Can We Use Targeted Next-Generation Sequencing an Alternative Method to the Conventional Tests in Haematological Malignancies?

Introduction: Advanced diagnostic methods give a huge advantage for identication of the abnormalities in myeloid malignancies. Researchers tried to show the potential importance of genetic tests both before the onset of the disease and during the remission. Large testing panels prevents false negative results in myeloid malignancies. But the important question is how can be merged with conventional cytogenetic and molecular cytogenetic techniques together with NGS technologies. Methods: In this paper, we draw an algorithm for evaluation of the malignancies. In order to evaluation of genetic abnormities we performed cytogenetics, molecular cytogenetics and NGS testing panels in hematologic malignancies. In this study, we analyzed 132 patients which are referred to Medical Genetics Laboratory within different type of hematologic malignancies. We highlighted possible algorithm for cytogenetically normal cases. Results: We analyzed cytogenetically normal patients by using NGS 141 gene panel and we detected two or more pathogenic variations in 20 out of 132 patients. Conclusions: Despite of long turnaround time conventional techniques is still golden standard for myeloid malignancies but sometimes cryptic gene fusions or complex abnormalities cannot easily be identied by conventional techniques that conditions advanced technologies are recommended.


Introduction
Myeloid malignancies are originating from a hematopoietic progenitor cells and characterized by defective differentiation of myeloid progenitor cells [1]. Advanced molecular diagnostic techniques have been change the diagnostic algorithm. Increased usage of next generation sequencing (NGS) can help to change the scope, timing and suitability of genetic testing in hematologic malignancies [2]. Despite the increased advances in NGS technology and rising of study ndings which supports the diagnostic and prognostic usage of mutational pro ling in myeloproliferative neoplasms (MPN), the clinical decisionmaking role is still not fully utilized [3].
Diagnostics algorithms of acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) has been changed in recent years [4,5]. Due to the advances in NGS technology various myeloid NGS panels are commercially available and generally analyse 25-50 genes which can be classi ed into several functional categories including the splicing machinery, epigenetic modi ers, transcription factors, signalling molecules and chromatin modi ers [6,7,8,9].
The increased knowledge of genetic abnormalities has been updated classi cation of the Acute Myeloid Leukaemia (AML). The World Health Organization [11] and European LeukemiaNet [12] added new subgroups of diseases and molecular genetic abnormalities have also been added in diagnostic criteria's.
Increased number of mutational, epigenetic and expression studies will be help identi cation of the novel markers in myeloid malignancies. National Comprehensive Cancer Network (NCCN) has been added mutations in FLT3, NPM1, CEBPA and KIT genes for risk evaluation [13] and according to the ELN guidelines TP53, RUNX1 and ASXL1 mutations should also be considered during the evaluation of risk. According to the studies; SF3B1, IDH1 and IDH2 have also be added as a marker [3,6,7,14,15]. Laboratories should consider the risks for using large panels to test for myeloid malignancies. There are signi cant differences in intensity and variant outputs between different laboratories. Therefore, there is an urgent need to develop some standard diagnostic kits. In addition, existing somatic variant classi cation rules do not include complex interactions between variants within gene pathways [3]. Using NGS based myeloid gene panels will help for identi cation of multiple recurrent somatic mutations in many AML patients, and additional molecular genetic mutations can be detected in most cases, even within de ned AML entities [2]. In myelodysplastic syndrome (MDS), NGS allows the detection of molecular mutations in approximately 90% of patients [6,16,17].
As a result, the data of NGS should be interpreted in the context of other laboratory ndings regarding cytomorphology, histopathology, immunophenotyping, conventional molecular genetics, cytogenetics, and clinical diagnostic parameters.
In this study, we analysed 149 patients which are referred to Medical Genetics Laboratory within different type of hematologic malignancies. We performed conventional cytogenetics, molecular cytogenetics and NGS analysis in these cases. According to the our results we highlighted possible algorithm for cytogenetically normal cases.

Patient samples
The present study included 132 patients (77 were men and 55 were women) from December 2017 to September 2018. Written informed consent was obtained from all cases. If patients are under 18 [9 children (≤ 15 years)], from a parent and/or legal guardian were obtained. Study was approved by the Ethics Committee of our university and conducted in accordance with the ethical principles established in the Declaration of Helsinki. The median age of cases was 56 years, ranging from 1 to 90 years and there were 8 children (≤ 15 years) in 132 adults. Patient characteristics were shown in Table 4. Our cohort consists of 132 patients whose diagnosed with MM (39), AML (24), CLL (14), MDS (13), Lymphoma subtypes (13), ALL (6), Pancytopenia (4), MPD (2), Castleman Disease (2), Acute leucosis (2) and 13 other hematologic malignancies. DNA was isolated from bone marrow (QIAamp DNA Blood Mini Kit (bone marrow = 132) (Qiagen, Germany) and peripheral blood (MagNA Pure system Roche Diagnostics). DNA was quanti ed using a Qubit uorometer (Thermo Fisher Scienti c).

