Recognition of Philadelphia chromosome-like acute lymphoblastic leukemia as part of routine diagnostic work-up.

INTRODUCTION: Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL) is a biologically and clinically challenging subtype of B-cell ALL which has been incorporated into the 2016 revision of the World Health classification of acute leukemia. It is independently associated with poor outcome. As it can only be reliably detected by expression profiling, it is difficult to diagnose with routine methods. Its recognition has become of greater importance due to prognostication and even more due to the new diagnostic options given by targeted therapies. There is still no standardized diagnostic test enabling its prompt recognition. Here, we introduce our approach how to detect it by combination of widely available techniques. METHODS: 179 ALL patients diagnosed in our center during the last 8 years were included. Data on immunophenotype and cytogenetics were used to select patients with potentially Ph-like ALL (65/179). CRLF2 gene rearrangement (CRLF2-r) was tested by FISH in 59/65 patients, and next-generation sequencing was done by Archer FusionPlex ALL kit in 34 patients. TSLPR expression was determined in 20 patients. RESULTS: Philadelphia chromosome-like aberrations were confirmed in 9 patients. In 10% of tested samples, CRLF2-r was confirmed. Due to a lack of material, NGS was done only in a half of potentially Ph-like cases. In 10%, other Ph-like fusions were found by NGS. CONCLUSIONS: The obtained frequencies, and genetic and patients' characteristics are in concordance with the literature data, ensuring a reliable detection of this challenging ALL subtype. The proposed algorithm allows detection of Ph-like ALL at reasonable cost and acceptable workload.

Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis.

High resolution melting analysis (HRM) is a powerful method for genotyping and genetic variation scanning. Most HRM applications depend on saturating DNA dyes that detect sequence differences, and heteroduplexes that change the shape of the melting curve. Excellent instrument resolution and special data analysis software are needed to identify the small melting curve differences that identify a variant or genotype. Different types of genetic variants with diverse frequencies can be observed in the gene specific for patients with a specific disease, especially cancer and in the CALR gene in patients with Philadelphia chromosome-negative myeloproliferative neoplasms. Single nucleotide changes, insertions and/or deletions (indels) in the gene of interest can be detected by the HRM analysis. The identification of different types of genetic variants is mostly based on the controls used in the qPCR HRM assay. However, as the product length increases, the difference between wild-type and heterozygote curves becomes smaller, and the type of genetic variant is more difficult to determine. Therefore, where indels are the prevalent genetic variant expected in the gene of interest, an additional method such as agarose gel electrophoresis can be used for the clarification of the HRM result. In some instances, an inconclusive result must be re-checked/re-diagnosed by standard Sanger sequencing. In this retrospective study, we applied the method to JAK2 V617F-negative patients with MPN.