Increased expression of cyclin‐D1 on trephine bone marrow biopsies independently predicts for shorter overall survival in patients with multiple myeloma treated with novel agents
Authors: Anna Tasidou, Maria Roussou, Evangelos Terpos, Efstathios Kastritis, Maria Gkotzamanidou, Maria Gavriatopoulou, Magdalini Migkou, Evangelos Eleutherakis‐Papaiakovou, Nikitas Nikitas, Dimitra Anagnostou, Meletios A. Dimopoulos
Journal: American Journal of Hematology (2012)

Multiple myeloma (MM) comprises 1% of all malignancies and 13% of hematological malignancies in the Caucasian population. Yearly incidence is 4/100,000 in the US and is higher in blacks and males [1]. The pathogenesis of the disease is relatively unknown; several chromosomal abnormalities have been related to the development of the disease, but none is characteristic of MM. Cyclin-D1 is a protein encoded by the CCND1 (bcl-1) gene on chromosome 11q13, and is an important regulator of G1 to S phase progression. Overexpression or overactivity of the CCND1 is common in malignancies including MM, mantle cell lymphoma, breast cancer, and hepatocellular cancer [2-4]. Previous studies have shown that CCND1 is expressed in 50% of patients with MM and that CCND1 overexpression represents an unfavorable prognostic factor in MM [5, 6]. However, a recent study in a small number of patients showed that overexpressed CCND1 in MM is an independent prognostic marker associated with a more durable response to bortezomib. The aim of our study was to evaluate the impact of the immunohistochemical expression of cyclin-D1 in the plasma cells of trephine biopsies on survival of newly-diagnosed patients with MM who were treated with novel agents. One hundred and thirty consecutive patients with newly diagnosed MM were evaluated (66 males and 64 females). All patients were diagnosed, treated, and followed-up in a single center (Department of Clinical Therapeutics, University of Athens Medical School, Athens, Greece). Clinical characteristics of the patients are depicted in Table I. The median age of the patients was 68 years (range: 35–85 years). Forty-five patients (35%) had advanced disease at diagnosis (Stage 3 according to international staging system (ISS)). One hundred and fifteen patients (pts) had symptomatic disease that required therapy: 29 (25%) received bortezomib-based regimens (15 patients VTD (Bortezomib, Thalidomide and Dexamethasone), 5 patients VMP, and 5 patients PAD (Bortezomib, Doxorubicin and Dexamethasone)), 31 (26%) received thalidomide-based regimens (21 patients melphalan prednisone and thalidomide (MPT) and 10 patients thalidomide, vincristine, doxorubicin, dexamethasone (T-VAD)), and 55 received conventional treatment (20 patients VAD and 35 patients melphalan and prednisone) as first-line therapy, while all patients received regimens containing bortezomib or an immunomodulatory agents at some point during the course of their disease. Nineteen patients (16.5%) received high-dose melphalan plus autologous stem cell transplantation (ASCT). The median follow-up time for these patients was 22 months. Among patients with symptomatic myeloma (N = 115), positive staining for cyclin-D1 (Fig. 1) was found in 35 (30%) patients, for CD56 in 45 (39%), for CD117 in 94 (81%), and for CD27 in 72 (62%) patients. In patients with asymptomatic/smoldering myeloma, positive staining for cyclin-D1 was found only in 1 (7%) patient, for CD56 in 9 (64%), and for CD117 in 6 (43%) (P < 0.01 for all comparisons compared to symptomatic patients). We also observed more patients (n = 41) with positive staining for CD56 and ISS-1 or ISS-2 (P = 0.01) and more patients (n = 43) with positive staining for CD117 and ISS-3 MM (P = 0.002). The median overall survival (OS) for patients with symptomatic MM was 57 months (95% CI: 22–120). In the univariate analysis, positivity for cyclin-D1 (41 vs. 62 months, P = 0.03) and for CD117 (50 vs. 75 months, P = 0.018) were associated with inferior survival, while positivity for CD56 (47 vs. 62 months, P = 0.286), MUM-1 (52.7 vs. 63.8 months, P = 0.528), and CD27 (57 vs. 50 months, P = 0.445) were not. Other factors associated with inferior OS, in the univariate analysis, included ISS-3 (median OS 37 months vs. 57 months for ISS-2 and 73 months for ISS-1, P = 0.005), Hb < 10 g/dL (56 vs. 73 months, P = 0.044), corrected serum calcium ≥11.5 g/dL (29 vs. 62 months, P = 0.02), serum lactate dehydrogenase above upper normal limit (31 vs. 61 months, P = 0.05), serum creatinine >2 mg/dL (26 vs. 64 months, P = 0.007), low platelet counts (<100,000/mL) (22 vs. 62 months, P = 0.031), and age >65 years (45 months vs. not reached for younger patients, P = 0.002). In the multivariate analysis, positivity for cyclin-D1 (HR: 2.6; P = 0.001), ISS stage (HR: 1.8; P = 0.001), and age >65 (hazard ratio 2.7, P = 0.003) were independently associated with inferior survival (Fig. 2). Patients with cyclin-D1 positivity had a median survival of 22 months in ISS-2 (vs. 64 months for the rest of ISS-2 patients, P = 0.01) and of 13 months in ISS-3 (vs. 47 months for the rest of ISS-3, P = 0.012). Positive control for cyclin D1 IHC (A) (lymph node from a patient with mantle cell lymphoma) and a positive (B) and a negative (C) IHC for cyclin D1 in bone marrow trephine biopsies. