Abstract 3824: Analysis of TOPO II and p53 by immunohistochemistry and qPCR in sarcoma patients with ChemFx assay to determine sensitivity against Adriamycin and Etoposide
Authors: Jacobsen Max Henry , Ni Lijun , Althouse Sandy , Prasad Nagendra , Sandusky George , Rushing Daniel 
Published: July 2017

Sarcomas are a heterogeneous group of tumors that account for about 200,000 cancers diagnosed each year and represent 15% of all pediatric malignant tumors. They comprise over fifty subtypes that include bone and soft tissue sarcomas. Bone sarcomas represent about 20% of all sarcomas. It is estimated that over 50% of human sarcomas have a TP53 mutation. Topoisomerase II (TOPO II) is the target for doxorubicin which is the most effective chemotherapeutic agent, although it is sometimes replaced with etoposide to decrease risk of cardiac toxicity.

In this study, 83 clinical sarcomas were evaluated from the IU Medical Center. Of these 83 clinical cases, 34 were primary tumors, 46 were metastatic tumors. Subtypes included in this study are: Liposarcoma, Leiomyosarcoma, Fibrosarcoma, Malignant Fibrous Histiosarcoma (MFH), Synovial Sarcoma, Osteosarcoma, and Ewings. Paraffin-embedded tissue blocks were obtained from the Indiana University Heath Pathology Laboratory under IRB approved protocols. Immunostainings with TOPOII and P53 antibodies (DAKO) were preformed using the DAKO platform with LSAB2 system at the IU Health Pathology Laboratory. The stained tissues were scanned using Aperio Imaging System and analyzed using the positive-pixel algorithm. In addition, qPCR (TaqMan® assays) was performed using the RNA isolated from the same specimens to analyze TOPO II by a second technique. Live tissue testing was also employed using ChemFX assay was performed on fresh specimens to determine Adriamycin and Etoposide (VP-16) sensitivity and/or resistance. Response to Adriamycin and Etoposide were combined to form a composite precision value and then compared with Clinical Response. This showed a 100% positive predictive value and 90% negative predictive value. We combined the two together to create an overall “Combined Response” Variable. This variable was comparted to Immunohistochemistry for both TOPO II and P53, and the expression levels of TOPOII RNA.

The results showed that the lung metastases expressed a higher level of TOPO II and P53 then did the primary tumors. qPCR and immunohistochemistry data did not correlate with each other. The positive pixel data in the primary tumors were 5.238% for TOPO II and 2.4818% for P53, compared to the metastatic lung lesions with 9.64% for TOPO II and 4.5692% for P53. The combined response versus the categorized TOPO II IHC (into categories of increasing intensity, 1,2,3) had a Chi-squared p-value of 0.656. The combined response to versus the categorized P53 IHC (&lt;5% and &gt;=5%) had a Fisher-Exact test p-value of 0.082. There were approximately 40 resistant and 16 sensitive combined responses available for comparison.

We conclude that P53 is a negative predictor for clinical response, with 12 of 40 resistance cases stained positive for P53, whereas 15 of the 16 sensitive cases were negative for P53.

Citation Format: Max Henry Jacobsen, Lijun Ni, Sandy Althouse, Nagendra Prasad, George Sandusky, Daniel Rushing. Analysis of TOPO II and p53 by immunohistochemistry and qPCR in sarcoma patients with ChemFx assay to determine sensitivity against Adriamycin and Etoposide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3824. doi:10.1158/1538-7445.AM2017-3824