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The online version of this article has a Supplementary Appendix. BackgroundThe identification of the JAK2V617F mutation is mandatory in the diagnostic work-up of Philadelphia chromosome-negative myeloproliferative neoplasms. Several molecular techniques to detect this mutation are currently available, but each of them has some limits. Design and MethodsWe set up a novel molecular method for the identification of the JAK2V617F mutation based on an allele-specific loop-mediated amplification, not polymerase chain reaction analysis. This innovative technique amplifies DNA targets under isothermal conditions with high specificity, efficiency and rapidity. The method does not require either a thermal cycler or gel separation and the DNA amplification reaction is visible to the naked eye and can be monitored by turbidimetry. This method was validated on DNA from cell lines as well as from patients with myeloproliferative neoplasms. The results were compared with those obtained by conventional polymerase chain reaction methods. ResultsThis assay detects, within 1 hour, the JAK2V617F mutation down to an allele burden of 0.1-0.01%. All samples positive by polymerase chain reaction (n=146) proved positive when tested by allele-specific loop-mediated amplification and none of the 80 negative controls gave false positive results. In addition, six patients with essential thrombocythemia previously diagnosed as being JAK2V617F negative by polymerase chain reaction analysis were found to be positive (at a low level) by allele-specific loop-mediated amplification. Furthermore, this assay discriminated the amount of JAK2V617F tumor allele within intervals of positivity, above 50%, between 50% and 10% and below 10%. ConclusionsAllele-specific loop-mediated amplification is a simple, robust and easily applicable method for the molecular diagnosis and monitoring of JAK2V617F mutation in patients with chronic myeloproliferative neoplasms.Key words: myeloproliferative neoplasms, JAK2V617F mutation, LAMP. neoplasms. Haematologica 2012;97(9):1394-1400. doi:10.3324/haematol.2011 A novel, highly sensitive and rapid allele-specific loop-mediated amplification assay for the detection of the JAK2V617F mutation in chronic myeloproliferative neoplasms © F e r r a t a S t o r t i F o u n d a t i o n © F e r r a t a S t o r t i F o u n d a t i o n Citation: Minnucci G, Amicarelli G, Salmoiraghi S, Spinelli O, Guinea Montalvo ML, Giussani U, Adlerstein D, and Rambaldi A. A novel, highly sensitive and rapid allele-specific loop-mediated amplification assay for the detection of the JAK2V617F mutation in chronic myeloproliferative

Acute lymphoblastic leukemia (ALL) is curable in about 40–50% of adult patients, however this is subject to ample variations owing to several host- and disease-related prognostic characteristics. Currently, the study of minimal residual disease (MRD) following induction and early consolidation therapy stands out as the most sensitive individual prognostic marker to define the risk of relapse following the achievement of remission, and ultimately that of treatment failure or success. Because substantial therapeutic advancement is now being achieved using intensified pediatric-type regimens, MRD analysis is especially useful to orientate stem cell transplantation choices. These strategic innovations are progressively leading to greater than 50% cure rates.

Until today, JAK2 alterations have been mainly associated with myeloid malignancies among which they play a key pathogenic role in chronic myeloproliferative neoplasms. More recently, aberrations involving the JAK2 gene have also been reported in lymphoid diseases, including acute leukemia and lymphomas. In addition, the constitutively activating JAK2V617F mutation has been identified in some patients affected by B-chronic lymphocytic leukemia with a concomitant myeloproliferative neoplasm. Interestingly, these cases could help us to better understand the pathogenesis of these myeloid and lymphoid diseases and reveal if they share a common ancestral progenitor or just coincide. The involvement of JAK2 in lymphoid neoplasms may suggest the possibility of new therapeutic approaches broadening the use of JAK1-2 inhibitors also to these malignancies.

The monitoring of minimal residual disease (MRD) in Philadelphia-negative acute lymphoblastic leukemia (ALL) requires the identification at diagnosis of immunoglobulin/T-cell receptor (Ig/TCR) rearrangements as clonality markers. Aiming to simplify and possibly improve the patients’ initial screening, we designed a capture-based next-generation sequencing (NGS) panel combining the Ig/TCR rearrangement detection with the profiling of relevant leukemia-related genes. The validation of the assay on well-characterized samples allowed us to identify all the known Ig/TCR rearrangements as well as additional clonalities, including rare rearrangements characterized by uncommon combinations of variable, diversity, and joining (V-D-J) gene segments, oligoclonal rearrangements, and low represented clones. Upon validation, the capture NGS approach allowed us to identify Ig/TCR clonal markers in 87% of a retrospective cohort (MRD-unknown within the Northern Italy Leukemia Group (NILG)-ALL 09/00 clinical trial) and in 83% of newly-diagnosed ALL cases in which conventional method failed, thus proving its prospective applicability. Finally, we identified gene variants in 94.7% of patients analyzed for mutational status with the same implemented capture assay. The prospective application of this technology could simplify clonality assessment and improve standard assay development for leukemia monitoring, as well as provide information about the mutational status of selected leukemia-related genes, potentially representing new prognostic elements, MRD markers, and targets for specific therapies.