Lineage Switch in an Infant B-Lymphoblastic Leukemia With t(1;11)(p32;q23); KMT2A/EPS15, Following Blinatumomab Therapy.

We report a 6 month-old infant girl with t(1;11)(p32;q23), KMT2A/EPS15-rearranged B-acute lymphoblastic leukemia (B-ALL) that was refractory to traditional ALL-directed chemotherapy. Following administration of blinatumomab, she experienced lineage switch from B-ALL to acute myeloid leukemia (AML). Myeloid-directed chemotherapy resulted in clearance of AML by flow cytometry, though a residual CD19+ B-ALL population persisted (0.14%). Following bridging blinatumomab, the patient achieved B-ALL and AML remission, as measured by flow cytometry. The patient subsequently underwent allogeneic hematopoietic stem cell transplant. Unfortunately, she relapsed with CD19+ B-ALL one-month post-transplantation. Next generation sequencing study of IGH/IGL using ClonoSEQ® analysis detected 3 dominant sequences all present in her original B-ALL, lineage switched AML, and post-transplant relapsed B-ALL, though the latter showed an additional 4 sequences, three of which were present at low abundance in the original diagnostic sample. The presence of the same clones throughout her disease course suggests cellular reprogramming and differentiation following chemotherapy and immunotherapy. This is the first reported case of lineage switch of B-ALL with t(1;11) and also the first report of a lineage switch case that used ClonoSEQ® to define the clonality of the original B-ALL, lineage switched AML, and relapsed B-ALL.

Brain tissue gadolinium retention in pediatric patients after contrast-enhanced magnetic resonance exams: pathological confirmation.

BACKGROUND: Retained gadolinium from gadolinium-based contrast agents (GBCAs) used in MR exams has been inferred based on signal changes on serial brain MRI and subsequently demonstrated pathologically in adults. Retention has been similarly inferred in children but pathological demonstration in pediatric patients is limited. The long-term effects of retained gadolinium are unknown but are potentially of greater concern in children given their increased vulnerability from continuing development and their expected longer period of exposure. Several factors can influence gadolinium retention. In adults as well as in children, greater accumulation has been demonstrated based on MR signal changes with linear compared with macrocyclic gadolinium chelates, attributed to lower chelate affinity with linear agents. Effects of age at exposure on retention are unknown, while differences in GBCA washout rates are still under investigation and might affect gadolinium retention relative to time of GBCA administration. OBJECTIVE: The purpose of this study was to confirm whether gadolinium brain deposits are present in pediatric patients who received GBCAs and to quantify the amounts present. MATERIALS AND METHODS: Brain autopsy specimens from 10 pediatric patients between 1 year and 13 years of age who underwent at least one contrast-enhanced MR exam were analyzed for elemental gadolinium using inductively coupled plasma mass spectrometry. Brain samples included white matter, basal ganglia (putamen, globus pallidus), thalamus, dentate nucleus and tumor tissue as available. Type and dose of contrast agent, number and timing of contrast-enhanced MR exams and renal function (estimated glomerular filtration rate [eGFR]) were documented for each child. RESULTS: Patient exposures ranged from 1 dose to 20 doses of GBCAs including both macrocyclic and linear ionic agents. Gadolinium was found to be present in brain tissue in all children and was generally highest in the globus pallidus. Those who received only macrocyclic agents showed lower levels of gadolinium retention. CONCLUSION: This study demonstrates pathological confirmation of gadolinium retention in brain tissue of a series of pediatric patients exposed to GBCAs including not only linear ionic agents but also macrocyclic agents with both nonionic and ionic compounds. The distribution and deposition levels in this small pediatric population are comparable with the findings in adults. While the clinical significance of these deposits remains unknown, at this point it would be prudent to exert caution and avoid unnecessary use of GBCAs in pediatric patients.

A fatal case of bortezomib-induced lung toxicity in a young adult heart transplant recipient.

