Capture-based next-generation sequencing improves the identification of immunoglobulin/t-cell receptor clonal markers and gene mutations in adult acute lymphoblastic leukemia patients lacking molecular probes

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\u2019 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

Chronic Myeloid Leukemia: Molecular Monitoring of Residual Disease by Genomic DNA Compared to Conventional mRNA Analysis in Follow-Ups up to 8 Years

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder resulting from the t(9;22)(q34;q11) balanced reciprocal translocation within a pluripotent stem cell (SC). The resulting Philadelphia (Ph) chromosome produces BCR-ABL1 fusion gene coding for a deregulated Abl tyrosine- kinase with constitutive and tumorigenic activity. The first line therapy of CML is imatinib mesylate, which targets Bcr-Abl protein, inhibiting proliferation pathways. Complete cytogenetic response can be achieved in 95% of patients treated in the early chronic phase (CP)1. Molecular monitoring of minimal residual disease is crucial to detect poor responses to imatinib and optimizing treatment with second generation tyrosine-kinase inhibitors or allogeneic stem cell transplantation. Residual leukemia is assessed by a quantitative manner evaluating levels of BCR-ABL1 transcripts by real-time reverse transcriptase PCR (qRT-PCR). Although qRT-PCR detects mRNA levels in a very sensitive manner, the negative result is difficult to interpret, because undetectable levels of chimeric transcript can reflect either an effective elimination of leukemia cells, or the presence of a quiescent leukemia SC transcriptionally silent

Identification of kitM541l somatic mutation in chronic eosinophilic leukemia, not otherwise specified and its implication in low-dose imatinib response

Activating mutations of KIT receptor tyrosine kinase have been reported in different neoplasms. The M541L KIT substitution (KITM541L) has been described to be associated with pediatric mastocytosis, to enhance growth rate of the affected cells and to confer higher sensitivity to imatinib therapy. We investigated the presence of KITM541L in five males with chronic eosinophilic leukemia, not otherwise specified (CEL, NOS), all negative for Platelet-derived growth factor-alpha (PDGFR) or PDGFRbeta abnormalities, which responded to imatinib therapy. To assess whether the mutation was constitutive or somatic in nature, we evaluated its presence analyzing either the neoplastic or normal cell population (epidermal cells or CD3-positive T lymphocytes). KITM541L substitution was found in 4 out of 5 patients and in all it was somatic in nature. All patients were treated with low dose imatinib (100 mg daily orally), achieving complete and persistent clinical and hematological remission (median follow-up 74 months). One patient relapsed after 50 months. Our study strongly suggests to search for the KITM541L in patients with CEL, NOS, negative for PDGFRalpha and PDGFRbeta abnormalities, to identify a subgroup of cases who may benefit from low dose imatinib therapy

Effects and outcome of a policy of intermittent imatinib treatment in elderly patients with chronic myeloid leukemia.

none25We report a study of an alternative treatment schedule of imatinib (IM) in chronic myeloid leukemia (CML). Seventy-six Philadelphia-positive (Ph+), BCR-ABL-positive patients aged 65 years or older who had been treated with IM for more than 2 years and who were in stable complete cytogenetic response (CCgR) and major molecular response (MMR) were enrolled in a single-arm study to test the effects of a policy of intermittent IM (INTERIM) therapy for 1 month on and 1 month off. With a minimum follow-up of 4 years, 13 patients (17%) lost CCgR and MMR and 14 (18%) lost MMR only. All these patients resumed continuous IM and all but one (lost to follow-up) regained CCgR and MMR. No patients progressed to accelerated or blastic phase or developed clonal chromosomal abnormalities in Ph+ cells or BCR-ABL mutations. In elderly Ph+ CML patients carefully selected for a stable CCgR (lasting >2 years), the policy of INTERIM treatment affected the markers of residual disease, but not the clinical outcomes (overall and progression-free survival). This trial was registered at www.clinicaltrials.gov as NCT 00858806.noneRusso D; Martinelli G; Malagola M; Skert C; Soverini S; Iacobucci I; De Vivo A; Testoni N; Castagnetti F; Gugliotta G; Turri D; Bergamaschi M; Pregno P; Pungolino E; Stagno F; Breccia M; Martino B; Intermesoli T; Fava C; Abruzzese E; Tiribelli M; Bigazzi C; Cesana BM; Rosti G; Baccarani M.Russo D; Martinelli G; Malagola M; Skert C; Soverini S; Iacobucci I; De Vivo A; Testoni N; Castagnetti F; Gugliotta G; Turri D; Bergamaschi M; Pregno P; Pungolino E; Stagno F; Breccia M; Martino B; Intermesoli T; Fava C; Abruzzese E; Tiribelli M; Bigazzi C; Cesana BM; Rosti G; Baccarani M

