Randall Y. Chan

Tumor-associated macrophages promote neuroblastoma via STAT3 phosphorylation and up-regulation of c-MYC

Tumor-associated macrophages (TAMs) are strongly associated with poor survival in neuroblastomas that lack MYCN amplification. To study TAM action in neuroblastomas, we used a novel murine model of spontaneous neuroblastoma lacking MYCN amplification, and observed recruitment and polarization of TAMs, which in turn enhanced neuroblastoma proliferation and growth. In both murine and human neuroblastoma cells, we found that TAMs increased STAT3 activation in neuroblastoma cells and transcriptionally up-regulated the MYC oncogene. Analysis of human neuroblastoma tumor specimens revealed that MYC up-regulation correlates with markers of TAM infiltration. In an IL6ko neuroblastoma model, the absence of IL-6 protein had no effect on tumor development and prevented neither STAT3 activation nor MYC up-regulation. In contrast, inhibition of JAK-STAT activation using AZD1480 or the clinically admissible inhibitor ruxolitinib significantly reduced TAM-mediated growth of neuroblastomas implanted subcutaneously in NOD scid gamma mice. Our results point to a unique mechanism in which TAMs promote tumor cells that lack amplification of an oncogene common to the malignancy by up-regulating transcriptional expression of a distinct oncogene from the same gene family, and underscore the role of IL-6-independent activation of STAT3 in this mechanism. Amplification of MYCN or constitutive up-regulation of MYC protein is observed in approximately half of high-risk tumors; our findings indicate a novel role of TAMs as inducers of MYC expression in neuroblastomas lacking independent oncogene activation.

Hepatoblastoma in a patient with methylmalonic aciduria.

Childhood malignant tumors and their treatment are not well described in the natural history of methylmalonic aciduria (MMA). Here we present a case of hepatoblastoma occurring in the native liver of a 19‐month‐old male with MMA. His tumor was unresectable at diagnosis and he received neoadjuvant chemotherapy with cisplatin, 5‐fluorouracil and vincristine. He developed metabolic acidosis and hyperglycemia during chemotherapy. In addition, he developed anemia, thrombocytopenia and febrile neutropenia. He underwent a combined liver‐kidney transplant for local control of his tumor and to treat MMA. He remains in remission more than five years after his transplant. In addition, his transplant has cured his MMA and he is able to tolerate a regular diet without developing metabolic crises.

Hypersegmented Neutrophils in an Adolescent Male With Heatstroke.

A 14-year-old male presented to the hospital after syncope during football practice on a hot summer day. On examination, temperature was 40.41C (104.71F) and heart rate was 180 beats per minute. He was unresponsive, intubated, and cooled in the pediatric intensive care unit. Laboratory values revealed acute kidney injury and ischemic hepatitis. Complete blood count showed a white blood cell count of 17!10/L, hemoglobin of 14.8 g/L, platelet count of 395!10/L, and mean corpuscular volume of 83.8 fL (normal, 81.4 to 91.9 fL). Peripheral blood smear showed hypersegmented neutrophils with “botryoid” nuclei (Supplemental Fig. 1 Supplemental Digital Content 1, http://links.lww.com/JPHO/A98). Hypersegmented neutrophils are classically seen with folate (vitamin B9) orcobalamin (vitamin B12) deficiency. These morphologic changes of the neutrophil nucleus occur due to impaired DNA synthesis from inadequate substrate or impaired replication from a toxin or medication effect. Arrest of nuclear maturation, impaired cell division, and unbalanced cell growth results in characteristic large cells with immature nuclei with relative cytoplasmic maturity. Red blood cell macrocytosis often accompanies hypersegmented neutrophils and can be seen in hypothyroidism, alcohol abuse, uremia, and myelodysplastic syndromes. Hypersegmented neutrophils without red blood cell macrocytosis, as in our patient, has been described in patients with hyperthermia, uremia, and concurrent megaloblastic and microcytic anemia from combined folate and/ or cobalamin deficiency along with iron deficiency or thalassemia. As the finding of hypersegmented neutrophils preceeds macrocytosis, neutrophil hypersegmentation without macrocytosismay represent early cobalamin and folate deficiency.1 The term “botryoid” refers to nuclei that appear like a cluster of grapes around a stem.2 Botryoid nuclei have been described in patients with hyperthermia due to cocaine and methamphetamine use,3 malignant hyperthermia, neuroleptic malignant syndrome,4 and autoimmune disorders such as rheumatoid arthritis, psoriatic arthritis, and systemic sclerosis.5 In comparison, the multilobed nuclei in cobalamin and folate deficiency appear disorganized. The pathogenesis of botryoid nuclei in hyperthermia has not been clearly elucidated. Hyperthermia may activate the intrinsic signaling pathway that initiates apoptosis since pyknosis, nuclear condensation in the setting of irreversible cell death, is seen in neutrophils in response to hyperthermia.6 In addition, microtubular decomposition may play a role.7 In vitro experiments have shown that radially segmented neutrophils are induced by applying heat.8 Botryoid changes in >50% of neutrophils on a peripheral blood smear may be sufficient to diagnose heatstroke.2 Fewer than 50% botryoid neutrophils is suggestive of heatstroke. Clinicians should be aware that hyperthermia can cause hypersegmented neutrophils so as to avoid unnecessary evaluations for other etiologies. Our patient’s mental status and renal function improved with cooling and intravenous fluids. He was extubated on the first day of hospitalization. Subsequent peripheral blood smears showed resolution of hypersegmented neutrophils by the second hospital day. He was discharged home after 3 days without any complications.

