The Prevalence of Cytogenetic Abnormalities Detected by Interphase FISH Method in Chronic Lymphocytic Leukemia

Normal Fish Cytogenetics and 13q Deletions Unveil Marked Biological and Clinical Heterogeneity In Chronic Lymphocytic Leukemia

SNP-Arrays: a Routine Cytogenetic Tool In Chronic Lymphocytic Leukemia ?.

Background:R‐ISS (Revised International Staging System) incorporates cytogenetic abnormalities for risk stratification of multiple myeloma. High‐risk cytogenetic abnormalities in R‐ISS include 17p deletion, t(4;14),t(14;16). Detection of these abnormalities by interphase FISH (Fluorescence in‐situ hybridisation) has become a popular modality owing to its targeted detection ability in non‐mitotic cells.Aims:The aim of this study was to observe the frequency of cytogenetic abnormalities in the Indian setting and their co‐relation with treatment outcomes.MethodsRetrospective single‐centre study conducted at a single tertiary care centre from North India. Multiple myeloma FISH panel comprising of 5 abnormalities ‐ 13q deletion, 17p deletion, t(4;14), t(11;14), t(14;16) were done in all patients with multiple myeloma, either at diagnosis or when they were referred for relapse or progression of disease. Records of patients in the last two years (January 2016 ‐July 2018) were analysed.Results:Over a period of 30months, myeloma FISH panel was done in 49 patients. Of these, abnormalities were detected in 52% (n = 25) patients. 90% patients belonged to ISS stage‐3. FISH panel was done at diagnosis, first relapse and disease progression in 77.5% (n = 38), 7.3% (n = 3) and 19.5% (n = 8), respectively. The most common abnormality was 13q deletion (36.7%). 17p deletion,t(4;14) and t(11;14) were seen in 8.2%(n = 4),4%(n = 2) and 2.4% (n = 1) patients, respectively. Response to treatment could be evaluated in 46 patients which was graded as per IMWG (International Myeloma Working Group) response criteria. The overall response rate (CR/VGPR/PR) of patients in different groups–Normal, 13q deletion, 17p deletion was 79.2%, 50%, 50% respectively. This implied that nearly half of the patients (n = 8) in 13q deletion group did not respond to therapy. Of these, 4 patients (50%) had 13q deletion at baseline. The possible reasons for this may be ‐ detection of 13q deletion in relapsed/refractory patients, concomitant t(4;14) on FISH panel, associated 13q deletion detection on conventional karyotyping and lack of 1q amp detection in our panel which could have let to an unfavourable outcome.Summary/Conclusion:IgH translocations are rare in Indian patients. 13q deletions, akin to the 17p group, are associated with high beta‐2 microglobulin levels (ISS‐3), poor responses to bortezomib based therapy and are seen on disease progression.

Interphase fluorescence in situ hybridization (FISH) can identify submicroscopic deletions adjacent to the breakpoints of rearrangements undetected by conventional cytogenetics. In this study, the characteristics and frequency of the IgH deletion identified by interphase FISH were investigated in patients with multiple myeloma (MM) and chronic lymphocytic leukemia (CLL). The study group included 29 patients with MM and eight patients with CLL. Interphase FISH was performed with the IgH dual color, break-apart rearrangement probe and the IgH/CCND1 dual color, dual fusion translocation probe. The IgH deletion was found in 14% (4/29) of patients with MM and 13% (1/8) of the patients with CLL. Four patients had deletions of the whole or variable region of IgH on the native chromosome 14, whereas one patient had a deletion of the IgH variable region on a der(11)t(11;14). In two patients, the IgH break-apart FISH showed both patterns with and without IgH deletions. In cases showing the same pattern by IgH break-apart FISH, the IgH/CCND1 FISH showed different patterns, and vice versa. A variety of patterns of the IgH deletion were identified by interphase FISH using IgH break-apart and IgH/CCND1 probes in patients with MM and CLL. The results of this study suggest that the integrated information obtained with IgH break-apart and IgH/CCND1 FISH was needed to interpret FISH results unambiguously.

Molecular cytogenetic abnormalities in patients with concurrent chronic lymphocytic leukemia and multiple myeloma shown by interphase fluorescence in situ hybridization: evidence of distinct clonal origin

To assess the value of fluorescence in situ hybridization (FISH) for the detection of genomic abnormalities among patients with chronic lymphocytic leukemia (CLL). Interphase FISH was performed on bone marrow samples derived from 105 patients with CLL at the time of diagnosis using probes for D13S319/13q14, ATM/11q22, P53/17p13 and CEP12. The abnormalities and prognostic factors were analyzed. Overall survival of the patients was calculated. The FISH assay has detected genomic abnormalities in 81 (77.1%) of the patients, among which D13S319/13q14 deletion was the most common (49/105, 46.67%). 24(22.86%) patients had trisomy 12, 21(20.00%) had ATM/11q deletion, and 12(11.43%) had P53/17p deletion. A significant correlation was found between Binet staging and the detected abnormalities (< 0.05). With a median follow-up time of 10 months, 11 patients (10.5%) had died. Compared with those with P53 deletion, patients with 13q deletion showed a better overall survival. However, the overall survival did not significantly differ between patients with various genomic abnormalities (> 0.05). FISH is capable of detecting common genomic aberrations among patients with newly diagnosed CLL. Deletion of D13S319/13q14 is the most common aberration in such patients. Genomic aberrations are significantly correlated with Binet staging but not the overall survival of CLL patients.

