Cell Cycle Checkpoints p16 and p21-Strong Predictors of Clinicopathologic Outcomes in High-Grade Osteosarcoma.

PURPOSE: In this study, we used a series of immunohistochemical measurements of 2 cell cycle regulators, p16 and p21, to evaluate their prognostic value, separately and in combination, for the disease outcomes. METHOD: A total of 101 patients with high-grade osteosarcoma were included in this study. Clinicopathologic data were collected, and immunohistochemistry for p16 and p21 was performed and interpreted by 3 independent pathologists. Statistical analysis was performed to assess the strength of each of these markers relative to disease outcome. RESULTS: Our results indicate that more than 90% expression (high) of p16 by immunohistochemistry on the initial biopsy has a strong predictive value for good histologic response to chemotherapy. The patients are also more likely to survive the past 5 years and less likely to develop metastasis than patients with less than 90% p16 (low) expression. The results for p21, on the other hand, show a unique pattern of relationship to the clinicopathologic outcomes of the disease. Patients with less than 1% (low) or more than 50% (high) expression of p21 by immunohistochemistry show a higher chance of metastasis, poor necrotic response to chemotherapy, and an overall decreased survival rate when compared with p21 expression between 1% and 50% (moderate). Our results also showed that the expression of p16 and combined p16 and p21 demonstrates a stronger predictive relationship to 5-year survival than tumor histologic necrosis and p21 alone. DISCUSSION: The results of this study, once proven to be reproducible by a larger number of patients, will be valuable in the initial assessment and risk stratification of the patients for treatment and possibly the clinical trials.

Investigating surface proteins and antibody combinations for detecting circulating tumor cells of various sarcomas.

Circulating tumor cells (CTCs) have gathered attention as a biomarker for carcinomas. However, CTCs in sarcomas have received little attention. In this work, we investigated cell surface proteins and antibody combinations for immunofluorescence detection of sarcoma CTCs. A microfluidic device that combines filtration and immunoaffinity using gangliosides 2 and cell surface vimentin (CSV) antibodies was employed to capture CTCs. For CTC detection, antibodies against cytokeratins 7 and 8 (CK), pan-cytokeratin (panCK), or a combination of panCK and CSV were used. Thirty-nine blood samples were collected from 21 patients of various sarcoma subtypes. In the independent samples study, samples were subjected to one of three antibody combination choices. Significant difference in CTC enumeration was found between CK and panCK + CSV, and between panCK and panCK + CSV. Upon stratification of CK+ samples, those of metastatic disease had a higher CTC number than those of localized disease. In the paired samples study involving cytokeratin-positive sarcoma subtypes, using panCK antibody detected more CTCs than CK. Similarly, for osteosarcoma, using panCK + CSV combination resulted in a higher CTC count than panCK. This study emphasized deliberate selection of cell surface proteins for sarcoma CTC detection and subtype stratification for studying cancers as heterogeneous as sarcomas.

Microfluidics-Enabled Isolation and Single-Cell Analysis of Circulating Tumor Cells.

Microfluidic platforms enable the enrichment and analysis of circulating tumor cells (CTCs), a potential biomarker for cancer diagnosis, prognosis, and theragnosis. Combined with immunocytochemistry/immunofluorescence (ICC/IF) assays for CTCs, microfluidics-enabled detection presents a unique opportunity to study tumor heterogeneity and predict treatment response, both of which can help cancer drug development. In this chapter, we detail the protocols and methods employed to fabricate and use a microfluidic device for the enrichment, detection, and analysis of single CTCs from the blood samples of sarcoma patients.

Results of a Randomized, Double-Blinded, Placebo-Controlled, Phase 2.5 Study of Saracatinib (AZD0530), in Patients with Recurrent Osteosarcoma Localized to the Lung.

