Molecular and cytogenetic characterization of variant Philadelphia positive (Ph+) Chronic Myeloid Leukaemia (CML) cases.Hessa Al hajiri, Zahida Parveen Khilji, Yara Anwar, Shabeer Padariyakam, Khitam Abu Khadija, Joy Mitchell, Zafar Nawaz and Ivone B. Otazu Cytogenetic and Molecular Cytogenetic Laboratory, Department of Laboratory Medicine and Pathology, Hamad Medical Corporation, Doha Qatar. Background Chronic Myeloid Leukaemia is a pluripotent hematopietic stem cell disorder defined by expression BCR/ABL1 fusion gene, a constitutively activated tyrosine kinase. The fusion gene is a result of t(9;22) (q34;q11.2) or related variant rearrangements leading to formation of Philadelphia chromosome (Ph). In less than 5-10% of the CML patients the bone marrow cells appear to have a karyogram with a complex translocation involving a Ph positive and with a third chromosome in addition to chromosomes 9 and 22. Although the BCR/ABL1 fusion gene can be localized by FISH on 22q11.2 and/or identified molecularly, the biological and clinical significance of the derivative and the mechanism behind their formation remain unclear and widely discussed, as does our understanding of the molecular events driving the disease evolution. Representative photographs and schemes of nuclei carrying BCR/ABL fusion gene by FISH using LSI BCR/ABL1 DF and ES translocation probes corresponding to the 5 Cases given in figure 3. 3 Molecular cytogenetic analysis of the whole chromosomes and flanking regions of BCR and ABL genes by array-CGH A summary of the array-CGH represents a whole chromosomes analysis in figure 6a, while Figure 6b represents a deletion flanking region of chromosome 9q34 and 22q11.2 resulting of a variant Philadelphia chromosome, respectively. Case 1 Case 2 6a 6b Case 5 Aims To discuss the 5 variant Ph+ cases at diagnosis involving a third chromosome and in order to asses underlying rearrangements concomitant 9q34 deletion status, as well as prognostic significance of these changes. Case 3 Materials & Methods Bone marrow specimens used for Conventional karyotype, iFISH/mFISH using the BCR/ABL1 fusion gene (Extra Signal (ES)/(Dual Fusion (DF), TEL/CEP probes and array-CGH (105K Agilent) analysis were used to asses whole chromosome regions involved of the variant for additional genomic rearrangements. Case 4 Summary Table of Results Case 5 Results Conventional Karyotype of the bone marrow/ CML samples A summary of the partial karyotypes/GTG banding of the variant translocations corresponding to 5 cases is given in figure 1. A summary of the patient’s karyotypes, FISH (DF) & (ES) and CGH results of the CML patients with mechanism of variant translocation and locus designation of the additional chromosome is given in table 1. Case KARYOTYPE AT DIAGNOSIS AND FOLLOW UP* ISCN BCR/ABL (DF) ISCN BCR/ABL (ES) ph Mechanism Array-CGH analysis 1 Masked Philadelphia chromosome resulting from a one–two ways translocation Schematic representation of the mechanism involved rearrangement masked Ph chromosome as occurred is given in figure 4a and 4b. ONE-STEP Mechanism Rearrangement 1 46,XX,t(8;9;22)(p11.2;q34q34;q11.2)[20] nuc ish (ABL1,BCR)x3 (ABL1 con BCRx2)[195/200] nuc ish (ABL1,BCRx3) (ABL1con BCRx2)[5/400] nuc ish (ABL1,BCRx2) (ABL1 con BCRx1) [19/350]/ nuc ish(ABL1x2) (BCRx2) (ABL1 con BCRx1) [13/350] nuc ish (ABL1,BCRx3) (ABL1 con BCRx2) [10/350]. nuc ish (ASS,ABL1,BCRx3) (ABL1 con BCRx1)[50/50] nuc ish (ASS,ABL1,BCRx3) (ABL1con BCRx1)[10/100] + two Normal 2* 46,X,t(X;9;22)(p22.1;q34;q11.2[10]/ 46,XX[56] + one Normal 3* 46,XX,t(9;22;10)(q34;q11.2;q26)[17]/ 46,XX[23] nuc ish (ASS,ABL1,BCRx3) (ABL1 con BCRx2) [33/50]/ nuc ish (ASS,ABL1,BCRx3) (ABL1 con BCRx1) [16/50] + one Normal 4a As ocurred in cases described previously. 