Prognostic value of bone marrow angiogenesis in patients with multiple myeloma undergoing high-dose therapy

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1 (2004) 34, & 2004 Nature Publishing Group All rights reserved /04 $ Prognostic value of bone marrow angiogenesis in patients with multiple myeloma undergoing high-dose therapy S Kumar, MA Gertz, A Dispenzieri, MQ Lacy, LA Wellik, R Fonseca, JA Lust, TE Witzig, RA Kyle, PR Greipp and SV Rajkumar Division of Hematology, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA Summary: Bone marrow (BM) angiogenesis is increased in multiple myeloma and is an important prognostic factor. However, prior studies were mainly done on patients receiving conventional chemotherapy and there is limited data on its prognostic value in patients undergoing high-dose therapy (HDT) as initial therapy. We studied BM angiogenesis in terms of microvessel density (MVD) in 88 newly diagnosed patients,who were uniformly treated,with 3 4 cycles of induction chemotherapy followed by HDT. We examined if MVD at diagnosis was predictive of response to induction therapy or to subsequent HDT. In addition, we also examined its prognostic value in these patients. The median MVD for primary refractory patients was 28 (range,2 84) compared to 27 (range,2 99) for responding patients (P ¼ 0.7). The median progression-free survival (PFS) was 21 months for those with high-grade angiogenesis compared to 42 months for those with low-grade angiogenesis, P ¼ The median overall-survival (OS) from diagnosis was 40 months for those with high-grade angiogenesis and not yet reached,for those with low-grade angiogenesis, P ¼ BM MVD at the time of initial diagnosis is an important prognostic factor for OS and PFS in patients undergoing autologous transplantation as frontline therapy for myeloma. (2004) 34, doi: /sj.bmt Published online 31 May 2004 Keywords: angiogenesis; multiple myeloma; stem cell transplantation; prognosis; refractory Multiple myeloma is characterized by the proliferation of monoclonal plasma cells resultingin anemia, bone pain or pathological fractures due to lytic bone involvement, hypercalcemia and often, renal failure. 1 The median age at diagnosis is 63 2 with nearly individuals diagnosed with this disorder in this country each year and dying Correspondence: Dr SV Rajkumar, Division of Hematology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; Rajkumar.vincent@mayo.edu Received 24 September 2003; accepted 16 March 2004 Published online 31 May 2004 of causes related to it each year. 3 Combination chemotherapy regimens, especially those containing alkylating agents and steroids are effective in controllingthe disease. Highdose chemotherapy with stem cell rescue was introduced to capitalize on the chemosensitivity of the disease and has been shown to improve survival compared to conventional chemotherapy in prospective clinical trials. 4,5 However, patients undergoing autologous transplants eventually relapse and salvage therapy options remain limited. Better understandingof the biology of the disease has led to development of potentially beneficial agents in the recent years. Identifyingpatients at high risk of relapse after highdose therapy (HDT) can lead to application of these new agents as well as immunomodulatory approaches to this group with the intent of decreasing the relapse risk. Angiogenesis or formation of new blood vessels from existingblood vessels (in contrast to vasculogenesis, which involves formation of de novo blood vessels) is an important component of normal development. In the context of solid tumors, ability of the tumor to initiate angiogenesis in the tumor tissue has been correlated with increased ability to invade and metastasize. 6,7 In the context of hematological malignancies increased bone marrow (BM) angiogenesis has been demonstrated in multiple myeloma, 8 11 chronic myelocytic leukemia (CML), 12 acute myeloid and lymphocytic leukemias, chronic lymphocytic leukemia (CLL) 16 as well as myelodysplastic syndromes 12 and has been found to have prognostic value. The endothelial cells of the new blood vessels are believed to play an important role in the survival and proliferation of the malignant cells in these disorders, possibly mediated through growth factors and cytokines. We have previously demonstrated that BM angiogenesis is increased in plasma cell proliferative disorders, 17 and increases as the disease progresses from MGUS to active multiple myeloma. 18 In addition, we have previously shown that the BM angiogenesis persists after conventional therapy 19 as well as after autologous transplantation in myeloma. 20 Although we have shown earlier that BM angiogenesis has prognostic value in patients with myeloma, these patients were not treated in a similar manner, and most patients did not receive stem cell transplantation as initial therapy. Thus, there is limited data on the prognostic effect of BM angiogenesis in patients receivinghdt with stem cell transplantation for newly diagnosed myeloma. 21 Given the superior therapeutic benefit with HDT in myeloma, we investigated the

