unmutated IgHV genes, 9 (26%) del(17p) and 4 (12%) del(11q). Responses were evaluated according to the 2008 NCI-WG criteria: 5 pts (15%) achieved CR (including 1 CRi) and 17 pts (50%) PR, for an ORR of 65%. Of the 5 CR pts, 1 was MRD-negative by flow cytometry. Two of the PR pts achieved marrow CR and only had persistent lymphadenopathy. Of the 22 responders, 13 are continuing on therapy with ongoing response (median time on therapy 14 months, range 8–20 months). Despite ongoing response, 7 pts discontinued therapy due to: transition to HSCT (3 pts; after 3, 6 and 6 months, respectively), pulmonary embolism (1 pt; after 4 months), recurrent zoster infection (1 pt; after 17 months of therapy) and physician choice (2 pts; after 9 and 16 months of therapy). Two pts discontinued therapy because of loss of response after 12 and 18 months of treatment, respectively. Thirty-one pts (91%) are alive. There have been no deaths on therapy. Three deaths occurred after discontinuation of therapy due to refractory disease (1), following HSCT (1) and due to progressive disease and adenocarcinoma of the lung (1). The most common grade 3–4 treatment-related hematological adverse events included neutropenia (15 pts, 44%), thrombocytopenia (3 pts, 9%) and anemia (1 pt, 3%). Thrombotic events occurred in 2 pts (6%). One pt experienced grade 3 pulmonary embolism and another patient grade 2 superficial vein thrombosis. In both cases with thrombotic event, ESAs were concomitantly administered. One pt (3%) experience a grade 3 infusion reaction during the first ofatumumab administration. Eight grade 3 infectious episodes occurred: pneumonia (3), neutropenic fever (2), parotitis (1), LE cellulitis (1) and CNS toxoplasmosis based on radiological findings (1). Intermittent grade 1–2 diarrhea was reported by 8 pts (24%). Grade 1–2 lenalidomide-associated tumor flare reaction was seen in 8 pts (24%). The median tolerated daily dose of lenalidomide was 5 mg/day (2.5-10 mg). In conclusion, the combination of ofatumumab and lenalidomide is well tolerated. Neutropenia was the most common toxicity. This combination induced responses in 65% of pts with relapsed CLL, all of whom had received prior chemoimmunotherapy. Responses are durable and ongoing in some pts. Disclosures:Ferrajoli:GlaxoSmithKline: Research Funding; Celgene: Research Funding. O'Brien:GlaxoSmithKline: Consultancy; Celgene: Consultancy. Wierda:GlaxoSmithKline: Research Funding.

Higher serum beta2-microglobulin levels are associated with better survival in chronic hemodialysis patients: a reverse epidemiology

beta2-Microglobulin (beta2-M) has been considered a surrogate marker of putative mid-molecular weight (MW) uremic toxins, compounds difficult to dialyze by low-flux dialysis membranes. This study was performed to evaluate the relationship between serum beta2-M and survival of chronic hemodialysis (CHD) patients and the association of beta2-M levels and factors associated with mortality. Part I of this study is a retrospective cohort evaluation that determined the relationship between beta2-M and mortality, and Part II is a cross-sectional study that evaluated the relationship between beta2-M and factors associated with mortality. Laboratory parameters, including beta2-M, albumin, prealbumin, creatinine, blood urea nitrogen (BUN), high-sensitivity C-reactive protein (hs-CRP), lipid battery, KT/V, and normalized protein nitrogen appearance (nPNA), were reviewed in Part I and measured in Part II. Clinical and demographic data, including age, sex, duration of hemodialysis, presence of cardiovascular disease, and presence of diabetes mellitus, were also recorded. Part I: During the follow-up period of 5 years, there were 95 all-cause deaths among the 289 patients. Comparison of survivors and non-survivors indicated that serum beta2-M was higher in survivors (36.8 ± 12.3 vs. 32.6 ± 13.2 µg/ml, p = 0.009). Kaplan-Meier analysis indicated that all-cause mortality in the lower beta2-M group was significantly higher compared to the higher beta2-M group (p < 0.0001). Multivariate Cox regression analyses indicated elevated beta2-M levels were significantly associated with lower mortality rate (relative risk: 0.608; 95% CI: 0.37 to 0.99; p = 0.046). Part II: The mean serum beta2-M concentration was 37.1 ± 14.4 µg/ml. Univariate analysis indicated that beta2-M was positively correlated with nPNA, duration of HD, BMI, and the concentrations of creatinine, albumin, BUN, and hs-CRP, but was negatively correlated with HDL-C concentration. Multiple regression analysis indicated that levels of nPNA (p < 0.001), duration of hemodialysis (p < 0.001), creatinine (p < 0.001), albumin (p = 0.006), BUN (p = 0.011), and HDL-C (p = 0.038) were independently associated with serum beta2-M concentration. Our results showed that higher serum beta2-M levels are associated with better survival in CHD patients and that nutritional status might be an independent predictor of serum beta2-M concentration in these patients.