2, 3, 4 and 5 4 46,XY,t(9;22;15)(q34;q11.2;p11.1)[30] nuc ish (ABL1,BCRx4) (ABL1 con BCRx1)[70/100]/ nuc ish (ABL1,BCRx3) (ABL1 con BCRx2)[28/100] nuc ish (ASS,ABL1,BCRx3) (ABL1 con BCRx1)[50/50] + one Normal 5 46,XY,t(9;22;10)(q34;q11.2;p13)[20] nuc ish(ABL1,BCRx2)(ABL1 con BCRx1) [140/150] nuc ish (ABL1,BCRx2) (ABL1 con BCRx1)[50/50] + one arr cgh 9q34.11 (131455699-13357471 3)x1 Arr cgh 22q11.23 (23871009-25593010) x1 BCR BCR q11 ABL Conclusion or ABL q34 derivative Chr+ (a) and/or Derivative chr+ (b) Ph 9 22 THIRD CHROMOSOME 9q+ Masked Philadelphia chromosome resulting from a one–two ways translocation 4b Molecular cytogenetic analysis of the BCR/ABL1 fusion gene applying LSI BCR/ABL Extra Signal (ES) and Dual fusion (DF) probes. Photograph and Schematic represents the interphase and metaphase FISH (iFISH/mFISH) patterns found showing the LSI BCR/ABL extra signal dual color (ES) probe and the BCR/ABL dual color dual fusion (DF) probe in figure 2. Note: probe color green is normal BCR gene, orange is normal ABL gene & yellow (green/orange) represents BCR/ABL fusion depicted by arrows in the interphase cells picture. TWO-STEP Mechanism Rearrangement As ocurred in case 1 t (8;9;22) (p11.2;q34q34;q11.2) p11.2 BCR q11 BCR ABL 22 Ph Ph ABL q34 9 9q+ 8 9q+ 8p+ 2 Normal Interphase Cell in BCR/ABL DF and ES Normal Metaphase cells in BCR/ABL DF and ES Molecular cytogenetic analysis of the regions flanking telomere’s of the chromosome 8 applying ToTelVysion probe Photographic representation of Metaphase FISH analysis of leukemic bone marrow depicted by arrows to show the complex variant translocation Ph involving a third chromosome 8 (In case 1) is shown in figure 5. • In this present study we report five CML cases carrying variant complex Ph+ translocations by cytogenetic, FISH and Array-CGH methods. • The characterization of these cases emphasizes application of cytogenetic studies on metaphase cells, mFISH/iFISH using DF and ES BCR/ABL fusion gene probe and telomeres probes confirming chromosomes of the variant involved. • Our findings support the difference between DF & ES probes used for BCR/ABL1 gene fusion analysis. The ES probe is more applicable to characterize variants and the molecular determination of the minor (m) and/or the Major (M) breakpoints pattern of the BCR locus region 22q11.2, respectively. • We suppose that, the on set of these variant translocation in this cases might have occurred at the early presentation of the disease despite the chronic phase. • Array-CGH analysis on these cases reveal an additional genomic rearrangements in case 5, a deletion 2.12Mb at 9q34.11 and also a deletion 1.72Mb at 22q11.23 cytogenetic bands close to the break point of BCR/ABL rearrangement. In this case a deletion occur at 5’; of the ABL breakpoint (ASS gene region) may be the cause of changes in DF and ES probes to a single fusion pattern. Altogether, the abnormalities in these case (deletion BCR/ABL flanking region) carries a probability of insufficient genetic heterogeneity of this subgroup of CML. • In conclusion, despite the low no of patients with the variant CML translocation the molecular cytogenetics characterization is a valuable tool to elucidate the pathogenetic routes of CML through the level of disease and over all survival with the typical t(9;22)(q34;q11.2) translocation after treatment would significantly yield a great knowledge in CML research. Positive BCR/ABL patterns by DF and ES m-bcr* Interphase cells Acknowledgements Positive BCR/ABL patterns by ES M-bcr * Metaphase cells Normal ABL 9q34 Normal BCR gene 5 Case 1 Derivative 9qter Derivative chromosome del 8pter Positive BCR/ABL patterns by ES M-bcr* Interphase cells ASS Gene Internal control in 9q34 Normal chromosome 8 We thank all the colleagues of the Cytogenetics laboratory and Ms. Najla Abdul Kareem, Research Technologist, Shareefa Ashiq Backer and Attiya Zainab, Senior Secretaries of DLMP, and we take this privilege to thank the HMC and NCCR organization for this opportunity. There are not conflicts of interest to disclose. BCR ABL fusion gene * LSI BCR/ABL1 fusion gene Dual color (DF), Extra Signal (ES) translocation considering the Minor breakpoint (m-bcr) and the Major breakpoint (M-bcr) Signal patterns to differentiate BCR gene locus region 22q11.2 FISH performed with ToTelVysion in case 1 correlating to TWO-STEP mechanism. Single cell with arrows indicating the chromosome 8 p-arm (green), q-arm (red) that indicates that 8pter has been translocated onto chromosome 9qter.