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Impact of Cyclin D1 expression on OS. Recent studies have confirmed the impact of several molecular markers on survival of MM patients. Immunohistochemical staining for proteins implicated in MM pathogenesis is useful for the evaluation of the activity of the relative genes. Previous studies have shown that the majority of MM patients have deregulated at least one of the cyclin genes, suggesting that this may have prognostic value [7, 8]. Moreover, a correlation between cyclin D1 expression, the percentage of bone marrow infiltration, and the degree of differentiation of neoplastic plasma cells (histologic grading) has been reported, mostly in cell lines [8, 9], but only in a small series of patients [10]. In our study, 30% of newly-diagnosed patients with symptomatic disease had positive myeloma cells for cyclin-D1 in their trephine biopsies compared to only 7% of patients with smoldering myeloma. This finding suggests that expression of cyclin-D1 increases with enhanced disease activity. In the multivariate analysis, cyclin-D1, ISS stage, and age >65years were independently associated with inferior survival. This result further supports the value of cyclin-D1 in the disease course. The reported studies to-date produced conflicting results. Cyclin-D1 overexpression has been linked to the t(11;14) translocation and polysomy of chromosome 11 [11] but the presence of a t(11;14) seems to be a prognostically "neutral" genetic abnormality [12, 13]. In our study, the patients were diagnosed with MM from Jan 1, 2000 until December 31, 2007 and we had no fluorescent In situ hybridization or caryotype data. The overexpression of cyclin-D1 correlated with durable responses and better prognosis to bortezomib therapy in small series of patients [14-16] Furthermore, in another small study with 45 patients who received conventional chemotherapy (MP) plus rituximab, the cyclin-D1 immunohistochemical expression correlated with better prognosis [14]. On the other hand, cyclin-D1 negativity did not convey any adverse effect on progression free survival (PFS) or OS in 94 patients (49 newly diagnose and 45 with relapsed/refractory disease) who received the T-VAD (thalidomide, vincristine, adriamycin, dexamethasone) regimen [13]. The discrepancy between the above results can be explained by the differences in the population, i.e., the immunoactivity of cyclin-D1 was evaluated in small series of refractory or relapsed MM patients and not in large population of newly diagnosed patients, as in our study. Moreover, the follow-up period in our cohort of patients was significantly longer than that of the previous studies. In a study that included 74 newly-diagnosed patients who received high-dose therapy with ASCT, patients who overexpressed cyclin-D1 had significantly longer duration of remission in comparison with patients who did not (41 vs. 26 months, respectively; P = 0.02). As a result, median event-free survival was longer in these patients (33 vs. 24 months, respectively; P = 0.055) [17]. The Arkansas group has also reported that the mRNA positive expression of cyclin-D1 correlated with better survival in patients who received "total" therapy approaches with tandem transplants [18]. However, in our study only 16.5% of patients received an ASCT. The small number of patients in our series who received ASCT may be a key explanation to the differences in the prognostic importance of cyclin D1 expression, since these patients may have a significant benefit with high-dose therapy approaches but in the absence of such a strategy this may be associated with a less favorable outcome. The use of novel agents in our patients may also have greater impact in patients without cyclin D1 overexpression, and actually the survival in our patients, which is mainly a non-transplant eligible population, is 57 months. The c-kit receptor (CD117) is a tyrosine kinase involved in cell growth and can be expressed in one-third of patients with symptomatic MM. The impact of CD117 on prognosis is controversial. Several studies have shown that the increased expression of CD117 on bone marrow, measured by flow cytometry, correlated with good prognosis in MM [19, 20]. In our study, in the univariate analysis, positivity