Interphase Fuorescence In Situ Hybridisation (iFISH) To Detect Cryptic Chromosome Defects in Adult B-Cell Acute Lymphoblastic Leukemia (B-ALL).
Authors: Bernasconi Paolo , Cavigliano Paola Maria , Boni Marina , Dambruoso Irene , Zappatore Rita , Giardi Ilaria , Calatroni Silvia , Rocca Barbara , Lunghi Monia , Castagnola Carlo , Astori Cesare , Lazzarino Mario 
Published: April 2020

Abstract
In B-ALL karyotype, age, leucocyte count, immunophenotype, mediastinal mass and leukemic cells in the central nervous system (CNS) are the most important prognostic parameter. The assessment of the chromosome pattern is mandatory for planning a risk-adjusted protocol-based therapy. However, even if the quality level of conventional cytogenetic (CC) analyses has progressively improved, there is a still significant failure rate in identifying karyotype defects in B-ALL. So, due to its considerable developments FISH on interphase cells has been used in addition to CC and has become the method of choice for the detection of high-risk chromosomal changes in B-ALL.
In the present study iFISH was performed in 31 adult B-ALL patients (18 females and 13 males; median age 38 years, range 16–76) who presented normal G- and Q-banded karyotypes on CC. They were part of a large series of 251 consecutive adult B-ALL patients who came to our observation in a ten years period (1994–2005). In this series CC presented a failure rate of 19.9% and detected a normal karyotype in a total of 66 patients (32%), 31 of whom had cells in fixative still available for the present iFISH study. This last, which was aimed at detecting the true incidence of the BCR-ABL, ETV6-AML1, MLL rearrangements and p16/INK4A deletion, was carried out with the following commercial probes: LSI BCR/ABL1 dual color single fusion, LSI TEL/AML1 ES, LSI MLL and LSI p16 (9p21)/CEP 9 dual color (Vysis, Downers Grove, IL, USA). Hybridization procedures were carried out according to manufacturers’ guidelines. Cut-off values were determined after having analysed two-hundred cells from ten normal controls and using a one-sided binomial distribution with a 95% confidence interval. So, the cut-off values were fixed at 10% and 6% for the BCR/ABL1 and MLL probes and at 3% for both the ETV6-AML1 and the LSI p16 (9p21)/CEP 9 probes.
Overall iFISH detected chromosome defects in 13/31 (41.9%) patients. The most common abnormality, which incidence was 25.8%, consisted in the loss of either one or two red signals corresponding to the LSI p16 (9p21)/CEP 9 dual color probe, caused by a cryptic del(9)(p21) deletion. Six patients presented a monosomy and 2 a nullisomy of the p16/INK4A locus. The ETV6-AML1 rearrangement was detected in no patient. In contrast, the amplification of the AML1 gene and the loss of one ETV6 gene were seen in 2 patients each. Both these defects were present in a small percentage of cells (ranging from 11% to 15%) which had escaped CC identification. A monosomy and an amplification of the MLL gene were observed in one patient each, one of whom with an already documented AML1 amplification. A cryptic BCR-ABL rearrangement was discovered in no patient.
In conclusion our data suggest that i) in B-ALL iFISH plays a pivotal role in the accurate definition of karyotype since it readily discovered genetic aberrations in about 40% of our adult patients with an apparently normal karyotype, ii) the loss of either one or two p16/INK4a genes, frequently seen in T-ALL, seems to be one of the commonest defect also in adult B-ALL, iii) the ETV6-AML1 and MLL rearrangements are extremely rare in adult B-ALL, iii) since iFISH did not detect the BCR-ABL translocation in any of our chromosomally normal patients, CC seems to be effective in identifying Ph1+ B-ALLs.