Background: Various genetic technologies have been employed in the identification of genomic complexity and refinement of prognostic classification of clinically heterogeneous disease of chronic lymphocytic leukemia (CLL). Objective: The present study of interphase cytogenetics and conventional karyotyping was undertaken to perform comprehensive analysis of CLL genetics with an approach to refine early prognostication of disease. Material & Methods: Retrospective analysis by fluorescence in situ hybridization (FISH) was carried out on total 671 patients of CLL at diagnosis between 2008 and 2015. Conventional cytogenetics studies were performed in 50 of 671 patients using CPG Oligonucleotide + IL-2 and TPA (12-O-Tetradecanyl Phorbol 13-acetate) for stimulation of lymphocytes cultures. Results: Interphase cytogenetics could detect recurrent abnormalities such as del(13q14), +12, del(17p13), del(11q22), del(6q23) in 71% of cases. The incidence of del(13q) was higher in Rai stage 0, I, II (p = 0.0005); whereas patients with ≥2 aberrations were more common in advance stage III, IV (p = 0.001). Frequency of IgH translocation was 7%. Morphology and immunophenotypic analysis revealed atypical CLL with higher frequency of t(14;19) than t(14;18). Conventional karyotype could detect abnormal karyotype in 97% of cases which displayed targeted FISH abnormalities along with additional non-targeted chromosomal abnormalities. Patients with negative FISH markers showed clonal non-recurrent numerical and structural changes. The complex karyotype was identified in 24% cases which included targeted FISH aberrations as well as non-targeted numerical and structural abnormalities like deletions, and unbalanced translocations. A significant association was observed between complex karyotype and coexistence of ≥2 FISH markers (p = 0.009) and del(11q22) &/or del(17p) (p = 0.03). Conclusion: Our data of interphase FISH with integration of conventional karyotyping revealed genomic complexity that helped identification of biological subclasses with clinical impact at diagnosis. Further, these cytogenetic subclasses along with molecular markers are likely to evolve more refined prognostic groups, which will help design risk-adapted therapies in B-CLL.
Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease with variable response to chemotherapy, survival period from less than one year to more than 15 years [
Although gold standard, targeted nature of interphase cytogenetics limits its contribution in the assessment of comprehensive genomic assessment, the detection of additional clonal abnormalities apart from recurrent aberrations by conventional karyotyping helps identification of different genetic subclasses with distinct prognostic classification [
Recently, complex karyotype with IgVH mutation has been found to be a high risk group in CLL [
The present study was undertaken 1) to evaluate the frequency of recurrent interphase FISH abnormalities which include del(13q14), +12, del(11q22), del(17p13), del(6q23) and correlate these markers with clinico-pa- thological parameters, 2) to evaluate frequency and characterization of IgH translocations and their association with typical & atypical CLL, 3) to investigate comprehensive analysis of genetic picture by conventional karyotyping, & 4) to correlate complexity and/or non-complexity of karyotypic picture with FISH results & clinical variables.
A total of 671 CLL patients (512 Males & 159 Females, Age Range: 24 - 92 years, Median Age: 58 years, M:F Ratio: 3.2) diagnosed by standard morphology and immunophenotypic criteria between May, 2008 and Dec, 2015 at the Department of Medical Oncology, Tata Memorial Hospital, were included in the present study. The diagnosis of atypical CLL included morphology (nuclear indentation) and immunophenotype (CD 22+ve, CD 23 dim/−ve, surface Ig weak and FAM7 +ve). FISH and conventional karyotyping studies were performed at diagnosis before the decision of treatment initiation. Those patients who needed treatment were treated with standard chemotherapy including Fludarabin, Chlorambucil and Cyclophosphamide.