Prognostic Impact of Cytogenetic Abnormalities On Outcomes of Newly Diagnosed Multiple Myeloma Patients Treated with Thalidomide-Dexamethasone Incorporated Into Double Autologous Stem-Cell Transplantation: An Analysis of 593 Patients

AbstractAbstract 3562Aim of the present study was to evaluate the clinical outcome of a large series of younger patients with symptomatic multiple myeloma (MM) who were enrolled in two subsequent clinical trials of thalidomide-dexamethasone (thal-dex) incorporated into double autologous stem-cell transplantation (ASCT) to support high-dose melphalan (200 mg/m2). In both studies, thal (100 mg/day for the first 14 days and then 200 mg/day) and pulsed dex (between 480 and 160 mg per cycle), were administered from the onset until the second ASCT.The analysis was performed on an intention-to-treat basis on a total of 593 patients who were followed for a median of 36 months. The best VGPR and CR rates were 69% and 35%, respectively. The median duration of CR was 66 months. Median TTP and PFS were 53 and 44 months, respectively. The 5-year projected rates of TTP and PFS were 46% and 38%, respectively, while the corresponding value for OS was 67%.More than 80% of the patients were screened at diagnosis for the presence of cytogenetic abnormalities by FISH analysis. Forty-five percent of patients had del(13q), while t(4;14) and del(17p) were found in 16 % and 7 % of patients, respectively. The presence of del(17p) and/or t(4;14) was associated with a significantly shorter 5-year projected TTP, PFS and OS in comparison with the absence of these abnormalities, indifferently from the presence or absence of del(13q) (TTP: 30% vs 53%, respectively P=0.0000; PFS: 28% vs 45%, respectively, P=0.0000; OS: 53% vs 69%, respectively, P=0.0000). OS and PFS curves of patients carrying del(13q) alone were almost superimposable to those of patients without cytogenetic abnormalities, while TTP was significantly shorter for patients with del(13q) alone (5-year projected rates: 40% vs 53%, respectively, P=0.04). Patients carrying del(17p) in the absence of t(4;14) had similar 5-year projected TTP and PFS as compared with t(4;14) positive but del(17p) negative patients. However, OS was significantly shorter for the subgroup with del(17p) and absence of t(4;14) in comparison with that of patients carrying t(4;14) without del(17p) (5 year projected rates: 18% vs 70%, respectively, P=0.03). In a multivariate analysis, presence of del(17p) and high beta2-m at baseline were the most important variables adversely influencing TTP (HR: 2.3, P=0.001 and HR: 1.8, P=0.002, respectively), PFS (HR: 2.0, P=0.001 and HR: 1.9, P=0.001, respectively), and OS (HR: 3.9, P=0.000 and HR: 2.0, P=0.005, respectively). Additional variables predicting for shorter TTP and PFS were the presence of t(4;14) (HR: 1.8, P=0.004) and of del(13q) (HR: 1.6, P= 0.009). Also the quality of best response to the overall treatment program influenced clinical outcomes. In particular, patients achieving CR had a significantly longer PFS and OS than those achieving a VGPR (PFS: median 68 vs 40 months, respectively, P=0.007; 5-year projected OS rates: 84% vs 70%, respectively, P=0.01).In conclusion, incorporation of thal-dex into double autotransplantation failed to overcome the poor prognosis conferred by del(13 q), t(4;14) and del(17p). In a multivariate Cox regression analysis, del(17p) and high levels of serum beta2-m at diagnosis were the strongest variables adversely influencing PFS and OS. In comparison with the presence of t(4;14) but absence of del(17p), patients carrying del(17p) without t(4;14) had a significantly shorter OS, possibly due to their worst outcome after relapse. Presence of del(13q) alone conferred a significantly shorter TTP, but did not have an adverse impact on OS due to the favorable role of effective salvage therapies incorporating either bortezomib or lenalidomide. Disclosures:Off Label Use: use of first line thalidomide in preparation for ASCT. Cavo:Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, no; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, no; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, no.

