Comprehensive Genetic Analysis by Integration of Conventional Karyotyping and Interphase FISH Helps Refinement of Biological Subclasses with Clinical Impact in Chronic Lymphocytic Leukemia

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 Corresponding author. P. S. Kadam Amare et al. 428 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.

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 [8] [9] [14] [22]- [24].
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-pathological 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.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 [17] [20].Minimum 200 cells were scored in each spe-cimen.The cut off threshold for trisomy and dual fusion probes was 2%.The cut off threshold for LSI 13q14 deletion, ATM deletion, and 6q deletion was 5%.The baseline value for TP53 deletion and break apart probe for IgH & C-MYC was 7%.Patients with ≥2 FISH markers were grouped under coexistence of ≥2 aberrations.IgH translocation studies were carried out in a cohort of 557 out of 671 patients (Age Range: 37 -88 Years).

Material & Methods
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) [7].After 72 hrs of stimulation, cultures were terminated and chromosome preparation was done as per standard protocol.The correlation of FISH markers with clinical variables and with conventional karyotyping findings was evaluated by Pearson's chi-square test (SPSS version 20).

Discussion
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 [10]- [12] [17] [22] [30] [31].As summarized in Table 1,  [31].Although reported incidence of del(17p) is 4% -15%, the frequency in our series of del(17p) was towards higher side 15% than del(11q22)(13%).The correlation of Rai staging with 17p deletion showed no significant association of del(17p) with stage of the disease in B-CLL which may indicate that del(17p) may not necessarily be associated with disease stage, rather it occurs frequently in progressive disease [11] [12] [18] [21] [32].In the present study, the identification of progressive vs non-progressive disease and treatment response with cytogenetic findings was not possible due to unavailability of clinical follow up.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 [11] [33] [34].
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 [10] [12] [17] [21] [31].The frequencies of all FISH markers as a sole aberration and in combination with other recurrent markers revealed that del(11q22) and del(17p) occur frequently in combination with other recurrent FISH marker/s rather than sole aberration.We found correlation of Abn(13q) with lower age and lower stage of disease.There was no association of high risk markers del(11q) and del(17p) with either variable age or stage of the disease, whereas group of coexistence of ≥2 abnormalities was associated with Rai stage III, IV.Study by Quijano et al. [11] found higher proliferative index in B-CLL patients with co-existence of del(13q) & del(17p).The review of literature emphasized that del(17p), a high risk progressive group commonly seen in association with high risk factors like unmutated IgVH, CD38 positivity rather than stage of disease [10] [11] [13] [14] [18] [22] [32] [35].
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 [9] [32] [35] [38].Recently, Thompson et al. [32] reported that complex karyotype is an independent powerful predictor of outcome of targeted Ibrutinib-based regimen as well as stronger predictor of biological behavior than del(17p).
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 [15] [16] [18] [19] [30] [39] [40].These mutation markers along with integrated cytogenetic findings will contribute in better refinement of prognostic groups in B-CLL.

Conclusion
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.

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.

Figure 3 .
Figure 3. Bone marrow morphology shows heterogeneous mixture of small and medium sized lymphocytes with indented nuclei.

Table 1 .
Frequency of FISH targeted markers by FISH in CLL in present study (n = 671) and incidence in reported literature.

Table 2 .
Correlation of FISH genetic groups with age and stage of the disease (n = 432).

Table 5 .
Karyotyping and FISH markers details in the present study (n = 31).

Table 6 .
Correlation of FISH markers with complex and non-complex karyotype (n = 31).