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Purpose: To investigate the molecular mechanism underlying intrinsic resistance to ABT-199.Experimental Design: Western blots and real-time RT-PCR were used to determine levels of Mcl-1 after ABT-199 treatment alone or in combination with cytarabine or daunorubicin. Immunoprecipitation of Bim and Mcl-1 were used to determine the effect of ABT-199 treatment on their interactions with Bcl-2 family members. Lentiviral short hairpin RNA knockdown of Bim and CRISPR knockdown of Mcl-1 were used to confirm their role in resistance to ABT-199. JC-1 assays and flow cytometry were used to determine drug-induced apoptosis.Results: Immunoprecipitation of Bim from ABT-199–treated cell lines and a primary patient sample demonstrated decreased association with Bcl-2, but increased association with Mcl-1 without corresponding change in mitochondrial outer membrane potential. ABT-199 treatment resulted in increased levels of Mcl-1 protein, unchanged or decreased Mcl-1 transcript levels, and increased Mcl-1 protein half-life, suggesting that the association with Bim plays a role in stabilizing Mcl-1 protein. Combining conventional chemotherapeutic agent cytarabine or daunorubicin with ABT-199 resulted in increased DNA damage along with decreased Mcl-1 protein levels, compared with ABT-199 alone, and synergistic induction of cell death in both AML cell lines and primary patient samples obtained from AML patients at diagnosis.Conclusions: Our results demonstrate that sequestration of Bim by Mcl-1 is a mechanism of intrinsic ABT-199 resistance and supports the clinical development of ABT-199 in combination with cytarabine or daunorubicin for the treatment of AML. Clin Cancer Res; 22(17); 4440–51. ©2016 AACR.

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PURPOSE Infant and young childhood medulloblastoma (iMB) is usually treated without craniospinal irradiation (CSI) to avoid neurocognitive late effects. Unfortunately, many children relapse. The purpose of this study was to assess salvage strategies and prognostic features of patients with iMB who relapse after CSI-sparing therapy. METHODS We assembled a large international cohort of 380 patients with relapsed iMB, age younger than 6 years, and initially treated without CSI. Univariable and multivariable Cox models of postrelapse survival (PRS) were conducted for those treated with curative intent using propensity score analyses to account for confounding factors. RESULTS The 3-year PRS, for 294 patients treated with curative intent, was 52.4% (95% CI, 46.4 to 58.3) with a median time to relapse from diagnosis of 11 months. Molecular subgrouping was available for 150 patients treated with curative intent, and 3-year PRS for sonic hedgehog (SHH), group 4, and group 3 were 60%, 84%, and 18% ( P = .0187), respectively. In multivariable analysis, localized relapse ( P = .0073), SHH molecular subgroup ( P = .0103), CSI use after relapse ( P = .0161), and age ≥ 36 months at initial diagnosis ( P = .0494) were associated with improved survival. Most patients (73%) received salvage CSI, and although salvage chemotherapy was not significant in multivariable analysis, its use might be beneficial for a subset of children receiving salvage CSI < 35 Gy ( P = .007). CONCLUSION A substantial proportion of patients with relapsed iMB are salvaged after initial CSI-sparing approaches. Patients with SHH subgroup, localized relapse, older age at initial diagnosis, and those receiving salvage CSI show improved PRS. Future prospective studies should investigate optimal CSI doses and the role of salvage chemotherapy in this population.

Fortunately > 80% of children diagnosed with cancer become long-term survivors; however, this population is at a significantly increased risk of morbidity and mortality as a result of their previous cancer therapy, and long-term follow-up (LTFU) is critical. Multiple barriers to receiving adequate LTFU care have been studied. We investigated whether lack of enrollment in a therapeutic clinical trial may be a barrier to receiving LTFU care. We conducted a review of 353 patient records at the Children’s Hospital of Michigan enrolled in our Children’s Oncology Group registry between January 1, 2005 and December 31, 2010. In total, 71 patients were excluded (death before follow-up, n= 61; currently receiving therapy, n= 5; known transfer of care, n =4; insufficient information, n =1). In total, 158 (56%) patients were enrolled in a therapeutic clinical trial. Follow-up rates at 1-, 2- and 5-years following completion of therapy for patients enrolled in a therapeutic clinical trial were 96.8% (153/158), 93.7% (148/158), and 81.7% (103/126), respectively, compared with 83.1% (103/124; P

Diffuse intrinsic pontine glioma (DIPG) remains a devastating disease. Panobinostat has been shown to have therapeutic efficacy both in vitro and in DIPG orthotopic xenograft models; however, clinical data in patients with DIPG are lacking. We present 2 cases of DIPG, who were treated with panobinostat at 22 to 25 mg/m/dose, 3 times weekly for 2 weeks in 3-week cycles and concomitant reirradiation after disease progression. Two episodes of asymptomatic thrombocytopenia were observed in 1 patient. Hyperacetylation of histone H4 of peripheral blood mononuclear cells was evident following treatment. In our experience, panobinostat administered with reirradiation was well tolerated at a relatively higher dose than that used in adult studies.