Combination of Ofatumumab and Lenalidomide In Patients with Relapsed Chronic Lymphocytic Leukemia: Initial Results of a Phase II Trial

AbstractAbstract 2464Frontline chemoimmunotherapies induce high response rates in patients with CLL. Once disease recurs, however, effective treatment options are limited and new therapeutic modalities and combinations are needed. Ofatumumab is a fully humanized anti-CD20 monoclonal antibody which produces an overall response rate (ORR) of 47%–58% in patients with fludarabine-refractory CLL (Wierda W. et al, 2010). Lenalidomide, an immunomodulatory agent, induces an ORR of 32–47% in patients with relapsed/refractory CLL, (Chanan-Khan A.A. et al. 2006; Ferrajoli A. et al. 2008). The rationale for combining ofatumumab and lenalidomide is based on their single agent efficacy, distinct and potentially complimentary mechanisms of action and non-overlapping toxicity profiles. Furthermore, the combination of lenalidomide and rituximab has shown significant activity in patients with relapsed disease (Ferrajoli et al. 2009). We, therefore, designed a phase II study to evaluate efficacy and tolerability of ofatumumab and lenalidomide given in combination in patients with relapsed CLL. Patients with active disease were eligible if they had received prior treatment with purine analog-based therapy, had an ECOG/WHO performance status of 0–2, adequate renal (creatinine clearance > 30ml/min) and hepatic function (total bilirubin < to 2 mg/dl and ALT < 2 × ULN). Patients with any neutrophil count were eligible, whereas patients with platelet counts < 30,000 mm3, positivity for HIV, active hepatitis B or C or recent history of tuberculosis were excluded from participation. In this trial ofatumumab is administered intravenously weekly for four consecutive weeks (300mg week 1, 1,000 mg week 2 and all subsequent doses), then monthly for months 2–6 and once every two months for months 7–24. Lenalidomide is given orally at the dose of 10 mg daily, starting on day 9 and continued daily. Allopurinol at the dose of 300mg daily is given during the first two weeks of treatment as tumor lysis prophylaxis. Treatment duration is 24 months, and responses are assessed after 3, 6, 12, 18 and 24 months of therapy. Thus far 26 of the 40 planned patients have been accrued to this study and we present an analysis of response and toxicity for the first 16 patients that have been on study for at least 3 months. The median age of the patients is 62 yrs (45–82). Eight patients (50%) had Rai stage III-IV disease. The median Beta-2M level was 4.4 mg/dL (2–6.1). The median number of prior treatments was 2 (1–8). Four patients (25%) were refractory to fludarabine and all pts had received prior rituximab. Nine patients (56%) had unmutated IGHV genes, 5 patients (31%) had chromosome 17p deletion and 3 patients (19%) had 11q deletion as detected by FISH analysis. Responses were evaluated according to the 2008 IWCLL criteria: 10 of the 16 evaluable patients achieved a response [2 CR (13%), 8 PR (50%)] for an ORR of 63%. Four patients with stable disease are continuing on treatment. One patient discontinued therapy and did not return for response assessment and another patient progressed. All patients are alive.The most common grade 3–4 treatment related adverse events observed were: neutropenia (8 pts, 50%) and anemia (2 pts, 13%). One patient (6%) developed grade 2 superficial vein thrombosis. Lenalidomide-associated tumor flare reaction was limited to grade 1 in 2 patients (13%) while a grade 3 infusion reaction was observed in 1 patient (6%) during the first ofatumumab administration. Three grade 3 infectious episodes occurred: 2 cases of pneumonia and 1 case of parotiditis. None of the patients received routine antibiotic prophylaxis. The median daily dose of lenalidomide tolerated was 5 mg/day (2.5–10 mg).In conclusion, our initial analysis indicates that the combination of ofatumumab and lenalidomide is therapeutically active in patients with relapsed CLL. This treatment is well tolerated. Neutropenia is the most common toxicity observed. Enrollment is ongoing, and updated results will be provided. Disclosures:Off Label Use: Ofatumumab and lenalidomide in patients with relapsed chronic lymphocytic leukemia. O'Brien: GlaxoSmithKline: Consultancy. Wierda: GlaxoSmithKline: Honoraria, Research Funding; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees. Estrov: Celgene Corporation: Consultancy. Keating: Celgene Corporation: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria. Ferrajoli: Celgene Corporation: Research Funding; GlaxoSmithKline: Research Funding.

