Hu14.18K.322A Causes Direct Cell Cytotoxicity and Synergizes with Induction Chemotherapy in High-Risk Neuroblastoma.

The disialoganglioside, GD2, is a promising therapeutic target due to its overexpression in certain tumors, particularly neuroblastoma (NB), with limited expression in normal tissues. Despite progress, the intricate mechanisms of action and the full spectrum of the direct cellular responses to anti-GD2 antibodies remain incompletely understood. In this study, we examined the direct cytotoxic effects of the humanized anti-GD2 antibody hu14.18K322A (hu14) on NB cell lines, by exploring the associated cell-death pathways. Additionally, we assessed the synergy between hu14 and conventional induction chemotherapy drugs. Our results revealed that hu14 treatment induced direct cytotoxic effects in CHLA15 and SK-N-BE1 cell lines, with a pronounced impact on proliferation and colony formation. Apoptosis emerged as the predominant cell-death pathway triggered by hu14. Furthermore, we saw a reduction in GD2 surface expression in response to hu14 treatment. Hu14 demonstrated synergy with induction chemotherapy drugs with alterations in GD2 expression. Our comprehensive investigation provides valuable insights into the multifaceted effects of hu14 on NB cells, shedding light on its direct cytotoxicity, cell-death pathways, and interactions with induction chemotherapy drugs. This study contributes to the evolving understanding of anti-GD2 antibody therapy and its potential synergies with conventional treatments in the context of NB.

Second Generation Small Molecule Inhibitors of Gankyrin for the Treatment of Pediatric Liver Cancer.

Background: Gankyrin, a member of the 26S proteasome, is an overexpressed oncoprotein in hepatoblastoma (HBL) and hepatocellular carcinoma (HCC). Cjoc42 was the first small molecule inhibitor of Gankyrin developed; however, the IC50 values of >50 µM made them unattractive for clinical use. Second-generation inhibitors demonstrate a stronger affinity toward Gankyrin and increased cytotoxicity. The aim of this study was to characterize the in vitro effects of three cjoc42 derivatives. Methods: Experiments were performed on the HepG2 (HBL) and Hep3B (pediatric HCC) cell lines. We evaluated the expression of TSPs, cell cycle markers, and stem cell markers by Western blotting and/or real-time quantitative reverse transcription PCR. We also performed apoptotic, synergy, and methylation assays. Results: The treatment with cjoc42 derivatives led to an increase in TSPs and a dose-dependent decrease in the stem cell phenotype in both cell lines. An increase in apoptosis was only seen with AFM-1 and -2 in Hep3B cells. Drug synergy was seen with doxorubicin, and antagonism was seen with cisplatin. In the presence of cjoc42 derivatives, the 20S subunit of the 26S proteasome was more available to transport doxorubicin to the nucleus, leading to synergy. Conclusion: Small-molecule inhibitors for Gankyrin are a promising therapeutic strategy, especially in combination with doxorubicin.

Small Molecule Cjoc42 Improves Chemo-Sensitivity and Increases Levels of Tumor Suppressor Proteins in Hepatoblastoma Cells and in Mice by Inhibiting Oncogene Gankyrin.

Objective: Relapsed hepatoblastoma (HBL) and upfront hepatocellular carcinoma (HCC) are notoriously chemoresistant tumors associated with poor outcomes. Gankyrin (Gank) is a known oncogene that is overexpressed in pediatric liver cancer and implicated in chemo-resistance. The goal of this study was to evaluate if the Gank-tumor suppressor axis is activated in chemoresistant hepatoblastoma patients and examine if an inhibitor of Gank, Cjoc42, might improve the chemosensitivity of cancer cells. Methods: Expression of Gank and its downstream targets were examined in fresh human HBL samples using immunostaining, QRT-PCR, and Western Blot. Cancer cells, Huh6 (human HBL) and Hepa1c1c7 (mouse HCC) were treated with Cjoc42 and with Cjoc42 in combination with cisplatin or doxorubicin. Cell proliferation, apoptosis, and chemoresistance were examined. To examine activities of Cjoc42 in vivo, mice were treated with different doses of Cjoc42, and biological activities of Gank and cytotoxicity of Cjoc42 were tested. Results: Elevation of Gank and Gank-mediated elimination of TSPs are observed in patients with minimal necrosis after chemotherapy and relapsed disease. The treatment of Huh6 and Hepa1c1c7 with Cjoc42 was not cytotoxic; however, in combination with cisplatin or doxorubicin, Cjoc42 caused a significant increase in cytotoxicity compared to chemotherapy alone with increased apoptosis. Examination of Cjoc42 in WT mice showed that Cjoc42 is well tolerated without systemic toxicity, and levels of tumor suppressors CUGBP1, Rb, p53, C/EBPα, and HNF4α are increased by blocking their Gank-dependent degradation. Conclusions: Our work shows that Cjoc42 might be a promising adjunct to chemotherapy for the treatment of severe pediatric liver cancer and presents mechanisms by which Cjoc42 increases chemo-sensitivity.

