Bertram Opalka

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.

The amount of BCR-ABL fusion transcripts detected by the real-time quantitative polymerase chain reaction method in patients with Philadelphia chromosome positive chronic myeloid leukemia correlates with the disease stage

Abstract The use of the real-time reverse-transcription polymerase-chain reaction (RT-PCR) method to quantify BCR-ABL transcripts before and after allogeneic transplant was prospectively studied in 65 patients with chronic myeloid leukemia (CML). The expression of the BCR-ABL transcript was determined and normalized using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene product as an endogenous reference. In the single step real-time PCR assay, tenfold serial dilutions of cDNA of the K5652 cell line remained positive down to 100 pg cDNA only. However, molecular relapses of CML after transplant were only safely detectable when a nested real-time PCR assay was performed, which was able to detect 1–10 pg cDNA from a tenfold serial dilution. The median normalized BCR-ABL transcript level was measured as 0.004% in 17 patients with a molecular relapse, 0.4% in 7 patients with a cytogenetic relapse, 2.6% in 36 patients with a stable phase of CML, and 36% in 5 patients with a relapse in a blast crisis. The analyzed median normalized amount of BCR-ABL transcript differed significantly (P<0.001) between the various disease stages. In ten CML patients with relapse, the real-time PCR method was used to monitor the response of various immunotherapies as donor leukocyte infusions, withdrawal of immunosuppression, or interferon-α application. The results of the quantitative evaluation of BCR-ABL transcripts reflected very well the clinical effect of the different applied immunotherapies. The new real-time PCR method seems to be a suitable technique for the early detection of relapse after allogeneic transplant in patients with the BCR-ABL transcript. Its ability to distinguish between molecular and cytogenetic relapse (P<0.001) allows early therapeutic decisions.

Pluripotent stem cells escape from senescence-associated DNA methylation changes

Pluripotent stem cells evade replicative senescence, whereas other primary cells lose their proliferation and differentiation potential after a limited number of cell divisions, and this is accompanied by specific senescence-associated DNA methylation (SA-DNAm) changes. Here, we investigate SA-DNAm changes in mesenchymal stromal cells (MSC) upon long-term culture, irradiation-induced senescence, immortalization, and reprogramming into induced pluripotent stem cells (iPSC) using high-density HumanMethylation450 BeadChips. SA-DNAm changes are highly reproducible and they are enriched in intergenic and nonpromoter regions of developmental genes. Furthermore, SA-hypomethylation in particular appears to be associated with H3K9me3, H3K27me3, and Polycomb-group 2 target genes. We demonstrate that ionizing irradiation, although associated with a senescence phenotype, does not affect SA-DNAm. Furthermore, overexpression of the catalytic subunit of the human telomerase (TERT) or conditional immortalization with a doxycycline-inducible system (TERT and SV40-TAg) result in telomere extension, but do not prevent SA-DNAm. In contrast, we demonstrate that reprogramming into iPSC prevents almost the entire set of SA-DNAm changes. Our results indicate that long-term culture is associated with an epigenetically controlled process that stalls cells in a particular functional state, whereas irradiation-induced senescence and immortalization are not causally related to this process. Absence of SA-DNAm in pluripotent cells may play a central role for their escape from cellular senescence.

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.

Impact of interferon alpha-induced cytogenetic improvement on survival in chronic myelogenous leukaemia

Summary. The objective of this study was to investigate the prognostic impact of the reduction of Philadelphia chromosome (Ph) positive metaphases by treatment of chronic myelogenous leukaemia (CML) with interferon (IFN) alpha. Therefore, we evaluated the outcome of patients with previously untreated chronic phase Ph‐positive CML, enrolled from 1984 to 1990 into two consecutive IFN trials at our institution. Of a total of 71 patients, 62 (87%) were evaluable for cytogenetic response. No cytogenetic improvement was seen in 16 patients (23%), 28 patients (38%) had a decrease in Ph‐positive bone marrow metaphases to levels ranging from 35% to 95%, and nine patients (13%) to levels between 5% and 34%. In nine patients (13%), Ph‐positive metaphases were no longer detectable. After a median follow‐up period of 33 months, the projected 5‐year survival is 55% for the 62 patients evaluable for cytogenetic response. In this patient population there was no significant difference in the survival probability according to patients’risk status as defined by the Sokal score. Categorization according to the extent of Ph reduction, however, allowed three groups with significantly different prognoses to be identified. Patients achieving a Ph reduction to less than 35% were found to constitute a low risk group with a median survival not yet known and a projected 5‐year survival of 90%. The 5‐year survival rate was 55% for patients with a Ph reduction to levels between 35% and 95%, and less than 10% for those without any cytogenetic improvement. Thus, this study demonstrates that cytogenetic improvement on IFN treatment is an important prognostic factor for survival.