Estimated glomerular filtration rate in the nephrotic syndrome

Plasma creatinine concentration and creatinine-based equations are most commonly used as markers of glomerular filtration rate (GFR). The abbreviated MDRD formula is considered the best available formula. Altered renal handling of creatinine, which may occur in the nephrotic syndrome, will invalidate creatinine-based formulas. We have evaluated the abbreviated MDRD formula in a large cohort of patients with proteinuria. Data on a cohort of patients with glomerular diseases were available from a large database. We have studied the relationship between estimated GFR (MDRD formula), and plasma cystatin C (CysC) and plasma beta-2-microglobulin (β2m) as markers of GFR. The final analysis included 142 patients (93 M/49 F), median age 48 years (±15), plasma creatinine 101 μmol/L (42-368), plasma albumin 28.0 g/L (10.0-47.0), proteinuria 6.4 g/day (0.03-37.9), eGFR-MDRD4 64 mL/min/1.73 m2 (15-165), β2m 3.43 mg/L (0.7-13.8) and CysC 1.14 mg/mL (0.56-4.00). As expected, we observed a hyperbolic relationship between eGFR and both β2m and CysC. In multivariable analysis, plasma albumin concentration proved to be the most important predictor of the relationship between eGFR and both CysC and β2m. In the presence of hypoalbuminaemia, eGFR was ~ 30-40% higher at equal levels of plasma CysC or β2m. Conclusions were similar when using the recently developed CKD-EPI formula. Plasma albumin concentration did not effect the relationship between eGFR estimated by the six-variable original MDRD formula and β2m. Our data point to discrepancies between eGFR using the six-variable MDRD formula and eGFR using the abbreviated MDRD formula as well as the CKD-EPI formula in patients with hypoalbuminaemia. One should be aware of possible limitations of creatinine-based eGFR formulas in patients with a nephrotic syndrome.

長期透析症例における破壊性脊椎関節症(DSA)の経年変化に関する検討(第1報)

This study was undertaken to evaluate destructive spondylarthropathy (DSA) in patients long-term dialysis. The patients were investigated by plain X-ray examinations twice with a three-year interval, and the changes in severity of the illness in 66 patients undergoing dialysis for over 5 years were defined. Group A (9 patients) consisted of patients with progression in grades during the three years, while group B (57 patients) consisted of patients without deterioration. There was no differences in the two groups in age, duration and the modality of dialysis. However, in group A, the level of plasma beta 2-microglobulin (beta 2M) was 33.5 +/- 14.6 micrograms/ml at the first observation, and 33.1 +/- 15.1 micrograms/ml at the second (NS). In group B, the level of plasma beta 2M was 45.6 +/- 8.13 micrograms/ml at the first observation, and 39.7 +/- 9.18 micrograms/ml at the second (p <0.05). No improvement in DSA was observed. The result of this study suggests that DSA will not improve once it has become overt, and the management for lowering the level of beta 2M may prevent the occurrence and progression of DSA.

Effective removal of leptin via hemodiafiltration with on-line endogenous reinfusion therapy

Leptin is a middle-molecular weight uremic toxin. Hemodiafiltration with on-line endogenous reinfusion (HFR) is a novel dialytic method combining the processes of diffusion, convection and adsorption. We performed a prospective crossover study of patients with end-stage renal disease to investigate the effect of HFR therapy on the level of leptin as compared to conventional low flux hemodialysis (LHD). Eleven stable hemodialysis patients were treated with LHD for 12 weeks and then treated with HFR (SG30 Plus; Sorin Group Italia S.r.1, Mirandola, Italy) for 12 weeks. After 12 weeks of HFR treatment, serum leptin levels significantly decreased (17.1 (2.66 - 39.5) at Week 12 vs. 12.3 (1.80 - 24.3) ng/ml at Week 24, p = 0.014). Although serum adiponectin levels also decreased (1.66 (1.44 - 1.86) at Week 12 vs. 1.12 (0.79 - 1.34) g/ml at Week 24, p = 0.001), the ratio of leptin to adiponectin did not increase after HFR treatment. Serum beta2-microglobulin (beta2M) levels significantly decreased (37.7 (29.8 - 42.6) at Week 12 vs. 28.3 (26.5 - 32.2) mg/dl at Week 24, p = 0.002). Dry weight, Kt/V(urea), normalized protein equivalent of nitrogen appearance, subjective global assessment, and serum albumin levels of the patients were not changed after HFR treatment. There was no difference in the serum levels of C-reactive protein or interleukin-6 between Week 12 and Week 24. The results of our study indicate that HFR may be a better therapy than LHD for removal of middle-molecular-weight uremic toxins such as leptin and b2M.

