Friedrich W. Cremer

Delineation of distinct subgroups of multiple myeloma and a model for clonal evolution based on interphase cytogenetics.

To delineate multiple myeloma (MM) subgroups and their clonal evolution, we analyzed 81 newly diagnosed patients by interphase fluorescence in situ hybridization using a comprehensive probe set for 10 chromosomes and two IGH rearrangements. A median of 5 probes per patient displayed aberrant signal numbers (range, 1–10). Additional copies most frequently found were for 15q22, 19q13, 9q34, 11q23, and 1q21. Losses commonly observed were of 13q14.3, 17p13, and 22q11. Predominance of gain or loss was quantified by a copy number score (CS) for each patient. Two peaks (CS = +3 and CS = 0) were found by plotting patient copy number scores over CS values corresponding to hyperdiploid and nonhyperdiploid MM. Cluster analysis revealed four major branches: (i) gain of 9q, 15q, 19q, and/or 11q; (ii) deletion of 13q and t(4;14); (iii) t(11;14); and (iv) gain of 1q. Statistical modeling of an oncogenetic tree indicated that early independent events were gain of 15q/9q and/or 11q, t(11;14); deletion of 13q followed by t(4;14); and gain of 1q. Aberrations of 17p13, 22q11, 8p12, and 6q21 were found as subsequent events. MM with gain of 1q was delineated as a subentity with significantly higher beta‐2‐microglobulin and lower hemoglobin levels, indicating a poor prognosis. From our results, we propose a model of MM for clonal evolution.

Expression of EGF-family receptors and amphiregulin in multiple myeloma. Amphiregulin is a growth factor for myeloma cells

A hallmark of plasma cells is the expression of syndecan-1, which has major functions in epithelial cells, in particular as the coreceptor of heparin-binding growth factors. We previously found that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a growth factor for malignant plasma cells. As amphiregulin (AREG) is another heparin-binding factor of the EGF family, we investigated its role in multiple myeloma (MM). Using Affymetrix DNA microarrays, we show here that the AREG gene was expressed by purified primary myeloma cells from 65 patients and that the expression was higher than in normal bone marrow (BM) plasma cells or plasmablastic cells. AREG stimulated IL-6 production and growth of BM stromal cells. Using real-time reverse transcriptase–polymerase chain reaction, we found that MM cells expressed ErbB receptors and that AREG promoted their growth. Furthermore, PD169540 (a pan-ErbB inhibitor) and IRESSA (an ErbB1-specific inhibitor) induced apoptosis of primary myeloma cells from 10/14 and 4/14 patients, respectively, and there was a synergistic effect with dexamethasone. Altogether, our data provide strong evidence that AREG plays an important role in the biology of MM and emphasize the advantages of using ErbB inhibitors, which might target myeloma cells as well as the tumor environment.

Heparan sulphate proteoglycans are essential for the myeloma cell growth activity of EGF-family ligands in multiple myeloma

The epidermal growth factor (EGF)/EGF-receptor (ErbB1-4) family is involved in the biology of multiple myeloma (MM). In particular, ErbB-specific inhibitors induce strong apoptosis of myeloma cells (MMC) in vitro. To delineate the contribution of the 10 EGF-family ligands to the pathogenesis of MM, we have assessed their expression and biological activity. Comparing Affymetrix DNA-microarray-expression-profiles of CD138-purified plasma-cells from 65 MM-patients and 7 normal individuals to those of plasmablasts and B-cells, we found 5/10 EGF-family genes to be expressed in MMC. Neuregulin-2 and neuregulin-3 were expressed by MMC only, while neuregulin-1, amphiregulin and transforming growth factor-α were expressed by both MMC and normal plasma-cells. Using real-time polymerase chain reaction, we found HB-EGF, amphiregulin, neuregulin-1 and epiregulin to be expressed by cells from the bone marrow-environment. Only the EGF-members able to bind heparan-sulphate proteoglycans (HSPGs) – neuregulin-1, amphiregulin, HB-EGF – promote the growth of MMC. Those ligands strongly bind MMC through HSPGs. The binding and the MMC growth activity was abrogated by heparitinase, heparin or deletion of the HS-binding domain. The number of HS-binding EGF ligand molecules bound to MMC was higher than 105 molecules/cell and paralleled that of syndecan-1. Syndecan-1, the main HSPG present on MM cells, likely concentrates high levels of HS-binding-EGF-ligands at the cell membrane and facilitates ErbB-activation. Altogether, our data further identify EGF-signalling as promising target for MM-therapy.