Loss of expression of the DRR 1 gene at chromosomal segment 3p21.1 in renal cell carcinoma

Consistent deletion of DNA sequences in chromosomal band 3p21 observed in a variety of human tumors suggests the presence of one or more tumor suppressor genes within this region. Previously, we reported on the construction of two distinct cosmid contigs and our identification of several new genes within 3p21.1. In our search for tumor suppressor genes from this region, we have cloned a gene that we have called DRR 1 (downregulated in renal cell carcinoma). The gene was first mapped to 3p21.1 by fluorescence in situ hybridization analysis. Further analysis of yeast artificial chromosome clones in 3p14.2–p21.1 refined its localization. DRR 1 spans about 10 Kb of genomic DNA with a 3.5‐Kb mature transcript. The putative protein encoded by this gene is 144 amino acids and includes a nuclear localization signal and a coiled domain. The gene showed loss of expression in eight of eight renal cell carcinoma cell lines, one of seven ovarian cancer cell lines, one of one cervical cancer cell line, one of one gastric cancer cell line, and one of one non–small‐cell lung cancer cell line. Southern blot analysis did not show any altered bands, indicating that gross structural changes or deletions did not cause the loss of expression. This gene was also found to have reduced expression in 23 of 34 paired primary renal cell carcinomas. Mutational analysis detected three polymorphic sites within the gene, but no point mutations were identified in the 34 primary tumors. However, we did detect base substitutions in 4 of 12 cell lines that had undetectable expression of the gene. We also transfected the gene into DRR 1–negative cell lines and observed clear growth retardation. Our results suggest that loss of expression of the DRR 1 gene may play an important role in the development of renal cell carcinoma and possibly other tumors. Genes Chromosomes Cancer 27:1–10, 2000.

Prognostic relevance of soluble human leukocyte antigen-G and total human leukocyte antigen class I molecules in lung cancer patients.

The aim of this study was to determine the prognostic significance of soluble human leukocyte antigen (HLA) class I (sHLA-I) and HLA-G molecules in lung cancer patients. A total of 23 small-cell lung cancer (SCLC) and 114 non-small-cell lung cancer (NSCLC) patients, including 55 adenocarcinoma, 46 squamous cell carcinoma (SCC), and 13 patients with undifferentiated carcinoma, were prospectively enrolled. Levels of sHLA-G and sHLA-I were analyzed by specific enzyme-linked immunosorbent assay. Median levels of sHLA-G and sHLA-I were significantly increased in patients compared with controls (34 ng/ml [3.6-160] vs 14 ng/ml [0-98], p < 0.0001; 2580 ng/ml [749-5770] vs 1370 ng/ml [274-2670], p < 0.0001, respectively). Regarding the different subgroups, patients with NSCLC or SCLC showed increased sHLA-I levels, whereas sHLA-G was exclusively elevated in NSCLC, especially in patients with SCC. Patients with sHLA-I<2800 ng/ml (p = 0.008) or sHLA-G<40 ng/ml (p = 0.073) showed prolonged overall survival (OS). Using these cut-offs in patients with SCC, a pronounced prognostic significance for sHLA-G (p = 0.003) and sHLA-I (p = 0.004) was observed for the prediction of OS. Here, multivariate analysis confirmed sHLA-G and sHLA-I in addition to disease stage as independent prognostic factors. The prognostic power was further enhanced by combining the two factors and comparing the OS of patients with low sHLA-I and low sHLA-G against the remaining ones. In conclusion, plasma levels of sHLA-G and sHLA-I are potent predictors for OS in lung cancer patients.

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.

Estimating the relapse stage in chronic myeloid leukaemia patients after allogeneic stem cell transplantation by the amount of BCR-ABL fusion transcripts detected using a new real-time polymerase chain reaction method

We have used a new single‐step real‐time reverse transcription polymerase chain reaction (RT‐PCR) method to quantify BCR‐ABL transcripts, thereby estimating the relapse stage in chronic myeloid leukaemia patients after allogeneic transplants. In 402 samples from 172 patients, BCR‐ABL expression was determined and normalized, using the GAPDH housekeeping gene product as an endogenous reference. In our real‐time RT‐PCR assay, serial dilutions of RNA of the K562 cell line remained positive down to 7·5 pg. The median normalized BCR‐ABL amount differed significantly (P < 0·001) between the various disease stages and was 0·06% (range 0·001–1·55%), 3·2% (range 1·4–5·6%) and 21·5% (range 6·8 −827%) in 17 patients with a molecular relapse, in eight patients with a cytogenetic relapse and in 10 patients with a haematological relapse respectively.

Combination therapy with interferon alpha‐2b plus low‐dose interferon gamma in pretreated patients with Ph‐positive chronic myelogenous leukaemia

Between March 1988 and July 1990, 28 adults with chronic myelogenous leukaemia (CML) were treated with a combination of recombinant human interferon (IFN) alpha‐2b s.c. (initial dose 4 x 106 U/m2) and recombinant human IFN gamma s.c. (50 μg totally) daily. All patients were in chronic phase disease and had been treated previously with chemotherapy or bone marrow transplantation. A complete haematologic remission was achieved in three patients (11%), a haematologic remission in 12 patients (43%), and a partial haematologic remission in seven patients (25%). Six patients did not respond to this schedule. Acute side‐effects were flu‐like symptoms, fever and chills. During long‐term treatment six patients developed poly‐arthralgia. Haematotoxicity WHO grade III occurred in three patients, and WHO grade IV in two patients. One patient developed psychosis, and in another patient an exacerbation of a pre‐existing sarcoidosis was observed. We conclude that this combination is tolerable and effective in inducing haematological remissions in pretreated CML patients.