Biology and Treatment of High-Risk CLL: Significance of Complex Karyotype

Several reports highlight the clinical significance of cytogenetic complexity, namely, complex karyotype (CK) identified though the performance of chromosome banding analysis (CBA) in chronic lymphocytic leukemia. Indeed, apart from a number of studies underscoring the prognostic and predictive value of CK in the chemo(immune)therapy era, mounting evidence suggests that CK could serve as an independent prognosticator and predictor even in patients treated with novel agents. In the present review, we provide an overview of the current knowledge regarding the clinical impact of CK in CLL, touching upon open issues related to the incorporation of CK in the clinical setting.

Karyotype asymmetry in Cuscuta L. subgenus Pachystigma reflects its repeat DNA composition

Cuscuta is a cytogenetically diverse genus, with karyotypes varying 18-fold in chromosome number and 89-fold in genome size. Each of its four subgenera also presents particular chromosomal features, such as bimodal karyotypes in Pachystigma. We used low coverage sequencing of the Cuscuta nitida genome (subgenus Pachystigma), as well as chromosome banding and molecular cytogenetics of three subgenus representatives, to understand the origin of bimodal karyotypes. All three species, C. nitida, C. africana (2n = 28) and C. angulata (2n = 30), showed heterochromatic bands mainly in the largest chromosome pairs. Eighteen satellite DNAs were identified in C. nitida genome, two showing similarity to mobile elements. The most abundant were present at the largest pairs, as well as the highly abundant ribosomal DNAs. The most abundant Ty1/Copia and Ty3/Gypsy elements were also highly enriched in the largest pairs, except for the Ty3/Gypsy CRM, which also labelled the pericentromeric regions of the smallest chromosomes. This accumulation of repetitive DNA in the larger pairs indicates that these sequences are largely responsible for the formation of bimodal karyotypes in the subgenus Pachystigma. The repetitive DNA fraction is directly linked to karyotype evolution in Cuscuta.

Multiplex ligation-dependent probe amplification identifies copy number changes in normal and undetectable karyotype MDS patients

Chromosomal abnormalities play an important role in classification and prognostication of myelodysplastic syndrome (MDS) patients. However, more than 50% of low-risk MDS patients harbor a normal karyotype. Recently, multiplex ligation-dependent probe amplification (MLPA) has emerged as an effective and robust method for the detection of cytogenetic aberrations in MDS patients. To characterize the subset of MDS with normal karyotype or failed chromosome banding analysis, we analyzed 144 patient samples with normal karyotype or undetectable through regular chromosome banding analysis, which were subjected to parallel comparison via fluorescence in situ hybridization (FISH) and MLPA. MLPA identifies copy number changes in 16.7% of 144 MDS patients, and we observed a significant difference in overall survival (OS) (median OS: undefined vs 27 months, p=0.0071) in patients with normal karyotype proved by MLPA versus aberrant karyotype cohort as determined by MLPA. Interestingly, patients with undetectable karyotype via regular chromosome banding indicated inferior outcome. Collectively, MDS patients with normal or undetectable karyotype via chromosome banding analysis can be further clarified by MLPA, providing more prognostic information that benefit for individualized therapy.

Chromosome banding analysis and genomic microarrays are both useful but not equivalent methods for genomic complexity risk stratification in chronic lymphocytic leukemia patients

Genome complexity has been associated with poor outcome in patients with chronic lymphocytic leukemia (CLL). Previous cooperative studies established five abnormalities as the cut-off that best predicts an adverse evolution by chromosome banding analysis (CBA) and genomic microarrays (GM). However, data comparing risk stratification by both methods are scarce. Herein, we assessed a cohort of 340 untreated CLL patients highly enriched in cases with complex karyotype (CK, 46.5%) with parallel CBA and GM studies. Abnormalities found by both techniques were compared. Prognostic stratification in three risk groups based on genomic complexity [0-2, 3-4 and ≥5 abnormalities] was also analyzed. No significant differences in the percentage of patients classified into each category were detected, but only a moderate agreement was observed between methods when focusing in individual cases (κ=0.507; p

Chromosome Banding and Mechanism of Chromosome Aberrations

Chromosome identification depends on the morphological features of the chromosome and therefore karyotype and its banding pattern analyses are the most suitable technique to identify each and every chromosome of a chromosome complement. Moreover, aberrations caused by breaks play an important role in the evolution of a chromosome set and chromosome complement by decreasing or increasing the chromosome number. Therefore, both the aspects are discussed in detail in the present chapter. At present, the chapter will highlight the karyotype and its components, karyotype trends, evolution and its role in speciation, banding pattern and techniques, chromosome differentiation and linearization, banding applications and their uses, detection and analysis of chromosomal aberrations, chromosome and chromatid types of aberrations and mechanism of the formation of chromosome aberrations and breaks for karyotype evolutionary trends.

Multiplex ligation-dependent probe amplification identifies copy number changes in normal and undetectable karyotype MDS patients

Background Chromosomal abnormalities play an important role in classification and prognostication of myelodysplastic syndromes (MDS) patients. However, more than 50% low risk MDS patients harbor a normal karyotype. Recently, multiplex ligation-dependent probe amplification (MLPA) has emerged as an effective and robust method for the detection of cytogenetic aberrations in MDS patients. Methods To characterize the subset of MDS with normal karyotype or failed chromosome banding analysis, we analyzed 144 patient samples with normal karyotype or undetectable through regular chromosome banding, which were subjected to parallel comparison via fluorescence in situ hybridization (FISH) and MLPA. Results MLPA identifies copy number changes in 16.7% of 144 MDS patients and we observed a significant difference in overall survival (OS) (median OS: undefined vs 27 months, p=0.0071) in patients with normal karyotype proved by MLPA, versus aberrant karyotype cohort as determined by MLPA. Interestingly, patients with undetectable karyotype via regular chromosome banding indicated inferior outcome. Conclusion Collectively, MDS patients with normal or undetectable karyotype via chromosome banding analysis can be further clarified by MLPA, providing more prognostic information that benefit for individualized therapy.

