Non-Thermal Plasma Application in Tumor-Bearing Mice Induces Increase of Serum HMGB1.

The application of cold atmospheric plasma (CAP) in cancer therapy could be one of the new anticancer strategies. In the current work, we used cold atmospheric plasma jet for the treatment of cultured cells and mice. We showed that CAP induced the death of MX-7 mouse rhabdomyosarcoma cells with the hallmarks of immunogenic cell death (ICD): calreticulin and heat shock protein 70 (HSP70) externalization and high-mobility group box 1 protein (HMGB1) release. The intensity of HMGB1 release after the CAP treatment correlated directly with the basal extracellular HMGB1 level. Releasing from dying cells, HMGB1 can act as a proinflammatory cytokine. Our in vivo study demonstrated that cold atmospheric plasma induces a short-term two-times increase in serum HMGB1 level only in tumor-bearing mice with no effect in healthy mice. These findings support our hypothesis that CAP-dependent HMGB1 release from dying cancer cells can change the serum HMGB1 level. At the same time, we showed a weak cytokine response to CAP irradiation in healthy mice that can characterize CAP as an immune-safety physical antitumor approach.

Fibrocytes represent a novel MDSC subset circulating in patients with metastatic cancer.

Fibrocytes are hematopoietic stem cell-derived fibroblast precursors that are implicated in chronic inflammation, fibrosis, and wound healing. Myeloid-derived suppressor cells (MDSCs) expand in cancer-bearing hosts and contribute to tumor immune evasion. They are typically described as CD11b⁺HLA-DR⁻ in humans. We report abnormal expansions of CD11b⁺HLA-DR⁺ myeloid cells in peripheral blood mononuclear fractions of subjects with metastatic pediatric sarcomas. Like classical fibrocytes, they display cell surface α smooth muscle actin, collagen I/V, and mediate angiogenesis. However, classical fibrocytes serve as antigen presenters and augment immune reactivity, whereas fibrocytes from cancer subjects suppressed anti-CD3-mediated T-cell proliferation, primarily via indoleamine oxidase (IDO). The degree of fibrocyte expansion observed in individual subjects directly correlated with the frequency of circulating GATA3⁺CD4⁺ cells (R = 0.80) and monocytes from healthy donors cultured with IL-4 differentiated into fibrocytes with the same phenotypic profile and immunosuppressive properties as those observed in patients with cancer. We thus describe a novel subset of cancer-induced myeloid-derived suppressor cells, which bear the phenotypic and functional hallmarks of fibrocytes but mediate immune suppression. These cells are likely expanded in response to Th2 immune deviation and may contribute to tumor progression via both immune evasion and angiogenesis.

Phase 2 trial of sorafenib in children and young adults with refractory solid tumors: A report from the Children's Oncology Group.

BACKGROUND: Sorafenib is an oral small molecule inhibitor of multiple kinases controlling tumor growth and angiogenesis. The purpose of the phase 2 study was to determine the response rate of sorafenib and gain further information on the associated toxicities, pharmacokinetics, and pharmacodynamics of sorafenib in children and young adults with relapsed or refractory tumors including rhabdomyosarcoma, Wilms tumor, hepatocellular carcinoma (HCC), and papillary thyroid carcinoma (PTC). PROCEDURE: Sorafenib, 200 mg/m(2) /dose, was administered every 12 hr continuously for 28 day cycles using a two-stage design in two primary strata (rhabdomyosarcoma and Wilms tumor) and two secondary strata (HCC and PTC). Correlative studies in consenting patients included determination of sorafenib steady state trough concentrations and assessments of VEGF and sVEGFR2. RESULTS: Twenty patients (median age of 11 years; range, 5-21) enrolled. No objective responses (RECIST) were observed in the 10 evaluable patients enrolled in each of the two primary disease strata of rhabdomyosarcoma and Wilms tumor. No patients with HCC or PTC were enrolled. Sorafenib was not associated with an excessive rate of dose-limiting toxicity (DLT). The mean ± SD steady state concentration during cycle 1 day 15 was 6.5 ± 3.9 µg/ml (n = 10). CONCLUSIONS: Sorafenib was well tolerated in children at 200 mg/m(2) /dose twice daily on a continuous regimen with toxicity profile and steady state drug concentrations similar to those previously reported. Single agent sorafenib was inactive in children with recurrent or refractory rhabdomyosarcoma or Wilms tumor.

