Person:

Hornicek, Francis

The molecular events in chordoma pathogenesis have not been fully delineated, particularly with respect to copy number changes. Understanding copy number alterations in chordoma may reveal critical disease mechanisms that could be exploited for tumor classification and therapy. We report the copy number analysis of 21 sporadic chordomas using array comparative genomic hybridization (CGH). Recurrent copy changes were further evaluated with immunohistochemistry, methylation specific PCR, and quantitative real-time PCR. Similar to previous findings, large copy number losses, involving chromosomes 1p, 3, 4, 9, 10, 13, 14, and 18, were more common than copy number gains. Loss of CDKN2A with or without loss of CDKN2B on 9p21.3 was observed in 16/20 (80%) unique cases of which six (30%) showed homozygous deletions ranging from 76 kilobases to 4.7 megabases. One copy loss of the 10q23.31 region which encodes PTEN was found in 16/20 (80%) cases. Loss of CDKN2A and PTEN expression in the majority of cases was not attributed to promoter methylation. Our sporadic chordoma cases did not show hotspot point mutations in some common cancer gene targets. Moreover, most of these sporadic tumors are not associated with T (brachyury) duplication or amplification. Deficiency of CDKN2A and PTEN expression, although shared across many other different types of tumors, likely represents a key aspect of chordoma pathogenesis. Sporadic chordomas may rely on mechanisms other than copy number gain if they indeed exploit T/ brachyury for proliferation.

Undifferentiated pleomorphic sarcoma/Malignant Fibrous Histiocytoma (MFH) is one of the most common subtypes of human soft tissue sarcoma. Using cross species genomic analysis, we define a geneset from the (LSL)-(Kras^{G12D}); (Trp53^{Flox/Flox}) mouse model of soft tissue sarcoma that is highly enriched in human MFH. With this mouse geneset as a filter, we identify expression of the RAS target FOXM1 in human MFH. Expression of (Foxm1) is elevated in mouse sarcomas that metastasize to the lung and tissue microarray analysis of human MFH correlates overexpression of FOXM1 with metastasis. These results suggest that genomic alterations present in human MFH are conserved in the (LSL)-(Kras^{G12D}); (p53^{Flox/Flox}) mouse model of soft tissue sarcoma and demonstrate the utility of this pre-clinical model.

Background: Reversing multidrug resistance (MDR) has been an important goal for clinical and investigational oncologists. In the last few decades, significant effort has been made to search for inhibitors to reverse MDR by targeting ATP-binding cassette (ABC) transporters (Pgp, MRP) directly, but these efforts have achieved little clinical success. Protein kinases play important roles in many aspects of tumor cell growth and survival. Combinations of kinase inhibitors and chemotherapeutics have been observed to overcome cancer drug resistance in certain circumstances. Methods: We screened a kinase specific inhibitor compound library in human osteosarcoma MDR cell lines to identify inhibitors that were capable of reversing chemoresistance to doxorubicin and paclitaxel. Results: We identified 18 small molecules that significantly increase chemotherapy drug-induced cell death in human osteosarcoma MDR cell lines U-2OSMR and KHOSR2. We identified A-770041 as one of the most effective MDR reversing agents when combined with doxorubicin or paclitaxel. A-770041 is a potent Src family kinase (Lck and Src) inhibitor. Western blot analysis revealed A-770041 inhibits both Src and Lck activation and expression. Inhibition of Src expression in U-2OSMR and KHOSR2 cell lines using lentiviral shRNA also resulted in increased doxorubicin and paclitaxel drug sensitivity. A-770041 increases the intracellular drug accumulation as demonstrated by calcein AM assay. Conclusions: These results indicate that small molecule inhibitor A-770041 may function to reverse ABCB1/Pgp-mediated chemotherapy drug resistance. Combination of Src family kinase inhibitor with regular chemotherapy drug could be clinically effective in MDR osteosarcoma. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-681) contains supplementary material, which is available to authorized users.