PURPOSE: Osteosarcoma is a rare cancer and a third of patients who have completed primary treatment will develop osteosarcoma recurrence. The Src pathway has been implicated in the metastatic behavior of osteosarcoma; about 95% of samples examined express Src or have evidence of downstream activation of this pathway. Saracatinib (AZD0530) is a potent and selective Src kinase inhibitor that was evaluated in adults in Phase 1 studies. The primary goal of this study was to determine if treatment with saracatinib could increase progression-free survival (PFS) for patients who have undergone complete resection of osteosarcoma lung metastases in a double-blinded, placebo-controlled trial. Patients and Methods. Subjects with recurrent osteosarcoma localized to lung and who had complete surgical removal of all lung nodules were randomized within six weeks after complete surgical resection. Saracatinib, or placebo, was administered at a dose of 175 mg orally, once daily, for up to thirteen 28-day cycles. RESULTS: Thirty-seven subjects were included in the analyses; 18 subjects were randomized to receive saracatinib and 19 to receive placebo. Intent-to-treat analysis demonstrated a median PFS of 19.4 months in the saracatinib treatment group and 8.6 months in the placebo treatment group (p=0.47). Median OS was not reached in either arm. CONCLUSIONS: Although saracatinib was well tolerated in this patient population, there was no apparent impact of the drug in this double-blinded, placebo-controlled trial on OS, and Src inhibition alone may not be sufficient to suppress metastatic progression in osteosarcoma. There is a suggestion of potential clinical benefit as evidenced by longer PFS in patients randomized to saracatinib based on limited numbers of patients treated.

Radiation Treatment for Ewing Sarcoma Family of Tumors in Adult Patients: A Single Institution's Experience Over 40 Years.

PURPOSE/OBJECTIVES: To report prognostic factors and long-term outcomes in adults with Ewing sarcoma treated with definitive radiotherapy. MATERIALS AND METHODS: We reviewed patients 18 years old and above with nonmetastatic Ewing sarcoma treated with radiotherapy +/- chemotherapy or surgery. Outcomes were stratified by age (30 and above vs. younger than 30 y), soft tissue extension, tumor size (≥8.5 vs. <8.5 cm), tumor location, resection (yes vs. no), and treatment era (1970-1992 vs. 1993-2012). Toxicities were scored using the RTOG criteria. RESULTS: Fifty-five patients (21 women) were treated with radiotherapy. Average age at diagnosis: 26.7 years (38 patients below 30 vs. 17 patients 30 y and above). A total of 43 had soft tissue extension (78%). Median tumor size: 8.5 cm. Most tumors were in the pelvis (40%), followed by the lower (27%) and upper (24%) extremities. All but 1 patient received chemotherapy; 13 underwent resection. Median dose: 55 Gy. Median follow-up: 3.6 years; 17.5 years for living patients. The 5-year overall (OS) and cause-specific survival (CSS) rates were both 46%. OS and CSS rates were unaffected by age (P=0.97), tumor size (P=0.12), or tumor location (P=0.99). Soft tissue extension portended a significantly poorer prognosis for 5-year OS and CSS: 37% vs. 82% (with and without, respectively; P=0.04). Patients who underwent resection had improved 5-year OS and CSS: 77% vs. 37%, respectively (P=0.01). Patients treated after 1993 had improved 5-year OS: 58% vs. 37% (P=0.0264). CONCLUSIONS: Adult patients with Ewing sarcoma experience similar treatment outcomes regardless of age at diagnosis. Soft tissue extension represents a poor prognostic factor. Aggressive trimodality therapy achieved the highest OS and CSS.

Outcome of Patients With Recurrent Osteosarcoma Enrolled in Seven Phase II Trials Through Children's Cancer Group, Pediatric Oncology Group, and Children's Oncology Group: Learning From the Past to Move Forward.

PURPOSE: The use of radiographic response as the primary end point in phase II osteosarcoma trials may limit optimal detection of treatment response because of the calcified tumor matrix. We performed this study to determine if time to progression could be used as an end point for subsequent studies. PATIENTS AND METHODS: We performed a retrospective analysis of outcome for patients with recurrent/refractory osteosarcoma enrolled in one of seven phase II trials conducted by the Children's Oncology Group and predecessor groups from 1997 to 2007. All trials used RECIST or WHO radiographic response criteria and the primary end point of response rate. The following potential prognostic factors-age, trial, number of prior chemotherapy regimens, sex, and race/ethnicity-were evaluated for their impact on event-free survival (EFS). We used data from a phase II study (AOST0221) of patients with osteosarcoma who were given inhaled granulocyte-macrophage colony-stimulating factor with first pulmonary recurrence who had an EFS as well as biologic end point to determine the historical disease control rate for patients with fully resected disease. RESULTS: In each included trial, the drugs tested were determined to be inactive on the basis of radiographic response rates. The EFS for 96 patients with osteosarcoma and measurable disease was 12% at 4 months (95% CI, 6% to 19%). There was no significant difference in EFS across trials according to number of prior treatment regimens or patient age, sex, and ethnicity. The 12-month EFS for the 42 evaluable patients enrolled in AOST0221 was 20% (95% CI, 10% to 34%). CONCLUSION: The EFS was uniformly poor for children with recurrent/refractory osteosarcoma in these single-arm phase II trials. We have now constructed baseline EFS outcomes that can be used as a comparison for future phase II trials for recurrent osteosarcoma.