Vascular endothelial growth factor serum levels in children with newly diagnosed rhabdomyosarcoma.

BACKGROUND: The adverse prognostic impact of elevated levels of circulating Vascular Endothelial Growth Factor (VEGF) is described in several malignancies. However, no information is available in childhood rhabdomyosarcoma (RMS). In the present study, serum VEGF-A (sVEGF-A) was measured at diagnosis in a series of patients with RMS. PROCEDURE: sVEGF-A was assessed retrospectively in 17 newly diagnosed RMS patients. sVEGF-A concentrations were determined by quantitative enzyme-linked immunoabsorbent ELISA kit and their possible associations with age at diagnosis, gender, histology, primary site, primary size, Intergroup Rhabdomyosarcoma Study (IRS) post-surgical group, and outcome were investigated. RESULTS: sVEGF-A median value in patients with RMS was significantly higher than in controls: 499.0 pg/ml, range: 2,648.0 versus 301.5 pg/ml, range: 716.0 (P = 0.013). Although not statistically significant probably due to the limited number of patients, sVEGF-A median levels resulted higher in unfavorable primary sites (277.0 vs. 539.0 pg/ml; P = 0.31), and advanced groups (390.0 vs. 715.0; P = 0.29). Patients with shorter 5-year overall survival (OS) and 5-year progression-free survival (PFS) times also had higher sVEGF-A levels, although again the difference was not statistically significant (P = 0.18 and P = 0.22, respectively). CONCLUSIONS: Circulating VEGF is significantly increased in pediatric patients with newly diagnosed RMS. Further studies in larger series of RMS patients are needed to understand whether measurements of circulating VEGF might have a role in assessing prognosis and modulating treatment.

Is the NBN gene mutation I171V a potential risk factor for malignant solid tumors in children?

NBN gene is considered as one of the low-to-moderate cancer susceptibility gene. At least 4 germline NBN mutations have been found in several malignancies in adults. In our studies, we observed the high incidence of germline mutation I171V of NBN gene in breast, colorectal, larynx cancer, and in multiple primary tumors. In this study, we would like to answer the question whether I171V germline mutation of NBN gene may constitute risk factor for solid tumors in children. The frequency of this mutation has been analyzed in patients with neuroblastoma (n=66), Wilms tumor (n=54), medulloblastoma (n=57), and rhabdomyosarcoma (n=82) hospitalized in Pediatric Oncology, Hematology and Bone Marrow Transplantation Department in the years between 1987 and 2010. About 2947 anonymous blood samples collected on Guthrie cards drawn from the newborn screening program of the Wielkopolska region have been used as controls. All the patients and controls came from the same geographical region. I171V mutation of the NBN gene has been observed in 5 controls. Among children with solid tumors only in 1 child with medulloblastoma I171V variant has been found. In conclusion, I171V germline mutation in contrary to adults cannot be considered as a risk factor for children malignancies. However, owing to low number of patients with solid tumors the possibility of a Type II error may exist.

Survival and prognostic analysis of preoperative indicators in patients undergoing surgical resections with rhabdomyosarcoma.

