Cytoplasmic Immunoglobulin Fluorescence In Situ Hybridization (cIg FISH) Enhances the Quantitative Detection of Chromosome Abnormalities In Plasma Cell Neoplasms Compared to Conventional FISH
Authors: Yang Hai-Su , Montella Josephine , Holt Laura , Stephenson Christine , Brenholz Pauline , Sun Guoxian , Berger Carol , Scholl Thomas , Chari Ajai , Mazumder Amitabha , Jagannath Sundar , Dong Henry Y. 
Published: October 2019

Abstract
Abstract 1198
Risk stratification of plasma cell neoplasms (PCNs) has become an integral part of clinical management along with the rapid advances in treatment. Cytogenetics is widely performed at diagnosis but has a low abnormality detection rate due to poor proliferation of plasma cells (PCs) in culture and inability to identify cryptic translocations. Conventional interphase FISH improves detection sensitivity, but lacks specificity since the percentage of PCs is diluted by the large number of normal bone marrow cells due to the indiscriminative analysis of all cells by FISH. The International Myeloma Working Group recommends all FISH tests for myeloma be PC specific by either PC enrichment or identifying PCs by cytoplasmic immunoglobulin staining (cIg FISH). However, the real benefits of cIg FISH compared to conventional FISH have never been demonstrated in a controlled study. In this objective comparative study, 75 paired samples from patients with PCNs were analyzed concurrently by conventional FISH and cIg FISH with probes for t(4;14), t(11;14), t(14;16), -13/13q (RB1/LAMP1), 17p- (TP53) and CEP3. For conventional FISH, a minimum of 200 nuclei were scored per probe set. For cIg FISH, 100 cIg positive cells were scored per probe with a minimum of 25 cIg+ cells required for conclusive reporting. In this study, 51/75 cases met the above criteria for direct comparison (see Table). cIg FISH results demonstrated an improved overall detection rate [-13/13q: 53% vs. 45%; 17p-: 14% vs. 12%; t(11;14): 25% vs. 24% and t(4;14): 14% vs.12%]. Impressively, cIg FISH quantitatively identified a much higher proportion of abnormal PCs in all cases. The median % of abnormal PCs with all probes was >50% by cIg FISH but no more than 10% by conventional FISH. The abnormal PCs detected by cIg FISH were >90% of cIg+ cells in 18 cases with at least one probe. In contrast, the abnormal PCs detected by conventional FISH were <5% of scored cells in 12 cases with all probes. Of 11 cases with discordant results between cIg FISH and conventional FISH, cIg FISH detected more abnormalities in 8/11 cases. In 2 cases from the same myeloma patient during follow up, t(11;14) was detected by both methods but del(13q) was detected only by conventional FISH, which was related to a concomitant myelodysplastic syndrome rather than myeloma, confirmed by cytogenetic study. In the third case, multiple abnormalities were detected by both methods but +3 was detected only by conventional FISH; its nature remains undetermined. Out of the remaining 24 cases, cIg+ cells were absent or sparse in 18 due to low PC counts. The results of conventional FISH were normal in all 18 cases. Six cases had only <25 cIg+ cells per probe, but most of the cIg+ cells were abnormal in 4/6 cases with probes consistent with the concurrent results of conventional FISH. The other 2 cases had a single abnormality only by conventional FISH at the level close to its detection threshold. Collectively, these results suggest the percentage of PCs in cIg FISH samples need to reach a threshold in order to ensure unequivocal results and performance consistency, which appears to be similar to the detection limit of conventional FISH. In summary, cIg FISH tends to have a greater detection rate and consistently identifies a higher proportion of abnormal PCs in all samples compared to conventional FISH. It can be used not only at diagnosis for accurate prognostic assessment, but also at relapse and during active disease for better monitoring expansion of abnormal clones and clonal evolution.Table 1Comparison of results of cIg FISH and conventional FISH for same PCN samplesSide-by-side Conventional FISH & cIg FISH N=51-13/13q RB1/LAMP117p- TP53+3 CEP3t(11;14) BCL1/IGHt(4;14) FGFR3IGHt(14;16) IGH/MAFNumber (%) of positive cases27 (53)7 (14)17 (33)13 (25)7 (14)1 (2)cIg FISH Conventional FISH23 (45)6 (12)17 (33)12 (24)6 (12)1 (2)Median % pos cells/case765456918378cIg FISH Conventional FISH79109610Range of % pos cells/case14-10019-9626-8663-10021-9678cIg FISH Conventional FISH4-806-114-461-461-2210

Disclosures:
Jagannath: Celgene: Honoraria; Millenium/Takeda Pharma: Honoraria; J&J Family: Honoraria; Onyx: Honoraria; Merck: Honoraria.