Detection of somatic TP53 mutations and 17p deletions in patients with chronic lymphocytic leukemia: a review of the current methods

ABSTRACT Chronic lymphocytic leukemia is the most common hematologic malignancy among adults in Western countries. Several studies show that somatic mutations in the TP53 gene are present in up to 50% of patients with relapsed or refractory chronic lymphocytic leukemia. This study aims to review and compare the methods used to detect somatic TP53 mutations and/or 17p deletions and analyze their importance in the chronic lymphocytic leukemia diagnosis and follow-up. In chronic lymphocytic leukemia patients with refractory or recurrent disease, the probability of clonal expansion of cells with the TP53 mutation and/or 17p deletion is very high. The studies assessed showed several methodologies able to detect these changes. For the 17p deletion, the chromosome G-banding (karyotype) and interphase fluorescence in situ hybridization are the most sensitive. For somatic mutations involving the TP53 gene, moderate or high-coverage read next-generation sequencing and Sanger sequencing are the most recommended ones. The TP53 gene mutations represent a strong adverse prognostic factor for patient survival and treatment resistance in chronic lymphocytic leukemia. Patients carrying low-proportion TP53 mutation (less than 20-25% of all alleles) remain a challenge to these tests. Thus, for any of the methods employed, it is essential that the laboratory conduct its analytical validation, documenting its accuracy, precision and sensitivity/limit of detection.

When karyotype is decisive for myelodysplastic syndromes diagnosis

ABSTRACT Introduction: The myelodysplastic syndromes (MDS) are a group of heterogeneous clonal hematopoietic stem cell disorders that results in peripheral blood (PB) cytopenias and bone marrow (BM) dysplasia. Dysplasia is the hallmark of the disorder, and must exceed the threshold of 10%. Conventional karyotype (KT) has a role in the classification and prognostication of subtypes. In daily practice, many cases are diagnosed in face of exuberant clinical complains, but cases with dismal evidences pose real difficulties to definitively conclude the case. Material and methods: The objective of this study is to detect cases in which no morphology evidence of dysplasia or increased blasts were observed but KT was decisive for MDS diagnosis. 666 cases were admitted to rule out MDS. Results: There were found 5 (0.75%) cases who presented no evident dysplasia morphology or whose dysplasia was borderline but the karyotype was decisive because showed clonal evidence. The karyotype was: case 1: 46,XY,del(5q)(q13q33),del(11)(q13q23)[7]/46,XY[13]; case 2: 46,XX,del(11)(q21q23)[20]; case 3: 46,XX,del(7)(q22q34)[4]/46,XX[8]; case 4: 47,XX,del(5)(q13q33),+mar[12]/46,XX[8] and case 5: 46,XXt(2;11)(p21;q24),del(4)(?q25),del(21)(q22)[14]/46,XX[6]. Conclusion: Patients with cytopenia and insufficient or borderline evidence of dysplasia may experience a long journey before a MDS diagnosis is made. Cytogenetics studies may abbreviate this pathway when clonal aberrations considered presumptive of MDS are detected. This study shows that karyotype should still be considered as a diagnostic tool.

JAK2-mutated acute myeloid leukemia: comparison of next-generation sequencing (NGS) and single nucleotide polymorphism array (SNPa) findings between two cases

JAK2 mutations are rare in de novo acute myeloid leukemia (AML), and JAK2-mutated acute myeloid leukemia (AML) patients usually have a previous history of myeloproliferative neoplasms (MPNs). Current advances in laboratory techniques, such as single nucleotide polymorphism array (SNPa) and next-generation sequencing (NGS), have facilitated new insight into the molecular basis of hematologic diseases. Herein, we present two cases of JAK2-mutated AML in which both SNPa and NGS methods added valuable information. Both cases had leukemogenic collaboration, namely, copy-neutral loss of heterozygosity (CN-LOH), detected on chromosome 9. One of the cases exhibited both JAK2 and IDH2 mutations, most likely having originated as an MPN with leukemic transformation, while the other case was classified as a de novo AML with JAK2, CEBPA, and FLT3 mutations.

Diagnosis and treatment of chronic lymphocytic leukemia: recommendations from the Brazilian Group of Chronic Lymphocytic Leukemia

ABSTRACT Chronic lymphocytic leukemia is characterized by clonal proliferation and progressive accumulation of B-cell lymphocytes that typically express CD19+, CD5+ and CD23+. The lymphocytes usually infiltrate the bone marrow, peripheral blood, lymph nodes, and spleen. The diagnosis is established by immunophenotyping circulating B-lymphocytes, and prognosis is defined by two staging systems (Rai and Binet) established by physical examination and blood counts, as well as by several biological and genetic markers. In this update, we present the recommendations from the Brazilian Group of Chronic Lymphocytic Leukemia for the diagnosis and treatment of chronic lymphocytic leukemia. The following recommendations are based on an extensive literature review with the aim of contributing to more uniform patient care in Brazil and possibly in other countries with a similar social-economic profile.