FISH was performed on interphase cells from bone marrow aspirate and/or peripheral blood with panel of probes that includes LSI D13S319(13q14.3)/LSI 13q34, CEP 12, LSI(17p13.1)/D17Z1, LSI ATM(11q22)/ D11Z1, LSI MYB(6q23)/D6Z1, LSI IgH break apart, LSI dual fusion IgH/CCND1, IgH/BCL2, IgH/BCL3 and C-MYC break apart probe (Abbott Molecular, Delkenheim, Germany and Kreatech Diagnostics, The Netherland). FISH protocol followed as described previously [
Conventional karyotyping was performed in 50 patients from a cohort of 671 patients. The target materials were bone marrow aspirate and/or peripheral blood which were cultured in HAM F10 with 10% fetal calf serum along with immunostimulatory CPG oligonucleotide DSP 30 (2 µM) (TIB MOL BIOL, Berlin, Germany) in combination with Interleukin-2 (IL-2) (200 units/ml) (Roche, Sydney, Australia). Simultaneously, cultures were also stimulated with TPA (12-O-tetradecanoyl phorbol 13-acetate) (50 ng/ml) [
A 13q biallelic deletion was detected in 30/288 (10%) cases (
The frequency of IgH translocations in a cohort of 557 cases was 7% (42/557). The translocations were characterized as t(14;18)(q32;q21)(41%:17/42), t(14;19)(q32;q13)(24%:10/42), t(8;14)(q32;q24)(2%:1/42) and variant IgH translocations (33%:14/42) (
Cytogenetic Marker | No. of Patients (%) | Incidence in Reported Literature |
---|---|---|
del(13q)/-13 | 288(43) | 40% - 60% |
Trisomy 12 | 127(19%) | 20% - 40% |
del(11q22) | 87(13%) | 10% - 25% |
del(17p13)/-17 | 101(15%) | 4% - 15% |
del(6q23) | 51(8%) | 5% - 9% |
FISH Genetic Groups | Age (years) | p value | Rai Stage | P value | ||
---|---|---|---|---|---|---|
<58 | >58 | 0, I, II | III, IV | |||
Positive (%) | Negative (%) | Positive | Negative | |||
del(13q)/-13 | 67(42) | 168(53) | 0.028 | 103(71) | 146(51) | 0.0005 |
92(58) | 150(43) | 43(29) | 140(49) | |||
Trisomy 12 | 31(43) | 204(50) | 0.253 | 36(59) | 213(57) | 0.814 |
41(57) | 201(50) | 25(41) | 158(43) | |||
del(11q22) | 14(61) | 221(49) | 0.155 | 10(48) | 239(58) | 0.341 |
9(39) | 223(51) | 11(52) | 172(42) | |||
del(17p13)/-17 | 18(62) | 217(48) | 0.155 | 15(60) | 234(58) | 0.806 |
11(38) | 231(52) | 10(40) | 173(42) | |||
del(6q23) | 8(62) | 227(49) | 0.370 | 6(50) | 243(58) | 0.587 |
5(38) | 237(51) | 6(50) | 177(42) | |||
≥2 aberrations | 65(50) | 170(49) | 0.766 | 54(45) | 195(63) | 0.001 |
64(50) | 178(51) | 67(55) | 116(37) |
IgH Translocation | Atypical CLL | Typical CLL |
---|---|---|
t(14;18) | 7(54%) | 6(46%) |
t(14;19) | 5(71%) | 2(29%) |
t(8;14) | 1(100%) | 0 |
Variant IgH translocations | 7(63%) | 4(37%) |
IgH translocations were most frequently accompanied by trisomy 12(53%:22/42) followed by del(13q) (28%: 12/42) (
Of total 50 specimens, successful cultures were obtained in 40(80%) specimens. Both CPG-oligonucleotide DSP 30 +IL-2 and TPA stimulation showed almost equal proliferative index. A minimum of 10 - 20 metaphase cells were karyotyped in each specimen. Of total 40, 31 preparations with 200 - 300 G-band resolution were karyotyped and of the total 30/31 specimens (97%) displayed abnormal karyotype (
Interestingly, 2/11 cases with no targeted abnormalities by FISH displayed clonal abnormalities del(13) (q14q22) (
IgH Translocation | Trisomy 12 | del(13q)/-13 | del(11q22) | Sole Aberration |
---|---|---|---|---|
t(14;18) | 46% | 31% | - | 23% |
t(14;19) | 71% | 28% | - | - |
t(8;14) | - | - | - | 100% |
Variant IgH Translocations | 45% | 9% | 9% | 36% |
Sr.No | Age | Sex | Rai Stage | Karyotype | FISH markers: del(13q)/-13, Abn(17p)/-17, del(11q), +12, del(6q) |