Artificial Intelligence Substantially Supports Chromosome Banding Analysis Maintaining Its Strengths in Hematologic Diagnostics Even in the Era of Newer Technologies

Background: Chromosome banding analysis (CBA) is one of the most important techniques in diagnostics and prognostication in hematologic neoplasms. CBA is still a challenging method with very labor-intensive wet lab processes and karyotyping that requires highly skilled and experienced specialists for tumor cytogenetics. Short turnaround times (TAT) are becoming increasingly important to enable genetics-based treatment stratification at diagnosis. Aim: Improve TAT and quality of CBA by automated wet lab processes and AI-based algorithms for automatic karyotyping. Methods: In the last 15 years the CBA workflow has gradually been automated with focus on the wet lab and metaphase capturing processes. Now, a retrospective unselected digital data set of 100,000 manually arranged karyograms (KG) with normal karyotype (NKG) from routine diagnostics was used to train a deep neural network (DNN) classifier to automatically determine the class/number and orientation of the respective chromosomes (AI based classifier normal, AI-CN). With a total of 6 Mio parameters, the DNN uses two distinct output layers to simultaneously predict the chromosome number (24 classes) and the angle that is required to rotate the chromosome in its correct, vertical position (360 classes). Training of the DNN took 16 days on a Nvidia RTX 2080 Ti graphic card with 4352 cores. AI-CN was implemented into the routine workflow (including ISO 15189) after 7 months of development and intensive testing. Results: The AI-CN was tested by highly experienced staff in an independent prospective validation set of 500 NKG: 22,675/23,000 chromosomes (98.6%) were correctly assigned by AI-CN. In 369/500 (73.8%) of cells all chromosomes were correctly assigned, in an additional 20% only 2 chromosomes were interchanged. The chromosomes accounting for the majority of misclassifications were chromosomes 14 and 15 as well as 4 and 5, which are difficult to distinguish in poor quality metaphases also for humans. The 1st AI-CN was implemented into routine diagnostics in August 2019 and the 2nd AI-CN - optimized for chromosome orientation - was used since November 2019. Since then more than 17,500 cases have been processed with AI-CN (>350,000 metaphases) in routine diagnostics resulting in the following benefits: 1) Reduced working time: an experienced cytogeneticist needs - depending on chromosome quality - between 1 and 3 minutes to arrange a KG, while AI-CN needs only 1 second and the cytogeneticist about 30 seconds to review the KG. 2) Shorter TAT: The proportion of cases reported within 5 days increased from 30% before AI-CN (2019) to 36% with AI-CN1 (2019) and 45% with AI-CN2 (2019/2020), while the proportion of cases reported >7 days was reduced to 28%, 21%, and 17%, respectively (figure). Using AI-CN for aberrant karyotypes results in correct assignment of normal chromosomes and thus also correct KG in cases with solely numerical chromosome abnormalities. Derivative chromosomes derived from structural abnormalities (SA) that differ clearly from any normal chromosome are not automatically assigned but are left out for manual classification. Thus, even in cases with SA, using AI-CN saves time. To allow AI based SA assignment, two additional classifiers normal/aberrant (CNA) were built: AI-CNA1 was trained on 54,634 KG encompassing 10 different SA (AKG) and 100,000 NKG and AI-CNA2 was trained on all AKG and an equal number of NKG. First validation tests are promising and optimization is ongoing. Once the CNA has been optimized, a standardized high quality of chromosome aberration detection is feasible. A fully automated separation of chromosomes is currently in progress and will reduce the TAT by another 12-24 hours. In a fully automated workflow the detection of small subclones can be further optimized by increasing today's standard of 20 metaphases to several hundred, even without any delay in TAT and need for additional personnel. Conclusions: Implementation of AI in CBA substantially improves the quality of results and shortens turnaround times even in comparison to highly trained and experienced cytogeneticists. In the majority of cases a complete karyotype analysis can be guaranteed within 3 to 7 days, allowing CBA based treatment strategies at diagnosis. This fully automated workflow can be implemented worldwide, is rapidly scalable, can be performed cloud based and requires in the near future fewer experienced tumor cytogeneticists. Figure Disclosures Hänselmann: MetaSystems: Current Employment. Lörch:MetaSystems: Current equity holder in private company.

Multiplex ligation-dependent probe amplification identifies copy number changes in normal and undetectable karyotype MDS patients

Background In myelodysplastic syndromes (MDS), cytogenetic aberrations play an important role for classification and prognostication. However, more than 50% low risk MDS patients harbor a normal karyotype. Recently, multiplex ligation-dependent probe amplification (MLPA) has emerged as an effective and robust method for the detection of cytogenetic aberrations in MDS patients.Methods To characterize the subset of MDS with normal karyotype or failed chromosome banding analysis, we analyzed 144 patient samples with normal karyotype or undetectable through regular chromosome banding, which were subjected to parallel comparison via fluorescence in situ hybridization (FISH) and MLPA.Results MLPA identifies copy number changes in 16.7% of 144 MDS patients and we observed a significant difference in overall survival (OS) (median OS: undefined vs 27 months, p=0.0071) in patients with normal karyotype proved by MLPA, versus aberrant karyotype cohort as determined by MLPA. Interestingly, patients with undetectable karyotype via regular chromosome banding indicated inferior outcome. Conclusion Collectively, MDS patients with normal or undetectable karyotype via chromosome banding analysis can be further clarified by MLPA, providing more prognostic information that benefit for individualized therapy.