Combination Therapy with Lenalidomide and Rituximab in Patients with Relapsed Chronic Lymphocytic Leukemia (CLL).

Abstract 206Lenalidomide is an immunomodulatory agent that has clinical activity in CLL. In patients (pts) with relapsed/refractory CLL treatment with single agent lenalidomide induces an overall response rate of 32–47% when used as monotherapy (Chanan-Khan A.A. et al. 2006; Ferrajoli A. et al. 2008). Rituximab has modest activity as monotherapy, but significally synergizes with chemotherapy agents when administered to pts with CLL. The addition of rituximab to lenalidomide resulted in clinical responses in a small number of pts with CLL that had progressed while on lenalidomide monotherapy (Chanan-Khan A.A. et al. 2006). Because lenalidomide stimulates NK cell proliferation (Wu et al. 2008) we hypothesized that lenalidomide will enhance the activity of rituximab.We, therefore, designed a phase II study to evaluate the combination of lenalidomide and rituximab in pts with relapsed CLL.Pts with CLL and active disease were eligible if they had received prior treatment with purine analog-based therapy. Standard inclusion criteria were required in terms of organ function and performance status, and pts with any ANC or platelet count were eligible.All pts received rituximab (375 mg/m2) intravenously on days 1, 8, 15 and 22 of cycle 1, and then once every 4 weeks during cycles 3–12. Lenalidomide was given orally at the dose of 10 mg/day starting on day 9 of cycle 1 and continued daily for 12 cycles. Each cycle consisted of 28 days of treatment. During the first two weeks of therapy, allopurinol at the dose of 300 mg daily was prescribed as prophylaxis for tumor lysis.Sixty pts were accrued between October 2008 and July 2009. Thirty-seven pts have received treatment for at least 6 cycles and are evaluable for response and toxicity. The median age is 59 yrs (range 44–83), 15 pts (41%) have Rai stage III-IV disease and the median beta-2M level was 3.6 mg/dL (1.5–9). The median number of prior treatments was 2 (1–9), 9 pts (24%) were refractory to fludarabine and all pts had received prior rituximab. Twenty-six pts (70%) had unmutated IgVH, 9 pts (24%) had chromosome 17p deletion and 10 pts (37%) had 11q deletion by FISH analysis.After 6 cycles of treatment, 25 pts achieved a response [6 nodular PR (16%), 19 PR (51%)] for an OR of 68% (according to 1996 NCI-WG criteria). Six pts (16%) attained stable disease or clinical improvement and are continuing on treatment, and 6 pts (16%) failed to respond, including one death that occurred on day 34 owing to infectious complications. Responses according to pts characteristics are summarized in the table: [Display omitted] Most common grade 3–4 treatment related adverse events observed were: neutropenia (16 pts, 43%), fatigue (6 pts,16%) and thrombocytopenia (4 pts, 11%). One pt (3%) developed grade 3 tumor lysis syndrome and 1 pt (3%) had grade 3 joint pain. Infectious complications occurred in 9 pts (24%): neutropenic fever (6 pts), pneumonia (2 pts) and urosepsis (1 pt). Lenalidomide-associated tumor flare reaction was limited to grade 1 (8 pts, 22%) and grade 2 (1 pt, 3%).We examined the effect of therapy with lenalidomide and rituximab on the distribution of circulating B, T, and NK cell subsets. When compared to the baseline, there were significant decreases in the percentage of CD19+CD20+ B cells along with significant increases in the percentages of CD4+ T, CD8+ T, CD4+CD25hiCD127− regulatory T, and CD3−CD16+CD56+ NK cells after 3 cycles of therapy (paired sample t test).In conclusion, our results suggest that the combination of lenalidomide and rituximab is superior to single agent lenalidomide, despite all our pts having received prior rituximab. Additionally, there was no increase in toxicity and lenalidomide-associate tumor flare reaction was less frequent and less severe with this combination compared to single agent lenalidomide. Disclosures:Ferrajoli:Celgene: Research Funding; Genentech: Research Funding. Off Label Use: The use of Lenalidomide for the treatment of CLL is considered investigational.. O'Brien:Celgene: Consultancy; Genentech: Research Funding. Wierda:Genentech: Honoraria; Celgene: Speakers Bureau. Keating:Celgene: Data Monitoring Committee, Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees.

