Findings In Acute Lymphoblastic Leukemia Research Articles

Flow cell sorting followed by PCR-based clonality testing may assist in questionable diagnosis and monitoring of acute lymphoblastic leukemia.

Multicolor flow cytometry (MFC) has highly reliable and flexible algorithms for diagnosis and monitoring of acute lymphoblastic leukemia (ALL). However, MFC analysis can be affected by poor sample quality or novel therapeutic options (e.g., targeted therapies and immunotherapy). Therefore, an additional confirmation of MFC data may be needed. We propose a simple approach for validation of MFC findings in ALL by sorting questionable cells and analyzing immunoglobulin/T-cell receptor (IG/TR) gene rearrangements via EuroClonality-based multiplex PCR. We obtained questionable MFC results for 38 biological samples from 37 patients. In total, 42 cell populations were isolated by flow cell sorting for downstream multiplex PCR. Most of the patients (n=29) had B-cell precursor ALL and were investigated for measurable residual disease (MRD); 79% of them received CD19-directed therapy (blinatumomab or CAR-T). We established the clonal nature of 40 cell populations (95.2%). By using this technique, we confirmed very low MRD levels (<0.01% MFC-MRD). We also applied it to several ambiguous findings for diagnostic samples, including those with mixed-phenotype acute leukemia, and the results obtained impacted the final diagnosis. We have demonstrated possibilities of a combined approach (cell sorting and PCR-based clonality assessment) to validate MFC findings in ALL. The technique is easy to implement in diagnostic and monitoring workflows, as it does not require isolation of a large number of cells and knowledge of individual clonal rearrangements. We believe it provides important information for further treatment.

A significant finding in acute lymphoblastic leukemia: bilateral serous retinal detachment

A significant finding in acute lymphoblastic leukemia: bilateral serous retinal detachment

SHIP1 Inhibition As Novel Therapeutic Approach in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is one of the most common B cell malignancies in the Western world. Malignant B cells are blocked from differentiating into immunoglobulin producing-plasma cells and clonally accumulate in the spleen, lymph nodes, bone marrow and peripheral blood. CLL is characterized by immunosuppression throughout all disease stages, which is mediated by increased numbers of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Jitschin and Braun et al., Blood 2014) and direct inhibitory effects of the malignant CLL cells on T cells (Christopoulos etal., Blood 2011).Over the past decade, significant improvement in understanding the pathogenesis of CLL has highlighted the importance of active B cell receptor (BCR) signaling. This has revealed promising targeted treatment options, including the small molecule inhibitors targeting the phosphatidylinositol-3-kinase (PI3K) signaling pathway. Idelalisib and Duvelisib are under clinical investigation for CLL treatment, however, treatment-related toxicities are limiting their application and none of these approaches are curative, highlighting the importance of functional anti-tumor immune responses in CLL for prolonged treatment efficacy.Here, we are testing a novel approach that aims to selectively target CLL B cells and simultaneously restore an appropriate immune cell function. The phosphatase SH2-domain-containing inositol 5ʹ-phosphatase 1 (SHIP1) serves as negative feedback molecule and downregulates PI3K signaling in B cells by dephosphorylating the 5'phosphate of Phosphatidylinositol (3,4,5)-trisphosphate. We hypothesize that CLL cells rely on such negative regulators for optimal survival and can only tolerate a maximum signaling level. We are therefore testing whether SHIP1 inhibition induces hypersignaling and thereby CLL cell death. Furthermore, we are investigating whether SHIP1 inhibition simultanously stimulates immune responses, as it has been shown to induce expansion of murine hematopoietic and mesenchymal stem cell compartments (Brooks et al., Stem cells 2014). 3α-Aminocholestane (3AC) is a small molecule inhibitor of SHIP1 and can be used for pharmacological inhibition.First, we investigated the expression and phosphorylation levels of SHIP1 in CLL. We found SHIP1 to be expressed at various levels in CLL peripheral blood and strongly phosphorylated compared to age-matched healthy donors. Besides, SHIP1 transcription is upregulated in lymph nodes as compared to peripheral blood, which is in line with the assumption of increased BCR signaling in secondary lymphoid organs. We then set out to investigate the consequences of SHIP1 phosphatase inhibition. Similarly, to recent findings in acute lymphoblastic leukemia (Chen et al., Nature 2015), pharmacological inhibition of SHIP1 lead to rapid cell death of CLL cells. We further investigated the mode of cell death and observed specific features of apoptosis, namely caspase 3/7 activation and phosphatidylserine exposure on the outer cell membrane. This has been tested on primary CLL patient samples and T cell leukemia/lymphoma 1 (TCL1)-driven murine CLL cells and was not observed or significantly less pronounced in other lymphoma cell lines or healthy primary B cells. To confirm the specificity of the observed effects, we genetically activated AKT with a GFP reporter in the TCL1-driven mouse model in vivo and in vitro. By tracking GFP-expressing CLL cells, we observed an initial expansion followed by rapid cell death in vitro. When we induced AKT activation in vivo, GFP+ CLL cells were not detectable in the peripheral blood, total CLL count declined upon induction and we found decreased tumor burden in the secondary lymphoid organs, particularly in the lymph nodes. In addition to the direct effects on CLL cells, we sought to investigate the impact of SHIP1 inhibition on other immune cell functions. We observed that SHIP1 inhibition lowers the activity threshold of T cells: When we stimulated a reporter cell line with suboptimal doses of anti-CD3, 3AC treatment significantly enhanced the response rate.Therefore, we propose SHIP1 as a novel interesting target in CLL. In contrast to kinase inhibition and downregulation of the BCR signaling strength, phosphatase inhibition and BCR signaling overaction provides an attractive new treatment strategy for elimination of malignant CLL cells and stimulation of immune responses. DisclosuresNo relevant conflicts of interest to declare.