Next generation sequencing
In order to evaluation of myeloid neoplasm speci c 141 genes The Human Myeloid Neoplasms QIAseq Targeted DNA Panel (Qiagen, Germany) was used. This panel covers exon/intron boundaries as shown in Figure 1 and covers genes has been listed oin Table 1 [18]. MiSeq sequencing-by synthesis bench top sequencer used for sequencing of ampli ed targets according to the manufacturer's protocol for pairedend sequencing (Illumina, San Diego, CA, USA). Data analysis and quality assessment for calling of single-nucleotide variants and analysis of short insertions and deletions evaluated by using Ingenuity Variant Analysis (IVA) programme. Amplicons were noted as a dropout and excluded from analysis if the coverage at any analysed position in any of the two paired-end sequences (minimal coverage) was 100x, with allele frequency >5%were included for subsequent investigation. Known hotspot or clinically actionable variants detected below these thresholds were veri ed using orthogonal methods such as Sanger sequencing.

Cytogenetic Assessment
Karyotyping: Marrow Max and Chang media were used for cultures for bone marrow and peripheral blood specimens in a CO 2 incubator. After 24, 48, or 72 h of incubation culteres were harvested. Colcemid was used for arresting of metaphase cells and chromosome slides were stained by using for G banding protocol. International System for Human Cytogenetic Nomenclature (ISCN 2016) [19] was used for reporting and a least 20 metaphases were analysed in each culture.
Fluorescence in situ hybridization (FISH): FISH was applied according to the manufacturer's recommendations. A total of 200 interphase cells were analysed for each sample and images captured/stored by using Applied Imaging/Cytovision system. Final results were reported using the cutoff established in the laboratory for each of the probes tested [20]. Speci c gene panels for FISH was applied for each malignancy. FISH panels for each of the malignancies are listed in below.

Results
Characteristics of the 132 patients are summarized in Table 2. Among these patients, 77 were men and 55 were women. The median age was 56 years, ranging from 1 to 90 years and there were 124 adults and 8 children (≤ 15 years). Cytogenetic and molecular cytogenetic evaluations were normal in all of the cases. By applying NGS using the 141 gene panel to the cytogenetically normal patients we found two or more pathogenic variations in 20 out of 132 patients. Detailed mutation list has been shown in Table 3.

Next generation sequencing
Next generation sequencing of hotspot regions of 141 gene has been performed in 132 bone marrow sample which are refereed from Department of Haematology. Variables with a depth of coverage > 100x and an allele frequency of > 5% were included in this study. Known hot spots or variants identi ed below the threshold that may require clinically intervention were con rmed by using the Sanger sequencing. Variables of unknown signi cance were excluded from the clinical bene t analysis. Variants were classi ed as pathogenic and possible pathogenic according to the gene and clinical effects. Pathogenic variations were higher in patients with AML, MM, CLL. Two or more pathogenic variations were identi ed in 20 out of 132 patients. List of the variants has been shown in Table 3. ASXL1 c.1934dupG mutation was detected in AML and Myelo brosis patients. NRAS c.181C > A was detected in 2 MM patients, IDH2 c.419G > A was identi ed in AML and CMML cases. TET2 c.945delC mutation detected in AML and primary myelo brosis cases, SRSF2 c.284C > A mutation was identi ed in AML and primary myelo brosis cases. MPL c.1544G > T mutation was detected in AML and primary myelo brosis cases, SF3B1 c.2098A > G were identi ed in AML, and MDS cases. The lowest allelic frequency was observed in one MM patient in % 5, % 20 for pathogenic variation of KRAS: c.183A > C. These results show us clonally could be observed in lowest percentages. In the literature, it is recommended that to be able to determine clonally it should be evaluated up to 5% allelic fraction [21].