Post‐transplantation tumour load in bone marrow, as assessed by quantitative ASO‐PCR, is a prognostic parameter in multiple myeloma

High‐dose therapy (HDT) and autologous transplantation prolongs remission duration and survival in multiple myeloma (MM), but relapse still occurs at a median of 2 years post‐HDT. In order to investigate whether the number of residual tumour cells in the bone marrow (BM) after transplantation can predict the duration of response, a quantitative allele‐specific oligonucleotide polymerase chain reaction (qASO‐PCR) assay was used to measure tumour load in BM at 3–6 months post‐HDT in 67 patients. The method of maximally selected log‐rank statistics was used to test for the existence of a cut‐off value in the BM tumour load data set. A cut‐off value with respect to progression‐free survival (PFS) was identified (P = 0·001). The estimated threshold for placing patients into a ‘good’ or ‘bad’ prognostic group was 0·015% (n = 22 and 38 respectively) with a median PFS of 64 months vs. 16. Multivariate analysis showed grouping by PCR result to be an independent prognostic factor for PFS (estimated hazard ratio after shrinkage, 3·91). This study identifies for the first time a threshold of the post‐HDT tumour load with prognostic significance for PFS in MM. Quantitative molecular assessment thus may help to identify those patients who are in need of further treatment early after autologous transplantation.

Microarray-based understanding of normal and malignant plasma cells

Summary:  Plasma cells (PCs) develop from B lymphocytes following stimulation by antigen and express a genetic program aimed at the synthesis of immunoglobulins. This program includes the induction of genes coding for transcription factors such as PRDM1, X‐box‐binding protein 1 and BHLHB3, cell‐surface molecules such as CD138/syndecan‐1, and for the unfolded protein response. We review how the microarray technology has recently contributed to the understanding of the biology of this rare but essential cell population and its transformation into premalignant and malignant PCs.

Evaluation of the serum-free light chain test in untreated patients with AL amyloidosis

The diagnostic value of serum electrophoresis and urinary light chain excretion in AL amyloidosis is poor due to the usually low amount of monoclonal protein. This study evaluates immunoglobulin light chains in the serum of a large series of untreated patients with systemic AL amyloidosis, and proves that free light chain levels reflect disease severity. We evaluated the Serum Free Light Chain (FLC) test in a series of 133 untreated patients with systemic AL amyloidosis. The FLC test detected the monoclonal gammopathy in 87% compared with 92% for immunofixation of serum and urine in combination. However, both tests proved complementary. The FLC test was also a valuable tool in patients with advanced renal failure in spite of uninvolved light chain retention. Higher FLC levels were associated with higher bone marrow plasmocytosis, poorer Karnofsky index and heart involvement, and therefore reflected disease severity.

Comprehensive Genetic Counseling for Families At Risk for HNPCC: Impact on Distress and Perceptions

The aim of the study was to explore distress and health beliefs before and after comprehensive interdisciplinary counseling in families at risk for hereditary non-polyposis colorectal cancer (HNPCC). Results reported here were derived from a consecutive sample of 65 counselees [31 patients with colorectal cancer (CRC) and 34 unaffected at-risk persons] who participated in interdisciplinary counseling provided by human geneticists, surgeons, and psycho-oncologists before genetic testing. Data were collected from self-administered questionnaires before, as well as 4-6 weeks after, counseling. Distress and perceptions specific to HNPCC were assessed at both timepoints using standardized as well as author-derived instruments. Distress declined after counseling, as did worries related to HNPCC. An increase was found in personal belief in control of cancer risk, for instance, in the perceived efficacy of early detection of CRC. We also observed a trend toward greater anticipated ability to cope with a positive gene test after counseling. Changes after counseling were generally more pronounced for persons at risk, as compared to patients with cancer. The decrease in distress was partly attributable to an increase in personal self-confidence. One-third of the sample reported enhanced communication specific to hereditary disease within the family after counseling. A substantial minority, however, said they experienced increased worry and physical symptoms after counseling. Overall, counselees demonstrated less stress and perceived cancer threat as well as enhanced beliefs regarding personal control over cancer, suggesting an overall beneficial impact of comprehensive counseling. Further research is needed to identify those individuals most at risk for increased fear and worry related to HNPCC so that they may be most appropriately counseled.