Clonal evolution in chronic lymphocytic leukemia detected by fluorescence in situ hybridization and conventional cytogenetics after stimulation with CpG oligonucleotides and interleukin-2: A prospective analysis

Multiple myeloma (MM) is a plasma cell dyscrasia characterized by frequent 13q deletions and IgH translocations that have clinical prognostic significance. We evaluated clonal plasma cells by interphase fluorescence in situ hybridization (FISH) and combined with immunofluorescence detection of cytoplasmic light chain (cIg-FISH) for the presence of 13q deletions and IgH translocations. The FISH results were compared with conventional cytogenetic analysis. Of the 25 bone marrow specimens from MM patients, 11 (44%) had 13q deletions. IgH translocations involving cyclin D1 (t(11;14)) and FGFR3 (t(4;14)) were found in 32 and 36%, respectively. P53 deletions were detected in 20% of the cases. One patient had coexistence of t(ll;14) and t(4;14), which has not been previously reported. Conventional cytogenetic analysis was performed in 15 cases and revealed complex numerical and structural changes in 7. Karyotype analysis failed to detect 3 of 6 cases with 13q deletions, and also missed most of the IgH translocations and p53 deletions detected by cIg-FISH. On the other hand, the complex numerical and structural changes shown by conventional cytogenetics were not demonstrated by interphase FISH. Since 13q deletions, IgH translocations and a hypodiploid karyotype are significant prognostic factors for MM, our study illustrates the importance of combining conventional cytogenetics with interphase FISH analysis in patients with MM.

Cohort Analysis of FISH Testing of CD138+ Cells in Relapsed Multiple Myeloma: Implications for Prognosis and Choice of Therapy

The most frequent chromosomal abnormalities in B-cell chronic lymphocytic leukemia (B-CLL) are deletions on 13q14 and 17p13, trisomy 12, and 14q32 rearrangement. Conventional cytogenetic analysis underestimates the frequency of specific chromosome aberrations in B-CLL because of the low rate of spontaneous mitoses and the poor response to mitogen stimulation. We used interphase fluorescence in situ hybridization (I-FISH) to explore the incidence of chromosomal changes in the peripheral blood cells of B-CLL patients. Probes for 13q14 (D13S319), 17p13 (p53), the centromere of chromosome 12 (CEP12), and 14q32 (IGHC/IGHV) were applied to detect chromosomal aberrations in peripheral blood samples from 83 B-CLL patients (60 men, 23 women). Molecular cytogenetic aberrations were found in 61 cases (73.5%), and 8 patients (9.6%) showed 2 kinds of abnormalities. The most frequent abnormality was deletion of 13q14 (41.0%), followed by +12 (19.3%), deletion of 17p13 (12%), and 14q32 rearrangement (9.6%). FISH results were analyzed for correlation with Binet stages. The percentages of patients who showed abnormalities by FISH were 73.0%, 73.3%, and 80% for Binet stages A, B, and C, respectively, and the percentages of patients with abnormalities who showed 2 anomalies were 7.9%, 27.3%, and 0% for Binet stages A, B, and C, respectively. We noted no consistent pattern among the various Binet stages in the distribution of either the types of FISH-detected anomalies or the numbers of FISH anomalies. I-FISH was found to be a rapid, exact, and sensitive technique for analysis of chromosome aberrations in CLL. FISH could provide accurate information regarding the molecular cytogenetic features of CLL.

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults and is characterized by a clonal accumulation of mature apoptosis-resistant neoplastic cells. It is also a heterogeneous disease with a variable clinical outcome. Here, we present a review of currently known (epi)genetic alterations that are related to the etiology, progression and chemo-refractoriness of CLL. Relevant literature was identified through a PubMed search (1994-2014) of English-language papers using the terms CLL, signaling pathway, cytogenetic abnormality, somatic mutation, epigenetic alteration and micro-RNA. CLL is characterized by the presence of gross chromosomal abnormalities, epigenetic alterations, micro-RNA expression alterations, immunoglobulin heavy chain gene mutations and other genetic lesions. The expression of unmutated immunoglobulin heavy chain variable region (IGHV) genes, ZAP-70 and CD38 proteins, the occurrence of chromosomal abnormalities such as 17p and 11q deletions and mutations of the NOTCH1, SF3B1 and BIRC3 genes have been associated with a poor prognosis. In addition, mutations in tumor suppressor genes, such as TP53 and ATM, have been associated with refractoriness to conventional chemotherapeutic agents. Micro-RNA expression alterations and aberrant methylation patterns in genes that are specifically deregulated in CLL, including the BCL-2, TCL1 and ZAP-70 genes, have also been encountered and linked to distinct clinical parameters. Specific chromosomal abnormalities and gene mutations may serve as diagnostic and prognostic indicators for disease progression and survival. The identification of these anomalies by state-of-the-art molecular (cyto)genetic techniques such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), single nucleotide polymorphism (SNP) microarray-based genomic profiling and next-generation sequencing (NGS) can be of paramount help for the clinical management of these patients, including optimal treatment design. The efficacy of novel therapeutics should to be tested according to the presence of these molecular lesions in CLL patients.

