Detection of BCR-ABL T315I mutation by peptide nucleic acid directed PCR clamping and by peptide nucleic acid FISH.

BACKGROUND: Mutations of the BCR-ABL1 fusion gene represent a well established cause of resistance to tyrosine kinase inhibitors. Among the different mutations identified T315I is of particular concern since it is not effectively targeted by the majority of Tyrosine Kinase Inhibitors so far available. We developed a novel assay based on peptide nucleic acid (PNA) technology coupled to immunofluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of BCR-ABL (T315I) mutation thus improving both, diagnostic resolution and the study of clonal prevalence. Furthermore we developed an additional method based on PNA directed PCR-clamping for the fast and easy detection of the mutation. RESULTS: The PNA directed PCR clamping allows to detect an amount of mutated template as low as 0.5 %. This method is highly sensitive, specific and cheap and could be applied even in laboratory not equipped for more sophisticated analysis. Furthermore, the PNA FISH method allows to identify a small amount of progenitor cells still present after therapy with specific inhibitors. CONCLUSIONS: We present here two different methods based on PNA for the detection of T315I useful for different purposes. PNA-FISH can be used to study clonal evolution. In addition, this method could help in the study of compound mutations being able to identify two different mutations in a single cell. PNA directed PCR clamping although not superior to sequencing can be applied worldwide even in laboratory not equipped to search for mutations.

Pathophysiology, clinical features and radiological findings of differentiation syndrome/all-trans-retinoic acid syndrome.

In acute promyelocytic leukemia, differentiation therapy based on all-trans-retinoic acid can be complicated by the development of a differentiation syndrome (DS). DS is a life-threatening complication, characterized by respiratory distress, unexplained fever, weight gain, interstitial lung infiltrates, pleural or pericardial effusions, hypotension and acute renal failure. The diagnosis of DS is made on clinical grounds and has proven to be difficult, because none of the symptoms is pathognomonic for the syndrome without any definitive diagnostic criteria. As DS can have subtle signs and symptoms at presentation but progress rapidly, end-stage DS clinical picture resembles the acute respiratory distress syndrome with extremely poor prognosis; so it is of absolute importance to be conscious of these complications and initiate therapy as soon as it was suspected. The radiologic appearance resembles the typical features of cardiogenic pulmonary edema. Diagnosis of DS remains a great skill for radiologists and haematologist but it is of an utmost importance the cooperation in suspect DS, detect the early signs of DS, examine the patients' behaviour and rapidly detect the complications.

A new HPLC-UV validated method for therapeutic drug monitoring of tyrosine kinase inhibitors in leukemic patients.

Development and validation of simple, rapid, and reliable high-performance liquid chromatography (HPLC)-UV method for quantification of major tyrosine kinase inhibitors, imatinib, dasatinib, and nilotinib, in human plasma is presented. Chromatographic separation of the drugs is achieved on an RP-C(18) column at flow rate of 0.9 mL/min at 35°C; eluate is monitored at 267 nm. Mean intra-day and inter-day precision for all compounds are 2.5 and 13.3%; mean accuracy is 13.9%; extraction recovery ranges within 40.24 and 81.81%. Calibration curves range from 10 to 0.005 µg/mL. Limits of detection are 10 ng/mL for imatinib and nilotinib, 50 ng/mL for dasatinib; limits of quantitation are 50 ng/mL for imatinib and nilotinib, 100 ng/mL for dasatinib. Although this method allows the detection of dasatinib, levels found in patients plasma are close to the limit of detection, then below the limit of quantitation. Quantification with HPLC-mass spectrometry, then, is required for dasatinib to give a correct evaluation. In conclusion, the sensitivity of this new method is sufficient to perform therapeutic monitoring and pharmacokinetic studies of imatinib and nilotinib but not dasatinib in CML patients.

Health-related quality of life in chronic myeloid leukemia patients receiving long-term therapy with imatinib compared with the general population.

The main objective of this study was to investigate whether patients with chronic myeloid leukemia (CML) in treatment with long-term therapy imatinib have a different health-related quality-of-life (HRQOL) profile compared with the general population. In total, 448 CML patients were enrolled, and the SF-36 Health Survey was used to compare generic HRQOL profiles. Symptoms were also assessed. HRQOL comparisons were adjusted for key possible confounders. The median age of patients was 57 years and the median time of imatinib treatment was 5 years (range 3-9 years). The largest HRQOL differences were found in younger patients. In particular, patients aged between 18 and 39 years had marked impairments in role limitations because of physical and emotional problems, respectively: -22.6 (P < .001), -22.3 (P < .001). Patients with CML age 60 or older had a HRQOL profile very similar to that reported by the general population. Women had a worse profile than men when each were compared with their peers in the general population. Fatigue was the most frequently reported symptom. The HRQOL of CML patients is comparable with that of population norms in many areas, however, younger and female patients seem to report the major limitations.

Dasatinib is safe and effective in unselected chronic myeloid leukaemia elderly patients resistant/intolerant to imatinib.