Bortezomib is approved for the treatment of multiple myeloma but increasingly used in heart transplant (HTx) recipients with antibody-mediated rejection (AMR). Severe pulmonary toxicity is a rare complication in multiple myeloma patients treated with bortezomib, but has not been described in a solid organ transplant recipient. A 20-year-old man 7 years post-HTx presented with acute rejection with hemodynamic compromise. Endomyocardial biopsy showed mixed rejection (ISHLT grade 2R-3R acute cellular rejection (ACR) and pAMR 1 (I+) with diffuse C4d staining). Two new high MFI circulating MHC class-II donor-specific antibodies (DSA) were detected. Treatment included corticosteroids, antithymocyte globulin, plasmapheresis, IVIG, rituximab, and bortezomib (1.3 mg/m2 ). Due to rebound in DSA, a second course of bortezomib was started. Thrombocytopenia and peripheral neuropathy prompted a 50% dose reduction during the 2nd course. Shortly after the 3rd reduced dose, the patient developed hypoxemic respiratory failure. Bronchoscopy revealed pulmonary hemorrhage with negative infectious studies. Chest CT showed bilateral parenchymal disease with bronchiectasis and alveolar bleeding. Despite treatment with high-dose steroids, severe ARDS ensued with multisystem organ failure. The patient expired 23 days after the final dose of bortezomib. Post-mortem lung histology revealed diffuse alveolar damage, pulmonary fibrosis, and hemorrhage. Cardiac histology showed resolving/residual ACR 1R and pAMR 1 (I+). While rare, bortezomib-induced lung toxicity (BILT) can occur in HTx recipients and can carry a high risk of mortality. Drug reaction and immediate drug withdrawal should be considered in patients who develop respiratory symptoms, though optimal management of BILT is unclear.

Consideration of Maternal Anti-enterocyte IgA Transfer With Resulting Infantile Alloimmune Enteropathy.

Autoimmune enteropathy is a rare cause of infantile diarrhea. Cases typically involve infants with a protracted course of diarrhea found to have underlying autoimmune disease or immune dysfunction, leading to chronic intestinal inflammation. We describe a case of immune-mediated enteropathy in an infant with no identifiable autoimmune disease. The patient was exclusively breastfed by his mother who had Crohn's disease, and he was found to have circulating anti-enterocyte immunoglobulin A (IgA) antibody. There was no circulating anti-enterocyte immunoglobulin G or immunoglobulin M. The patient's disease and symptoms resolved with cessation of breastfeeding, and no immunomodulatory medications have been needed in 20 months of follow-up. The case raises suspicion for alloimmune disease, and it is hypothesized that intestinal injury was mediated by maternally transmitted anti-enterocyte IgA antibody.

Evaluation of the Utility of Bone Marrow Morphology and Ancillary Studies in Pediatric Patients Under Surveillance for Myelodysplastic Syndrome.

OBJECTIVES: To evaluate the utility of flow cytometry, karyotype, and a fluorescence in situ hybridization (FISH) panel in screening children for myelodysplastic syndrome (MDS). METHODS: Bone marrow morphology, flow cytometry, karyotype, and FISH reports from 595 bone marrow specimens (246 patients) were analyzed. RESULTS: By morphology, 8.7% of cases demonstrated at least unilineage dysplasia and/or increased blasts. Flow cytometry identified definitive abnormalities in 2.8% of cases, all of which had abnormal morphology. Of the 42 cases (7.2%) with acquired karyotypic abnormalities, 26 had no morphologic dysplasia. With a 98.2% concordance between karyotype and MDS FISH, FISH only identified two additional cases, both with low-level (<4%) abnormalities. Peripheral blood count evaluation only identified the absence of thrombocytopenia to correlate with an absence of abnormal ancillary tests. CONCLUSIONS: The combination of morphologic evaluation and karyotype with judicious use of flow cytometry and MDS FISH is sufficient to detect abnormalities for these indications.

Improving Time to Antibiotics for Pediatric Oncology Patients With Suspected Infections: An Emergency Department-Based Quality Improvement Intervention.

OBJECTIVE: Studies in pediatric patients with fever and neutropenia demonstrate that shorter time to antibiotics is associated with a decrease in pediatric intensive care unit admissions and in-hospital mortality. In 2012, a 2-phase quality improvement intervention was implemented in a pediatric emergency department (ED) to improve care for this high-risk patient population.The objective was to determine if the introduction of (1) a rapid absolute neutrophil count (ANC) test and (2) a standardized prearrival process decreased time to antibiotics for febrile hematology/oncology(heme/onc) patients presenting to the ED. METHODS: The rapid ANC test introduced in February 2012 decreased turn-around-times in the laboratory from 60 to 10 minutes. The standardization of the prearrival communication between the heme/onc team and ED was implemented in August 2012 as part of a clinical standard work pathway for heme/onc patients who presented to the ED with fever and possible neutropenia. Time from arrival to the ED to administration of first antibiotic was measured.Data from January 2011 to December 2013 were analyzed using statistical process control. RESULTS: Seven hundred eighteen encounters for 327 patients were included. After the rapid ANC test, the proportion of patients who received antibiotics within 60 minutes of arrival increased from 47% to 60%. There was further improvement to 69% with implementation of the clinical standard work pathway. Mean time to antibiotics decreased from 83 to 65 minutes (21% decrease). CONCLUSION: This 2-phase quality improvement intervention increased the proportion of patients who received antibiotics within 60 minutes of arrival to the ED. Similar processes may be implemented in other pediatric EDs to improve timeliness of antibiotic administration.