The molecular mechanisms underlying chordoma pathogenesis are unknown. We therefore sought to identify novel mutations to better understand chordoma biology and to potentially identify therapeutic targets. Given the relatively high costs of whole genome sequencing, we performed a focused genetic analysis using matrix-assisted laser desorption/ionization-time of flight mass spectrometer (Sequenom iPLEX genotyping). We tested 865 hotspot mutations in 111 oncogenes and selected tumor suppressor genes (OncoMap v. 3.0) of 45 human chordoma tumor samples. Of the analyzed samples, seven were identified with at least one mutation. Six of these were from fresh frozen samples, and one was from a paraffin embedded sample. These observations were validated using an independent platform using homogeneous mass extend MALDI-TOF (Sequenom hME Genotyping). These genetic alterations include: ALK (A877S), CTNNB1 (T41A), NRAS (Q61R), PIK3CA (E545K), PTEN (R130), CDKN2A (R58*), and SMARCB1 (R40*). This study reports on the largest comprehensive mutational analysis of chordomas performed to date. To focus on mutations that have the greatest chance of clinical relevance, we tested only oncogenes and tumor suppressor genes that have been previously implicated in the tumorigenesis of more common malignancies. We identified rare genetic changes that may have functional significance to the underlying biology and potential therapeutics for chordomas. Mutations in CDKN2A and PTEN occurred in areas of chromosomal copy loss. When this data is paired with the studies showing 18 of 21 chordoma samples displaying copy loss at the locus for CDKN2A, 17 of 21 chordoma samples displaying copy loss at PTEN, and 3 of 4 chordoma samples displaying deletion at the SMARCB1 locus, we can infer that a loss of heterozygosity at these three loci may play a significant role in chordoma pathogenesis.

Osteosarcoma is the most common bone cancer in children and adolescents. Previously, we have found that cyclin-dependent kinase 11 (CDK11) signaling was essential for osteosarcoma cell growth and survival. Subsequently, CDK11 siRNA gene targeting, expression profiling, and network reconstruction of differentially expressed genes were performed between CDK11 knock down and wild type osteosarcoma cells. Reconstructed network of the differentially expressed genes pointed to the AR as key to CDK11 signaling in osteosarcoma. CDK11 increased transcriptional activation of AR gene in osteosarcoma cell lines. AR protein was highly expressed in various osteosarcoma cell lines and patient tumor tissues. Tissue microarray analysis showed that the disease-free survival rate for patients with high-expression of AR was significantly shorter than for patients with low-expression of AR. In addition, AR gene expression knockdown via siRNA greatly inhibited cell growth and viability. Similar results were found in osteosarcoma cells treated with AR inhibitor. These findings suggest that CDK11 is involved in the regulation of AR pathway and AR can be a potential novel prognostic marker and therapeutic target for osteosarcoma treatment.

The clinical significance of Cluster of Differentiation 44 (CD44) remains controversial in human ovarian cancer. The aim of this study is to evaluate the clinical significance of CD44 expression by using a unique tissue microarray, and then to determine the biological functions of CD44 in ovarian cancer. In this study, a unique ovarian cancer tissue microarray (TMA) was constructed with paired primary, metastatic, and recurrent tumor tissues from 26 individual patients. CD44 expression in TMA was assessed by immunohistochemistry. Both the metastatic and recurrent ovarian cancer tissues expressed higher level of CD44 than the patient-matched primary tumor. A significant association has been shown between CD44 expression and both the disease free survival and overall survival. A strong increase of CD44 was found in the tumor recurrence of mouse model. Finally, when CD44 was knocked down, proliferation, migration/invasion activity, and spheroid formation were significantly suppressed, while drug sensitivity was enhanced. Thus, up-regulation of CD44 represents a crucial event in the development of metastasis, recurrence, and drug resistance to current treatments in ovarian cancer. Developing strategies to target CD44 may prevent metastasis, recurrence, and drug resistance in ovarian cancer.

Background: Multi-drug resistance (MDR) remains a significant obstacle to successful chemotherapy treatment for osteosarcoma patients. One of the central causes of MDR is the overexpression of the membrane bound drug transporter protein P-glycoprotein (P-gp), which is the protein product of the MDR gene ABCB1. Though several methods have been reported to reverse MDR in vitro and in vivo when combined with anticancer drugs, they have yet to be proven useful in the clinical setting. Results: The meta-analysis demonstrated that a high level of P-gp may predict poor survival in patients with osteosarcoma. The expression of P-gp can be efficiently blocked by the clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 system (CRISPR-Cas9). Inhibition of ABCB1 was associated with reversing drug resistance in osteosarcoma MDR cell lines (KHOSR2 and U-2OSR2) to doxorubicin. Materials and Methods We performed a meta-analysis to investigate the relationship between P-gp expression and survival in patients with osteosarcoma. Then we adopted the CRISPR-Cas9, a robust and highly efficient novel genome editing tool, to determine its effect on reversing drug resistance by targeting endogenous ABCB1 gene at the DNA level in osteosarcoma MDR cell lines. Conclusion: These results suggest that the CRISPR-Cas9 system is a useful tool for the modification of ABCB1 gene, and may be useful in extending the long-term efficacy of chemotherapy by overcoming P-gp-mediated MDR in the clinical setting.

