Chromosome Banding Analysis Defines Subclasses of CLL with 13q14 Deletion and Identifies a New Mechanism of Submicroscopic 13q14 Deletions Occurring in the Breakpoint Region of Reciprocal Translocations.
Authors: Schoch Claudia , Dicker Frank , Schnittger Susanne , Kern Wolfgang , Haferlach Torsten 
Published: October 2019

Abstract
13q14 deletions are the most frequent abnormality in CLL and are overall associated with a favourable prognosis. However, the clinical course of the disease is heterogeneous within this subgroup of CLL. In order to characterize this subgroup, which is identified in routine diagnostics by interphase FISH, in more detail we performed chromosome banding analysis in addition. By improving the cultivation technique using the immunostimulatory CpG-oligonucleotide, DSP30, and IL-2 we reached a high success rate in routine diagnostics. Since August 2005 416 CLL were analyzed in parallel with chromosome banding analysis (CBA) and interphase-FISH. The FISH panel included probes for the detection of trisomy 12, IGH-rearrangements, and deletions of 6q21, 11q22.3 (ATM), 13q14 (D13S25 and D13S319), and 17p13 (TP53). 411/416 (98.8%) cases could be successfully stimulated for metaphase generation. 348/411 (84.7%) cases showed chromosomal aberrations in CBA while abnormalities were detected by FISH in 332 of 416 (79.8%) successfully evaluated cases. In 229 cases (55%) a 13q14 deletion was detected by FISH, including 58 patients with a homozygous deletion. CBA was not evaluable in 4/229 cases. A normal karyotype was observed in 9/229, due to a small size of the aberrant clone missed by CBA (20% of interphase nuclei) in 1 case and due to the small size of the deletion not visible in CBA in 8 cases (growth of the aberrant clone was confirmed by FISH on metaphases). In 108 cases a deletion 13q was the only abnormality detected in CBA. 29 cases showed one other abnormality in addition to del(13q) (del(11q) n=13, +12 n=2, der(17p) n=3, other abnormalities not detectable by the used FISH panel n=11). In 51 cases 2 or more abnormalities were observed in addition to the 13q deletion. Interestingly, 28 cases did not show a 13q-deletion but a reciprocal translocation or insertion with a breakpoint in 13q14. In all these cases FISH on metaphases was performed with a whole chromosome painting probe for chromosome 13 and a probe for either D13S25 or D13S319, demonstrating a loss of one D13S25/D13S319 signal from the derivative chromosome 13 and the partner. In 9 cases D13S25/D13S25 was also lost from the homologous chromosome 13 (homozygous 13q14 deletion). The translocation partner was confirmed in a second FISH analysis also confirming the reciprocal nature of the abnormality. The breakpoints of the partner chromosomes were distributed all over the genome (1p13, 1q23, 1q24, 1q42, 1q42, 3q21, 3q21, 4p16, 4q23, 5q13, 5q15, 6q11, 6q23, 7p21, 8p23, 8q21, 8q22, 9p22, 9q21, 9q33, 10p15, 10q24, 11p15, 11q23, 13q34, 15q15, 16q24, 16q24). In conclusion, CBA offers important information in addition to interphase FISH in CLL. 1) CBA detects chromosome abnormalities in addition to 13q14 deletion which can not be detected with a standard interphase FISH panel. 2) CBA provides new biological insights into different mechanisms leading to loss of 13q14. Prospective clinical trials have to evaluate the prognostic impact of the different subclasses of CLL with 13q14 deletion that now can be identified by chromosome banding analysis.