We present a case of inadvertent high-dose therapy with temozolomide in a child with recurrent diffuse intrinsic pontine glioma followed by a rapid clinical response. The patient was a 7-year-old boy who initially presented with a history of left facial palsy, double vision, headache, and ataxia. His symptoms were completely resolved following radiotherapy but recurred 3 months after. Following recurrence, he received temozolomide in a dose >3 times higher than prescribed inadvertently but tolerated well with a rapid clinical response. He eventually deteriorated after he was substituted with a lower dose of temozolomide and died.

Purpose: To investigate the molecular mechanism underlying intrinsic resistance to ABT-199. Experimental Design: Western blots and real-time RT-PCR were used to determine levels of Mcl-1 after ABT-199 treatment alone or in combination with cytarabine or daunorubicin. Immunoprecipitation of Bim and Mcl-1 were used to determine the effect of ABT-199 treatment on their interactions with Bcl-2 family members. Lentiviral short hairpin RNA knockdown of Bim and CRISPR knockdown of Mcl-1 were used to confirm their role in resistance to ABT-199. JC-1 assays and flow cytometry were used to determine drug-induced apoptosis. Results: Immunoprecipitation of Bim from ABT-199–treated cell lines and a primary patient sample demonstrated decreased association with Bcl-2, but increased association with Mcl-1 without corresponding change in mitochondrial outer membrane potential. ABT-199 treatment resulted in increased levels of Mcl-1 protein, unchanged or decreased Mcl-1 transcript levels, and increased Mcl-1 protein half-life, suggesting that the association with Bim plays a role in stabilizing Mcl-1 protein. Combining conventional chemotherapeutic agent cytarabine or daunorubicin with ABT-199 resulted in increased DNA damage along with decreased Mcl-1 protein levels, compared with ABT-199 alone, and synergistic induction of cell death in both AML cell lines and primary patient samples obtained from AML patients at diagnosis. Conclusions: Our results demonstrate that sequestration of Bim by Mcl-1 is a mechanism of intrinsic ABT-199 resistance and supports the clinical development of ABT-199 in combination with cytarabine or daunorubicin for the treatment of AML. Clin Cancer Res; 22(17); 4440–51. ©2016 AACR.

Tumor biopsy is rarely performed in diffuse intrinsic pontine glioma (DIPG) due to the presumed risk of surgical complications, although data on the surgery related morbidity of DIPG biopsy is sparse. We performed a retrospective review on 22 consecutive cases of DIPG diagnosed from 2002 to 2012 at Children's Hospital of Michigan, 15 of which underwent biopsy. Transient new or worsening neurological deficits were observed in three of 15 cases following surgery. No surgery related mortality or permanent deficit was observed, and the mean overall survival was 10.4 ± 3.8 months. Undergoing biopsy did not adversely affect the outcome. Pediatr Blood Cancer 2015;62:163–165. © 2014 Wiley Periodicals, Inc.

Background. Cancer stem cell theory suggests that the presence of tumor initiating stem-like cells in cancers may be responsible for cancer progression and relapse. CD133 cell surface maker expression has been used to identify stem-like cells in cancer cell lines. Our goal was to identify such cells in neuroblastoma cell lines and to study the cytotoxicity of common anticancer drugs for those cells. Materials and Methods. CD133þ cells from SK-N-SH and SK-N-BE cell lines were isolated using magnetic microbeads. Cytotoxicity of four anticancer drugs was studied on CD133þ and CD133� populations. The percentage of live, apoptotic, and dead cells in each population after drug treatment was estimated by MTT and PI/ Annexin-binding assays. Western blot analyses were used to identify differences in the expression of kinases. Results. Eight to 10% of SKN-SH and 3–5% of SK-N-BE cells were CD133þ. These cells were more resistant than CD133� cells to all four chemotherapeutic agents tested in the MTT assay. Decreased apoptosis was observed in CD133þ cells compared to CD133� cells by PI/Annexin V-binding assay. Western blot analysis showed that CD133þ cells expressed less MKP-1. Phosphorylated forms of both ERK and P-38 kinases were expressed at higher levels in CD133þ cells than in CD133� cells. Conclusions. This study suggests that CD133þ cells are more resistant to anticancer drugs than CD133� cells. Differences in the expression and phosphorylation of kinases could be partially responsible for this difference. Targeting CD133-expressing cells could be a strategy to develop more effective treatments for neuroblastoma. Pediatr Blood Cancer 2010;54:361–368. 2009 Wiley-Liss, Inc.