Several preoperative blood and biochemical parameters are associated with postoperative survival in many kinds of tumors. The aim of this study is to study the predictive value of several routine preoperative blood and biochemical parameters on the prognosis patients with rhabdomyosarcoma (RMS).We retrospectively recruited 55 patients diagnosed with RMS and had surgery at West China Hospital, Sichuan University between January 2010 and December 2018. Baseline characteristics of the patients, tumor features, surgery details, and values of several examinations were extracted. A long-term follow-up was conducted by phone call. A novel statistical analysis was subsequently carried out to look for the relationship of preoperative parameters and patients' prognosis.The ROC analysis showed an area under curve (AUC) of 0.608, 0.620, 0.626, 0.591, and 0.518 for neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), monocyte to lymphocyte ratio (MLR), lactic dehydrogenase (LDH), and alkaline phosphatase (ALP) respectively, and the cut-off value of 2.843, 162.961, and 0.239 for NLR, PLR, and MLR respectively. The survival analysis showed that certain blood and biochemical parameters could cause differences in overall survival (OS) (P = .005 for NLR, P = .005 for PLR, and P = .007 for MLR) and progression free survival (PFS) (P = .029 for NLR, P = .008 for PLR, and P = .013 for MLR).Several preoperative blood and biochemical parameters are novel prognostic factors in RMS patients. Specifically, a higher NLR, PLR, and MLR value will predict a statistically shorter OS and PFS.In the future, surgeons should care more about NLR, PLR, and MLR values and several other parameters in patients' preoperative normal blood and biochemical tests to predict the postoperative conditions.

Increased midkine serum levels in pediatric embryonal tumor patients.

Serum levels of midkine (MK), a heparin-binding growth factor, are elevated in adult cancer patients. We analyzed sera of pediatric tumor patients in comparison to a large number of children and adolescents without malignant disease. MK was studied in sera of 152 noncancer patients and 29 embryonal tumor patients (14 nephroblastoma, 10 neuroblastoma, and 5 rhabdomyosarcoma) using an enzyme-linked immunosorbent assay. Noncancer patients underwent elective surgical procedures or suffered from an endocrinologic disease. They had no evidence of inflammation or injury. MK serum levels were significantly higher in tumor patients (median 0.621 ng/mL) than in noncancer patients. About 86% of tumor patients were identified using a cut-off value of 0.176 ng/mL. MK values did neither correlate with tumor size nor with stage or histology, but decreased in half of the nephroblastoma patients after chemotherapy and surgery. MK values were found to be elevated in only 2 out of 5 rhabdomyosarcoma patients. MK may serve as an additional marker for the detection of pediatric embryonal tumors, but its clinical relevance for the evaluation of response to therapy needs further study.

Cell-Free DNA in Pediatric Rhabdomyosarcoma: Potential and Challenges.

Rhabdomyosarcoma is an aggressive solid tumor that may disseminate hematogenously giving metastasis which represents the most important prognostic factor. Chances of an effective cure in childhood cancer rely on the capacity to make an early and accurate diagnosis, detect metastatic disease or relapse, and predict the response to treatment.Liquid biopsy is a very promising blood test for cancer detection and noninvasive disease monitoring. This method has a great advantage to use blood and plasma, a more accessible biological material, quick and easy to obtain with minimal pain and risk for patients. In particular, circulating free DNA (cfDNA) represents a tumor biomarker detected in plasma that gives information on biology and genetic background of tumor.Moreover, cfDNA mutation detection could be a reliable method to monitor the efficacy of treatment and to discover novel targets for a personalized treatment in pediatric solid tumor. Here, we describe an optimized protocol to cfDNA isolation from small amount of plasma, as well as a method to assess the quantity and quality of cfDNA. Finally, we propose ddPCR as a reliable method to detect mutations at low frequency in cfDNA obtained from pediatric rhabdomyosarcoma samples.

Flow cytometric phenotype of rhabdomyosarcoma bone marrow metastatic cells and its implication in differential diagnosis with neuroblastoma.