Role of Residual Kidney Function and Convective Volume on Change in β2-Microglobulin Levels in Hemodiafiltration Patients

Removal of beta2-microglobulin (beta2M) can be increased by adding convective transport to hemodialysis (HD). The aim of this study was to investigate the change in beta2M levels after 6-mo treatment with hemodiafiltration (HDF) and to evaluate the role of residual kidney function (RKF) and the amount of convective volume with this change. Predialysis serum beta2M levels were evaluated in 230 patients with and 176 patients without RKF from the CONvective TRAnsport STudy (CONTRAST) at baseline and 6 mo after randomization for online HDF or low-flux HD. In HDF patients, potential determinants of change in beta2M were analyzed using multivariable linear regression models. Mean serum beta2M levels decreased from 29.5 +/- 0.8 (+/-SEM) at baseline to 24.3 +/- 0.6 mg/L after 6 mo in HDF patients and increased from 31.9 +/- 0.9 to 34.4 +/- 1.0 mg/L in HD patients, with the difference of change between treatment groups being statistically significant (regression coefficient -7.7 mg/L, 95% confidence interval -9.5 to -5.6, P < 0.001). This difference was more pronounced in patients without RKF as compared with patients with RKF. In HDF patients, beta2M levels remained unchanged in patients with GFR >4.2 ml/min/1.73 m2. The beta2M decrease was not related to convective volume. This study demonstrated effective lowering of beta2M levels by HDF, especially in patients without RKF. The role of the amount of convective volume on beta2M decrease appears limited, possibly because of resistance to beta2M transfer between body compartments.

Low-flux hemodialysis suppresses interferon-gamma production: the possible role of beta2-microglobulin

Interferon-gamma (IFN-gamma) is required for the acquired immune response involving maturation and activation of antigen-presenting cells (APC); both IFN-gamma production and activation of APC are impaired in ESRD patients on low-flux hemodialysis (HD). High levels of uremic toxins including beta2-microglobulin (beta2M) are thought to play a role in immunosuppression. To test this hypothesis, we conducted an A-B-A crossover study to examine the influence of high-flux HD (A) versus low-flux HD (B) in ESRD patients (n = 14) using biocompatible synthetic dialyzer membranes (Polyamix(R), Gambro, Hechingen, Germany) and ultrafiltered bicarbonate-buffered dialysis fluid. Each study period lasted 6 months and at the end of each period, Kt/V urea, plasma levels of albumin, C-reactive protein (CRP) and beta2M were determined. In addition, production of IFN-gamma induced by heat-killed Staphylococcus epidermidis (Staph epi) was performed in whole blood cultures. Kt/V urea (mean +/- SEM) was 1.45 +/- 0.06 in Period A, 1.36 +/- 0.07* in Period B, and 1.51 +/- 0.07 in Period A' (*p < 0.01). beta2M resting levels increased from 26.6 +/- 1.42 mg/l in Period A to 34.7 +/- 2.30* mg/l in Period B, and decreased to 21.6 +/- 1.07 mg/l at the end of Period A' (*p < 0.0001). Albumin and CRP levels did not differ significantly. Staph epi-induced IFN-gamma production was 1.2 +/- 0.39 ng/1,000 PBMC at the end of Period A, 0.34 +/- 0.10 ng/1,000 PBMC* in Period B, and 2.71 +/- 0.67 ng/1,000 PBMC at the end of Period A' (*p < 0.05). There was an inverse correlation between b2M levels and whole blood IFN-gamma production (R2 = 0.26). High levels of uremic toxins such as beta2M suppress IFN-gamma production in ESRD patients under low-flux HD. High-flux HD reduces beta2M levels by 30% and improves IFN-gamma production suggesting an improved cellular immune response in ESRD patients.