External-beam radiotherapy for pediatric and young adult desmoid tumors.

BACKGROUND: To report long-term outcomes following radiotherapy for desmoid tumors in children and young adults and identify variables impacting local-regional control and treatment complications. PROCEDURE: From 1978 to 2008, 30 patients <30 years old were treated with radiotherapy for a pathologically confirmed desmoid tumor. The median age at radiotherapy was 23.7 years old (range, 10.3-29.9). Fifteen patients underwent definitive radiotherapy, 14 received radiotherapy after gross total resection, and 1 received preoperative radiotherapy. Sixteen patients received 1.8 Gy once daily and 14 received 1.2 Gy twice daily. Variables analyzed for prognostic value included gender, age at diagnosis, primary or recurrent presentation, age at radiotherapy, tumor site, tumor size, extent of resection, fractionation schedule, and radiotherapy dose. RESULTS: The actuarial 15-year overall survival and local-regional control rates were 96% and 55%, respectively. Local-regional control in patients <18 years old at the time of radiotherapy was 20% versus 63% in those 18-30 years old (P = 0.08). Local-regional control rates for tumors receiving ≥ 55 Gy and < 55 Gy were 79% and 30%, respectively (P = 0.02). No other factors had a statistically significant association with local-regional control by univariate analysis. Twelve of 30 patients experienced grade 3-4 complications, including pathologic fractures, impaired range of motion, pain, and in-field skin cancers. CONCLUSIONS: The role of radiotherapy in managing young patients with desmoid tumors remains unclear. Younger patient age is associated with inferior local-regional control following RT. In children and young adults, doses ≥55 Gy were associated with improved tumor control, but also lead to increased risk of complications.

Adjuvant radiotherapy for pediatric and young adult nonrhabdomyosarcoma soft-tissue sarcoma.

PURPOSE: To evaluate the prognostic factors, outcomes, and complications in patients aged ≤30 years with resectable nonrhabdomyosarcoma soft-tissue sarcoma treated at the University of Florida with radiotherapy (RT) during a 34-year period. METHODS AND MATERIALS: A total of 95 pediatric or young adult patients with nonrhabdomyosarcoma soft-tissue sarcoma were treated with curative intent with surgery and RT at the University of Florida between 1973 and 2007. The most common histologic tumor subtypes were synovial sarcoma in 22 patients, malignant fibrous histiocytoma in 19, and malignant peripheral nerve sheath tumor in 11 patients. The mean age at RT was 22 years (range, 6-30). Of the 95 patients, 73 had high-grade tumors; 45 had undergone preoperative RT and 50 postoperative RT. The prognostic factors for survival, local recurrence, and distant recurrence were analyzed. RESULTS: The median follow-up was 7.2 years (range, 0.4-30.5). The actuarial 5-year local control rate was 88%. A microscopically negative margin was associated with superior local control. Although 83% of local recurrence cases initially developed in the absence of metastases, all patients with local failure ultimately died of their disease. The actuarial estimate of 5-year overall survival and disease-free survival was 65% and 63%, respectively. Of all the deaths, 92% were disease related. An early American Joint Committee on Cancer stage, tumor<8 cm, and the absence of neurovascular invasion were associated with superior disease-free survival. The National Cancer Institute Common Toxicity Criteria, version 3, Grade 3-4 treatment complication rate was 9%. No secondary malignancies were observed. CONCLUSION: In the present large single-institution study, we found positive margins and locally advanced features to be poor prognostic factors for both local progression and survival. The results from the present study have helped to characterize the therapeutic ratio of RT in pediatric and young adult sarcoma patients and have provided a basis for identifying high-risk patients for whom treatment intensification might be justified.

Chest wall Ewing sarcoma family of tumors: long-term outcomes.

