Identification of a novel cryptic variant chromosomal rearrangement involving 9q34, 22q11.2, and 5q22 resulting in ins(9;22) and t(5;22) in chronic myeloid leukemia: a case report.

Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic stem cells characterized by the aberrant production and uncontrolled proliferation of mature granulocytes with normal cell differentiation. The Philadelphia (Ph) chromosome resulting from reciprocal translocation between chromosomes 9 and 22 is the main genetic molecular hallmark of CML seen in more than 90% of the patients. However, about 5-10% of CML patients show a variant genetic rearrangement, involving one or more chromosomes in addition to 9 and 22. Herein, we describe the results of hematological, cytogenetic, fluorescence in situ hybridization (FISH), and high-end molecular analysis in a 77-year-old man diagnosed with CML. The combination of conventional cytogenetic analysis along with metaphase FISH and whole chromosomal paint revealed a novel cryptic variant chromosomal rearrangement involving 9q34, 22q11.2, and 5q22, resulting in ins(9;22) and t(5;22). At the molecular level, using PCR, myeloid NGS panels, and whole transcriptome analyses, we showed that this complex rearrangement indeed resulted in the formation of the BCR::ABL1 e13a2 major fusion transcript. No additional somatic mutations or kinase domain mutations were identified, thereby suggesting that the current case is indeed genetically homogeneous. This study provided strong evidence to support the idea that insertion-derived BCR::ABL1 fusions often involve complex chromosomal abnormalities that are overlooked by conventional cytogenetics but can be identified by a combination of conventional, molecular cytogenetics, and high-end NGS studies.

Validation of a novel NGS based BCR::ABL1 kinase domain mutation detection assay in Indian cohort.

The efficacy and treatment outcome of a CML patient are heavily dependent on BCR::ABL1 kinase domain (KD) mutation status. Next-generation sequencing technology is a bright alternative to the previously used sanger sequencing method due to its global presence in diagnostic setups, massive parallel sequencing ability, and far better sensitivity. In the present study, we have demonstrated a new protocol for kinase domain mutation analysis using the next-generation sequencing (NGS) method using the ion torrent sequencing platform. This protocol uses RNA as the starting material, followed by nested PCR to amplify the fusion transcript, which is subsequently used as a template for NGS. Initial validation and comparison of this assay with the sanger sequencing (SS) method yielded 95.23% agreement. CML samples (n = 121) with a failure to TKI response were subjected to this newly developed NGS-based assay to detect KD mutations, from which samples were found to have mutations with a sensitivity ranging from 2.32 to 93.41%. A total of 34.71% of samples (n = 42) were found to be positive for one or more KD mutations, whereas 65.29% of samples (n = 81) were found to be negative. Nine samples out of 42 positive samples, i.e., 21.42%, were found to have compound mutations. This is one of the first studies from India, which includes more than 160 samples and is analyzed by the NGS approach for KD mutation analysis.

Identification, screening and optimization of significant parameters for stir cast hybrid aluminium metal matrix composite.

Different industries have explicit applications of Aluminum Metal Matrix Composites (AMMCs). However, porosity, wettability and Ultimate Tensile Strength (UTS) are important factors that need to consider while fabricating AMMCs. This paper focuses on identification of the most significant parameters for low porosity and high ultimate tensile strength (UTS) of hybrid aluminium metal matrix composite. Significant as well as non-significant factors were identified from the literature survey and trial experiments. Without considering the significance of given responses, identified factors were represented graphically by the fishbone diagram. FMEA (Failure Mode and Effect Analysis) was used to find out significant parameters from all the parameters that represented by fishbone diagram. Afterwards, Plackett-Burman design was used to screen the most significant parameters out of the significant parameters. Stirring speed, stirring time, preheating temperature and reinforcement amount was found the most significant parameters to attain high UTS and low porosity. However, because of the unfavourable combination of parameters % porosity increased beyond 7% during screening design based experiments. Subsequently, these parameters were optimized by factorial design. The result shows optimized parameters stirring speed 650 rpm and stirring time 12 minutes provides low porosity and high UTS. UTS improved up to 310 MPa due to an optimized range of stirring speeds and stirring time. However, porosity increased beyond 3% due to excess stirring in cast composites during factorial design based experiments. Moreover, studies were carried out to understand the effect of stirring, fluxing, degassing and moulding methods on porosity, UTS, clustering and surface finish of the cast composite. It was observed from a supplementary study that permanent mould reduces surface roughness below 3µm, compared to the sand casting process. The porosity was reduced below 3% by degassing and fluxing. Low agglomeration was observed in specimen prepared with automatic stirring process compared to the manually stirred specimen. Compared to others, in specimen 4, UTS was improved beyond 150 MPa because of permanent mould, automatic stirring, degassing and fluxing.