Ulrike Buttkereit

Serum Free Light Chain Analysis and Urine Immunofixation Electrophoresis in Patients with Multiple Myeloma

Purpose: Retrospective studies have shown that immunoassays measuring free light chains (FLC) in serum are useful for diagnosis and monitoring of multiple myeloma. This study prospectively evaluates the use of FLC assays and, for the first time, investigates the relationship between serum FLC concentrations and the presence and detectability of Bence Jones (BJ) proteins in the urine. Patients and Methods: Three hundred seventy-eight paired samples of serum and urine were tested from 82 patients during the course of their disease. The sensitivities of serum FLC analysis and urine immunofixation electrophoresis (IFE) in detecting monoclonal FLC were compared. Serum FLC concentrations required for producing BJ proteins detected by IFE were determined. Results: Abnormal FLC were present in 54% of serum samples compared with 25% by urine tests. In abnormal serum samples for κ or λ, the sensitivity of IFE to detect the respective BJ proteins in urine were 51% and 35% and the median serum FLC concentrations required to produce detectable BJ proteins were 113 and 278 mg/L. Renal excretions of monoclonal FLC increased with serum concentrations, but excretions significantly decreased at high serum concentrations combined with renal dysfunction. Conclusion: Serum FLC assays are significantly more sensitive for detecting monoclonal FLC than urine IFE analysis. They also have the advantage of FLC quantification and are more reliable for monitoring disease course and response to treatment.

Immune parameters in multiple myeloma patients: influence of treatment and correlation with opportunistic infections.

The present study evaluated cellular and humoral immune parameters in myeloma patients, focusing on the effect of treatment and the risk of opportunistic infections. Peripheral blood lymphocyte subsets and serum levels of nonmyeloma immunoglobulins (Ig) were analysed in 480 blood samples from 77 myeloma patients. Untreated myeloma patients exhibited significantly reduced CD4+/45RO+, CD19+, CD3+/HLA-DR+, and natural killer (NK) cells, as well as nonmyeloma IgA, IgG and IgM. Conventional-dose chemotherapy resulted in significantly reduced CD4+ and even further decline of CD4+/CD45RO+ and CD19+ cells, most notably in relapsed patients. Additional thalidomide treatment had no significant effects on these parameters. Following high-dose chemotherapy (HD-CTX), prolonged immunosuppression was observed. Although CD8+, NK, CD19+ and CD+/CD45RO+ cells recovered to normal values within 60, 90, 360 and 720 days, respectively, CD4+ counts remained reduced even thereafter. Nine opportunistic infections were observed, including five cytomegalovirus (CMV) diseases, one Pneumocystis carinii pneumonia (PCP) and three varicella zoster virus infections with CMV diseases and PCP occurring exclusively after HD-CTX. Opportunistic infections were correlated with severely reduced CD4+, as well as CD4+/CD45RO+ and CD19+ counts. Thus, myeloma patients display cellular and humoral immunodeficiencies, which increase following conventional as well as HD-CTX, and constitute an important predisposing factor for opportunistic infections.

Thalidomide in combination with vincristine, epirubicin and dexamethasone (VED) for previously untreated patients with multiple myeloma

Abstract:  The present study aimed to evaluate the side‐effects and efficacy of thalidomide in combination with an anthracycline‐containing chemotherapy regimen in previously untreated myeloma patients. Thalidomide (400 mg/d) was combined with bolus injections of vincristine and epirubicin and oral dexamethasone (VED). Chemotherapy cycles were repeated every 3 wk until no further reduction in myeloma protein was observed, whereas the treatment with thalidomide was continued until disease progression. Thirty‐one patients were enrolled, 12 patients were exclusively treated with thalidomide in combination with VED and 19 patients additionally received high‐dose melphalan, for consolidation. Adverse events and response to therapy were assessed prior to treatment with high‐dose chemotherapy. Response to thalidomide combined with VED was complete remission in six patients (19%), partial remission in 19 patients (61%), stable disease in five patients (16%), and progressive disease in one patient (3.2%). Grade 3 and 4 adverse events consisted of leukocytopenia in 10 patients (32%), and thrombocytopenia and anemia in one patient each (3.2%). Neutropenic infections grade 3 and 4 occurred in seven (23%) and three patients (9.7%), respectively, including two patients (6.5%) who died from septic shock. Deep vein thrombosis occurred in eight patients (26%), constipation in 20 patients (65%), and polyneuropathy in 20 patients (65%). The probability of event‐free survival and overall survival in the whole group of patients at 36 months were 26 and 62%, respectively. In conclusion, the combination of thalidomide with VED appears to be highly effective in previously untreated patients with multiple myeloma, but it is associated with a high rate of thrombotic events, polyneuropathy, and neutropenic infections.

