Detection of NPM1 exon 12 mutations and FLT3 – internal tandem duplications by high resolution melting analysis in normal karyotype acute myeloid leukemia

<p>Abstract</p> <p>Background</p> <p>Molecular characterisation of normal karyotype acute myeloid leukemia (NK-AML) allows prognostic stratification and potentially can alter treatment choices and pathways. Approximately 45–60% of patients with NK-AML carry <it>NPM1 </it>gene mutations and are associated with a favourable clinical outcome when <it>FLT3</it>-internal tandem duplications (ITD) are absent. High resolution melting (HRM) is a novel screening method that enables rapid identification of mutation positive DNA samples.</p> <p>Results</p> <p>We developed HRM assays to detect <it>NPM1 </it>mutations and <it>FLT3</it>-ITD and tested diagnostic samples from 44 NK-AML patients. Eight were <it>NPM1 </it>mutation positive only, 4 were both <it>NPM1 </it>mutation and <it>FLT3</it>-ITD positive and 4 were <it>FLT3</it>-ITD positive only. A novel point mutation Y572C (c.1715A>G) in exon 14 of <it>FLT3 </it>was also detected. In the group with <it>de novo </it>NK-AML, 40% (12/29) were <it>NPM1 </it>mutation positive whereas <it>NPM1 </it>mutations were observed in 20% (3/15) of secondary NK-AML cases. Sequencing was performed and demonstrated 100% concordance with the HRM results.</p> <p>Conclusion</p> <p>HRM is a rapid and efficient method of screening NK-AML samples for both novel and known <it>NPM1 </it>and <it>FLT3 </it>mutations. <it>NPM1 </it>mutations can be observed in both primary and secondary NK-AML cases.</p

Myelodysplastic Syndrome with t(1;7) Associated with Marked Dysmegakarypoiesis & Severe Thrombocytopenia: A Case Report and Review of the Literature

We present the case of a 70-year-old woman who had a bone marrow examination performed to investigate marked thrombocytopenia in the context of a recent history of metastatic glucagonoma. Surprisingly this identified marked dysmegakaryopoiesis and fulfilled diagnostic criteria for refractory cytopenia with multilineage dysplasia, with a relatively uncommon associated cytogenetic lesion t(1;7). We present the case and review the literature of this cytogenetic lesion

The sensitivity of CD138 immunostaining of bone marrow trephine specimens for quantifying marrow involvement in MGUS and myeloma,including samples with a low percentage of plasma cells

This is a publisher's version of an article published in Haematologica 2006 published by European Hematology Association. This version is reproduced with permission from European Hematology Association. http://www.haematologica.org/Accurate quantification of plasma cells in bone marrow samples is essential for the diagnosis, classification and prognosis of plasma-cell dyscrasias. Published comparisons between aspirate/trephine morphology, flow cytometry and immunohistochemistry are lacking. Bone marrow plasma cells from 100 patients with plasma cell myeloma or monoclonal gammopathy of undetermined significance were quantified by a 500-cell differential count on Romanowsky-stained aspirate slides, flow-cytometry gating of CD38bright+/CD138+cells,hematoxylin and eosin trephine section examination and CD138 trephine immunohistology. The results of quantification by the different methods were compared. Compared to other methods, CD138 trephine immunohistology consistently demonstrated greater plasma-cell infiltration. Immunohistology is the most sensitive method for assessment of plasma-cell infiltration at diagnosis or post-therapy,especially in patients with minimal bone marrow involvement

Is serial testing required to diagnose imported malaria in the era of rapid diagnostic tests?