Clinical heterogeneity of pediatric hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is often a chemoresistant neoplasm with a poor prognosis. Pediatric HCC may reflect unique biological and clinical heterogeneity. PROCEDURE: An IRB-approved retrospective institutional review of patients with HCC treated between 2004 and 2015 was undertaken. Clinical, radiographic, and histologic data were collected from all patients. RESULTS: Thirty-two patients with HCC, median age 11.5 years (range 1-20) were identified. Seventeen patients had a genetic or anatomic predisposition. Histology was conventional HCC (25) and fibrolamellar HCC (7). Evans staging was 1 (12); 2 (1); 3 (10); 4 (9). Sixteen patients underwent resection at diagnosis and five patients after neoadjuvant chemotherapy. Surgical procedures included liver transplantation (LT, 11), hemihepatectomy (9), and segmentectomy (1). Eighteen patients had medical therapy (13 neoadjuvant, 5 adjuvant). Most common initial medical therapy included sorafenib alone (7) and cisplatin/doxorubicin-based therapy (8). Overall, 14 (43.8%) patients survived with a median follow-up of 58.8 months (range 26.5-157.6). Cause of death was most often linked to lack of primary tumor surgery (11). Of the survivors, Evans stage was 1 (11), 2 (1), and 3 (2, both treated with LT). Four of 18 patients (22%) who received medical therapy, 8 of 17 patients with a predisposition (47%), and 14 of 21 patients (66%) who underwent surgery remain alive. CONCLUSIONS: Genetic and anatomic predisposing conditions were seen in over half of this cohort. Evans stage 1 or 2 disease was linked to improved survival. LT trended toward improved survival. Use of known chemotherapy agents may benefit a smaller group of pediatric HCC and warrants formal prospective study through cooperative group trials.

A General Pediatrician's Approach to Anemia in Childhood.

Anemia may be defined as a reduction in red blood cell mass or blood hemoglobin concentration. Physiologically, this represents a hemoglobin level that is too low to meet cellular oxygen demands. Practically, the lower limit of normal is set at 2 standard deviations below the mean based on age, gender, and ethnicity/race. Anemia can lead to impaired growth, development, and poor neurocognitive outcome. As such, it is essential for pediatricians to recognize and conduct appropriate testing for a child with anemia. [Pediatr Ann. 2020;49(1):e10-e16.].

Internalizing symptoms in AYA survivors of childhood cancer and matched comparisons.

OBJECTIVE: As the number of pediatric cancer survivors increases, so does our need to understand behavioral late effects. Prior studies show mixed results, with some noting increased emotional distress and psychiatric diagnoses in cancer survivors and others suggesting resilience. The purpose of our study was to evaluate internalizing symptoms such as anxiety and depression in young adult survivors of childhood cancer compared with matched classroom matched peers. METHOD: We completed a multisource, cross-sectional examination of internalizing symptoms using a semistructured psychiatric interview with 18-year-olds with a history of pediatric cancer compared with age, race, and gender matched classroom peers who had been identified during the survivor's first year of treatment and their primary caregivers. RESULTS: Fifty-seven young adult survivors of childhood cancer and 60 comparison peers participated. There were no significant differences between survivors and their peers on the basis of self- or parent-reported depressive or anxiety symptoms or number of psychiatric diagnoses. CONCLUSIONS: Young adult survivors of childhood cancer and their parents did not report increased rates of anxiety or depression compared with their former classroom peers. Despite experiencing a major life challenge, this group of young adults with cancer did not report more current or past symptoms of internalizing psychopathology.