BACKGROUND: The goal of this study was to develop a flow cytometric (FCM) method for assessing the presence of metastatic cells in bone marrow (BM) and peripheral blood (PB) obtained from rhabdomysarcoma (RMS) patients. Myogenin (Myf4), a specific molecular RMS marker, was also investigated in the same samples. Since neuroblastoma (NB) metastasizes to the BM, the potential application of cytometry in differential diagnosis was explored. PATIENTS AND METHODS: CD45, CD56, CD90 and CD57 antibodies were used in 7 paired BM and PB samples (from 7 RMS stage IV patients at presentation), 23 BM samples (from 13 RMS stage I and II patients at presentation), and ten paired BM and PB samples taken at presentation and five BM samples taken at recurrence from 13 NB stage 4 patients. RESULTS: All seven BM samples from RMS stage IV (but not those from patients with localized disease) showed both the CD45- CD56+ phenotype and the Myf4 transcript. Four cases also showed CD90 and two CD57 positivity. Neither the CD45- CD56+ phenotype, nor Myf4 were recorded in the BM and PB samples from patients with localized disease. All the NB BM samples (15/15) showed the CD45- CD56+ CD90+ phenotype and 10/15 also showed CD57 positivity. Only 3/10 blood samples from the NB patients revealed tumor cells. CONCLUSION: CD45, CD56, CD90 and CD57 antibodies can be used in FCM for marrow metastasis detection in both, RMS and NB patients.

A case of extremely chemoresistant pure pleomorphic rhabdomyosarcoma of the uterus associated with a high serum LDH level.

BACKGROUND: Pleomorphic rhabdomyosarcoma (RMS) of gynecologic origin is an exceedingly rare, highly malignant tumor. Only a few cases have been reported in the last decades. CASE REPORT: A 60-year-old postmenopausal woman presented with a high LDH level of unknown origin. Ultimately, she was diagnosed with pleomorphic RMS. She underwent total hysterectomy, bilateral salpingo-oophorectomy, left pelvic and paraaortic lymphadenectomy and partial omentectomy. Surgery was followed by systemic chemotherapy and pelvic irradiation. Unfortunately, the patient did not respond to treatment. Her disease course correlated with the fluctuation of plasma LDH levels. Ultimately she died within 20 months of the diagnosis. CONCLUSION: It is important to have better insight and to set a standard multimodal treatment for adult RMS. In addition, plasma LDH levels can be considered as a prognostic marker for RMS, particularly in advanced stage.

Evidence for an important role of neutrophils in the efficacy of photodynamic therapy in vivo.

To investigate the role of neutrophils in the efficacy of photodynamic therapy (PDT) in rhabdomyosarcoma-bearing rats, the number of these circulating phagocytes was decreased or increased before interstitial PDT by use of rabbit anti-rat neutrophil serum or granulocyte colony-stimulating factor, respectively. After administration of the antiserum, the number of circulating neutrophils decreased by 99.9%. However, the number of monocytes, lymphocytes, and platelets decreased as well (by 100%, 80%, and 25%, respectively). Under these conditions, PDT did not retard tumor growth at all. However, after cessation of the antiserum treatment 5 days after PDT, a striking decrease in the growth rate occurred subsequent to an increase above the normal range of the number of circulating neutrophils. Administration of the granulocyte colony-stimulating factor led to a specific 4-fold increase in the number of circulating neutrophils. In these rats, the tumor growth at day 2 after PDT was retarded as compared with PDT-treated rats that received saline only. Statistical evaluation of both experimental conditions showed that the efficacy of PDT, expressed as the percentage of change in tumor volume at day 2 after treatment, was dependent on the number of circulating neutrophils present at the day of PDT (P = 0.001; r2 = 0.482). Apparently, neutrophils are indispensable for successful PDT in vivo.

Serum interleukin-2 receptor levels in Hodgkin disease and other solid tumors of childhood.