2 236 prognostic value of BM angiogenesis in a group of patients uniformly treated with autologous stem cell transplantation for multiple myeloma. We also examined if increased angiogenesis was associated with refractoriness to conventional chemotherapy or HDT by correlatingthe microvessel density (MVD) with the response to initial induction therapy as well as to transplant. Patients and methods We identified patients from the institutional myeloma transplant database, who had undergone autologous stem cell transplantation and in whom archived BM biopsies were available from the time of diagnosis. Only those patients, who had proceeded to an immediate transplant followinginitial cytoreductive therapy, irrespective of the response, were included in this study in order to obtain a uniform group of patients. Complete follow-up was available for all the patients. Microvessel density determination Microvessels were identified by immunohistochemical stainingfor CD34, performed on slides made from paraffin-embedded BM biopsy blocks by a labeled streptavidin biotin peroxidase method as previously described. 18,19 Paraffin sections of well-vascularized tonsil were included with each batch to serve as a positive control; a section stained with nonimmune rabbit immunoglobulin was used as a negative control for each sample tested. BM angiogenesis was estimated by microscopic evaluation in terms of MVD, as previously described. 18,19 This technique has been evaluated in the past and found to be reproducible, with excellent interobserver concordance. 20 Slides stained with antibodies to CD34, first were scanned under low power ( 100) to identify three hot spots or areas with the greatest number of microvessels. These three areas then were evaluated at 400 magnification using an 40 objective and an 10 ocular lens. To ensure consistency, the same individual usingthe same microscope (SK) assessed all slides. To eliminate bias, slides were examined under blinded conditions, with no prior knowledge of the clinical details. Interobserver variation was determined in a random subset of 20 patients in which MVD was assessed independently by another individual (SVR). Large vessels and vessels within the bony spicules and under the periosteum were excluded. Areas of stainingwithout discrete breaks were counted as single vessels. The presence of a lumen or red cells was not required for the identification of microvessels; any highlighted endothelial cell or cell cluster separate from the adjacent microvessels was counted as a distinct vessel. The number of vessels in one 400 field was determined for each hot spot; the average was taken as the MVD and expressed as microvessels per field. On the basis of the MVD, the patients were grouped into high- and low grades using the median MVD as the cutoff. The methodology of hot-spot determination has been studied head to head with more objective methods such as computerized image analysis and has been found to be accurate and reproducible. Stem cell transplantation The initial induction therapy consisted of one of three regimens namely, dexamethasone as a single agent (32%), combination of vincristine, adriamycin, and dexamethasone (VAD) (42%) and thalidomide with or without dexamethasone (26%). Patients received 3 4 cycles of induction therapy and proceeded to stem cell harvest irrespective of the initial response. Primary refractory disease was defined as failure to achieve at least a 50% reduction in the monoclonal protein. Stem cell harvest was performed usinga combination of cyclophosphamide with growth factors, either GM-CSF or G-CSF. The conditioning therapy was high-dose melphalan (200 mg/m 2 ) alone in 64 patients (73%) and melphalan (140/m 2 ) with total-body irradiation in the remaining25 patients. Statistical analysis The Fisher exact test was used to compare differences in nominal variables. Student s t-test was used for comparing continuous variables. Correlation between continuous variables was studied usingthe Spearman rank correlation. Overall survival was defined as the time from diagnosis to death with those still alive censored at the time of the last follow-up. Progression-free survival (PFS) post transplant was defined as the time from transplant to disease progression with patients censored at the time of the last follow-up. Overall survival was estimated usingthe Kaplan Meier method; 22 survival curves were tested for statistical significance using the log-rank test. All tests were two-tailed; Po0.05 was considered significant for all comparisons. This study was approved by the Mayo Foundation Institutional Review Board, and was conducted in accordance with United States federal regulations and the Declaration of Helsinki. Results In all, 88 patients were identified who satisfied the inclusion criteria and had BM biopsies available for microvessel examination. The median age of the patient group was 58 years (range; 29 72); 55 (63%) were males. In total, 13 patients (14%) were part of earlier publications on BM angiogenesis. 8,18 Overall, 39 (44%) patients have had disease progression following transplant and 68 patients were alive at the time of last follow-up. The median followup for those alive was 27 months (range months) from diagnosis and 20 months (range months) from transplant. The median MVD at the diagnosis for the entire group was 28 (mean, 30; range, 2 99). There was a high degree of correlation in the slides assessed for interobserver variation (rho ¼ 0.874; Po0.001). Based on the median MVD, patients were grouped into two groups; low (median MVDo27; 43 patients) and high MVD (median MVDX27; 45 patients). There was no significant difference between the two groups (high- and low MVD) in terms of the pre-transplant