A model for the functional assessment of elderly with myeloid neoplasms

Objective: Myeloid neoplasms are heterogeneous diseases that are more incident in the elderly. The goals of this study were to aggregate a geriatric approach to the patient assessment, to show the impact of gender, age, hemoglobin concentration and comorbidities on the functionality of elderly with myeloid neoplasms and to better understand how the instruments of functional assessment work according to the aggressiveness of the disease. Methods: Elderly patients (≥60 years old) with myeloid neoplasms were assessed using the Karnofsky scale, Eastern Cooperative Oncologic Group scale, and basic and instru- mental activities of daily living scales. The hematopoietic cell transplantation-comorbidity index assessed the comorbidities. A mixed logistical regression model was fitted to estimate the impact of gender, age, hemoglobin concentration and the hematopoietic cell transplantation-comorbidity index on patients' functionality. Results: Eighty-two patients with a mean age of 72.8 years (range: 60-92 years) were evaluated. Eighty percent had good Karnofsky and Eastern Cooperative Oncologic Group scales and 39% were independent according to the daily living activity scales. All of the patients with poor Karnofsky and Eastern Cooperative Oncologic Group scales were classified as dependent by the daily living activity scales. The mixed logistic regression models showed that age, gender, hemoglobin concentration and the comorbidity index impacted on the daily living activity scales. Karnofsky and Eastern Cooperative Oncologic Group scales were affected by hemoglobin and the comorbidity index. The model hypothesized the hemoglobin concentration at which there was a higher risk of poor Karnofsky and Eastern Cooperative Oncologic Group scales. This hemoglobin concentration depended on comorbidities and on the aggressiveness of the myeloid neoplasm. Conclusion: The geriatric approach improved the sensitivity and specificity ...

Identifying the similarities and differences between single nucleotide polymorphism array (SNPa) analysis and karyotyping in acute myeloid leukemia and myelodysplastic syndromes

Objective: To standardize the single nucleotide polymorphism array (SNPa) method in acute myeloid leukemia/myelodysplastic syndromes, and to identify the similarities and differ- ences between the results of this method and karyotyping. Methods: Twenty-two patients diagnosed with acute myeloid leukemia and three with myelodysplastic syndromes were studied. The G-banding karyotyping and single nucleotide polymorphism array analysis (CytoScan(r) HD) were performed using cells from bone marrow, DNA extracted from mononuclear cells from bone marrow and buccal cells (BC). Results: The mean age of the patients studied was 54 years old, and the median age was 55 years (range: 28-93). Twelve (48%) were male and 13 (52%) female. Ten patients showed abnormal karyotypes (40.0%), 11 normal (44.0%) and four had no mitosis (16.0%). Regarding the results of bone marrow single nucleotide polymorphism array analysis: 17 were abnor- mal (68.0%) and eight were normal (32.0%). Comparing the two methods, karyotyping identified a total of 17 alterations (8 deletions/losses, 7 trissomies/gains, and 2 translocations) and single nucleotide polymorphism array analysis identified a total of 42 alterations (17 losses, 16 gains and 9 copy-neutral loss of heterozygosity). Conclusion: It is possible to standardize single nucleotide polymorphism array analysis in acute myeloid leukemia/myelodysplastic syndromes and compare the results with the abnormalities detected by karyotyping. Single nucleotide polymorphism array analysis increased the detection rate of abnormalities compared to karyotyping and also identified a new set of abnormalities that deserve further investigation in future studies. .

Diversity of breakpoints of variant Philadelphia chromosomes in chronic myeloid leukemia in Brazilian patients

Background: Chronic myeloid leukemia is a myeloproliferative disorder characterized by the Philadelphia chromosome or t(9;22)(q34.1;q11.2), resulting in the break-point cluster regionAbelson tyrosine kinase fusion gene, which encodes a constitutively active tyrosine kinase protein. The Philadelphia chromosome is detected by karyotyping in around 90% of chronic myeloid leukemia patients, but 5-10% may have variant types. Variant Philadelphia chromosomes are characterized by the involvement of another chromosome in addition to chromosome 9 or 22. It can be a simple type of variant when one other chromosome is involved, or complex, in which two or more chromosomes take part in the translocation. Few studies have reported the incidence of variant Philadelphia chromosomes or the breakpoints involved among Brazilian chronic myeloid leukemia patients. Objective: The aim of this report is to describe the diversity of the variant Philadelphia chromosomes found and highlight some interesting breakpoint candidates for further studies. Methods: the Cytogenetics Section Database was searched for all cases with diagnoses of chronic myeloid leukemia during a 12-year period and all the variant Philadelphia chromosomes were listed. Results: Fifty (5.17%) cases out of 1071 Philadelphia-positive chronic myeloid leukemia were variants. The most frequently involved chromosome was 17, followed by chromosomes: 1, 20, 6, 11, 2, 10, 12 and 15. Conclusion: Among all the breakpoints seen in this survey, six had previously been described: 11p15, 14q32, 15q11.2, 16p13.1, 17p13 and 17q21. The fact that some regions get more fre- quently involved in such rare rearrangements calls attention to possible predisposition that should be further studied. Nevertheless, the pathological implication of these variants remains unclear. .