Hematoxylin Bodies in Pediatric Bone Marrow Aspirates and their Utility in the Diagnosis of Systemic Lupus Erythematosus.

In our recent case report, the finding of lupus erythematosus (LE) cells in a bone marrow aspirate led to the diagnosis of systemic lupus erythematosus (SLE) and appropriate treatment, although the patient was not clinically suspected to have SLE. To determine whether LE cells are present in the bone marrow aspirates of SLE patients, but overlooked in routine bone marrow morphology review, bone marrow aspirates from 30 pediatric patients (15 with SLE and 15 with other diagnoses) evaluated by rheumatologists were reviewed. LE cells were found in the bone marrow aspirates of only 1 SLE patient and none in non-SLE patients. However, hematoxylin bodies were identified in 53% (8/15) of SLE patients. Neither hematoxylin bodies nor LE cells were found in the aspirates from patients with other disorders. Three additional pediatric patients identified prospectively were found to have hematoxylin bodies in the bone marrow aspirates. Although the diagnosis was not initially suspected, 2 of the 3 patients were subsequently diagnosed with SLE. All patients with hematoxylin bodies and SLE had antinuclear antibody titers ≥1:640 with a homogeneous staining pattern. In addition, bone marrow aspirates of 9 adult patients were reviewed, and neither LE cells nor hematoxylin bodies were identified. In summary, hematoxylin bodies were present in the bone marrow aspirates of many pediatric SLE patients, while LE cells were rare. The finding of hematoxylin bodies in pediatric bone marrow aspirates is a helpful and specific diagnostic clue that may lead to the diagnosis of SLE when other clinical features are nonspecific.

Is Identification of Lupus Erythematosus Cells Still Useful? A Case Report.

A 13-year-old girl presented with significant weight loss, depression, anemia, and neutropenia. The preliminary diagnosis was anorexia nervosa combined with depression. Due to peripheral cytopenia, a bone marrow biopsy was performed to rule out leukemia. Lupus erythematosus (LE) cells were found in the bone marrow aspirate, which prompted autoantibody testing, although clinically it was not suspected the patient had systemic lupus erythematosus (SLE). Further testing demonstrated very high levels of antinuclear antibodies (ANA) (>12 U) and anti-double strand DNA (dsNDA) (>1000 IU/mL), which confirmed the diagnosis of SLE. The patient was treated with steroids for SLE, and symptoms improved quickly. In conclusion, although the identification of LE cells as one of the diagnostic criteria for SLE has been obsolete, careful examination of bone marrow to identify LE cells is still very important in the diagnosis of unsuspected SLE.

Isolated appendiceal typhlitis masquerading as perforated appendicitis in the setting of acute lymphoblastic leukemia.

Abdominal pain is common during chemotherapy for childhood leukemia. Clinically differentiating typhlitis from appendicitis can be difficult. We present an 8-year-old boy with abdominal pain in the setting of acute lymphoblastic leukemia and neutropenia. Following appendectomy for presumed appendicitis, pathology revealed appendiceal typhlitis. Diagnostic and treatment considerations are discussed.

Clinical validation and implementation of a multiplexed immunosuppressant assay in dried blood spots by LC-MS/MS.