Chordomas are primary malignant tumors of the notochord that are resistant to conventional chemotherapy. Expression of programmed cell death ligand 1 (PD-L1), prevalence of tumor-infiltrating lymphocytes (TILs), and their clinical relevance in chordoma remain unknown. We evaluated PD-L1 expression in three chordoma cell lines and nine chordoma tissue samples by western blot. Immunohistochemical staining was performed on a chordoma tissue microarray (TMA) that contained 78 tissue specimens. We also correlated the expression of PD-L1 and TILs with clinical outcomes. PD-L1 protein expression was demonstrated to be induced by IFN-γ in both UCH1 and UCH2 cell lines. Across nine human chordoma tissue samples, PD-L1 protein was differentially expressed. 94.9% of chordoma samples showed positive PD-L1 expression in the TMA. The expression score of PD-L1 for metastatic chordoma tumors was significant higher as compared with non-metastatic chordoma tumors. Expression of PD-L1 protein significantly correlates with the presence of elevated TILs, which correlates with metastasis. In summary, our study showed high levels of PD-L1 are expressed in chordoma, which is correlated with the prevalence of TILs. The current study suggests targeting PD-L1 may be a novel immunotherapeutic strategy for chordoma clinical trials.

Background: Chordomas are rare malignancies that primarily affect adults, but also rarely affect pediatric patients. We compared the imaging appearance, demographic and anatomic distributions of adult and pediatric chordomas in a large cohort. Methods: We performed a retrospective review of medical records of 220 subjects with histologically confirmed chordomas of the axial skeleton and pre-treatment magnetic resonance imaging studies. Age, sex, type of chordoma (conventional, chondroid or dedifferentiated), the anatomic location of the chordoma, as well as whether the lesion was primarily extra-osseous were recorded. Pediatric subjects were less than 21 years at the time of diagnosis. Binomial two-sample tests of proportions and Fisher’s exact tests were used to compare proportions between the pediatric and adult subjects. Results: Fifty six pediatric subjects (58.9 % female) and 164 adult subjects (42.1 % female) were identified. The proportion of female subjects with chordomas was significantly higher in the pediatric cohort compared to the adult cohort (P = 0.04). Most chordomas occur in Caucasians, however African-Americans were more represented in the pediatric cohort than in the adult cohort (P = 0.01). 69.6 % (39/56) of the pediatric chordomas involved the clivus/skull base and cervical spine compared to 29.3 % (48/164) of the adult chordomas (P = 1.99 × 10−7). Only 1.8 % (1/56) of the pediatric chordomas was in the sacrococcygeal region compared to 36.0 % (59/164) of the adult chordomas (P = 2.55 × 10−8). In cases where pre-treatment imaging was available, 93.8 % (16/17) of pediatric chordomas were predominantly extra-osseous compared to 76.7 % (46/60) of adult chordomas (P = 0.17). Conclusions: Pediatric chordomas more often affect females and occur most frequently at the craniocervical junction with decrease in incidence distally in the spine, whereas adult chordomas most frequently involve the craniocervical and sacrococcygeal regions.

Osteosarcoma is the most common primary bone malignancy in children and adolescents. Herein, we investigated the role of cluster of differentiation 44 (CD44), a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration in osteosarcoma. We constructed a human osteosarcoma tissue microarray with 114 patient tumor specimens, including tumor tissues from primary, metastatic, and recurrent stages, and determined the expression of CD44 by immunohistochemistry. Results showed that CD44 was overexpressed in metastatic and recurrent osteosarcoma as compared with primary tumors. Higher expression of CD44 was found in both patients with shorter survival and patients who exhibited unfavorable response to chemotherapy before surgical resection. Additionally, the 3′-untranslated region of CD44 mRNA was the direct target of microRNA-199a-3p (miR-199a-3p). Overexpression of miR-199a-3p significantly inhibited CD44 expression in osteosarcoma cells. miR-199a-3p is one of the most dramatically decreased miRs in osteosarcoma cells and tumor tissues as compared with normal osteoblast cells. Transfection of miR-199a-3p significantly increased the drug sensitivity through down-regulation of CD44 in osteosarcoma cells. Taken together, these results suggest that the CD44-miR-199a-3p axis plays an important role in the development of metastasis, recurrence, and drug resistance of osteosarcoma. Developing strategies to target CD44 may improve the clinical outcome of osteosarcoma.