---|---|---|---|---|---|
1 | 66 | M | II | 46, XX, del(18)(p11) [ | Negative |
2 | 68 | M | I | 47, XY, +12 [ | nuc ish(CEP12X3) |
3 | 70 | M | III | 46, XY, del(17)(p13) [ | nuc ish(D17Z1X4-5, TP53X2) |
4 | 45 | M | I | 47, XY, del(7)(q32), +12, del(13)(q14.1q14.3) [ | nuc ish(CEP12X3) (D13S319X1, 13q34X2) |
5 | 66 | F | I | 46, XX, del(13)(q14q22) [ | Negative |
6 | 62 | M | I | 46, XY, t(1;14)(q42;q32) [ | Negative |
7 | 68 | M | IV | 47, XY, del(19)(p11), +21 [ | Negative |
8 | 56 | M | 0 | 47, XY, t(?;7)(?;p15), +12 [ | nuc ish(CEP12X3) |
9 | 52 | M | I | 45, XY, −8, del(13)(q14q22), i(17q), −18 [ | nuc ish(D17Z1X2, TP53X1), (D17Z1X1, TP53X1) |
10 | 70 | M | III | 46, XY, del(6)(q22), del(11)(q22), del(15)(q24), −18 [ | nuc ish(D11Z1X2, ATMX1) |
11 | 50 | F | II | 47, XY, +12 [ | nuc ish(CEP12X3) (D13S319X1, 13q34X2) |
12 | 64 | M | III | 47, XY, +1, i(1p), −2, +12 [ | nuc ish(D11Z1X2, ATMX1) |
13 | 71 | F | III | 46, XX, del(13)(q14) [ | nuc ish(D13S319X1, 13q34X2), (D13S319X1, 13q34X1) |
14 | 67 | M | 0 | 46, XY, del(13)(q14.1q14.3) [ | nuc ish(D13S319X1, 13q34X2) |
15 | 42 | M | II | 46, XY, t(13;14)(q22;q32) [ | nuc ish(D11Z1X2, ATMX1) |
16 | 58 | M | IV | 47, XY, del(15)(q11.2q15) [ | Negative |
17 | 66 | M | 0 | 47, XY, +9, del(12)(p13), del(15)(q15q22) [ | Negative |
18 | 57 | M | I | 45 - 46, XY, +9 [ | nuc ish(D11Z1X2, ATMX1) |
19 | 69 | M | 0 | 46, XY, +12 [ | nuc ish(D11Z1X2, ATMX1) |
20 | 64 | M | 0 | 45, XY, +11, del(11)(q22), −17, −18 [ | nuc ish(D11Z1X2, ATMX1), (D17Z1X1, TP53X1) |
21 | 60 | M | I | 47, XY, +12 [ | nuc ish(CEP12X3) |
---|---|---|---|---|---|
22 | 62 | M | I | 47, XY, del(5)(q12q13), del(6)(q23), del(11)(q23) [ | nuc ish(D11Z1X2, ATMX1), (D6Z1X2, MYBX1) |
23 | 45 | M | 0 | 46, XY [ | Negative |
24 | 52 | F | III | 46, XX, del(15)(q22.3q24) [ | Negative |
25 | 72 | M | II | 47, XY, +12 [ | nuc ish(CEP12X3) |
26 | 45 | F | II | 46, XY, t(14;18)(q32;q21) [ | Negative |
27 | 64 | M | IV | 46, XY, del(6)(q21q23) [ | nuc ish(D6Z1X2, MYBX1) |
28 | 61 | F | IV | 46, XX, del(13)(q12q14) [ | nuc ish(D13S319X1, 13q34X2) |
29 | 54 | F | III | 46, XX, t(?;14)(?;q32) [ | Negative |
30 | 55 | M | II | 46, XY, del(14)(q11.2q24) [ | Negative |
31 | 71 | M | III | 45, XY, −13 [ | nuc ish(D13S319X1, 13q34X1) |
In a cohort of 31 cases, 7 cases (24%) had complex karyotype. All cases with complex karyotype had targeted FISH aberrations like del(13q), +12, del(11q), del(17p), del(6q) and also additionally displayed numerical as well as structural abnormalities like deletions, and unbalanced translocations (
The incidence of targeted FISH abnormalities was 71% in our series of 671 patients of B-CLL and that was consistent with the range (60% - 80%) of reported studies [
FISH Genetic Groups | Complex Karyotype (%) | Non-Complex Karyotype (%) | P value | ||
---|---|---|---|---|---|
Positive | Negative | Positive | Negative | ||
del(13q)/-13 | 0 | 13(37) | 4(100) | 22(63) | 0.281 |
Trisomy 12 | 1(13) | 12(39) | 7(88) | 19(62) | 0.229 |
del(11q22) | 1(33) | 12(33) | 2(67) | 24(67) | 1.0 |
del(17p13)/-17 | 2(100) | 11(30) | 0 | 26(70) | 0.105 |
del(11q)&/or Abn(17p) | 5(71) | 8(25) | 2(29) | 24(75) | 0.03 |
del(6q23) | 0 | 13(34) | 1(100) | 25(66) | 1.0 |
≥2 aberrations | 4(100) | 9(26) | 0 | 26(74) | 0.009 |
Negative | 0 | 13(45) | 10(100) | 16(55) | 0.01 |
del(13q)/-13(43%) was most common, followed by trisomy 12(19%), del(17p)(15%), del(11q)(13%) and del(6q)(8%) [