BACKGROUND: Therapeutic drug monitoring of immunosuppressive drugs is important in transplant patients. We developed and validated liquid chromatography-mass spectrometry (LC-MS/MS) assay for simultaneous quantitation of tacrolimus (TaC), sirolimus (SrL), and cyclosporin A (CsA) in dried blood spots (DBSs) to offer patients home sample collection, avoiding travel for blood draws. METHODS: After extraction, samples were analyzed by LC-MS/MS in multiple reaction monitoring mode. RESULTS: The assay was linear between 1.2-40 ng/ml for TaC and SrL, and 30-1000 ng/ml for CsA. Inter- and intra-assay CVs were ≤14.8% for all 3 drugs. This method correlated well with the existing clinical whole blood assay, with coefficients of determination >0.95 for all 3 drugs. DBS quality control samples were stable for at least 30 days at -20, 4, and 25°C. Stability of patient DBS samples was at least 5 days at temperatures up to 60°C, except for SrL where degradation was observed at 60°C within 24 h. No effect of hematocrit level, blood spot volume or punch location was observed. CONCLUSION: Immunosuppressant levels measured in DBS correlate with whole blood LC-MS/MS assay and may contribute to successful outcome of organ transplant and patient satisfaction.

Implementation of filmarray respiratory viral panel in a core laboratory improves testing turnaround time and patient care.

The FilmArray respiratory virus panel detects 15 viral agents in respiratory specimens using polymerase chain reaction. We performed FilmArray respiratory viral testing in a core laboratory at a regional children's hospital that provides service 24 hours a day 7 days a week. The average and median turnaround time were 1.6 and 1.4 hours, respectively, in contrast to 7 and 6.5 hours documented 1 year previously at an on-site reference laboratory using a direct fluorescence assay (DFA) that detected 8 viral agents. During the study period, rhinovirus was detected in 20% and coronavirus in 6% of samples using FilmArray; these viruses would not have been detected with DFA. We followed 97 patients with influenza A or influenza B who received care at the emergency department (ED). Overall, 79 patients (81%) were given oseltamivir in a timely manner defined as receiving the drug in the ED, a prescription in the ED, or a prescription within 3 hours of ED discharge. Our results demonstrate that molecular technology can be successfully deployed in a nonspecialty, high-volume, multidisciplinary core laboratory.

Chronic myelogenous leukemia presenting in blast phase with nodal, bilineal myeloid sarcoma and T-lymphoblastic lymphoma in a child.

A 14-year-old boy presented with chronic myelogenous leukemia (CML) in blast phase with segregated extramedullary (nodal) myeloid sarcoma and T-lymphoblastic lymphoma. Immunohistochemical stains performed on the lymphadenectomy sample demonstrated T lymphoblasts in the lymph nodes and myeloblasts in the adjacent soft tissue. Fluorescence in situ hybridization performed on paraffin sections confirmed that both T-lymphoblast and myeloblast populations were positive for the t(9;22) BCR/ABL1 translocation. Subsequent flow cytometry analysis of the bone marrow showed expanded populations of abnormal myeloblasts and T lymphoblasts diagnostic of blast phase CML. To the best of our knowledge, bilineal blast phase of CML with segregated extramedullary T lymphoblasts and myeloblasts has not been reported.

Laboratory and clinical predictors of thrombosis and hemorrhage in 29 pediatric extracorporeal membrane oxygenation nonsurvivors.

Extracorporeal membrane oxygenation (ECMO) is a life-saving therapy for infants and children with cardiac and respiratory failure. However, thrombosis and hemorrhage are common complications. To determine clinical and laboratory predictors of thrombosis and hemorrhage resulting from ECMO, records and slides were reviewed from 29 consecutive autopsies from 2004 through 2008 of pediatric patients who received ECMO at our institution. Laboratory results, including prothrombin time, activated partial thromboplastin time, platelet count, fibrinogen level, and activated clotting time, were analyzed, as was heparin dosing. Thrombosis and hemorrhage were very common, with 1 or both seen in 86% of patients. Sixty-nine percent had thrombosis, and 52% had hemorrhage after ECMO initiation, including intracranial hemorrhage in 33% of the patients in whom brain examination was permitted. Hemorrhage and thrombosis coexisted in 31% of patients. Thrombosis was significantly more common in patients with congenital cardiac disease. Duration of ECMO therapy, being on ECMO at death, sepsis, and patient age and sex did not predict hemorrhage or thrombosis at autopsy. Laboratory tests were poor predictors of thrombosis and hemorrhage, with no correlation between these complications and prothrombin time, partial thromboplastin time, platelet count, fibrinogen level, activated clotting time, or heparin dose. In conclusion, thrombosis and hemorrhage continue to be frequent complications among patients who die during or after ECMO therapy. Patients with congenital cardiac disease appear especially susceptible to thrombosis on ECMO. Prothrombin time, partial thromboplastin time, platelet count, fibrinogen level, activated clotting time, and heparin dose are poor predictors of thrombosis or hemorrhage for pediatric patients who die after ECMO.

