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.
Here, we describe the immunoglobulin and T cell receptor (Ig/TCR) molecular rearrangements identified as a leukemic clone hallmark for minimal residual disease assessment in relation to TP53 mutational status in 171 Ph-negative Acute Lymphoblastic Leukemia (ALL) adult patients at diagnosis. The presence of a TP53 alterations, which represents a marker of poor prognosis, was strictly correlated with an immature DH/JH rearrangement of the immunoglobulin receptor (p < 0.0001). Furthermore, TP53-mutated patients were classified as pro-B ALL more frequently than their wild-type counterpart (46% vs. 25%, p = 0.05). Although the reasons for the co-presence of immature Ig rearrangements and TP53 mutation need to be clarified, this can suggest that the alteration in TP53 is acquired at an early stage of B-cell maturation or even at the level of pre-leukemic transformation.
SUMMARY While adult acute lymphoblastic leukemia (ALL) is curable in 40–50% of the patients, the individual prognosis is rather unpredictable due to associated biological and clinical risk factors. In both B- and T-precursor ALL, minimal residual disease (MRD) represents the most sensitive prognostic marker, useful to support critical treatment decisions, ranging from allogeneic stem cell transplantation in patients with inadequate MRD response to chemotherapy only in MRD responsive ones. This optimized risk-adapted strategy allows to spare transplant-associated morbidity and mortality in patients curable by chemotherapy. Further progress is expected from the integration of the MRD-based strategy with improved pediatric-type regimens and novel targeting agents for discrete ALL subsets. These changes are increasing the cure rate to above 50%.
Background and Aim of the Study For both childhood and adult Acute Lymphoblastic Leukemia (ALL) patients, clinical risk factors such as age, white cell count, response to steroids, time to complete remission, as well as biologic characteristics such as immunophenotype and cytogenetic at diagnosis are important but not sufficient in predicting clinical outcome. Aberrations of TP53 play a crucial role in the molecular pathogenesis of leukemias and lymphomas in which their presence is associated to disease progression and represents a strong predictor of poor clinical outcome. In childhood ALL, hereditary and acquired TP53 mutations are involved both in the pathogenesis and progression of the disease. In adult ALL, TP53 mutations are frequent in patients negative for recurrent fusion genes and correlate with poor response to induction therapy (Chiaretti S. et al, Haematologica 2013). The aim of this study was to evaluate the impact of TP53 alterations, analyzed by Next Generation Sequencing (NGS), on the outcome of a cohort of T (n= 57) and B (n= 114) precursor, Philadelphia (Ph) negative, adult ALL patients enrolled into the NILG-ALL 09/2000 clinical trial (ClinicalTrials.gov identifier: NCT00358072, Bassan R. et al, Blood 2009) in which molecular minimal residual disease was used to guide post-remissional therapy. Patients and Study design Among the 171 patients who were investigated for TP53 mutations, 16 proved also positive for t(4;11) and 3 for t(1;19). We analyzed DNA isolated from mononuclear cells obtained from bone marrow or peripheral blood samples containing at least 30% of blasts at diagnosis. The TP53 gene was sequenced using 454 ultra-deep sequencing (Roche Diagnostics) for alterations in exons 4 to 11, following the protocol developed in the IRON-II consortium. The sequencing data were analyzed by the Roche Diagnostics GS Run Browser and GS Amplicon Variant Analyzer software. The probabilities of survival were estimated using the Kaplan Meier method. The log-rank test was used to compare survival probabilities between subgroups of patients. Results and Discussion The data obtained by NGS allowed to identify 15 coding mutations detected in the DNA binding domain region (exons 5 to 8). These alterations were observed at diagnosis in 14 patients (8%), (11 B-precursor ALL and 3 T-ALL). In 12 cases these aberrations were single nucleotide changes, in 2 cases we found a duplication (one of 4 and the other of 8 nucleotides) and in one case there was an 11 base pair DNA insertion. Remarkably, all of these DNA alterations led to missense or frame-shift mutations that introduced a premature stop codon. Moreover, they were detected with a wide range of allele burden (from 5% to 97%) pointing out that TP53 mutations can be present at diagnosis in different proportions within the leukemic clones. All patients carrying a TP53 alteration reached complete remission after induction therapy but 13 out of 14 suffered an early relapse. Frequency of relapses was significantly higher in mutated than in wild-type cases (p=0.019). Relapse DNA samples were available in 3 patients and in all of them we detected the same TP53 mutation found at diagnosis, indicating the presence of a stable mutated clone. The univariate analysis enlightens a clear relationship between TP53 mutation with an increasing age (p= 0.0003) but no correlation with other clinical features such as gender, hemoglobin, white blood count, platelets, percentage of blasts and cytogenetics at diagnosis. Moreover, patients with mutated TP53 showed a Disease Free Survival (DFS) and Overall Survival (OS) dramatically shorter than wild-type patients. The 2 years DFS was 43% in the TP53 non-mutated subjects compared to 7% in the mutated (p=0.0007). Similarly, the 2 years OS was of 50% in wild-type patients and of 7% in mutated patients (p=0.0011) (Figure 1). Conclusions In adult ALL, response to induction chemotherapy is not different in patients with a wild-type or a TP53 mutated gene, but in these latter cases the leukemia relapse rate is dramatically higher. The frequency of these mutations observed at diagnosis and the poor clinical outcome indicate the need of their identification during the diagnostic work up of adult ALL to guide treatment strategies. The use of a highly sensitive deep sequencing approach is crucial to identify also minor leukemic clones carrying TP53 mutations that may lead to the rapid emergence of a treatment resistant disease. Disclosures Kohlmann: AstraZeneca: Employment.
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