Achieving a Molecular Remission before Allogeneic Stem Cell Transplantation in Adult Patients with Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia: Impact on Relapse and Long Term Outcome

Allogeneic stem cell transplantation (alloHSCT) in first complete remission (CR1) remains the consolidation therapy of choice in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). The prognostic value of measurable levels of minimal residual disease (MRD) at time of conditioning is a matter of debate. We analyzed the predictive relevance of MRD levels before transplantation on the clinical outcome of Ph+ ALL patients treated with chemotherapy and imatinib in 2 consecutive prospective clinical trials. MRD evaluation before transplantation was available for 65 of the 73 patients who underwent an alloHSCT in CR1. A complete or major molecular response at time of conditioning was achieved in 24 patients (37%), whereas 41 (63%) remained carriers of any other positive MRD level in the bone marrow. MRD negativity at time of conditioning was associated with a significant benefit in terms of risk of relapse at 5 years, with a relapse incidence of 8% compared with 39% for patients with MRD positivity (P\u2009=\u2009.007). However, thanks to the post-transplantation use of tyrosine kinase inhibitors (TKIs), disease-free survival was 58% versus 41% (P\u2009=\u2009.17) and overall survival was 58% versus 49% (P\u2009=\u2009.55) in MRD-negative compared with MRD-positive patients, respectively. The cumulative incidence of nonrelapse mortality was similar in the 2 groups. Achieving a complete molecular remission before transplantation reduces the risk of leukemia relapse even though TKIs may still rescue some patients relapsing after transplantation

Treatment-free remission in chronic myeloid leukemia patients treated front-line with nilotinib: 10-year followup of the GIMEMA CML 0307 study

We report the final analysis, with a 10-year follow-up, of the phase II study GIMEMA CML 0307 (NCT 00481052), which enrolled 73 adult patients (median age 51 years; range, 18-83) with newly diagnosed chronic-phase chronic myeloid leukemia to investigate the efficacy and the toxicity of front-line treatment with nilotinib. The initial dose was 400 mg twice daily; the dose was reduced to 300 mg twice daily as soon as this dose was approved and registered. The 10-year overall survival and progression-free survival were 94.5%. At the last contact, 36 (49.3%) patients were continuing nilotinib (22 patients at 300 mg twice daily, 14 at lower doses), 18 (24.7%) patients were in treatment-free remission, 14 (19.2%) were receiving other tyrosine-kinase inhibitors and four (5.5%) patients have died. The rates of major and deep molecular responses by 10 years were 96% and 83%, respectively. The median times to major and deep molecular response were 6 and 18 months, respectively. After a median duration of nilotinib treatment of 88 months, 24 (32.9%) patients discontinued nilotinib while in stable deep molecular response. In these patients, the 2-year estimated treatment-free survival was 72.6%. The overall treatment-free remission rate, calculated on all enrolled patients, was 24.7% (18/73 patients). Seventeen patients (23.3%), at a median age of 69 years, had at least one arterial obstructive event. In conclusion, the use of nilotinib front-line in chronic phase chronic myeloid leukemia can induce a stable treatment-free remission in a relevant number of patients, although cardiovascular toxicity remains of concern

Mechanisms of Resistance to Targeted Therapies in Chronic Myeloid Leukemia

Patients with newly diagnosed chronic myeloid leukemia (CML) usually received as first-line treatment a first- or second-generation tyrosine kinase inhibitor (TKI). Although initial responses are high, therapy fails in up to 40% of patients and initial response is lost within 2 years in approximately 25% of patients. In the last few years, intensive efforts have been spent to explain treatment failure, and different mechanisms of resistance have been identified, ranging from BCR-ABL1 kinase domain mutations to lack of adherence to therapy. In this review, we briefly summarize the clinical efficacy of approved TKIs and describe the main mechanisms of TKI resistance

Durable molecular response despite F317L and E255K mutations: Successful treatment of chronic myeloid leukemia with sequential imatinib, nilotinib and dasatinib.

No abstract availabl