Congenital diaphragmatic hernia and microtia in a newborn with mycophenolate mofetil (MMF) exposure: phenocopy for Fryns syndrome or broad spectrum of teratogenic effects?

A newborn female infant born to a woman on immunosuppressive medications including mycophenolate mofetil (MMF) for a renal graft secondary to lupus nephritis presented with congenital diaphragmatic hernia (CDH) and additional findings of microtia, esophageal atresia with tracheoesophageal fistula, cleft palate, congenital heart defect, digital anomalies, and dysmorphic facial features. Pulmonary hypoplasia resulted in death at day 2 of life. She was presumed to have Fryns syndrome based on diagnostic criteria established for this recessive disorder with prominent features including CDH, facial anomalies, and nail hypoplasia. In retrospect, this infant's findings are more likely the result of teratogenic exposure to MMF, as more recent data have emerged linking aural atresia, digital anomalies, and dysmorphic features to this drug. To date, this is the only human report of CDH in an infant with prenatal exposure to MMF, although the manufacturer's package insert alludes to animal studies with a broad spectrum of malformations, including CDH. Thus, a teratogenic exposure can mimic a known Mendelian genetic syndrome, and caution is urged in presuming a genetic etiology for infants with potential teratogenic exposure to relatively new drugs with limited published animal data.

Calretinin immunohistochemistry versus acetylcholinesterase histochemistry in the evaluation of suction rectal biopsies for Hirschsprung Disease.

Diagnosis of Hirschsprung disease (HSCR) relies on histologic and/or histochemical staining of sections from suction rectal biopsies. Acetylcholinesterase histochemistry (AChE) facilitates diagnosis but is not universally employed, in part because it requires special tissue handling. Calretinin immunohistochemistry (IHC) may be a useful alternative, because loss of calretinin immunoreactive nerves reportedly correlates spatially with aganglionosis. We investigated the patterns of calretinin IHC in suction rectal biopsies from HSCR and non-HSCR patients and compared the diagnostic value of calretinin IHC with a widely used rapid AChE method. In suction rectal biopsies that contain ganglion cells, small nerves in the lamina propria, muscularis mucosae, and superficial submucosa contain granular aggregates of calretinin immunoreactivity. Immunolabeling of these nerves is completely absent in the aganglionic biopsies of HSCR patients. Multiple observers independently reviewed calretinin IHC and AChE sections of suction rectal biopsies from 14 HSCR patients and 17 non-HSCR controls. Five observers, blinded to the correct diagnosis, scored each patient's calretinin IHC and AChE slides as HSCR, not HSCR, or equivocal. The frequencies of major and minor discrepant diagnoses were compared. Calretinin IHC yielded no misdiagnoses or major discrepancies between observers. In contrast, 2 misdiagnoses and significantly more interobserver disagreement resulted from the AChE-stained sections. Calretinin IHC appears to be a reasonable, and potentially superior, alternative to AChE as an adjunctive diagnostic method for evaluating suction rectal biopsies for HSCR.

Chimeric maternal cells with tissue-specific antigen expression and morphology are common in infant tissues.

Maternal microchimerism (MMc) has been purported to play a role in the pathogenesis of autoimmunity, but how a small number of foreign cells could contribute to chronic, systemic inflammation has not been explained. Reports of peripheral blood cells differentiating into tissue-specific cell types may shed light on the problem in that chimeric maternal cells could act as target cells within tissues. We investigated MMc in tissues from 7 male infants. Female cells, presumed maternal, were characterized by simultaneous immunohistochemistry and fluorescence in situ hybridization for X- and Y-chromosomes. Maternal cells constituted 0.017% to 1.9% of parenchymal cells and were found in all infants in liver, pancreas, lung, kidney, bladder, skin, and spleen. Maternal cells were differentiated: maternal hepatocytes in liver, renal tubular cells in kidney, and beta-islet cells in pancreas. Maternal cells were not found in areas of tissue injury or inflammatory infiltrate. Maternal hematopoietic cells were found only in hearts from patients with neonatal lupus. Thus, differentiated maternal cells are present in multiple tissue types and occur independently of inflammation or tissue injury. Loss of tolerance to maternal parenchymal cells could lead to organ-specific "auto" inflammatory disease and elimination of maternal cells in areas of inflammation.