Atypical Presentation of Childhood Leukemia.

To, The Editor, Leukemia is the most common childhood cancer worldwide [1]. Acute lymphoblastic leukemia (ALL) usually manifests with pallor, hepatosplenomegaly, lymphadenopathy, fever, bone pain, and bleeding [2]. Presence of frank osteolytic lesions with hypercalcemia is infrequent [3].We describes here a case of paediatric ALL who presented with multiple lytic lesions, hypercalcemia and absence of blasts in the peripheral blood film. A 10-year-old boy presents with generalised weakness, pain in right hypochondriac region which was non radiating, aggravated by coughing, associated with vomiting which was non bilious and non projectile for last 15 days. On examination, mild pallor was present with no icterus and lymphadenopathy. Liver was palpable 2 cm below the right costal margin. On investigations, complete hemogram showed Hb of 9.2g/ dl, WBC count of 8.0×109 /L with neutrophils 54%, lymphocyte 36%, monocyte 7%, eosinophil 2%, myelocyte 1% and platelet count 271 ×109 /L. Peripheral blood film showed mild anisocytosis with microcytes and hypochromia. His serum biochemistry studies were as follows: sodium: 134.8 mmol/L, potassium: 4.2 mmol/L, Crealinium: 0.93mg/dl, Calcium: 14.2 mg/dl, Phosphorous 6.4 mg/dl, uric acid: 7.6 mg/dl, LDH: 654 IU/L, Alkaline phosphatase: 110U/L, Albumin: 3.6g/dl. His ESR was 100 mm/h. Serum vitamin D3 level was 16.3 (15-30ng/ml). His serum parathyroid hormone (PTH) level was 7.2 (13.7-77.2 pg/ml). His abdominal ultrasound was normal except for hepatomegaly. The skeletal survey showed multiple lytic lesions in skull [Table/Fig-1] and ill defined translucent areas in pelvic bone. In view of anemia and multiple lytic lesions, bone marrow examination was done. Bone marrow examination revealed hypercellular marrow with 55% blasts which were positive for CD 10, CD 19, CD 22 and negative for myeloperoxidase confirming to diagnosis of precursor B acute lymphoblastic leukemia. Cytogenetic analysis of bone marrow showed 46XY karyotype. Molecular analysis for t (9; 22), t (12; 21), t (1; 19), and t (4; 11) using PCR method were negative. Patient was started on aggressive hydration, bisphosphonate and frusemide with monitoring of tumour lysis markers. For ALL, he was started with chemotherapy as per BFM 2002 protocol. His serum calcium levels decreased to 11.0 mg/dl after 48 hrs. Currently he is undergoing chemotherapy and his Day +15 marrow was hypocellular with 3% blasts (M1 marrow). Hypercalcemia is a rare finding in paediatric ALL ranging in frequency from 0.6- 4.8% [4]. Hypercalcemia of malignancy can occur due to two mechanisms. First is localized bone destruction by invasive cancer cells and second mechanism involves osteoclastic bone resorption after the release of humoral derived factors from tumour cells. Although hypercalcemia is assumed to be linked with high tumour bulk, a series of 22 patients showed no difference in event free survival in children with hypercalcemia at presentation. It has been reported that t (17;19), which is associated with poor prognosis, is frequently seen in patients with hypercalcemia due to humoral factors [5]. In our patient, hypercalcemia was due to direct bone invasion and cytogenetics was normal. The presence of multiple lytic lesions and hypercalcemia with no blasts in the peripheral blood are uncommon findings in ALL, which has prompted us to report this case. [Table/Fig-1]: X-ray Skull showing multiple lytic lesions

