Development of a Reflex FISH Assay Panel for Lymphoid Neoplasms Resulted Negative by Cytogenetics and Current FISH Panel and Positive by Hematopathology.
Authors: Mitter Navnit S , Lanno Stephen , Blackson Jason , Donskoy Michelle , Ehrenpreis Ralph 
Published: October 2019

Abstract
Abstract 4722
Fluorescence in situ hybridization (FISH) panel for detecting lymphoid neoplasms in interphase nuclei currently used at most of the laboratories initially utilizes the IGH (14q32.3) locus specific probe and based on negative or positive results obtained, additional testing is done with either the BCL6 (3q27), MYC (8q24) and MALT1 (18q21) locus specific probes, or the MYC/IGH (8q24/14q32.3), CCND1/IGH (11q13/14q32.3) and IGH/BCL2 (14q32.3/18q22) locus specific probes, respectively. Although these probes detect a large number of lymphoma-related abnormalities, approximately 10% of cases are still resulted negative by this FISH panel, as well as cytogenetics analysis on lymphoid mitogens stimulated bone marrow/peripheral blood cultures, but positive by hematopathology. Apparently, a need exists for either an expansion of this FISH panel or development of a reflex FISH panel to be used only when the current FISH panel fails to detect any abnormality in such cases. We selected three additional probes, TCR a/d, MYB and ALK to test further in a pilot study including twenty-five such patients, based on a high number of studies describing involvement of specific loci in gene rearrangements in lymphoid neoplasms. TCR a/d (14q11) locus is frequently involved in gene rearrangements in T-cell lymphomas and leukemias. These rearrangements include t(1;14)(q32;q11), t(1;14)(q34;q11), t(7;14)(p15;q11),
t(7;14)(q34-35;q11), t(8;14)(q24;q11), t(10;14)(q24;q11), t(11;14)(p13;q11), t(11;14) (p15;q11) and inv(14)(q11;q32)/t(14;14)(q11;q32), among others. MYB (6q23) locus shows loss of heterozygosity in a high proportion of patients with peripheral T-cell and NK/T-cell lymphomas. ALK (2p23) locus has also been well documented as involved in gene fusion with multiple partner loci, including 1q21, 2q11-13, 2q35, 3q21, 5q35, 17q23, 19p13.1 and Xq11-12 in Ki-1-positive anaplastic large cell lymphoma, a subtype of non-Hodgkin lymphoma involving Ki-1 antigen. A known negative control for each probe and a known MYB-positive control were used in a blind-coded set-up with the criteria of a positive result with abnormal probe signal pattern in at least 5% (10/200) of cells examined with the validated standard FISH protocols. This experimental reflex panel succeeded in detecting abnormalities in three of the twenty-five (12%) patients included in this pilot study. Two cases were positive for the loss of heterozygosity for the MYB locus, while one case was positive for involvement of TCR a/d locus in a translocation. The ALK probe did not detect any additional abnormality, but none of the patients had a hematopathology diagnosis of Ki-1+ anaplastic large cell lymphoma. Only four of the twenty-five patients studied had a hematopathology diagnosis of a T-cell disorder. Further, some of the patients included in this study had a low percentage of cells found abnormal by hematopathology. These factors could have affected the observed rate of abnormalities by this reflex FISH panel. Still, with 12% of patients showing abnormalities with this reflex FISH panel, this pilot study clearly demonstrates the usefulness of incorporation of this reflex FISH panel in the standard protocols for FISH studies in lymphoid neoplasms. We plan to continue investigation on additional patients meeting the criteria for inclusion in this study.

Disclosures:
No relevant conflicts of interest to declare.