Clinical Utility of Quantitative PCR for Chimerism and Engraftment Monitoring after Allogeneic Stem Cell Transplantation for Hematologic Malignancies

Although quantitative PCR (qPCR) has been explored for chimerism monitoring after allogeneic stem cell transplantation (SCT), evidence regarding its clinical utility compared with standard short tandem repeat (STR) is still limited. We retrospectively studied commercial qPCR and STR chimerism with respective positivity thresholds of .1% and 1% in 359 peripheral blood (PB) and 95 bone marrow (BM) samples from 30 adult patients after first HLA-matched SCT for myeloid malignancies or acute lymphatic leukemia. Concordance between the 2 methods was 79.5%, with all discordant samples positive in qPCR but negative in STR. Of the latter, sporadic qPCR positivity without clinical correlates was seen mostly in BM samples early post-transplant. In 7 of 21 patients with available follow-up samples in the first months after transplantation, qPCR but not STR revealed low levels (<1%) of sustained host chimerism in PB, reflecting delayed engraftment or persistent mixed chimerism (PMC). These conditions were associated with donor-recipient cytomegalovirus (CMV) serostatus and early CMV reactivation but not with immunosuppressive regimens or clinical outcome. qPCR predicted all 8/8 relapses with samples in the 6 months before onset by sustained positivity in both PB and BM compared with 1/8 relapses predicted by STR mainly in BM. The response kinetics to donor lymphocyte infusions for the treatment of PMC or relapse was shown by qPCR but not STR to be protracted over several months in 3 patients. Our results demonstrate the superior clinical utility of qPCR compared with STR for monitoring subtle changes of host chimerism associated with different clinical conditions, making a case for its use in the clinical follow-up of transplant patients.

Sensitive detection of rare antigen-specific T cells directed against Wilms’ tumor 1 by FluoroSpot assay

Relapse after hematopoietic stem cell transplantation remains a major cause of mortality for patients with acute myeloid leukemia (AML). The generation of adoptive immunotherapies for treating relapse in AML patients is an emerging field. In AML, the Wilms’ tumor 1 (WT1) antigen – which is highly expressed in AML blasts but only at low level in healthy tissue – has been identified as a promising target for immunotherapy [1,2]. Our study focused on the identification and functional characterization of WT1-specific T cells that could lyse AML blasts. To detect these rare WT1-specific T cells, the Enzyme Linked Immuno Spot (ELISpot) assay was chosen because it is highly sensitive and detects cytokine secreting cells on a single-cell level. The standard assay uses a precipitating substrate and measures single cytokines; whereas a more recent, modified form, the FluoroSpot assay, determines fluorescence and simultaneously detects the secretion of different cytokines [3]. In the current study, we compared results of standard ELISpot and FluoroSpot assays in AML patients. To characterize the pro-inflammatory response, we measured the number of single and double positive cells secreting interferon (IFN)-c and/or granzyme B. After the study was approved by the Institutional Review Board and participants provided written informed consent, T cells of AML patients and healthy controls were isolated from peripheral blood mononuclear cells (PBMC) by microbeads and magnetic-assisted cell sorting (MACS) columns (Pan T Cell Isolation Kit, Miltenyi Biotech, Bergisch Gladbach, Germany). 150,000 freshly isolated T cells were cultivated in duplicates with and without a WT1 peptide pool (1 lg/mL Peptivator WT1, Miltenyi Biotech). This pool of 15-mer peptides covers the complete sequence of the WT1 protein. After overnight incubation in RPMI 1640 medium plus 10% pooled human serum (Transfusion Medicine, University Hospital Essen, Germany) using round-bottom plates, cells were transferred in parallel either to standard ELISpot plates containing polyvinylidene fluoride (PVDF) membranes or to Immobilon Plate Fluorescence Low (IPFL) plates (Human IFN-c/Granzyme B FluoroSpot kit, Mabtech, Nacka Strand, Sweden). T cells with and without WT1 stimulation were incubated on PVDF and IPFL ELISpot plates for further 48 h at 37 C. Processing of the PVDF plates followed a protocol described in detail previously [4]. FluoroSpot assays were performed according to the manufacturer’s instructions. In brief, after activation with 35% ethanol, IPFL plates were washed and coated overnight with 15 lg/mL of monoclonal antibodies against IFN-c and granzyme B. After further washing steps, the FluoroSpot plates were blocked with 200lL of RPMI 1640 medium plus 10% pooled human serum for 30min. Then, medium was removed and T cells with and without WT1 stimulation were transferred to FluoroSpot plates. After 48 h of incubation, the wells were washed to remove the cells and 100 lL/well of secondary detection antibodies, tagged with fluorescein isothiocyanate (FITC) for IFN-c and cyanine 3 (Cy3) for granzyme B, diluted in D-PBS and 0.1% bovine serum albumin buffer, were added to the wells at a concentration of 2 lg/mL and incubated for 2 h at room temperature. Thereafter, 100lL of diluted antifluorescent antibodies was added to the wells and plates were incubated for another hour at room temperature without light exposure. After further washing, 50 lL of fluorescence enhancer solution was added to each well and left there for 15min. After drying the plates, spot numbers on PVDF and IPFL plates were analyzed by an ELISpot plate reader (AID FluoroSpot, Autoimmun Diagnostika GmbH, Strassberg, Germany). Double positive cells emerge from a computerized overlay of spot images. Mean numbers of duplicate analyses were considered. Comparative analyses in 20 AML patients after hematopoietic stem cell transplantation showed that the FluoroSpot was more sensitive than the standard ELISpot in detecting the WT1-specific release of IFN-c and granzyme B (Figure 1(A)). Using the FluoroSpot assay, mean frequencies of 0.0023% (3.5/150,000) IFN-c and of 0.013%

αβ-T-cell depleted donor lymphocyte infusion for leukemia relapse after allogeneic stem cell transplantation

αβ-T-cell depleted donor lymphocyte infusion for leukemia relapse after allogeneic stem cell transplantation