The Reliability and Specificity of c-kit for the Diagnosis of Acute Myeloid Leukemias and Undifferentiated Leukemias

We document findings on c-kit (CD117) expression in 1,937 pediatric and adult de novo acute leukemia cases, diagnosed in five single European centers. All cases were well characterized as to the morphologic, cytochemical, and immunologic features, according to the European Group for the Immunological Classification of Leukemias (EGIL). The cases included 1,103 acute myeloid leukemia (AML), 819 acute lymphoblastic leukemia (ALL), 11 biphenotypic acute leukemia (BAL), and 4 undifferentiated (AUL). c-kit was expressed in 741 (67%) AML cases, regardless of the French-American-British (FAB) subtype, one third of BAL, all four AUL, but only in 34 (4%) of ALL cases. The minority of c-kit+ ALL cases were classified as: T-cell lineage (two thirds), mainly pro-T–ALL or T-I, and B lineage (one third); cells from 62% of these ALL cases coexpressed other myeloid markers (CD13, CD33, or both). There were no differences in the frequency of c-kit+ AML or ALL cases according to age being similar in the adult and pediatric groups. Our findings demonstrate that c-kit is a reliable and specific marker to detect leukemia cells committed to the myeloid lineage, and therefore should be included in a routine basis for the diagnosis of acute leukemias to demonstrate myeloid commitment of the blasts. c-kit expression should score higher, at least one point, in the system currently applied to the diagnosis of BAL, as its myeloid specificity is greater than CD13 and CD33. Findings in ALL and AUL suggest that c-kit identifies a subgroup of cases, which may correspond to leukemias either arising from early prothymocytes and/or early hematopoietic cells, both able to differentiate to the lymphoid and myeloid pathways.

The Reliability and Specificity of c-kit for the Diagnosis of Acute Myeloid Leukemias and Undifferentiated Leukemias

AbstractWe document findings on c-kit (CD117) expression in 1,937 pediatric and adult de novo acute leukemia cases, diagnosed in five single European centers. All cases were well characterized as to the morphologic, cytochemical, and immunologic features, according to the European Group for the Immunological Classification of Leukemias (EGIL). The cases included 1,103 acute myeloid leukemia (AML), 819 acute lymphoblastic leukemia (ALL), 11 biphenotypic acute leukemia (BAL), and 4 undifferentiated (AUL). c-kit was expressed in 741 (67%) AML cases, regardless of the French-American-British (FAB) subtype, one third of BAL, all four AUL, but only in 34 (4%) of ALL cases. The minority of c-kit+ ALL cases were classified as: T-cell lineage (two thirds), mainly pro-T–ALL or T-I, and B lineage (one third); cells from 62% of these ALL cases coexpressed other myeloid markers (CD13, CD33, or both). There were no differences in the frequency of c-kit+ AML or ALL cases according to age being similar in the adult and pediatric groups. Our findings demonstrate that c-kit is a reliable and specific marker to detect leukemia cells committed to the myeloid lineage, and therefore should be included in a routine basis for the diagnosis of acute leukemias to demonstrate myeloid commitment of the blasts. c-kit expression should score higher, at least one point, in the system currently applied to the diagnosis of BAL, as its myeloid specificity is greater than CD13 and CD33. Findings in ALL and AUL suggest that c-kit identifies a subgroup of cases, which may correspond to leukemias either arising from early prothymocytes and/or early hematopoietic cells, both able to differentiate to the lymphoid and myeloid pathways.

