Jyotinder N. Punia

Mixed-phenotype acute leukemia (MPAL) exhibits frequent mutations in DNMT3A and activated signaling genes

Mixed-phenotype acute leukemia (MPAL) is a heterogeneous group of poor-prognosis leukemias with immunophenotypic features of at least two cell lineages. The full spectrum of genetic mutations in this rare disease has not been elucidated, limiting our understanding of disease pathogenesis and our ability to devise targeted therapeutic strategies. Here, we sought to define the mutational landscape of MPAL by performing whole-exome sequencing on samples from 23 adult and pediatric MPAL patients. We identified frequent mutations of epigenetic modifiers, most notably mutations of DNMT3A, in 33% of adult MPAL patients. Mutations of activated signaling pathways, tumor suppressors, and transcription factors were also frequent. Importantly, many of the identified mutations are potentially therapeutically targetable, with agents currently available or in various stages of clinical development. Therefore, the mutational spectrum that we have identified provides potential biological insights and is likely to have clinical relevance for patients with this poor-prognosis disease.

Mapping Radiation Injury and Recovery in Bone Marrow Using 18F-FLT PET/CT and USPIO MRI in a Rat Model

We present and test the use of multimodality imaging as a topological tool to map the amount of the body exposed to ionizing radiation and the location of exposure, which are important indicators of survival and recovery. To achieve our goal, PET/CT imaging with 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) was used to measure cellular proliferation in bone marrow (BM), whereas MRI using ultra-small superparamagnetic iron oxide (USPIO) particles provided noninvasive information on radiation-induced vascular damage. Methods: Animals were x-ray–irradiated at a dose of 7.5 Gy with 1 of 3 radiation schemes—whole-body irradiation, half-body shielding (HBS), or 1-leg shielding (1LS)—and imaged repeatedly. The spatial information from the CT scan was used to segment the region corresponding to BM from the PET scan using algorithms developed in-house, allowing for quantification of proliferating cells, and BM blood volume was estimated by measuring the changes in the T2 relaxation rates (ΔR2) collected from MR scans. Results: 18F-FLT PET/CT imaging differentiated irradiated from unirradiated BM regions. Two days after irradiation, proliferation of 1LS animals was significantly lower than sham (P = 0.0001, femurs; P < 0.0001, tibias) and returned to sham levels by day 10 (P = 0.6344, femurs; P = 0.3962, tibias). The degree of shielding affected proliferation recovery, showing an increase in the irradiated BM of the femurs, but not the tibias, of HBS animals when compared with 1LS (P = 0.0310, femurs; P = 0.5832, tibias). MRI of irradiated spines detected radiation-induced BM vascular damage, measured by the significant increase in ΔR2 2 d after whole-body irradiation (P = 0.0022) and HBS (P = 0.0003) with a decreasing trend of values, returning to levels close to baseline over 10 d. Our data were corroborated using γ-counting and histopathology. Conclusion: We demonstrated that 18F-FLT PET/CT and USPIO MRI are valuable tools in mapping regional radiation exposure and the effects of radiation on BM. Analysis of the 18F-FLT signal allowed for a clear demarcation of exposed BM regions and elucidated the kinetics of BM recovery, whereas USPIO MRI was used to assess vascular damage and recovery.

The use of BRAF V600E mutation-specific immunohistochemistry in pediatric Langerhans cell histiocytosis