A biallelic 13q deletion with incidence of 9% - 10% has been observed in other studies. We did not find correlation between mono/biallelic 13q deletion with age and stage of disease. The clinical impact of an biallelic 13q deletion is controversial [
The predominant clone of abn(13q) in cases with coexistence of ≥2 abnormalities is consistent with reported studies, which confirmed that abn(13q) is a most common genetic event [
The frequency of IgH translocation in our study was 7%, is consistent with reported studies 4% - 9% [
Conventional karyotyping with stimulated cultures with CPG Oligonucleotide with IL-2 as well as TPA found to be very efficient strategy as compared to FISH, enabled to detect targeted FISH aberrations as well as non-targeted comprehensive chromosomal aberrations in 97% of cases. Overall, 71% of cases had heterogeneous chromosomal abnormalities with affected loci other than FISH targeted loci which were present as either sole or as additional abnormalities along with FISH markers. We could efficiently identify large 13q deletion, del(13)(q12q14) & del(13)(q14q22) in two cases, where FISH failed to detect these non-targeted large deletions. Large deletions covering 13q12q14/q14q22 which fall in class II deletion cover RB1 and many additional genes, which may drive disease progression probably through involvement of novel genes pathways [
The complex karyotype displayed FISH targeted recurrent aberrations like del(13q14), +12, del(17p13), del(11q22), del(6q22) as well as non-targeted numerical and structural abnormalities, which were also detected in non-complex abnormal karyotypes. The significant correlation of complex karyotype with del(11q22) and/or del(17p) and also with FISH subgroup, coexistence of ≥2 abnormalities supports and reflects the fact that B-CLL with complex genome may be a consequence of progressive genomic instability with poor prognosis. This is supported by recent findings that showed complex karyotype with association of poor prognostic markers like del(17p), unmutated IgVH, decreased event free survival, chemo refractory to standard Fludarabine and also targeted Ibrutinib-based regimens [
Recently, Sanger sequencing and next generation sequencing have identified new genomic abnormalities such as NOTCH1, SF3B1, and MYD88 & BIRC3 mutations along with TP53 deletion/point mutation. NOTCH1 mutation is associated with higher risk of Richter Syndrome transformation. SF3B1 mutation is associated with chemo resistance to alkylating/Fludarabine therapy. BIRC3 mutation is associated with chemorefractory, relapsed disease and found to be very high risk, independent prognostic marker [
In conclusion, our study shows that interphase FISH with integration of conventional karyotyping is powerful strategy, able to identify not only recurrent targeted abnormalities but also genomic complexity with clinical significance that helped identification of additional prognostic subclasses. The point mutation markers along with integrated cytogenetic findings will contribute in better refinement of prognostic groups and help design risk-adapted treatment strategies in B-CLL.
P. S. Kadam Amare,S. Kakade,K. Chopra,M. Sengar,H. Menon,H. Jain,B. Bagal,P. G. Subramanian,S. Gujral, (2016) Comprehensive Genetic Analysis by Integration of Conventional Karyotyping and Interphase FISH Helps Refinement of Biological Subclasses with Clinical Impact in Chronic Lymphocytic Leukemia. Journal of Cancer Therapy,07,427-438. doi: 10.4236/jct.2016.76045