3 induction therapy (P ¼ 0.23). There was no correlation between response to induction chemotherapy and BM MVD. Median MVD for primary refractory patients was 28 (range, 2 84) compared to 27 (range, 2 99) for those with chemosensitive disease (P ¼ 0.7). However, there was a significant difference between the high- and low MVD groups in terms of the conditioning regimen they received; 40% of patients with low MVD received Mel/TBI compared to 18% with high MVD, P ¼ The median BM plasma cell percentage was 34%, the labelingindex was 41% in 25 patients (31%) and the b-2 microglobulin was more than 4 mg/l in 28 patients (32%). In univariate analysis, MVD was correlated with b-2 microglobulin (rho ¼ 0.311, Pp0.005), and hemoglobin (rho ¼ 0.396; Po0.01); no significant correlation was seen between MVD and labelingindex, LDH, BM plasma cell percentage, cytogenetics status or creatinine. A formal multivariate analysis was not performed includingmvd and other known prognostic factors due to one or more missingvariables in a number of patients. Figure 1 Kaplan Meier survival plot demonstratingthe relapse-free survival from diagnosis for the two groups (high- vs low-grade angiogenesis). 237 Response and survival There was no difference in MVD between patients achievinga CR from the transplant (median MVD 22) compared to those not attaininga CR (median MVD 31) (P ¼ 0.08). However, patients with high MVD had a significantly higher risk of relapse following HDT compared to those with low MVD (RR: 2.2; 95% CI: 1.1, 4.3; P ¼ 0.02). No effect of the induction regimen or conditioningtherapy was observed on survival. The median PFS was significantly different based on baseline angiogenesis status, 21 months for patients with high MVD compared to 42 months for those with low MVD, P ¼ (Figure 1). Patients with increased angiogenesis also had a shorter post transplant overall survival as well (Figure 2). The median overall survival from diagnosis was 40 months for those with high MVD and not yet reached, for those with low MVD group, P ¼ Discussion In this study of 88 patients with newly diagnosed multiple myeloma treated uniformly with HDT and autologous stem cell transplantation, we have once again demonstrated increased BM angiogenesis and its prognostic value. Results from this study confirms our previous observations regarding BM angiogenesis in myeloma 8,10,23 and extends the results previously observed in a group of patients undergoing different therapeutic interventions. 8 BM angiogenesis appears to be an important biologic feature of multiple myeloma and is seen in all stages of the disease. In a series of 400 patients with MGUS, smolderingmm and active MM, a progressive increase was observed in the BM angiogenesis from MGUS to MM. 18,24 Additionally, increased angiogenesis has also been demonstrated in related conditions like solitary plasmacytoma, where it has predictive value for subsequent progression to multiple myeloma. 25 Although subjective, estimation of BM MVD Figure 2 Kaplan Meier survival plot demonstratingthe overall survival from transplant for the two groups (high- vs low-grade angiogenesis). in MM has shown high interobserver correlation in our studies, 20 and its prognostic value has been reproducible in studies by us and others. 8 11,18,21 This study shows that BM MVD is a significant predictor of PFS and overall survival in patients undergoing autologous stem cell transplantation for newly diagnosed myeloma. There was no significant difference in the distribution of patients based on induction regimen between the high- and low MVD groups that could confound this analysis. Although more patients in the low MVD group had received Mel/TBI as the conditioning regimen, given the results of randomized trials comparing conditioningregimens, we do not believe that this imbalance affected the results of this study. The fact that angiogenesis comes out as a significant prognostic factor in spite of the relatively small sample size speaks to its large effect on outcome. Our results are consistent with prior data that angiogenesis has significant prognostic value in MM, and extend the results to patients treated with upfront autologous stem cell transplantation. Just as studies are lookingat ways to overcome the poor prognostic effect of known plasma cell markers such as cytogenetic deletion 13, trials also need to address strategies to overcome the poor prognostic effect of a bone microenvironment that is hospitable to residual plasma cells.