Discussion
Genetic and epigenetic alterations plays an important role during leukomogenesis [22]. Because of the limitations of the techniques, a variety of methods, from cytogenetic to complete genome sequencing, are needed to be able to prevent incomplete picture of genetic deviations in malignant homeopathies [23].
Advances in next generation sequencing (NGS) technology helps transformation of gene sequencing as a considerably faster and less expensive test, making it more practical in clinical practice. The validation of NGS panels are very critical and generally two-step approach is recommended for validation. The rst one is related with optimization and analysis for relevant errors during the testing and the second step is related with establishment of thresholds of depth of coverage and VAF (low variant allele frequency of variations) for each type of identi ed variant [24].
Recent years, NGS has been used for identi cation of T cell clonality, recurrent cytogenetic translocations, identi cation of Philadelphia chromosome in Acute Lymphoblastic Leukaemia [2]. In addition of these conditions, in lymphoproliferative diseases NGS has been also used identi cation of clonal IGH and TCR rearrangements in MRD (Minimal Residual Disease) [21]. NGS technology can be used for identi cation of mutant or clonal DNA in several circulating tumor cells and with the increasing number of molecular markers, it is also essential for clinical trials based on substantially parallel next generation sequencing (NGS) [25].
Increased number of studies in this eld will help to nd out new mutations and gives chance to be updated WHO classi cation for myeloid malignancies. Moreover, those studies will be help for development of novel targeted therapeutic agents and novel therapeutic targets [26]. The discovery of new mutations in myeloid neoplasms enables us to understand the variable prognosis and pathogenesis of these diseases. The use of cytogenetic-based techniques allows the identi cation of "gross" chromosomal abnormalities such as translocations, ampli cations, and deletions [22]. However the limitation of technique is based on size of the abnormality because genes can undergo changes in various ways (mutations, methylation, etc.) that may be critical for the onset and / or progression of malignant homeopathies. The major advance in NGS is identi cation of the molecular basis of leukemia because we can now classify malignant homeopathies at a molecular level that is more informative than the cytological classi cation [27].
We suggested to use this panel in routine clinical testing for myeloid malignancies which are cytogenetically and molecular cytogenetically reported as a normal. In this report, we identi ed variations in different genes which are related with epigenetic modi cations, RNA modi cations, transcription factors, DNA repair and cohesin complex. This shows us in patients which are cytogenetically or FISH were reported as a normal has to check in NGS panels. Thus, this will help to prevent false negative results in those cases and can help clinicians to be able to apply true treatment strategies. Our suggested possible algorithm was shown in Fig. 2. In this work, we analyzed pathogenic / possible pathogenic variations in different types of hematologic malignancies which were reported as a normal in cytogenetic and molecular cytogenetic analysis. This shows us only cytogenetic analysis does not su cient for evaluation of diseases. In addition, NGS gives chance to analyze the genomic copy number alteration of interest gene which triggers different questions for conventional cytogenetics during the evaluation of myeloid neoplasms. The questions are; is it essential for both NGS-based tests and cytogenetics for evaluation of newly diagnosed myeloid neoplasm patients? Which technique is the best for monitoring patients during the treatment or during relapse?
To be able to answer these questions, we examined the similarities and differences between a conventional karyotype and a targeted NGS panel.
1-Conventional cytogenetic gives information for both numerical and structural abnormalities of chromosomes but NGS is target speci c and can give only numerical abnormalities. We cannot used NGS for identi cation of insertion or translocations.
2-Karyotype produces a genomic information in single cell resolution, but NGS provides information for a "mean" copy number changes for all cells in a sample. Also, karyotype gives chance to be able to identi cation of the low percentage clones which contains different aberrations. On the other side, NGS panels identi es only numerical alterations which are shared with a signi cant portion of the cells in the sample (typically > 30%).
3-Third, the resolution of karyotype analysis is usually > 10 Mb while NGS panels offers resolution at the gene or exon level. At the same time, cytogenetic analysis cannot be identi ed submicroscopic abnormalities (10 Mb) but NGS panels can do. 4-If the aneuploidy is high, the ability of karyotype analysis becomes limited. For example, in the case of a complex karyotype which contains a small"marker chromosomes", it is di cult to identify the chromosomal origin of this abnormality. But targeted NGS panels identi es these abnormalities rapidly and reliably. 5-NGS panels measures copy numbers in relative and inaccurate proximity. The triploidy or tetraploid genome cannot be easily distinguished from the diploid genome. This can be easily determined by karyotype analysis.
These differences demonstrate that the advantages and disadvantages of targeted NGS and karyotype / targeted FISH techniques which supports the idea that complementary technologies are best used to evaluate the numerical and structural properties of neoplastic genomes.
NGS-based panel testing is widely accepted in clinical practice, and this can facilitate the construction of well-designed comprehensive NGS panels, especially during initial diagnosis. Albeit, the targeted NGS panels can evaluate the genome wide numerical imbalances, it cannot identify structural abnormalities, lack of single cell resolution and low target density, necessitate simultaneous cytogenetic analysis capable of presenting a complete picture of the genomic pro le. After the diagnostic evaluation, it may be gives advantage to use NGS panel testing to perform cytogenetic analysis for patients whose NGS results show signi cant clonal evolution.
In this study, we provided an information about the potential uses of NGS in a routine clinical setting and emphasized the importance of the method for clinical studies and improving outcomes of haematology patients. NGS gives chance to new perspective for personalized diagnostics and targeted therapy opportunity for myeloid malignancies. The advanced sensitivity and accuracy makes NGS as a potential diagnostic test in cytogenetically normal myeloid malignancies. Identi cation of novel speci c mutations can be help to nd out new targeted therapy options which would be a cornerstone of personalized medicine in hematologic malignancies. Finally, this study provides evidence on potential uses of NGS in the routine clinical setting, and provides a framework for cytogenetically normal cases.

Declarations Ethical approval
All procedures performed in the study involving human participants were in accordance with the ethics committee of the Trakya University Faculty of Medicine and followed Declaration of Helsinki.

Consent for publication
Informed consent was obtained from all individual participants included in the study.

Availability of data and material
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Competing Interests Statement
The authors declare that they have no con ict of interest.

Figure 2
Suggested algorithm for cytogenetically normal cases.