Irene Sadek

Multiplex Ligation-Dependent Probe Amplification Versus Multiprobe Fluorescence in Situ Hybridization To Detect Genomic Aberrations in Chronic Lymphocytic Leukemia: A Tertiary Center Experience

Cytogenetic abnormalities play a major role in the prognosis of patients with chronic lymphocytic leukemia (CLL). Several methods have emerged to try to best identify these abnormalities. We used fluorescence in situ hybridization (FISH) to determine the frequency of cytogenetic changes in our CLL patient population. We also evaluated the effectiveness of multiplex ligation-dependent probe amplification (MLPA) in detecting these abnormalities. Sixty-two B-CLL patients and 20 healthy controls were enrolled, and FISH and MLPA analyses were performed on peripheral blood samples. Using FISH, genomic aberrations were found in 73% of patients and presented as follows: single 13q14.3 deletion (60%), trisomy 12 (7%), ATM deletion (6%), 17p13.1 deletion (2%). MLPA analyses done on 61/62 patients showed sensitivity and specificity values of 90% and 100% respectively. MLPA revealed several additional copy number changes, the most common being 19p13 (LDLR and CDKN2D). Moreover, the cost for MLPA analysis, including technical time and reagents, is 86% less than FISH. In conclusion, cytogenetic abnormalities are a common finding in CLL patients, and MLPA is a reliable approach that is more cost effective and faster than FISH. Despite MLPA limitations of sensitivity, it can be used as a first-line screen and complementary test to FISH analysis.

Acute promyelocytic leukemia: A novel PML/RARα fusion that generates a frameshift in the RARα transcript and ATRA resistance

Acute promyelocytic leukemia (APL) is characterized by increased promyelocytes in the marrow that harbor a t(15;17) and promyelocyte leukemia (PML)/RARα fusion gene. The oncogenic gene product is believed to act through disruption of the transcription-modulating function of RARα. Differentiation of promyelocytes and remission is achieved with all transretinoic acid (ATRA) therapy usually in combination with chemotherapy. This report describes a patient with the t(15;17) who did not respond typically to ATRA and IDAC induction chemotherapy, although achieved and remains in complete remission five years following induction and one consolidation with high dose cytarabine (HIDAC). RT-PCR and sequencing revealed a novel fusion of RARα exon 3 to PML exon 5 that creates a frameshift and premature stop codon in the RARα portion of the transcript. Since none of the RARα functional domains are maintained, this case highlights the possibility that PML/RARα may directly affect promyelocyte differentiation through disruption of PML function.

Prolonged complete remission of myelodysplastic syndrome treated with danazol, retinoic acid and low-dose prednisone

Myelodysplastic syndrome (MDS) is a diverse group of clonal hematologic neoplasms. Different medications have been tried in MDS; however, no effective treatment has been yet established. We report a patient with MDS who achieved a complete remission in response to combination therapy of danazol, retinoic acid, and prednisone. A 53‐year‐old female presented with pancytopenia, macrocytosis, and hypercellular bone marrow with erythroid hyperplasia and dysplasia and 10% ringed sideroblasts. Cytogenetic studies revealed the presence of two abnormal clones. She was diagnosed as having MDS‐refractory anemia and was given blood transfusions to maintain blood cell counts at acceptable levels. At the same time, she was started on a combination of danazol (600 mg/day), retinoic acid (100 mg/day), and prednisone (10 mg every other day). Fourteen months later, the patient was in complete hematologic remission; she had normal peripheral blood count, and the blood smear showed normal morphology. Bone marrow studies revealed normal trilineage hematopoiesis. She was continued on the same combination treatment for 86 months, and she remained in complete clinical remission. Eighty‐eight months from diagnosis, she relapsed with acute myeloid leukemia. This is the first reported case of MDS‐RA that sustained a complete hematologic remission for a prolonged period in response to this combination treatment. This report indicates that restoration of normal hematopoiesis, prolongation of disease‐free survival, and delay in the transformation to acute leukemia may be achieved by this combination of treatment in a subset of patients with MDS, especially refractory anemia with severe thrombocytopenia. Am. J. Hematol. 64:306–310, 2000.