Heterogeneity of V delta 2 TCR delta gene rearrangements in acute myeloid leukemia.

In a previous study, we described the occurrence of T-cell receptor (TCR)delta gene rearrangements in 9/100 acute myeloid leukemia (AML) cases. In this study, we further characterized these rearrangements by Southern Blot hybridization using a V delta 2 specific probe and polymerase chain reaction (PCR). Southern Blot analysis revealed that rearrangements involved the V delta 2 gene segment in four patients. Interestingly the restriction fragments detected by the V delta 2 probe differed markedly in size. PCR analysis revealed a complete V delta 2(Dn)J delta 1 gene rearrangement, an incomplete V delta 2D delta 3 rearrangement and a large amplification product, which cannot be explained with normal VDJ recombinatorial processes, in one case each. Furthermore although V delta 2 and J delta were rearranged, no comigration of rearranged fragments was observed and no PCR product was obtained in one case. Obtained results in AML differ from findings in acute lymphoblastic leukemia (ALL) of B-cell lineage, where a more homogeneous pattern of rearrangements has been described. This heterogeneity might be related to the illegitimate occurrence of TCR delta gene rearrangements in AML.

Isolated Extramedullary Relapse of Acute Lymphoblastic Leukemia Presenting as an Intraspinal Mass

Leptomeningeal relapse is a common finding in acute lymphoblastic leukemia (ALL), but a solid intraspinal mass with cord compression as the sole site of extramedullary relapse is unusual. We report an adult patient with ALL, after 2 years of complete remission, who developed a spinal cord compression due to an intraspinal mass as a first manifestation of extramedullary relapse. Manifestation of first relapse in ALL presenting as an intraspinal mass is rare and may be added to other unusual extramedullary sites of relapse.

Prognostic and biological importance of chromosome findings in acute lymphoblastic leukemia

At least 25 structural chromosomal abnormalities are now found in acute lymphoblastic leukemia (ALL). Many of the abnormalities are associated with particular clinical or blast cell features. Chromosomal translocation breakpoints in ALL are among those that define regions of the genome of oncogenic potential, the recognition of which has led to an improved under-standing of the mechanisms of leukemogenesis. The prognostic importance of chromosome findings in ALL concerns demonstration of long-term survival in patients with high hyperdiploid leukemic clones and identification of patients with certain translocations who are at high risk of treatment failure and for whom alternative therapy such as bone marrow transplantation may be desirable. This review summarizes the more recent chromosomal findings in childhood and adult ALL and discusses how increasing recognition of structural change and adoption of alternative therapy for high-risk chromosomal groups may change the prognostic role of cytogenetics in this type of leukemia.

Correlation of Immunophenotypes with Chromosomal Findings in Acute Lymphoblastic Leukemia

Correlation of Immunophenotypes with Chromosomal Findings in Acute Lymphoblastic Leukemia

Prognostic implications of chromosomal findings in acute lymphoblastic leukaemia at diagnosis.

Chromosomes were studied on diagnostic bone-marrow samples from 39 children with acute lymphoblastic leukaemia (ALL). The patients were classified, according to the chromosomal characteristics of the major proportion of their leukaemia cells, into five categories; hyperdiploid, pseudodiploid, diploid, hypodiploid, and mixed. Patients in the hyperdiploid category had significantly longer first remissions than those in all other categories, and those in the pseudodiploid category had the shortest. Neither the absence of any normal cells nor the presence of detectable clones appeared to be an adverse feature. We suggest that the proportion of hyperdiploid cells, determined by conventional chromosomal staining techniques, may be used as an additional prognostic feature in childhood ALL.