for CD117 correlated with inferior survival (50 vs. 70 months, P = 0.018). However, this was not confirmed in the multivariate analysis. This may be explained by the positive correlation between cyclin-D1 and CD117 that excluded from the multivariate model CD117 in favor of cyclin-D1. CD56 is an adhesion molecule which is expressed in 70–80% of patients with MM. Several studies have shown association between the absence of expression in MM patients and extramedullary myeloma and shorter PFS but not different OS [20-22]. We also confirm that there is no prognostic impact of CD56 on MM patients' OS. MUM-1 (also known as Interferon Regulatory Factor-4) is a transcription factor, which regulates the differentiation of mature B-cells into antibody secreting plasma cells. The role of MUM-1 on MM pathogenesis is not clear because high expression of MUM-1 is also observed in normal plasma cells [23]. We found no influence of MUM-1 on OS in our series of patients. Finally, CD27 is a member of tumor necrosis factor receptor family and is an essential molecule for normal plasma cell differentiation through interaction with its ligand (CD70), whereas some myeloma cells lack its expression [24-26] Recent microarray studies revealed that the CD27 gene was the most underexpressed gene in MM along with that of stromal cell-derived factor-1 [27]. Two studies reported the negative impact of the lack of CD27 on the biology of MM patients [28, 29]. However in our study the expression of CD27 did not influence OS. A possible explanation is that in the study by Moreau et al., the authors have used flow cytometry and not immunohistochemical analysis. In the second study [28], the low expression of CD27 was associated with plasmacytoma progression to MM and as in our study there was no correlation with OS. The main advantage of our study is the homogeneous, newly diagnosed, large population of MM patients. Our findings suggest that the immunohistochemical expression of cyclin-D1 in the bone marrow trephine biopsies has independent prognostic value in patients with MM, even in the era of novel agents. This marker can easily be assessed in patients who undergo a trephine biopsy and offers significant prognostic information that may alter the treatment decision for these patients. Furthermore, novel agents targeting cyclin-D1 may be of therapeutic value in MM. We evaluated formalin-fixed, paraffin-embedded, bone marrow sections of 130 consecutive patients with newly-diagnosed MM before any kind of therapy administration. Informed consent was obtained from all patients and the study was conducted according to the Declaration of Helsinki guidelines. Bone marrow trephine were paraffin embedded and decalcified with EDTA buffer (EDTA/HCL) low pH. Immunochemistry staining was performed in automatic instrument (XT Benchmark, Ventana, Tucson, AZ) using an anti-cyclin-D1 monoclonal antibody (Cell Marque Corp., Rocklin, CA), an anti-CD56 monoclonal antibody (Cell Marque Corp., Rocklin, CA), an anti-CD27 monoclonal antibody (Novocastra, Newcastle upon Tyne, UK), an anti-CD117 monoclonal antibody, and an anti-MUM-1 monoclonal antibody (DAKO A/S, Glostrup, Denmark) and the dilution was 1:20. The percentage of immunochemical markers-positive plasma cells was expressed as their ratio to the total number of plasma cells which have characteristically morphology on microscopy. According to the immunoreactivity of cyclin-D1 (Fig. 1), CD56, and MUM-1 by plasma cells, we defined as positive group the group of patients with ≥20% positive plasma cells in the trephine biopsies for the above mentioned antigens. As positive control for Cyclin D1 Immunohistochemistry, lymph node sections from mantle cell lymphoma patients were used. Similarly, for CD117 and CD27 we defined as positive group the group of patients with ≥10% positive plasma cells in the trephine biopsies for these antigens. Differences between patients and controls were evaluated using the Mann–Whitney test. The Spearman rank correlation test was used to examine relationships between various parameters. Survival probabilities were calculated by the Kaplan–Meier method and comparisons made using the log-rank test to identify potential prognostic factors. For multivariate analysis Cox proportional hazards models were used. All P values are two sided and confidence intervals refer to 95% boundaries. Anna Tasidou*, Maria Roussou , Evangelos Terpos , Efstathios Kastritis , Maria Gkotzamanidou , Maria Gavriatopoulou , Magdalini Migkou , Evangelos Eleutherakis-Papaiakovou , Nikitas Nikitas , Dimitra Anagnostou*, Meletios A. Dimopoulos , * Department of Hematopathology, "Evangelismos" General Hospital, Athens, Greece, Department of Clinical Therapeutics, University of Athens School of Medicine, Athens, Greece.