Immune activation and antibody responses in non‐progressing elite controller individuals infected with HIV‐1

An extremely rare subset of patients infected with HIV-1 designated as "non-progressing elite controllers" appears to be able to maintain stable CD4(+) T-cell counts and a median plasma viremia below the detection limit of current ultrasensitive assays (<50-80 copies/ml of plasma) for >10 years in the absence of antiretroviral therapy. Lymphocyte subsets (CD4(+), CD8(+)), immune activation markers (HLA-DR(+), CD38(+), Beta-2-microglobulin), and HIV-specific antibody responses were longitudinally examined in four non-progressing elite controllers over more than 5 years. Two control groups of seronegative healthy individuals and untreated patients infected with HIV-1 presenting detectable viremia were also included. None of the non-progressing elite controllers displayed the high T-cell activation levels generally seen in the seropositive individuals, keeping them within the normal range. Three non-progressing elite controllers showed no significant immune system abnormalities when compared to seronegative individuals, displaying a low proportion of HIV-1-specific binding antibodies and low avidity index, similar to those observed for individuals infected recently with HIV-1. One non-progressing elite controller exhibited CD8(+) T-cell counts and beta2-M levels above normal ranges and developed a low but "mature" (high-avidity) HIV-1-specific antibody response. Thus, the non-progressing elite controllers are able to maintain normal T-cell activation levels, which may contribute to prevent, or greatly reduce, the damage of the immune system typically induced by the HIV-1 over time. They are, however, immunologically heterogeneous and very low levels of antigen exposure seem to occur in these patients, sufficient for sustaining a low, but detectable, HIV-1-specific immunity.

Human-like mouse models for testing the efficacy and safety of anti-beta2-microglobulin monoclonal antibodies to treat myeloma.

We showed recently that anti-beta2-microglobulin (beta2M) monoclonal antibodies (mAb) have remarkably strong apoptotic effects on myeloma cells in vitro and in SCID-hu mice. However, whether the mAbs will be therapeutic and safe in the treatment of myeloma patients, in whom every tissue expresses low densities of MHC class I molecules and elevated levels of soluble beta2M are present, remains to be determined. In this study, human-like myeloma mouse models (HLA-A2-transgenic NOD/SCID mice) were developed, which express mature and functional human MHC class I (HLA-A2 and human beta2M) on murine organs and present high levels of circulating human beta2M derived from human myeloma cells. Myeloma-bearing mice were treated intraperitoneally with anti-beta2M mAbs, and the distribution and effects of the mAbs on normal organs and established tumors were examined. Our results show that anti-beta2M mAbs were effective in suppressing myeloma growth in treated mice. The therapeutic efficacy of the mAbs in these mice are comparable with those observed in myeloma-bearing nontransgenic NOD/SCID mice in which no human MHC class I is expressed on murine organs. Furthermore, although the mAbs can be detected on different organs, no tissue damage or cell apoptosis was observed in the mice. Based on the antimyeloma efficacy and low toxicity in the mice, our study suggests that anti-beta2M mAbs may be safe and the tissue-expressing and soluble beta2M may not compromise their therapeutic effects in myeloma patients. This study provides further support for the future application of the mAbs as therapeutic agents for multiple myeloma.

CSF follow-up in HIV-1 infection: intrathecal production of HIV-specific and unspecific IGG, and beta-2-microglobulin increase with duration of HIV-1 infection.

Ninety-nine sequential cerebrospinal fluid (CSF) samples from 28 human immunodeficiency virus-1 (HIV-1)-infected patients were analyzed during the follow-up of 9 months to 4 years. Intrathecal synthesis of HIV-antibodies and IgG (p < 0.01), and the levels of beta-2-microglobulin (beta 2m) in the CSF (p < 0.05) and serum (p < 0.01) increased with duration of HIV-1 infection. No effect of duration of HIV-1 infection was observed on the individual CSF white cell counts and the levels of blood-brain-barrier (BBB) permeability. In 13 patients with HIV-1-associated central nervous system (CNS) disease, the effect of duration was seen as an increase of the individual beta 2m levels in serum (p < 0.01). Moreover, 7 of 9 patients who developed neurological disease or showed its progression during the study increased the level of beta 2m in the CSF. All of them increased the level of beta 2m in serum. In 15 neurologically healthy subjects, the effect of duration was expressed as an increase of the level of individual beta 2m in CSF (p < 0.05) and intrathecal IgG synthesis (p < 0.01). In the AIDS group, the level of beta 2m in the CSF increased, but in less severe stages the dependency of the individual CSF parameters on disease duration was not found. Our results indicate that elevated levels of beta 2m in CSF and serum appear to predict progression of neurological and systemic diseases, respectively. Elevated beta 2m in the CSF of clinically intact individuals may indicate subclinical neurological disease caused by HIV-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble syndecan-1 (sCD138) as a prognostic factor independent of mutation status in patients with chronic lymphocytic leukemia.

