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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients’ tumors into mice and testing responses to corresponding drugs.

Abstract

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Introduction

Patient-derived xenografts (PDXs), which are generated from the implantation of patient tumor pieces into immunodeficient mice, have emerged as a powerful preclinical model to aid personalized anti-cancer care. PDX models have been successfully developed for a variety of human malignancies. These include breast and ovarian cancers, malignant melanoma, colorectal cancer, pancreatic adenocarcinoma, and non-small cell lung cancer1,2,3,4,5. Tumor tissue can be implanted orthotopically or heterotopically. The fo....

Protocol

Fresh tissues from consenting patients with ovarian cancer were collected at the time of debulking surgery according to a protocol approved by Mayo Clinic Institutional Review Board (IRB). All animal procedures and treatments used in this protocol were approved by Mayo Clinic Institutional Animal Care and Use Committee (IACUC) and followed animal care guidelines.

1. Mate pair sequencing and analyses

NOTE: Either fresh or flash frozen tissue must be used for mate pair .......

Representative Results

Tissue from resected ovarian tumors at the time of debulking surgeries were collected in accordance with IRB guidance and used for 1) genomic characterization and 2) engraftment in immunocompromised mice (Figure 1). Mate-pair sequencing protocol36,37 was used to identify structural alterations in DNA including losses, gains and amplifications. A representative genome plot illustrating a landscape of genomic changes in one tumor (desi.......

Discussion

We describe the approach and protocols we used to conduct a “clinical trial” in PDX models that takes advantage of molecular characteristics of the tumor as obtained by genomic profiling to determine the best choice of drugs for testing. Multiple sequencing platforms are currently used for genomic characterization of primary tumors including whole genome sequencing, RNAseq and customized gene panels. For high grade serous ovarian carcinoma, MPseq to identify structural alterations, DNA rearrangements and copy.......

Acknowledgements

We thank the members of the Mayo Clinic Center for Individualized Medicine (CIM) Dr. Lin Yang and Faye R. Harris, MS, for the help in conducting experiments. This work was supported by Mr. and Mrs. Neil E. Eckles’ Gift to the Mayo Clinic Center for Individualized Medicine (CIM).

....

Materials

NameCompanyCatalog NumberComments
3M Vetbond3M, Co.1469SB
anti-AKT antibodyCell Signaling Technologies, Inc.9272
Anti-GAPDH antibody(G-9)Santa Cruz Biotech. Inc.sc-365062
Anti-MAPK antibodyCell Signaling Technologies, Inc.9926
Anti-phospho-AKT antibodyCell Signaling Technologies, Inc.9271
Anti-mTOR antibodyCell Signaling Technologies, Inc.2972
Anti-Phospho-mTOR antibodyCell Signaling Technologies, Inc.2971
Anti-Phospho-S6 antibodyCell Signaling Technologies, Inc.4858
Anti-Rictor antibodyCell Signaling Technologies, Inc.2114
Anti-S6 antibodyCell Signaling Technologies, Inc.2217
CaptisolChemScene, Inc.cs-0731
CarboplatinNOVAPLUS, Inc.61703-360-18
DMEMMediatech, Inc.10-013-CV
Easy-A Hi-Fi PCR Cloning EnzymeAgilent, Inc.600404-51
LubricantCardinal Healthcare82-280
MatrigelCorning, Inc.356234
McCoy's mediaMediatech, Inc.10-050-CV
MK-2206ApexBio, Inc.A3010
MK-8669ARIAD Pharmaceuticals, Inc.AP23573
Nair Sensitive SkinChurch & Dwight Co.Nair Hair Remover Shower Power Sensitive
NOD/SCID miceCharles River, Inc.NOD.CB17-Prkdcscid/NCrCrl
PaclitaxelNOVAPLUS, Inc.55390-304-05
PEG400Millipore Sigma, Inc.88440-250ML-F
PerjetaGenetech, Co.Pertuzumab
RituximabGenetech, Co.Rituxan
RPMI1640Mediatech, Inc.10-040-CV
SCID miceHarlan Laboratories, Inc.C.B.-17/IcrHsd-PrkdcscidLystbg
SLAx 13-6MHz linear transducerFUJIFILM SonoSite, IncHFL38xp
SonoSite S-series Ultrasound machineFUJIFILM SonoSite, IncSonoSite SII
Tween 80Millipore Sigma, Inc.P4780-100ML

References

  1. Tentler, J. J., et al. Patient-derived tumour xenografts as models for oncology drug development. Nature Reviews Clinical Oncology. 9, 338-350 (2012).
  2. Marangoni, E., et al. A new model of patien....

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