BRAF p.V600E mutations are detected in greater than 50% of pediatric Langerhans cell histiocytosis (LCH) lesions. However, the use of mutation‐specific BRAF V600E immunohistochemistry (IHC) as a surrogate for molecular testing in pediatric LCH is unknown. We tested the mutation‐specific BRAF V600E monoclonal antibody (clone VE1) in formalin‐fixed, paraffin‐embedded LCH samples from 26 pediatric patients (14 males and 12 females, ages 7 mo–17 y) using allele‐specific real‐time polymerase chain reaction (PCR) with a limit of detection of 0.5% as the comparative gold standard. BRAF VE1 staining was scored for both intensity (0‐3+) and percentage of immunoreactive tumor cells (0%‐100%). BRAF VE1 immunoreactivity was determined using both lenient (≥1+, ≥1%) and stringent (≥2+, ≥10%) scoring criteria. Using lenient‐scoring criteria, we found that the sensitivity and specificity of IHC compared with allele‐specific real‐time PCR were 100.0% and 18.2%, respectively. The poor specificity of lenient IHC analysis was attributable to weak, 1+ staining in both BRAF‐mutated and wild‐type LCH. Using stringent‐scoring criteria, we found that specificity improved to 100.0% at the expense of sensitivity that decreased to 80.0%. Stringent scoring generated 3 false‐negative results, but in all cases, neoplastic tissue comprised less than 5% of the stained section and/or the specimen was decalcified. In conclusion, highly sensitive molecular assays remain the gold standard for BRAF mutation analysis in LCH paraffin‐embedded lesions. To avoid false‐positive results, unequivocal VE1 staining of 2+ intensity in greater than or equal to 10% neoplastic histiocytes is required. However, negative VE1 results require additional studies to exclude false‐negatives, and stringent‐scoring criteria may not be optimal for scant or decalcified specimens.

Are micromegakaryocytes specific for refractory cytopenia of childhood (RCC)? A study of 38 pediatric patients with thrombocytopenia unrelated to RCC☆

BACKGROUND Micromegakaryocytes (microMKs) are considered the most reliable dysplastic feature for myelodysplastic syndrome (MDS), particularly refractory cytopenia of childhood (RCC); there is no minimal threshold for the diagnosis of RCC. Since most RCC patients present with thrombocytopenia, the presence of microMKs should raise concern for MDS/RCC. This study attempted to investigate the prevalence of microMKs and associated marrow fibrosis in patients with thrombocytopenia unrelated to MDS/RCC and the need for establishing a threshold for microMKs for the diagnosis of MDS/RCC. DESIGN Bone marrow biopsies of pediatric patients with thrombocytopenia unrelated to RCC were examined for microMKs and fibrosis by CD61 immunohistochemical and reticulin stains respectively. RESULT Thirty eight patients (1-18 years old) were included: 33 immune thrombocytopenia (ITP), 3 chronic thrombocytopenia, and 2 inherited macrothrombocytopenia. Fourteen cases (37%) had microMKs; four cases showed increased marrow fibrosis associated with microMKs (two had ITP and two had macrothrombocytopenia). All patients are alive and none developed MDS (follow up: 3months to 4 years). CONCLUSION MicroMKs can be seen in pediatric patients with thrombocytopenia unrelated to RCC. Hence the mere presence of microMKs is insufficient for the diagnosis of RCC in the pediatric population, and a quantitative threshold needs to be established.

Investigating the Abscopal Effects of Radioablation on Shielded Bone Marrow in Rodent Models Using Multimodality Imaging

The abscopal effect is the response to radiation at sites that are distant from the irradiated site of an organism, and it is thought to play a role in bone marrow (BM) recovery by initiating responses in the unirradiated bone marrow. Understanding the mechanism of this effect has applications in treating BM failure (BMF) and BM transplantation (BMT), and improving survival of nuclear disaster victims. Here, we investigated the use of multimodality imaging as a translational tool to longitudinally assess bone marrow recovery. We used positron emission tomography/computed tomography (PET/CT), magnetic resonance imaging (MRI) and optical imaging to quantify bone marrow activity, vascular response and marrow repopulation in fully and partially irradiated rodent models. We further measured the effects of radiation on serum cytokine levels, hematopoietic cell counts and histology. PET/CT imaging revealed a radiation-induced increase in proliferation in the shielded bone marrow (SBM) compared to exposed bone marrow (EBM) and sham controls. T2-weighted MRI showed radiation-induced hemorrhaging in the EBM and unirradiated SBM. In the EBM and SBM groups, we found alterations in serum cytokine and hormone levels and in hematopoietic cell population proportions, and histological evidence of osteoblast activation at the bone marrow interface. Importantly, we generated a BMT mouse model using fluorescent-labeled bone marrow donor cells and performed fluorescent imaging to reveal the migration of bone marrow cells from shielded to radioablated sites. Our study validates the use of multimodality imaging to monitor bone marrow recovery and provides evidence for the abscopal response in promoting bone marrow recovery after irradiation.