Exclusion of malaria traditionally requires three negative serial thick and thin blood films. However, many clinical laboratories now routinely perform rapid diagnostic tests (RDTs) in addition to blood films when malaria is suspected. We sought to determine whether serial testing is necessary in this setting. We examined 388 cases of malaria diagnosed during 1999–2010 at three laboratories in Melbourne, Australia. For each case, we ascertained whether the diagnosis was made on initial or follow-up testing. Nine cases (3.5%) were diagnosed after a negative initial blood film and RDT: 7 Plasmodium vivax, 1 P. ovale, and 1 P. falciparum. Of four case-patients with P. vivax in which clinical data were available, all had recent exposure to antimalarial medication. Our data suggest that among patients who have not received recent anti-malarial therapy, and when RDTs are performed and blood films are prepared, most malaria diagnoses are made by using the first set of tests

Clinicopathological features and outcomes of progression of CLL on the BCL2 inhibitor venetoclax

The BCL2 inhibitor venetoclax achieves responses in ∼79% of patients with relapsed or refractory chronic lymphocytic leukemia/small lymphocytic lymphoma (RR-CLL/SLL), irrespective of risk factors associated with poor response to chemoimmunotherapy. A limitation of this targeted therapy is progressive disease (PD) in some patients. To define the risk factors for progression, the clinicopathological features of PD, and the outcomes for patients after venetoclax failure, we analyzed 67 heavily pretreated patients on 3 early phase clinical trials. Investigations at progression included positron emission tomography scan and biopsy. Twenty-five (37%) patients manifested PD on therapy: 17 with Richter transformation (RT) and 8 with progressive CLL/SLL. RT occurred significantly earlier (median 7.9 months) than progressive CLL (median 23.4 months) (P = .003). Among patients who received the recommended phase 2 dose of venetoclax or higher (≥400 mg/d), fludarabine refractoriness and complex karyotype were associated with progression (hazard ratio 7.01 [95% confidence interval 1.7-28.5]; P = .002 and 6.6 [1.5-29.8]; P = .005, respectively), whereas del(17p) and/or TP53 mutation were not (P = .75). Median postprogression survival was 13 (<1-49.9) months. Bruton tyrosine kinase inhibitors were active in progressive CLL, but outcomes were mixed. Patients with disease that is fludarabine refractory or who have complex cytogenetics should have occult RT excluded before initiating venetoclax therapy

Mentoring in the management of hematological malignancies

Aim: The Mentoring in Management of Haematological Malignancies (MMHM) project aimed to improve treatment outcomes, coordinate care and provide best practice for patients with hematological cancers, by developing a program of mentoring and multidisciplinary care between a regional and a metropolitan centre. Methods: A regular multidisciplinary meeting conducted by teleconference was established between a tertiary metropolitan site and a regional practice to discuss cases of patients with hematological malignancies. Information from multidisciplinary team meetings was recorded to capture adherence to process and clinician outcomes. An educational program was developed. A gap analysis was performed to identify differences in routine practice between the two centers. Clinician satisfaction with mentoring and educational interventions was assessed by structured survey. Results: The MMHM project developed a formal mentoring system to improve the management of patients by building on established links and developing an innovative model of web-based multidisciplinary care. The project established a novel multidisciplinary meeting between a metropolitan and regional site. Common treatment policies were adopted between the two sites. Development of an educational framework and mentoring for health-care professionals in regional areas was achieved by tutorials and workshops. Most participating clinicians indicated their high level of satisfaction with the mentoring project. Conclusion: The MMHM project was a successful pilot of a mentoring program in hematological cancers between metropolitan and regional centers that resulted in improved referral links, facilitated better care coordination, updated treatment policies and guidelines and increased clinician satisfaction and knowledge.7 page(s

Detection of BRAF mutations in patients with hairy cell leukemia and related lymphoproliferative disorders

Hairy cell leukemia has been shown to be strongly associated with the BRAF V600E mutation. We screened 59 unenriched archived bone marrow aspirate and peripheral blood samples from 51 patients with hairy cell leukemia using high resolution melting analysis and confirmatory Sanger sequencing. The BRAF V600E mutation was detected in 38 samples (from 36 patients). The BRAF V600E mutation was detected in all samples with disease involvement above the limit of sensitivity of the techniques used. Thirty-three of 34 samples from other hematologic malignancies were negative for BRAF mutations. A BRAF K601E mutation was detected in a patient with splenic marginal zone lymphoma. Our data support the recent finding of a disease defining point mutation in hairy cell leukemia. Furthermore, high resolution melting with confirmatory Sanger sequencing are useful methods that can be employed in routine diagnostic laboratories to detect BRAF mutations in patients with hairy cell leukemia and related lymphoproliferative disorders