Hyperviscosity syndrome in splenic marginal zone lymphoma

![Figure][1] A 57-year-old man presented with 2 months of weakness, fatigue, gingival bleeding, and cachexia. Examination revealed right mydriasis, bilateral retinal hemorrhages, diffuse lung crackles, a tense abdomen, and normal lymph nodes. Increased blood viscosity prevented laboratory

Hyperglycemic Hyperosmolar State During Induction Chemotherapy for Acute Lymphoblastic Leukemia

Abstract We present the case of a 16-year-old boy who presented with fatigue, polyuria, and polydipsia while on chemotherapy for his relapsed acute lymphoblastic leukemia (ALL). Blood gas examination confirmed the diagnosis of hyperosmolar hyperglycemic state. The etiology for his hyperglycemia was most likely a result of oral glucocorticoid therapy combined with asparaginase therapy—both are a cornerstone of induction chemotherapy for ALL. The patient was aggressively rehydrated with saline, and medications were administered to correct his hyperkalemia. He was then slowly brought to euglycemia with a continuous infusion of insulin. Although hyperosmolar hyperglycemic state is rare during the treatment of ALL, frontline providers should be aware of this diagnosis because of the significant risk of hypovolemic shock and death if correction of hyperglycemia occurs prior to complete fluid resuscitation.

Delayed‐onset Thrombocytopenia in a Pediatric Burn Patient

A 14-year-old boy presented with 36% total body surface area (TBSA) flame burns to his face, neck, and torso. On presentation to the hospital, he was endotracheally intubated for possible inhalation injury. Fluid-responsive shock was managed with Lactated Ringer’s solution and albumin. Pain and agitation were managed with opiates and benzodiazepines. Aztreonam and clindamycin were started due to concern of septic shock in the setting of a history of penicillin allergy. Thermal burns are a significant cause of injury. Prehospital care involves cooling the burn with cool (but not cold) running water and covering the burn with nonadherent dressings (cling wrap) to minimize heat loss. At the hospital, initial resuscitation includes fluid resuscitation using the Parkland formula, airway protection for patients with concern for inhalation injury, pain control, and transfer to an intensive care unit. He underwent multiple escharotomies, debridements, and skin grafts during the first 2 weeks of hospitalization. His burns were dressed with topical silver sulfadiazine prior to skin debridement on day #11 (Fig. 1). He was started on vancomycin when he was found to have clindamycin-resistant Streptococcus pneumoniae on respiratory culture. During his hospitalization, he developed thrombocytopenia at day #3 with a platelet nadir of 137 3 10/L at day #3 followed by normalization of the platelet count by day #6. The most common causes of thrombocytopenia in a pediatric patient with severe burn injury include burn-induced thrombocytopenia, sepsis, disseminated intravascular coagulation (DIC), drug-induced thrombocytopenia, heparin-induced thrombocytopenia (HIT), and post-transfusion purpura (PTP) (Supporting Information Table I). Burn-induced thrombocytopenia is the most common immediately after injury. Our patient’s thrombocytopenia followed by normalization within a week is consistent with the presentation of burn-induced thrombocytopenia. DIC and sepsis were unlikely since our patient was well-appearing and clinically improving at the onset of thrombocytopenia. On day #13, he underwent split-thickness skin grafting of the torso. On the same day, his platelet count again started to decline despite improvement in his wounds and resolution of pneumonia. By day #19, he had developed severe thrombocytopenia with a platelet count of <5 3 10/L. Microscopic examination of his blood smear revealed absence of platelets and normal red and white blood cell morphology. Severe thrombocytopenia, defined as platelet count <50 3 10/L, is unusual in a patient with burn-induced thrombocytopenia or sepsis. Therefore, other etiologies such as PTP, drug-induced thrombocytopenia, thrombocytopenia due to viral infection, HIT, and ITP were considered. PTP occurs 7–10 days after blood product transfusion. However, our patient did not receive his first transfusion until day 20. Drug-induced thrombocytopenia is typically associated with recovery of the platelet count 1 week after discontinuation of the offending agent. We considered several medications such as vancomycin and sulfa drugs (specifically, silver sulfadiazine); none of the medications were temporally related to the thrombocytopenia as illustrated in Fig. 1. HIT was unlikely since thrombocytopenia typically develops 5–10 days after exposure to heparin in patient who has not been previously exposed to heparin and recovers by 1 week after discontinuation of heparin. Our patient was not receiving heparin at the time of onset of thrombocytopenia. In addition, platelet counts in HIT rarely drop below 20 3 10/L. Therefore, ITP was our primary concern. Platelet transfusion was given on day #21 since thrombocytopenia. A 1-hr post-transfusion platelet count showed no change in platelet count. Intravenous immunoglobulin (IVIG) (0.8 g kg) was given for suspected ITP and heparin was discontinued. Antibody analysis was negative for HIT. Platelet transfusions are usually avoided in ITP unless a patient develops severe bleeding. However, our patient was given platelets since his severe thrombocytopenia was preventing routine wound care including dressing changes and debridement. In a patient with unexplained thrombocytopenia, platelet count should be assessed 1 hr after transfusion, since a platelet count refractory to transfusion may suggest immune-mediated platelet destruction. Refractoriness to platelet transfusion raised concern for drug-induced thrombocytopenia or allo-immunization from exposure to RBC antigens from previous transfusions. However, the time course made drug-induced thrombocytopenia unlikely and no allo-antibodies were identified by our blood bank.

