Variability in Cytogenetic Testing for Multiple Myeloma: A Comprehensive Analysis From Across the United States

Abstract

Purpose: Multiple myeloma (MM) treatment has changed tremendously, with significant improvement in patient out-comes. One group with a suboptimal benefit is patients with high-risk cytogenetics, as tested by conventional karyotyping or fluorescence in situ hybridization (FISH). Methodology for these tests has been published, but not necessarily standardized.

Methods: We address variability in the testing and reporting methodology for MM cytogenetics in the United States using the ongoing African American Multiple Myeloma Study (AAMMS). We evaluated clinical and cytogenetic data from 1,221 patients (1,161 with conventional karyotyping and 976 with FISH) tested between 1998 and 2016 across 58 laboratories nationwide.

Results: Interlab and intralab variability was noted for the number of cells analyzed for karyotyping, with a significantly higher number of cells analyzed in patients in whom cytogenetics were normal (P 5.0025). For FISH testing, CD138-positive cell enrichment was used in 29.7% of patients and no enrichment in 50% of patients, whereas the remainder had unknown status. A significantly smaller number of cells was analyzed for patients in which CD138 cell enrichment was used compared with those without such enrichment (median, 50 v 200; P, .0001). A median of 7 loci probes (range, 1-16) were used for FISH testing across all laboratories, with variability in the loci probed even within a given laboratory. Chromosome 13-related abnormalities were the most frequently tested abnormality (n5956; 97.9%), and t(14;16) was the least frequently tested abnormality (n 5 119; 12.2%).

Conclusions: We report significant variability in cytogenetic testing across the United States for MM, potentially leading to variability in risk stratification, with possible clinical implications and personalized treatment approaches.

Fig 1.

(A) Laboratory distribution for the patients tested by conventional karyotyping (1,161 patients from 52 laboratories). (B) Laboratory distribution for the patients tested by fluorescence in situ hybridization (976 samples from 47 laboratories).

Fig 2.

Intralab and interlab variability in number of cells analyzed for reporting metaphase karyotyping. Data on 18 of a total of 52 laboratories (1,089 samples, representing 93.8% of the total study samples with karyotypes), which are the labs with variations in the evaluated cell numbers. They reported at least 2 of the 3 reporting categories of less than, equal to, or greater than 20 cells in patients analyzed. Karyotyping or cytogenetic testing in general evaluates on average 20 cells to identify the patient karyotypes. The other 34 unlisted labs included 28 labs with a single sample (20 labs testing 20 cells, 3 labs testing < 20 cells, 3 labs testing > 20 cells, and 2 labs with missing numbers), 3 labs with 2 samples all testing 20 cells, 2 labs with 3 samples all testing 20 cells.

Fig 3.

Type and number of cells evaluated by fluorescence in situ hybridization (FISH) across the laboratories. (A) Bone marrow evaluation included a median of 50 cells for CD138-positive (+)–enriched samples and a median of 200 cells without enrichment. In general, 50 CD138+-enriched cells were used, whereas 200 bone marrow cells without enrichment were used for FISH. (B) Intralab and interlab variability in using CD138+-enriched versus nonenriched samples for reporting FISH testing. Data on 21 of a total of 47 laboratories (943 samples, representing 96.6% of the total study samples with variations in the cell numbers), which reported at least 2 of the 3 methods of CD138+-enriched, not enriched, and unknown. (C) CD138-enriched cell type used for FISH over time. Data for years 2001-2003 were excluded because fewer than 2 tests were available for those years. Test years shown on the x-axis. Primary y-axis (left) shows the number of tests for each cell type (CD138 enriched and not enriched; box-plot) in each year, whereas the secondary y-axis (right) shows the percentage of tests for that year using CD138 enriched cells (line plot). (D) Intralab and interlab variability in the number of loci probed for FISH testing. Data on 24 of a total of 47 laboratories that had variability in the number of FISH probes tested per patient at that laboratory are shown. These patients represent 951 samples (97.4% of samples with FISH tests) included in the study. (E) Number of probes used per FISH test over time. Data for years 2001-2003 were excluded because fewer than 2 tests were available for those years. Test years shown on x-axis. Primary y-axis (left) shows the number of tests for each quartile (Q; Q1, 1-3 probes; Q2, 4-6 probes; Q3, 7-8 probes; Q4, 9-16 probes; box-plot) in each year, whereas the secondary y-axis (right) shows the percentage of the sum of Q3 and Q4 (7-16 probes; line-plot).

Fig 4.

Variability in the probes used for a given target locus for fluorescence in situ hybridization testing among various laboratories. Each pie diagram represents a target locus tested. (A) Chromosome (chr) 1p, (B) chr 1q, (C) chr 3, (D) chr 5, (E) chr 7, (F) chr 9, (G) chr 11, (H) chr 12, (I) chr 13t, (J) t(4;14), (K) t(11;14), (L) t(14;16), (M) chr 15, (N) chr 17p, (O) chr 17p3, (P) chr 14, and the number of laboratories reporting each variation of the probes for that locus.