Update on Genomic Profiling in Chronic Lymphocytic Leukemia

Recurrent Rearrangement of the PHF1 Gene in Ossifying Fibromyxoid Tumors

Chronic lymphocytic leukemia (CLL) is characterized by multiple copy number abnormalities (CNVs) with prognostic value. Identifying these structural variations is central to defining CLL pathogenesis, risk stratification, and therapeutic approaches. Fluorescence in situ hybridization (FISH) is the clinical gold standard in detecting prognostic CNVs in CLL. However, next-generation sequencing (NGS) techniques have become more readily available for clinical genomic applications and can also be used to identify CNVs. Here we present bioinformatic methods to accurately identify CNVs in CLL using NGS data. We used the CNV-calling algorithm PatternCNV to detect clinically relevant CNVs: deletion 17p13 [del(17p)], deletion 11q23 [del(11q)], deletion 13q14 [del(13q)], and trisomy 12. PatternCNV was run on 2274 samples (1500 somatic and 774 germline samples) from six different sequencing batches, screened using a targeted sequencing panel that covers all exons of 59 recurrently CLL mutated genes and additional amplicons covering the minimal affected regions of relevant CNVs. To correct for potential batch effects, PatternCNV was initially run to quantify exon coverage behavior without the chromosomes containing recurrent CNV events, 11, 12, 13, and 17. Principal component analyses and correlation matrices were analyzed, grouping the samples into four distinct clusters that contain similar exon coverage patterns. Samples in each of the four clusters were then independently re-run through PatternCNV using all chromosomes. Visual analysis of CNV plots revealed a bias in normalization. To correct this, the log2ratios were corrected to center the log ratio on the median coverage. Sample noisiness was calculated from the difference in the median absolute deviation (DiffMAD) and samples with a DiffMAD score greater than 0.3 were excluded. All CNV analyses were blinded to clinical FISH results. The effectiveness of our CNV calling was evaluated in 522 CLL patients who had FISH conducted within three months of the sample date. We excluded samples with low tumor metrics identified by FISH (less than 20% of cells with either del(17p), del(11q), trisomy 12 or del(13q)). When we compared our CNV analyses with the FISH data, we found high concordance 99.6% for del(17p), 97.5% for del(11p), 99.1% for trisomy 12, and 93.7% for del(13q). N=46 total discordant pairs were identified, with the highest discordance for del(13q), N=28, followed by del(11q), N=12. These novel bioinformatic methods allow for accurate detection of CNVs across NGS sequencing batches. The high concordance in detecting CNVs between targeted NGS and the gold standard of FISH, suggest NGS is an accurate tool for calling CNVs in CLL. Further, NGS can infer clinically relevant CNVs in genomic locations not targeted by FISH. Citation Format: Chantal E. McCabe, Erik Jessen, Daniel R. O'Brien, Julia E. Wiedmeier-Nutor, Susan L. Slager, Esteban Braggio. Identifying copy number variations in chronic lymphocytic leukemia using targeted next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3352.

e19558 Background: Multiple myeloma is characterized by the accumulation of malignant plasma cells within the bone marrow and regarded as incurable, but remissions may be induced with steroids, chemotherapy, thalidomide and stem cell transplants. The clinical heterogeneity of myeloma is dictated by the cytogenetic aberrations present in the clonal plasma cells. Fluorescence in situ hybridization (FISH) overcomes the limitations of standard cytogenetics and allows for the detection of numerical and structural chromosomal abnormalities in both metaphase spreads and interphase nuclei. Methods: We evaluated the chromosome abnormalities in 34 MM patients using conventional cytogenetics and interphase FISH with 6 probes such as IGH/CCND1, IGH/FGFR3, IGH/MAF, DS13S319/LAMP1, IGH/BAP, and p53/CEP17. Results: Cytogenetic abnormalities were found in 24 (70.6%) of the 28 MM patients. 10 (35.7%) patients had abnormal metaphases by conventional cytogenetics. Interphase FISH results were abnormal in 21 (61.8%) patients and 11 (52.3%) patients had abnormal interphase FISH but normal metaphases. The evidence of the loss of D13S319 with or without loss of LAMP1 was found in 6 (21.4%) patients, and loss of p53±CEP17 for 2 patients, IGH-BAP for 9 (26.5%) patients, IGH/FGFR3 for 2 patients, and IGH/CCND1 for 7 (20.6%) patients, respectively. However, there were none positive for IGH/MAF. Chromosome 13 abnormalities and IGH rearrangement is correlated with poor clinical outcome. Conclusions: Interphase FISH can provide useful information to evaluate the presence of prognostic chromosome abnormalities in addition to metaphase cytogenetics. And it should be used in the routine evaluation of multiple myeloma. No significant financial relationships to disclose.