Autografting with CD34+ peripheral blood stem cells: retained engraftment capability and reduced tumour cell content

The efficacy of an immunomagnetic purging method and the Isolex 300 devices were assessed for selecting CD34+ cells from leukapheresis products of 29 patients with non‐Hodgkins lymphoma (NHL), 39 with multiple myeloma and 34 with breast cancer. The mean purity of the CD34+ cell population was 93.6% and the mean recovery was 67.7%. Following enzymatic cleavage by chymopapain the expression of Thy‐1 and Leu‐8 was significantly reduced without affecting haematological recovery. The population of selected CD34+ cells of 4/8 patients with follicular lymphoma became PCR‐negative. A 2.5 log reduction of tumour cells could be achieved in four patients with multiple myeloma as shown by a quantitative PCR assay. There were no tumour cells detectable in any of the 19 CD34+ cell preparations of patients with breast cancer. In 64 patients who received 94 cycles of high‐dose therapy, a mean number of 4.7 × 106 CD34+ cells/kg were autografted. The time needed for platelet reconstitution was different when a comparison was made with 156 patients, who had received unmanipulated leukapheresis products (10 v 12 d, P = 0.006). No significant differences with regard to neutrophil recovery were noted. Five patients had a graft failure. Two of them died (on day 78 and 88 following PBSCT), and three patients were rescued with unmanipulated back‐up transplants. In conclusion, the immunomagnetic selection of CD34+ cells provides autografts with reduced tumour cell content and an engraftment ability similar to that of unmanipulated autografts.

First and second apheresis in patients with multiple myeloma: no differences in tumor load and hematopoietic stem cell yield

Autologous peripheral blood stem cells (PBSC) are now widely used to support myeloablative therapy in patients with multiple myeloma (MM). The presence of malignant cells in these autografts has been demonstrated. Characteristic kinetics with differential and concomitant mobilization of CD34+ and malignant cells after high-dose (HD) chemotherapy and hematopoietic growth factor administration have been reported. We determined the amounts of tumor cells and PBSC in leukapheresis products (LP) collected on day 1 (LP1) and 2 (LP2) from 16 MM patients harvested after HD chemotherapy and G-CSF. Furthermore, LP from six patients collected on day 5 (LP5) could be examined. The content of clonotypic cells was quantitated by an allele-specific oligonucleotide (ASO)-PCR assay based on limiting dilutions. CD34+ PBSC were determined by flow cytometry. The percentages of malignant cells in the leukapheresis products were in the range of 0% to 0.713% (mean 0.047%). CD34+ cells ranged between 0.06% and 5.4% (mean 1.23%). Comparing LP1 with LP2, no differences in the quantity of tumor cells (mean 0.0538% vs 0.0448%; P = 0.96) and CD34+ cells (mean 1.49% vs 1.33%; P = 0.50) were seen. The calculated number of tumor cells per CD34+ cell did not differ significantly (mean 0.0420 vs 0.0249; P = 0.65). Analyzing LP5 revealed no changes in the number of tumor cells per CD34+ cell (0.0511 vs 0.1044; P = 0.46) indicating a relatively constant ratio of PBSC to tumor cells during the course of PBSC harvesting. These results offer the possibility of combining LP harvested over several days without increasing the tumor load per CD34+ cell.

Effect of CD34 cell dose on hematopoietic reconstitution and outcome in 508 patients with multiple myeloma undergoing autologous peripheral blood stem cell transplantation.

Background: We analyzed the hematopoietic reconstitution and outcome of 508 patients with multiple myeloma (MM) with respect to the number of CD34+ cells reinfused at our center. Patients and methods: Each cohort of 390 patients (unselected CD34+ cell transplant) and 118 patients (CD34+ selected transplant) was divided into four subgroups. Among the 390 transplantations, 86 patients received a high dose (HD−) of ≥6.50 × 106 unselected CD34+ cells/kg, 116 patients a low dose (LD−) of <3.00 × 106 CD34+ cells/kg. Among the patients treated with CD34+ selected PBSC, 34 received ≥6.50 × 106 CD34+ cells/kg (HD+) and 16 <3.00 × 106 CD34+ cells/kg (LD+). Results: HD− patients experienced a reduced median time to leukocyte (13 d vs. 14 d) (P < 0.001) and platelet reconstitution >20 × 109/L (10 d vs. 12 d) (P < 0.001). Similarly, HD+ showed a reduced median time to leukocyte (12 d vs. 15 d) (P < 0.001) and platelet recovery >20 × 109/L (10 d vs. 11 d) (P = 0.058). CD34+ cell‐dose was significant for long‐term platelet recovery at day 360 (unselected transplant P = 0.015, selected transplant P = 0.023). Number of transplanted CD34+ cells had no significant impact on transplant related mortality, overall survival or CR/PR rates within 100 d. In terms of supportive care the differences of high‐/low‐dose grafts were minimal. Conclusions: These results confirm that high doses of CD34+ PBSC shorten hematopoietic reconstitution and reduce hospitalization. Nevertheless secure engraftment results from transplantation of 2.00–3.00 × 106 CD34+ cells/kg. As 60% of our pretreated patients are able to collect ≥5.00 × 106 CD34+ cells/kg within a single leukapheresis, division into two or more freezing bags allows safe tandem transplantation in the majority of MM patients.