Inherited Bone Marrow Failure Syndromes, Myeloid Neoplasms with Germline Predisposition and Myeloid Proliferations Associated with Down Syndrome

The inherited bone marrow failure syndromes (IBMFS) are a group of genetic disorders associated with inadequate production of one or more blood cell lineages (Table 3.1). These disorders are rare and heterogeneous, but are clinically important due to cytopenias and propensity to progress to severe aplastic anemia, myelodysplastic syndromes, acute leukemia, and other malignancies. Broadly, IBMF syndromes can be divided into multilineage cytopenias (Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, congenital amegakaryocytic thrombocytopenia), unilineage cytopenia (Diamond-Blackfan anemia, severe congenital neutropenia, thrombocytopenia with absent radii), and congenital dyserythropoietic anemia. The incidence of IBMFS may be difficult to assess due to the absence of large-scale epidemiologic studies. However, the current literature and large-center studies suggest an annual incidence of 65 per million live births [1].

Therapy-related Acute Leukemia With Mixed Phenotype and Novel t(1: 6)(q25;p23) After Treatment for High-risk Neuroblastoma

Neuroblastoma is the most common extracranial malignancy of childhood. Patients with high-risk disease receive multimodal treatment including chemotherapy combinations containing alkylating agents and topoisomerase inhibitors with potential for inducing therapy-related malignancy later in life. Most commonly, cytogenetic changes of pediatric therapy-related myelodysplastic syndrome/acute myeloid leukemia involve chromosome 5 or 7. Here we report a novel case of therapy-related myelodysplastic syndrome/acute myeloid leukemia 30 months after treatment for high-risk neuroblastoma with biphenotypic cell surface markers and a not yet described translocation t(1;6)(q25;p23).

A Rare Case of Pediatric Chronic Myelogenous Leukemia Presenting with Severe Thrombocytosis without Leukocytosis.

Pediatric chronic myelogenous leukemia (CML) is uncommon. We report a pediatric patient with CML presenting with a normal white blood cell count and no circulating immature myeloid cells. The patient presented with extreme thrombocytosis (platelet count range: 2,175-3,064 x 109/L) noted incidentally. No splenomegaly was found. Examination of the bone marrow aspirate (BMA) revealed normal cellularity and normal myeloid: erythroid ratio with marked megakaryocytic hyperplasia. Molecular studies on the BMA detected both the major BCR/ABL1p210 fusion transcript (9,280 copies; p210/ABL1 ratio: 38.2%) and the minor p190 transcript (below limit of quantitation). The platelet count normalized within 2 weeks after treatment with the second-generation tyrosine kinase inhibitor dasatinib. Follow-up 3 months later revealed a 1.87 log reduction in p210 transcripts compared to diagnosis and no detectable p190 transcripts. This case highlights the need to include BCR/ABL1 fusion testing to accurately diagnose pediatric patients presenting with isolated thrombocytosis.

Detection of Lymphoid and Myeloid Lineages in Infantile B-Cell Acute Lymphoblastic Leukemia With Mixed-Lineage Leukemia Rearrangement by Use of Flow Cytometry and Cytogenetic Analysis

Switch between lymphoid and myeloid cell lineage by a leukemic clone is an event with particularly poor prognostic significance. Lineage switch is not common and has previously been described by isolated case reports. Recently, a large, single-institution series drew attention to the association of this phenomenon with MLL (myeloid/lymphoid or mixed-lineage leukemia) abnormalities and highlighted the importance of differentiating between progression of original disease and evolution of treatment-associated malignancy. In this series, all patients who exhibited lineage switch died of disease, underlining the critical need for early recognition and stratification when making treatment decisions among this group. Flow cytometric monitoring has proven to be more sensitive and accurate than routine morphologic screening in evaluation for minimal residual disease and in tracking progression of leukemic clones. The present case illustrates how this methodology can augment detection and monitoring of leukemia that converts in lineage. This report emphasizes the importance of early recognition of the entity.