Serum levels of interleukin-2 receptor (IL2R) were determined in children with newly diagnosed Hodgkin disease (n = 68), Wilms tumor (n = 20), osteosarcoma (n = 18), rhabdomyosarcoma (n = 18), or Ewing sarcoma (n = 15). Measurements of soluble IL2R were positively correlated with disease stage in Hodgkin disease but not in other tumors. Very high levels of soluble IL2R (> or = 5000 U/ml) were significantly associated with a poorer treatment outcome in Hodgkin disease (p = 0.006) and retained significance in a multivariate analysis (p = 0.03). The addition of soluble IL2R measurements to existing prognostic models may improve risk assignment in children with Hodgkin disease.

Alveolar rhabdomyosarcoma associated with disseminated intravascular coagulation and a unique factor VIII antigen.

Disseminated intravascular coagulation (DIC) has been described in association with many tumors. We describe a patient with alveolar rhabdomyosarcoma in whom DIC developed with the initiation of chemotherapy. The patient achieved complete remission of his tumor for 14 months. An unusual factor VIII antigen was identified on crossed immunoelectrophoresis that was present at initial diagnosis, disappeared with remission, and returned with relapse of the tumor.

Dose-effect relationships for glucose-induced tumour acidification and its erythrocyte flux.

Preclinical investigations were performed with glucose administration in WAG/Rij rats carrying the rhabdomyosarcoma BA1112 in two sites per animal: one in the subcutis of the flank (for pH measurements in the tumour tissue) and one in the transparent "sandwich" chamber for measuring the erythrocyte flux in the tumour tissue as an indication for changes in tumour blood flow. A glucose solution (20%) was slowly infused intravenously in a range of dose levels, similar to those reported for inducing long-term hyperglycaemia in man. The eventual aim of such investigations is to sensitise tumours for hyperthermic treatment. This approach is not new, but the present experiments were performed with the aim to explore the level of the minimal amount of glucose which would nonetheless yield a likely therapeutic effect. Endpoints in this study were the blood glucose level and pH and the relative erythrocyte flux in the tumour tissue. Obviously, as one would expect, many significant changes in the various parameters were found as a response to administration of glucose. However, the changes in the blood glucose level, the induced decrease in tumour pH and the influence of the tumour volume did not show a well-defined relationship which was reliable enough to predict the exact influence of the various parameters on the magnitude of the desired changes in individual animals and/or tumours. This was probably caused by interfering differences in physiological feedback mechanisms. Nonetheless, the data indicate that the optimal effect was not obtained with the highest treatment level, but with moderate doses of glucose, i.e. 2.4-3 g.kg/h which induced a satisfactory tumour acidification of 0.25 pH units. This may turn out to a clinically useful pH drop for enhancing the cytotoxic effect of hyperthermia. The erythrocyte flux through the tumour tissue does not appear to be influenced to a sizeable extent by such a treatment.

MRI measurements correctly predict the relative effects of tumor oxygenating agents on hypoxic fraction in rodent BA1112 tumors.

PURPOSE: We evaluate whether magnetic resonance imaging (MRI) with blood oxygenation level-dependent (BOLD) contrast correctly predicts the relative effects of tumor-oxygenating agents on hypoxic fraction in BA1112 rhabdomyosarcomas in WAG/Rij rats. METHODS AND MATERIALS: The response of ten tumors to carbogen (95% O(2)/5% CO(2)), a perfluorocarbon emulsion (PFC), and the combination of PFC + carbogen was studied with high spectral and spatial resolution MR imaging of the water resonance at 4.7 Tesla. Decreases in MR signal linewidth indicate increases in tumor blood oxygen levels. RESULTS: Average MR signal linewidth was decreased 2.0% by carbogen, 2.5% by PFC + air, and 4.9% by PFC + carbogen. PFC + carbogen caused a larger linewidth decrease than either treatment alone (p < 0.04 by ANOVA). Maps of pixels responding to treatment indicate that combining PFC with carbogen significantly enlarges the area of the tumor in which oxygen levels are increased (p < 0.01 by ANOVA). CONCLUSION: MRI predicts that PFC + carbogen will increase radiosensitivity more than either treatment alone; this agrees with the known effects of these treatments on hypoxic fraction. Utilizing MRI to choose the treatment that maximizes the size and extent of increases in tumor oxygenation could reduce hypoxic fraction.