Abstract 400: MEK inhibition enhances immune checkpoint blockade treatment of murine models of neuroblastoma

Background: The tumor microenvironment (TME) plays an important role in neuroblastoma (NBL) biology. Infiltrations of tumor-associated macrophages with alterations in patterns of pro-inflammatory genes are associated with poor prognosis in NBL. However, the role of regulatory T-cells in NBL remains unknown. As several genes in MAPK pathway are among recurrent mutations in neuroblastomas, and the production of pro-inflammatory mediators in immune cells is MAPK-dependent, we hypothesized targeted kinase inhibitor in combination with check-point blockade could exert synergistic effects on the TME9s response to tumor cells. Therefore, we assessed the efficacy of combined therapy using a transgenic MYCN non-amplified neuroblastoma murine model driven by SV40 large T antigen (NB-Tag). Methods: Three human neuroblastoma cell lines and two NB-Tag derived mouse cell-lines were used for in vitro cell proliferation assay. For in vivo tumor growth models, combinations of cyclophosphamide, topotecan, trametinib, anti-CTLA4, and anti-PD1 therapies were studied in NB-Tag transgenic and transplantable subcutaneous (NB-SQ) murine models. Results: In vitro studies demonstrated trametinib had the highest anti-proliferative activity compared to other kinase inhibitors, and it effectively blocked cell cycle arrest in G1 phase. These anti-proliferative effects could not be rescued by co-culturing tumor cells with murine or human macrophages. In NB-Tag mice, which develop neuroblastoma spontaneously at 12 weeks, daily oral administration of trametinib (0.6mg/Kg) at pre-tumoral age (10 wk) significantly impaired tumor growth by 17 weeks (1424 mm3 in controls vs. 43 mm3 in treated mice). Treatment of 15 week-old NB-Tag mice (visible tumor by MRI) with trametinib after chemotherapy administration (5-days of Cyclophosphamide + Topotecan) also significantly impaired tumor regrowth (volume four weeks post-chemo, 491 vs. 42 mm3, p = 0.037), and more importantly, treatment increased the survival of NB-Tag mice compared to control (median survival: control = 24.5 wk, treated = 35 wk, p Conclusions: Our results provide strong evidence that MEK inhibition combined with checkpoint blockade significantly inhibited tumor formation in a syngeneic subcutaneous model. These findings indicate opportunities to enhance antitumor immunity with the potential to produce durable clinical responses in children with neuroblastomas. Citation Format: Sakunthala Muthugounder, Long Hung, Randall Chan, Jin Kim, Soheila Shirinbak, Hiroyuki Shimada, Shahab Asgharzadeh. MEK inhibition enhances immune checkpoint blockade treatment of murine models of neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 400. doi:10.1158/1538-7445.AM2015-400