4 238 Recent studies lookingat the interaction between the malignant plasma cell and the BM microenvironment have led to a better understandingof the biological basis for the prognostic value of BM angiogenesis. A close relationship has been demonstrated between the malignant plasma cells, the marrow stromal cells and the vascular endothelial cells. The malignant plasma cells can secrete various cytokines, includingvascular endothelial growth factor (VEGF), 26 basic fibroblast growth factor 27 and hepatocyte growth factor, 28 all known angiogenic factors. VEGF plays an important role in tumor angiogenesis, acting as a potent inducer of vascular permeability and servingas a specific endothelial cell mitogen. 26,29 Myeloma cells as well as BM stromal cells are capable of secretingvegf in response to a variety of different signals including cytokines such as TGF-b, tumor necrosis factor-a, IL-1b, IL-6, TNF-a, 30 and CD40-mediated signaling. 31,32 Stimulation of endothelial cells and BM stromal cells by VEGF results in increased interleukin (IL)-6 secretion by these cells, 26,32 which in turn can stimulate myeloma cell growth and inhibit plasma cell apoptosis. Existence of these autocrine and paracrine pathways explains, at least in part, the increased plasma cell proliferation seen in association with increased marrow MVD. 33 Given the supportive role that the endothelial cells play in plasma cell survival, it is possible that increased angiogenesis in the marrow may confer the plasma cells with some degree of resistance to chemotherapy. In this study, we examined if some of the prognostic value of angiogenesis in myeloma could be related to resistance to conventional chemotherapy. However, we failed to demonstrate any correlation between the response to initial chemotherapy and the marrow MVD at diagnosis. It seems likely that mechanisms other than drugresistance are responsible for the biologic basis of the prognostic value for marrow angiogenesis. Autologous stem cell transplantation has demonstrated survival benefit for patients with multiple myeloma. 4,5 Majority of the patients undergoing HDT for myeloma eventually relapse with a median survival of nearly 5 years. Several pre and post transplant factors have been identified, which have been useful in prospectively identifyingpatients at a high risk of relapse. These have included cytogenetic abnormalities, failure to achieve CR after transplant, high b 2 M as well as high proliferative rate of the plasma cells as indicated by the PCLI. In this study, we have demonstrated that increased marrow angiogenesis is an important prognostic factor for patients undergoing HDT. In a previous study evaluating BM angiogenesis in patients undergoing tandem transplantation for multiple myeloma, MVD was found to have independent prognostic value. 21 Unfortunately, due to the relatively small number of patients included in the study, we could not evaluate the importance of MVD in relation to other prognostic factors. BM MVD at the time of initial diagnosis is an important prognostic factor in patients with multiple myeloma and autologous transplantation does not overcome the poor predictive prognosis of high MVD. There does not appear to be a direct relationship between increased BM MVD and resistance to conventional therapy or HDT. Although our study answers important biological questions, given other available prognostic factors in this setting and the relatively small sample size in this study, additional research is needed to clarify the clinical utility of these observations. Acknowledgements Supported in part by Grants CA , CA 93842, CA85818, CA107476, and CA62242 from the National Cancer Institute, MD, USA. Dr Rajkumar is a Leukemia and Lymphoma Society of America Translational Research Awardee and is also supported by the Goldman Philanthropic Partnerships, IL, USA, and the Multiple Myeloma Research Foundation, CT, USA. References 1 Bataille R, Harousseau JL. Multiple myeloma. N Engl J Med 1997; 336: Kyle RA, Gertz MA, WitzigTE et al. Review of 1027 patients with newly diagnosed multiple myeloma. Mayo Clin Proc 2003; 78: Jemal A, Murray T, Samuels A et al. Cancer statistics, CA Cancer J Clin 2003; 53: Child JA, Morgan GJ, Davies FE et al. 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