Syndecan-1 (sCD138) is a transmembrane heparan sulfate-bearing proteoglycan expressed in epithelial cells as well as hematopoietic cells that demonstrate plasmacytoid differentiation. Higher levels of sCD138 correlate with poor outcome in myeloma. We examined the association of circulating sCD138 levels in plasma with clinical behavior in 104 patients with chronic lymphocytic leukemia. sCD138 levels were significantly higher in patients (median, 52.8 ng/ml; range, 13.4-252.7 ng/ml) than in healthy control subjects (median, 19.86; range, 14.49-33.14 ng/ml) (P < 0.01). Elevated sCD138 (>median, 52.8 ng/ml) was associated with significantly shorter survival (P = 0.0004); this association was independent of IgVH mutation status, beta2-microglobulin (beta2-M) level, and treatment history. Patients with mutated IgVH but high sCD138 levels (>52.8 ng/ml) had significantly shorter survival than those with mutated IgVH and lower levels of sCD138. Similarly, patients with unmutated IgVH but high sCD138 levels had significantly shorter survival than those with lower sCD138 levels and unmutated IgVH (P = 0.007). In a multivariate Cox regression model, only Rai stage, beta2-M, and sCD138 remained predictors of survival. These data suggest that sCD138 when combined with beta2-M and Rai stage, may replace the need for testing IgVH mutation status.

Expression of PRAME and WT1 in Multiple Myeloma Patients during High Dose Chemotherapy and Auto-SCT

High dose therapy enables further improvement in the outcome of multiple myeloma (MM) patients. However, it is still necessary to determine prognostic factors that may influence treatment results and provide additional criteria for the precise selection of treatment approaches. There are several tumor associated genes (MAGE, LAGE, GAGE, PRAME) that are over-expressed in different malignancies including MM. These genes are believed to modulate cancer properties and should be taken into account during treatment. Their significance as prognostic factors is under investigation. The aim of our study was to analyze the expression levels of PRAME and WT1 genes in MM patients during high dose chemotherapy following by auto-SCT. After having informed consent 25 primary MM patients were included into this study. The median age was 48 years (range, 31–62). All patients were treated by 3 cycles of VAD, Cyclophosphamide 6g/m2 + G-CSF to mobilize Stem cells, EDAP, melphalan 200 mg/m2 followed by auto-SCT. As second line therapy we used bortezomib+dexamethasone. Quantitative PRAME and WT1 gene expression analysis was performed by means of RQ-PCR. Results were normalized against expression of ABL gene which was used as internal control. Investigation was performed before treatment (n=25), after three VAD cycles (n=12), and before (n=5) and after auto-SCT (n=4). In primary MM patients: PRAME gene expression was found in 68% (n=17), WT1 in 24% (n=6) of patients, all of whom were PRAME-positive. Median expression levels were 0.1% (0.001–132%) for PRAME and 0.01% (0.002–0.07%) for WT1. PRAME and WT1 expression did not correlate with tumor bulk and was independent of the levels of M-protein, beta-2M and albumin. The expression of PRAME significantly decreased after 3 VAD cycles to 0.001–207% (n=8), at the moment of auto-SCT it was 0.05–6.1% (n=3) and after auto-SCT it was 0.013–4.9% (n=3). However for WT1, we observed increased of WT1 expression after 3 VAD cycles to 0.004-0.05% (n=4)and at the moment of auto-SCT it was 0.035–0.4% (n=3) and after auto-SCT – 0.019–2.03% (n=3). In the patients with high primary PRAME expression (>median expression) the frequency of CR+PR was significantly lower then in PRAME-negative primary patients and in patients with low (<median expression) primary PRAME expression (55% vs 84, p = 0.04). It was found also that WT1-positive primary patients were bad responders and they achieved only minimal response after 3 VAD cycles. It should be stressed that during treatment in a small number of initially negative PRAME and WT1 gene patients, we demonstrated detection by PCR. We detected the appearance of gene expression at low levels in 1 of 8 initially negative PRAME (6.1%) and in 5 of 19 initially negative WT1 (range of level 0.01–0.4%). The detection of gene expression did not correlate with disease status. All these patients achieve CR+ VGPR. In one of these secondary positive patients (acquired PRAME and WT1) relapse occurred.Conclusion: Expression of PRAME gene was found in 68% primary patients and the level of PRAME decreased with tumor reduction. High expression level of PRAME turned out to be a factor of unfavorable prognosis. Expression of WT1 was found in 24% of MM patients all of whom were PRAME-positive. WT1 expression increased during treatment in a small group of pts. Some initially negative pts acquired PRAME and WT1 expression during treatment, but clinical relevance of it is not clear so far.