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. 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 we identified provides potential biological insights and is likely to have clinical relevance for patients with this poor-prognosis disease.

Background Systemic forms of EBV‐associated T‐cell lymphoproliferative disorders of childhood (S‐EBV‐T‐LPD) comprise three major forms: EBV‐positive hemophagocytic lymphohistiocytosis (EBV‐HLH), systemic EBV‐positive T‐cell lymphoma (S‐EBV‐TCL), and systemic chronic active EBV infection (S‐CAEBV). These disorders occur rarely in children in Western countries. Here, we described eight children of such entities. Design Eight cases (six clinical and two autopsy) with S‐EBV‐T‐LPD of childhood were retrospectively identified from 1990 to 2015. Clinicopathologic parameters including histomorphology, immunophenotype, EBV studies, and T‐cell receptor gene rearrangement studies were recorded. Results Patients include five females and three males of Hispanic, Asian, and Caucasian origins with an age range of 14 months to 9 years. Fever, hepatosplenomegaly, cytopenias, abnormal EBV serologies, and very high EBV viral loads were common findings. Histologic findings showed EBV+ T‐cell infiltrates with variable degrees of architectural distortion and cytologic atypia ranging from no to mild cytologic atypia to overt lymphoma and tissue hemophagocytosis. All showed aberrant CD4+ or CD8+ T cells with dim to absent CD5, CD7, and CD3, and bright CD2 and CD45 by flow cytometry or loss of CD5 by immunohistochemistry. TCR gene rearrangement studies showed monoclonal rearrangements in all clinical cases (6/6). Outcomes were poor with treatment consisting of chemotherapy per the HLH‐94 or HLH‐2004 protocols with or without bone marrow transplant. Conclusion In this large pediatric clinicopathologic study of S‐EBV‐T‐LPD of childhood in the United States, EBV‐HLH, S‐EBV‐TCL, and S‐CAEBV show many overlapping features. Diagnosis is challenging, and overall outcome is poor using current HLH‐directed therapies.

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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.

The rarity of mixed phenotype acute leukemia (MPAL) has precluded adequate data to incorporate minimal residual disease (MRD) monitoring into therapy. Fluidity in MPAL classification systems further complicates understanding its biology and outcomes; this includes uncertainty surrounding the impact of shifting diagnostic requirements even between iterations of the World Health Organization (WHO) classification. Our primary objective was to address these knowledge gaps. To do so, we analyzed clinicopathologic features, therapy, MRD, and survival in a centrally-reviewed, multi-center cohort of MPAL uniformly diagnosed by the WHO classification and treated with acute lymphoblastic leukemia (ALL) regimens. ALL induction therapy achieved an EOI MRD negative (<0.01%) remission in most patients (70%). EOI MRD positivity was predictive of 5-year EFS (HR=6.00, p<0.001) and OS (HR=9.57, p=0.003). Patients who cleared MRD by EOC had worse survival compared to those EOI MRD negative. In contrast to adults with MPAL, ALL therapy without transplantation was adequate to treat most pediatric patients. Earlier MRD clearance was associated with better treatment success and survival. Prospective trials are now necessary to validate and refine MRD thresholds within the pediatric MPAL population and to identify salvage strategies for those with poor predicted survival.

Young males have a unique but rare predilection to develop mediastinal nonseminomatous germ cell tumors (NSGCTs) and concomitant acute megakaryoblastic leukemia (AMKL). Common cytogenetic and molecular abnormalities such as isochromosome 12p and somatic Tumor Protein P53(TP53) and Phosphatase And Tensin Homolog (PTEN) mutations have been reported in the presumed mutual neoplastic clones of origin. We report the case of a 17-year-old male who presented with a mediastinal NSGCT with high-grade sarcomatous transformation and a diagnosis of AMKL approximately 4 months later. Next-generation sequencing revealed identical KRAS Proto-Oncogene, GTPase (KRAS) p.Ala146Thr, TP53 p.Leu257Pro, and PTEN p.Leu181Pro missense mutations at similar variant allele frequencies in both the NSGCT and AMKL samples. Cytogenetic and microarray analyses detected shared copy gains in all chromosomes except chromosomes 9, 13, and Y. Multiple additional clonal chromosomal alterations were detected in the AMKL sample when compared with the NSGCT. This case emphasizes the shared clonal origins of these malignancies and identifies KRAS and other copy number alterations as potential molecular drivers in a subset of these combined diseases.

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′-18 Ffluorothymidine ( 18 F-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 radiationinduced 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 T 2 relaxation rates (DR 2 ) collected from MR scans. Results: 18 F-FLT PET/CT imaging differentiated irradiated from unirradiated BM regions. Two days after irradiation, proliferation of 1LS animals was significantly lower than sham (P 5 0.0001, femurs; P , 0.0001, tibias) and returned to sham levels by day 10 (P 5 0.6344, femurs; P 5 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 5 0.0310, femurs; P 5 0.5832, tibias). MRI of irradiated spines detected radiation-induced BM vascular damage, measured by the significant increase in DR 2 2 d after wholebody irradiation (P 5 0.0022) and HBS (P 5 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 18 F-FLT PET/CT and USPIO MRI are valuable tools in mapping regional radiation exposure and the effects of radiation on BM. Analysis of the 18 F-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.

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