Gankyrin Promotes Tumor-Suppressor Protein Degradation to Drive Hepatocyte Proliferation.

Background & Aims: Uncontrolled liver proliferation is a key characteristic of liver cancer; however, the mechanisms by which this occurs are not well understood. Elucidation of these mechanisms is necessary for the development of better therapy. The oncogene Gankyrin (Gank) is overexpressed in both hepatocellular carcinoma and hepatoblastoma. The aim of this work was to determine the role of Gank in liver proliferation and elucidate the mechanism by which Gank promotes liver proliferation. Methods: We generated Gank liver-specific knock-out (GLKO) mice and examined liver biology and proliferation after surgical resection and liver injury. Results: Global profiling of gene expression in GLKO mice showed significant changes in pathways involved in liver cancer and proliferation. Investigations of liver proliferation after partial hepatectomy and CCl4 treatment showed that GLKO mice have dramatically inhibited proliferation of hepatocytes at early stages after surgery and injury. In control LoxP mice, liver proliferation was characterized by Gank-mediated reduction of tumor-suppressor proteins (TSPs). The failure of GLKO hepatocytes to proliferate is associated with a lack of down-regulation of these proteins. Surprisingly, we found that hepatic progenitor cells of GLKO mice start proliferation at later stages and restore the original size of the liver at 14 days after partial hepatectomy. To examine the proliferative activities of Gank in cancer cells, we used a small molecule, cjoc42, to inhibit interactions of Gank with the 26S proteasome. These studies showed that Gank triggers degradation of TSPs and that cjoc42-mediated inhibition of Gank increases levels of TSPs and inhibits proliferation of cancer cells. Conclusions: These studies show that Gank promotes hepatocyte proliferation by elimination of TSPs. This work provides background for the development of Gank-mediated therapy for the treatment of liver cancer. RNA sequencing data can be accessed in the NCBI Gene Expression Omnibus: GSE104395.

Surgical management of children and adolescents with upfront completely resected hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive malignant neoplasm that is often chemoresistant. Complete surgical resection remains the mainstay of therapy. The role of liver transplantation (LT) in pediatric HCC is in evolution, as is the role of adjuvant chemotherapy for stage I disease. METHODS: A retrospective review of patients < 18 years of age with completely resected HCC treated with surgical intervention alone at our institution from 2004 to 2015 was conducted. RESULTS: Twelve patients with a median age of 12 years (range = 1-17; number of females = 7) with upfront resected HCC (Evans stage I) were identified. Four patients had HCC without identifiable risk factors (fibrolamellar-HCC = 2; early HCC arising in focal nodular hyperplasia = 1, well-differentiated [wd] HCC = 1). Four patients had early or wd-HCC in the context of portosystemic shunts (Abernethy = 2; mesocaval shunt and portal vein thrombosis = 2). Four patients had moderate to wd-HCC in the context of pre-existing liver disease with cirrhosis (progressive familial intrahepatic cholestasis type-2 = 2, alpha-1 antitrypsin deficiency = 1, Alagille syndrome = 1). Seven patients underwent LT (multifocal = 5; solitary = 2); five exceeded Milan criteria (MC) by imaging. Five patients underwent complete resection (segmentectomy = 2; hemihepatectomy = 3). Ten patients received no adjuvant chemotherapy. All patients are alive without evidence of disease with a median follow-up of 54.1 months (range = 28.1-157.7 months). CONCLUSIONS: Pediatric and adolescent patients with upfront, completely resected HCC can be effectively treated without chemotherapy. LT should be considered for nonmetastatic HCC, especially in the context of pre-existing chronic liver disease, even when the tumor exceeds MC. Distinct pediatric selection criteria are needed to identify patients most suitable for LT.

Do amount of variant differentiation and mitotic rate in bladder cancer change with neoadjuvant chemotherapy?