Assessment of cardiotoxicity during haemopoietic stem cell transplantation with plasma brain natriuretic peptide.

Cardiac failure is a known complication of haemopoietic stem cell transplantation (HSCT) and is often difficult to diagnose as patients may have multiple medical problems. Since brain natriuretic peptide (BNP) is largely a hormone of cardiac ventricular origin and is released early in the course of ventricular dysfunction, we have examined the value of serial plasma BNP levels for detecting cardiac failure in patients undergoing cytotoxic conditioning for HSCT. Fifteen patients undergoing HSCT were evaluated (10 undergoing autologous HSCT; five undergoing allogeneic HSCT). BNP was measured by radioimmunoassay prior to therapy and weekly for 5 weeks. Seven patients had a significant rise in BNP level (above a previously established threshold of 43 pmol/l associated with cardiac failure), occurring 1-4 weeks post commencement of conditioning. In three of these patients, cardiac failure was subsequently diagnosed clinically 3, 9 and 23 days after a BNP level of 43 pmol/l had been detected. These three patients had the highest peak BNP levels for the group and in each case elevation in BNP level occurred for a period exceeding 1 week. Although numbers were relatively small, a BNP >43 pmol/l was significantly associated with the inclusion of high-dose cyclophosphamide in the preparative regimen (P = 0.02). BNP levels showed no relationship to febrile episodes. In conclusion, these results show that plasma BNP may be used as a marker for early detection of cardiac dysfunction in patients undergoing HSCT, particularly if levels are increased for periods exceeding 1 week. Measurement of BNP during HSCT may be helpful in patients at risk of cardiac failure, in complex clinical situations and in monitoring the cardiotoxicity of preparative regimens.

Neurofibromatosis, factor IX deficiency, and rhabdomyosarcoma.

A paratesticular rhabdomyosarcoma occurred in a child with factor IX deficiency and neurofibromatosis, illustrating the need to consider carefully the various etiologic possibilities of a soft-tissue mass in a child with neurofibromatosis and/or a bleeding disorder.

Unexpected interactions between nicotinamide and CPT-11 in a rhabdomyosarcoma tumor model.

We investigated the potential chemosensitizing effect of nicotinamide on CPT-11, and the relationship between nicotinamide and CPT-11, intratumoral drug uptake in syngeneic rhabdomyosarcoma tumors in rats. Pretreatment with nicotinamide, known to improve tumor oxygenation, perfusion and radiotherapy effect, only caused a minor increase in tumor growth delay. To our surprise, intratumoral uptake of CPT-11 and its active metabolite SN-38 decreased significantly between 19% and 43%. This discrepancy suggests that the potential chemosensitizing effect of nicotinamide, seen in other studies, is based on a direct effect on tumor cells rather than on an increased delivery of anticancer drugs. A second finding is that plasma levels of CPT-11 and SN-38 respectively increase and decrease after nicotinamide exposure, suggesting inhibition of carboxylesterase, which is necessary for the conversion of CPT-11 to its active metabolite SN-38. Great care is required when combining nicotinamide with anticancer drugs, since unexpected pharmacokinetic and pharmacodynamic alterations might occur.

Hypercalcemia and elevated serum parathyroid hormone level in association with rhabdomyosarcoma.

Pseudohyperparathyroidism to sarcomas is very rare. A 16-year-old boy had a rhabdomyosarcoma and severe hypercalcemia associated with an elevated serum parathyroid hormone (PTH) level and osteolytic bone metastases. The four parathyroid glands were normal. Both the hypercalcemia and the serum PTH level responded to chemotherapy.