Background: Multiple myeloma (MM) has been studied by conventional cytogenetics and fluorescence in situ hybridization using DNA probes (FISH) on interphase nuclei and metaphase cells, but the relative clinical significance of proliferating (metaphase) versus non-proliferating (interphase) cells at diagnosis is poorly understood. We investigated a consecutive series of 154 patients (pts) with newly diagnosed untreated MM, and compared results for metaphase and interphase cells with survival.
Methods: Bone marrow within 30 days of diagnosis was analyzed by standard cytogenetics. Leftover cells were stored at −70°C. Interphase and metaphase FISH was done using probes for IGH, FGFR3, CCND1, c-MAF, D13S319, LAMP1, p53 and D17Z1 to detect t(4;14), t(11;14), t(14;16) and chromosome 13 or 17 anomalies, respectively. We analyzed <10 metaphases and 200 unselected interphase nuclei for each FISH probe set for each pt. Survival rates were estimated using Kaplan-Meier and differences in survival curves compared using log-rank test.
Results: 61 (39.6%) pts had abnormal metaphases by cytogenetics (43 pts) and/or FISH (46 pts): 12 (19.7%) had t(11;14) by cytogenetics (3 pts) and/or FISH (11 pts), 11 (18.0%) had t(4;14) by FISH but not cytogenetics, and 3 (4.9%) had t(14;16) by FISH but not cytogenetics. Metaphases with 13q- were seen in 30 pts (49.2%). Interphase FISH results were abnormal for 133 (86.3%) pts: mean percent neoplastic nuclei was 26.9 ± 21.6 (3.9 to 99.0). Mean percent plasma cells was 54.7 ± 24.6 (3.0 to 99.0). Each of the 61 pts with abnormal metaphases was also abnormal in interphase nuclei. Median survival for pts with ≥50% (20 pts) abnormal nuclei was 202 days vs. 1351 days for pts with <50% (134 pts) (p<0.001). Median survival for pts with abnormal metaphases (61 pts) was 587 days vs. 1404 days for pts with normal metaphases (93 pts) (p=0.002). Median survival was 381 days for 30 pts with 13q- metaphases compared with 1404 days for 38 pts with normal metaphases and 13q- interphase nuclei (p<0.001). Metaphase and interphase analysis along with other potential risk factors (hemoglobin <10, serum calcium ≥11, serum creatinine ≥2, plasma cell labeling index and percent bone marrow plasma cells) were evaluated by multivariate analysis using a stepwise selection in a Cox proportional hazards model. Only the observation of 13q- in metaphases or ≥50% abnormal interphase nuclei were significant predictors of survival.
Conclusions: Analysis of metaphases at diagnosis is important to detect proliferating disease which is a strong indicator of poor prognosis. Metaphase FISH is a necessary adjunct to conventional cytogenetics to detect cryptic t(4;14) and t(14;16), and accurately detect prognostic chromosome anomalies such as 13q-. Observing a 13q- in metaphase cells is a strong predictor of survival compared with observing 13q- in only interphase nuclei. At diagnosis, analysis of interphase nuclei is efficient, identifies an abnormal clone in 86% of pts and defines a subset of patients with ≥50% nuclei that have a poor prognosis. Since analysis of metaphases gives better prognostic information, interphase FISH is not a substitution in the cytogenetic work up of patients with MM.