Multiple pre- and post-analytical lean approaches to the improvement of the laboratory turnaround time in a large core laboratory

BACKGROUND Core laboratory (CL), as a new business model, facilitates consolidation and integration of laboratory services to enhance efficiency and reduce costs. This study evaluates the impact of total laboratory automation system (TLA), electric track vehicle (ETV) system and auto-verification (AV) of results on overall turnaround time (TAT) (phlebotomy to reporting TAT: PR-TAT) within a CL setting. METHODS Mean, median and percentage of outlier (OP) for PR-TAT were compared for pre- and post-CL eras using five representative tests based on different request priorities. Comparison studies were also carried out on the intra-laboratory TAT (in-lab to reporting TAT: IR-TAT) and the delivery TAT (phlebotomy to in-lab TAT: PI-TAT) to reflect the efficiency of the TLA (both before and after introducing result AV) and ETV systems respectively. RESULTS Median PR-TATs for the urgent samples were reduced on average by 16% across all representative analytes. Median PR-TATs for the routine samples were curtailed by 51%, 50%, 49%, 34% and 22% for urea, potassium, thyroid stimulating hormone (TSH), complete blood count (CBC) and prothrombin time (PT) respectively. The shorter PR-TAT was attributed to a significant reduction of IR-TAT through the TLA. However, the median PI-TAT was delayed when the ETV was used. Application of various AV rules shortened the median IR-TATs for potassium and urea. However, the OP of PR-TAT for the STAT requests exceeding 60min were all higher than those from the pre-CL era. CONCLUSIONS TLA and auto-verification rules help to efficiently manage substantial volumes of urgent and routine samples. However, the ETV application as it stands shows a negative impact on the PR-TAT.

Clonal heterogeneity in plasma cell myeloma

A 52-year-old man presented to the emergency department with fevers and chills in December, 2014, and was admitted with a diagnosis of pneumonia. CT to exclude pulmonary embolus showed diff use osseous lytic lesions, and he was also found to have anaemia and an IgG kappa monoclonal protein of 55·8 g/L on protein electrophoresis (a single band). After a bone marrow biopsy he was given a diagnosis of plasma cell myeloma and was treated with four cycles of CyBorD chemotherapy (cyclophosphamide, bortezomib, and dexamethasone) in preparation for an autologous stem cell transplantation. The aspirate from the bone marrow sampling showed a substantial increase in plasma cells (fi gure). The bone marrow core biopsy sample showed a hypercellular marrow with increased plasma cells, estimated to occupy 80% of marrow cellularity by CD138 immunohistochemistry. We saw that the neoplastic plasma cells had two distinct immunophenotypes: one population expressed CD138, kappa, and CD56; the other expressed CD138, kappa, CD20, and CD79a. Morphological diff erences were visible on routine haematoxylin and eosin staining, with prominent nucleoli in the CD56 area (fi gure). We could not create a more extensive immunophenotype because of tissue limitations, and too few events were captured by fl ow cytometry to allow characterisation using this technique. This case shows clear phenotypic heterogeneity in plasma cell myeloma, which might be due to multiple neoplastic clones (clonal heterogeneity). Immunophenotyping is routinely used in haematology to infer clonality, although variation is not defi nitive proof of clonality. Clonal heterogeneity in plasma cell myeloma is the foundation of work in the area of clonal tiding. Clonal tiding has been described in plasma cell myeloma with diff erent clones competing for dominance dependent on variable resistance to chemotherapy. These diff erent clones are typically shown using molecular techniques, but our image provides a practical illustration of this concept. Whether intratumoural phenotypic variation correlates to true clonal heterogeneity, and whether there is a role for identifi cation of this easily assessed entity in routine clinical practice, is unclear.

Effective enforcement of plasma transfusion policy

Plasma transfusions are used to replace deficient/dysfunctional coagulation factor(s) when the patient is significantly bleeding or whenever significant bleeding is anticipated and a specific coagulation factor concentrate is not available for use or in emergency situations when precise laboratory diagnosis is not possible. Most of coagulation factors need to be at a level of 25-30% to maintain adequate coagulation system.1 Plasma transfusion at a dose of 10-15 mL per kg body weight is expected to increase coagulation factors by 1020%.2‒4 After plasma transfusion, coagulation factor levels increase temporary and then decline to reach the baseline level within 6 to 24 hours.5 The effect of plasma transfusion on international normalized ratio (INR) is transient; for the same volume of transfused plasma, a greater reduction in INR is observed when INR is more than 1.8.6,7 Despite implemented policies and guidelines, published studies showed that plasma transfusion may be unjustified in up to 34% of the studied populations.8‒10