To highlight dasatinib role in the elderly, 125 unselected patients with CP-CML aged >60 years resistant/intolerant to imatinib were retrospectively evaluated. Grade 3-4 haematological and extra-haematological toxicities were reported in 39 (31.2%) and 34 (27.2%) patients; grade 3-4 haematological toxicity was higher in patients with 140 mg starting dose (50.0% vs 19.6%, p=0.001). Grade 3-4 pleuro-pericardial effusions occurred in 10 patients (8.0%). Dose reductions were more common in patients with 140 mg (88.4% vs 26.7%, p<0.001). Of 122 evaluable patients, 72 (59.1%) had cytogenetic response [12 (9.8%) partial, 60 (49.3%) complete]. Overall, 38/60 patients in complete CyR also achieved a molecular response. Cumulative OS at 24 and 48 months were 93.1% (95% CI 88.4-97.8) and 84.2% (95% CI 74.6-93.7). Dasatinib, at the recommended dose of 100mg/day, is effective and safe also in unselected elderly subjects.

JAK2 V617F mutation negative erythrocytosis (or how to more simply perform diagnosis and treat a patient with increased hematocrit).

SUMMARY: This case report focuses on a 71-year old patient affected by unknown dyspnea and erythrocytosis referred by his general practitioner to our center for specialist advice after a hematological examination had excluded polycythemia vera on grounds of negative test for JAK2 V617F/exon 12 mutation. An accurate clinical history and physical examination accompanied by respiratory function tests resulted in diagnosis of JAK2 V617F mutation negative erythrocytosis, and treatment could be started. The discussion examines decisional algorithms when a polyglobulic patient is referred for diagnosis.

Imatinib resistance in CML.

Imatinib is, at present, the first-choice treatment for patients with chronic myeloid leukaemia in chronic phase. Despite the impressive rate of complete haematological response and complete cytogenetical remissions, some cases show primary resistance or relapse after an initial response (secondary or acquired resistance). The most common mechanisms responsible for this resistance are BCR/ABL kinase domain mutations, BCR/ABL amplification and over-expression and clonal evolution with activation of additional oncogenic pathways. Here, we describe the molecular basis of imatinib resistance, the significance of molecular monitoring and the current efforts to overcome imatinib resistance, ranging from the development of new drugs to the stimulation of an immune response against the disease.

Acute myeloid leukemia induced by mitoxantrone treatment for aggressive multiple sclerosis.

Acute myeloid leukemia (t-AML) is one of the worst adverse events of mitoxantrone treatment, but the exact risk in multiple sclerosis (MS) patients is not yet known. We describe a case wherein the patient developed t-AML 11 months after mitoxantrone had been discontinued. The patient was treated by polychemotherapy and autologous bone marrow transplantation with complete recovery of t-AML and stabilization of the neurological disease.

New therapeutic approaches and prognostic factors in chronic myeloid leukemia.

Imatinib mesylate is now the first-choice treatment for all newly diagnosed CML patients, but despite the impressive percentage of responding patients, some CML cases show primary resistance or relapse after an initial response. Although some clinical and biological findings have been found to be associated with a lower probability of response to imatinib, at present no precise markers allowing to predict outcome for individual patients exist. The most common mechanisms of resistance to imatinib include BCR-ABL kinase domain mutations, BCR-ABL amplification and overexpression, and clonal evolution with activation of additional transformation pathways. These mechanisms are caused by the genomic instability, which characterises the Ph-positive clone. Several approaches to overcome resistance have been proposed, including novel tyrosine kinase inhibitors (TKIs) that have now reached the clinical or pre-clinical phase of evaluation. Clinical trials with these novel TKIs have shown good rates of hematologic and cytogenetic responses that appear also durable in time, but still cases of resistance are also observed, supporting the notion that genomic instability represents the major determinant affecting the final outcome of the CML patients when treated with TKIs. Understanding exactly the mechanisms leading to genomic instability of the Ph-positive cells represents therefore the real challenge for the near future.

Molecular monitoring in patients with chronic myelogenous leukemia.

Imatinib has revolutionized the treatment of chronic myelogenous leukemia (CML). Given the high rates of complete cytogenetic remission achieved with imatinib therapy, molecular monitoring of BCR-ABL transcript levels by real-time quantitative polymerase chain reaction has become the method of choice to assess the amount of residual disease below the cytogenetic threshold. BCR-ABL transcript levels measured at specific times during therapy may predict durable cytogenetic remission and prolonged progression-free survival or, on the contrary, failure and suboptimal response, thus directing clinical decisions. Recently, recommendations have been established for harmonizing the methodologies used to measure BCR-ABL transcripts in patients with CML, allowing results to be expressed on a standardized comparable international scale. Rising levels of BCR-ABL transcripts indicate the need for an analysis of kinase mutations, the major mechanism of imatinib resistance. The early detection and the characterization of these mutations may allow timely and appropriate treatment to overcome resistance.