PURPOSE: To review the 40-year University of Florida experience treating Ewing sarcoma family of tumors of the chest wall. METHODS AND MATERIALS: Thirty-nine patients were treated from 1966 to 2006. Of the patients, 22 were treated with radiotherapy (RT) alone, and 17 patients were treated with surgery with or without RT. Of 9 patients with metastatic disease, 8 were treated with RT alone. The risk profiles of each group were otherwise similar. The median age was 16.6 years, and the most frequent primary site was the rib (n=17). The median potential follow-up was 19.2 years. RESULTS: The 5-year actuarial overall survival (OS), cause-specific survival (CSS), and local control (LC) rates were 34%, 34%, and 72%, respectively. For the nonmetastatic subset (n=30), the 5-year OS, CSS, and LC rates were 44%, 44%, and 79%, respectively. LC was not statistically significantly different between patients treated with RT alone (61%) vs. surgery+RT (75%). None of the 4 patients treated with surgery alone experienced local failure. No patient or treatment variable was significantly associated with local failure. Of the patients, 26% experienced Common Toxicity Criteria (CTC) Grade 3+ toxicity, including 2 pulmonary deaths. Modern intensive systemic therapy helped increase the 5-year CSS from 7% to 49% in patients treated after 1984 (p=0.03). CONCLUSIONS: This is the largest single-institution series describing the treatment of chest wall Ewing tumors. Despite improvements in survival, obtaining local control is challenging and often accompanied by morbidity. Effort should be focused on identifying tumors amenable to combined-modality local therapy and to improving RT techniques.

Prognostic significance of interleukin-6 single nucleotide polymorphism genotypes in neuroblastoma: rs1800795 (promoter) and rs8192284 (receptor).

PURPOSE: Neuroblastoma is a childhood cancer of the sympathetic nervous system and many patients present with high-risk disease. Risk stratification, based on pathology and tumor-derived biomarkers, has improved prediction of clinical outcomes, but overall survival (OS) rates remain unfavorable and new therapeutic targets are needed. Some studies suggest a link between interleukin (IL)-6 and more aggressive behavior in neuroblastoma tumor cells. Therefore, we examined the impact of two IL-6 single nucleotide polymorphisms (SNP) on neuroblastoma disease progression. EXPERIMENTAL DESIGN: DNA samples from 96 high-risk neuroblastoma patients were screened for two SNP that are known to regulate the serum levels of IL-6 and the soluble IL-6 receptor, rs1800795 and rs8192284, respectively. The genotype for each SNP was determined in a blinded fashion and independent statistical analysis was done to determine SNP-related event-free survival (EFS) and OS rates. RESULTS: The rs1800795 IL-6 promoter SNP is an independent prognostic factor for EFS and OS in high-risk neuroblastoma patients. In contrast, the rs8192284 IL-6 receptor SNP revealed no prognostic value. CONCLUSIONS: The rs1800795 SNP [-174 IL-6 (G > C)] represents a novel and independent prognostic marker for both EFS and OS in high-risk neuroblastoma. Because the rs1800795 SNP [-174 IL-6 (G > C)] has been shown to correlate with production of IL-6, this cytokine may represent a target for development of new therapies in neuroblastoma.

Clear cell sarcoma of the stomach.

Clear cell sarcoma (CCS) is a high grade soft tissue sarcoma with a distinct molecular profile. Gastrointestinal CCS is very rare and most reported cases are in adults. We describe a 10-year-old female with a 4-month history of anemia who later developed fever, weight loss and abdominal pain. She was subsequently found to have a large infiltrative gastric mass. A diagnosis of CCS was confirmed by molecular and cytogenetic studies. This case illustrates the necessity of a multimodal approach, particularly the use of molecular studies, in the diagnostic evaluation of rare tumors presenting in unusual sites.

Familiarity of pediatricians with different commercially available neonatal and infant formulas.

To assess the familiarity of pediatricians with commercially available formulas and their use for cow's milk protein allergy and colic, a list of formulas was generated by visiting several grocery stores. Pediatricians were ask to indicate their familiarity with these and other "specialized" formulas with regard to protein and carbohydrate sources, energy content, hypoallergenicity, and indication for infant colic. The participants answered an average of 46% of the questions correctly. Respondents were very familiar with 27% of the formulas, and unfamiliar with 35%. The highest score was 70%, and 10% of the responders correctly answered 65% or more of the questions. Fifty-one percent correctly identified the protein source of the formulas; 32% correctly identified the carbohydrate source. The energy content of the formulas was correctly identified by 54%. These data suggest that pediatricians have a poor understanding of the content and appropriate use of neonatal and infant formulas.