Tumor biopsy is rarely performed in diffuse intrinsic pontine glioma (DIPG) due to the presumed risk of surgical complications, although data on the surgery related morbidity of DIPG biopsy is sparse. We performed a retrospective review on 22 consecutive cases of DIPG diagnosed from 2002 to 2012 at Children's Hospital of Michigan, 15 of which underwent biopsy. Transient new or worsening neurological deficits were observed in three of 15 cases following surgery. No surgery related mortality or permanent deficit was observed, and the mean overall survival was 10.4 ± 3.8 months. Undergoing biopsy did not adversely affect the outcome.

Type 2B VWD is caused by gain-of-function mutations in exon28 of the VWF gene encoding the binding site for platelet GPIb,leading to increased affinity of VWF to platelet GPIb withsubsequent increased clearance of HMWM in plasma [1–3]. Themultimeric structure of VWF allows it to function as a molecularbridge between the subendothelial matrix and the platelet GPIbreceptor complex [3]. One of the characteristic findings in type 2BVWD is the relative loss of VWF HMWM in plasma, which isbelievedtobeduetoincreasedclearanceofVWF–plateletcomplex.Here we describe a patient with type 2B VWD with characteristicloss of VWF HMWM and a normal platelet count initially.Interestingly, his HMWM appeared when he developed ITP, anddisappeared again when his platelet count increased. Possiblecauses leading to the restoration of his HMWM are discussed.

We review a unique case of NUT midline carcinoma that presented in a young girl with an initial diagnosis of tonsillar abscess. We stress the importance of assaying poorly differentiated carcinomas in young patients for the t(15;19) translocation. Our patient presented with tonsillar enlargement and cervical lymphadenopathy mimicking acute tonsillitis. The clinical suspicion for malignancy arose after an aspirate from the tonsil did not yield any pus, and biopsy of a cervical lymph node demonstrated undifferentiated carcinoma. Further analysis by fluorescence in situ hybridization was positive for rearrangements in both BRD4 and NUT genes consistent with NUT carcinoma. In addition, fluorodeoxyglucose—positron emission tomography (FDG-PET) revealed a very high standard uptake value in both the primary tumor and metastatic foci, suggesting that FDG-PET could be a useful tool in the staging and follow-up of NUT midline carcinoma.

Cancer stem cell theory suggests that the presence of tumor initiating stem-like cells in cancers may be responsible for cancer progression and relapse. CD133 cell surface maker expression has been used to identify stem-like cells in cancer cell lines. Our goal was to identify such cells in neuroblastoma cell lines and to study the cytotoxicity of common anticancer drugs for those cells.CD133+ cells from SK-N-SH and SK-N-BE cell lines were isolated using magnetic microbeads. Cytotoxicity of four anticancer drugs was studied on CD133+ and CD133- populations. The percentage of live, apoptotic, and dead cells in each population after drug treatment was estimated by MTT and PI/Annexin-binding assays. Western blot analyses were used to identify differences in the expression of kinases.Eight to 10% of SK-N-SH and 3-5% of SK-N-BE cells were CD133+. These cells were more resistant than CD133- cells to all four chemotherapeutic agents tested in the MTT assay. Decreased apoptosis was observed in CD133+ cells compared to CD133- cells by PI/Annexin V-binding assay. Western blot analysis showed that CD133+ cells expressed less MKP-1. Phosphorylated forms of both ERK and P-38 kinases were expressed at higher levels in CD133+ cells than in CD133- cells.This study suggests that CD133+ cells are more resistant to anticancer drugs than CD133- cells. Differences in the expression and phosphorylation of kinases could be partially responsible for this difference. Targeting CD133-expressing cells could be a strategy to develop more effective treatments for neuroblastoma.