Neoadjuvant chemotherapy (NAC) is currently recommended to all candidate patients with muscularis propria-invasive bladder cancer. However, NAC is effective in only a subset of patients, and predictors of response are lacking. Our study aimed to characterize tumoral changes with NAC usage and to identify features at bladder biopsy/transurethral resection (Bx/TUR) that may predict response. A retrospective search was performed to identify patients with bladder cancer that were pT2 at Bx/TUR upon whom a radical cystectomy (RC) was performed from 2007 to 2010. A blinded slide review of the Bx/TUR and RC was conducted. Presence, type, percent of tumor variant morphology, and tumoral mitotic rate were assessed. Ninety RC patients with slides available were identified (46 NAC, 44 non-NAC). In NAC-treated patients, there was a significantly higher percentage of nonurothelial variant differentiation in the RC compared with Bx/TUR, whereas there was no difference in the non-NAC subgroup. Percent variant differentiation at Bx/TUR was not a predictor of response. There was a significant decrease in mitotic rate between Bx/TUR and RC in NAC patients, whereas there was no difference in the non-NAC subgroup, although mitotic rate was not a predictor of response. In conclusion, percent variant differentiation and mitotic rate changed significantly from Bx/TUR to RC with NAC usage, although neither predicted response. Pathologists should be aware that variant differentiation is common in bladder cancer, with increased presence after NAC, in order to improve recognition and documentation of these findings.

Clinicopathologic characteristics and overall survival in patients with bladder cancer involving the gastrointestinal tract.

Involvement of the gastrointestinal (GI) tract by bladder cancer is rare and documented in only a few case reports with no prognostic information available. The aim of this study was to clinicopathologically characterize patients with pathologically proven bladder cancer in the GI tract. We reviewed pathology reports from cystectomy patients at our institution from 2006 to 2011, identifying those with GI involvement at or after cystectomy. Overall survival (OS) was analyzed using Kaplan-Meier curves and Cox proportional hazard regression models. Twelve patients had surgical pathology specimens with GI involvement (anus, rectum, colon, and small bowel) at (n = 11) or within 4 months (n = 1) of cystectomy. These patients were noted to be pathologically staged inconsistently. GI involvement was a negative predictor of survival, with a 1.5-year OS of 25 versus 62 % without GI involvement (P < 0.001), similar to our pT4 patients (OS 26 %). In node-negative patients, there was a significantly worse 1.5-year OS with GI involvement compared to those without tumor in the GI tract (P = 0.005). We provide the first case series of patients with bladder cancer in the GI tract. GI involvement is a strong negative predictor of survival and behaves comparable to pT4 patients. However, we recommend that pathologists adhere to the current pT staging guidelines, in which GI involvement is not a criterion, until further research is conducted illustrating if and how it should be incorporated.

Retrospective analysis of survival in muscle-invasive bladder cancer: impact of pT classification, node status, lymphovascular invasion, and neoadjuvant chemotherapy.

A range of studies have analyzed prognostic factors in bladder cancer. However, prior cohorts have included heterogeneous pT classification at biopsy and others were derived from large, multi-institutional clinical trials over the span of many years. Our objective was to analyze prognostic factors in a recent radical cystectomy (RC) cohort at a single institution and evaluate outcomes based on current practice patterns. A retrospective analysis of overall survival (OS) was conducted on 180 RC patients with biopsy proven pT2 disease between 2007-2010. Increasing pT classification was a negative predictor of survival. pT was grouped into three categories with pT0/a/is/1/2a surviving longer than pT2b/3a/3b, and pT4 having the worst prognosis. Subclassifying pT2 and pT3 showed no statistically significant difference in survival. Lymphovascular invasion (LVI) and node positivity correlated with decreased OS. Patients treated with neoadjuvant chemotherapy (NAC) had a higher incidence of pT0, yet pN1+ was more common and NAC was not associated with improved OS. This investigation provides reference OS values for patients with pathologically diagnosed muscle-invasive bladder cancer based on current medical guidelines outside the context of a clinical trial. pT4 was the strongest negative predictor of survival, followed by pN1+, the group pT2b/3a/3b, and presence of LVI. NAC patients were noted to have a higher frequency of low pT classification, yet more frequent node positivity, suggesting that pT classification in NAC patients may not accurately reflect remaining tumor burden.