Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

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 cancer^1,2,3,4,5. 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 .......

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.......

Materials

Name	Company	Catalog Number	Comments
3M Vetbond	3M, Co.	1469SB
anti-AKT antibody	Cell Signaling Technologies, Inc.	9272
Anti-GAPDH antibody(G-9)	Santa Cruz Biotech. Inc.	sc-365062
Anti-MAPK antibody	Cell Signaling Technologies, Inc.	9926
Anti-phospho-AKT antibody	Cell Signaling Technologies, Inc.	9271
Anti-mTOR antibody	Cell Signaling Technologies, Inc.	2972
Anti-Phospho-mTOR antibody	Cell Signaling Technologies, Inc.	2971
Anti-Phospho-S6 antibody	Cell Signaling Technologies, Inc.	4858
Anti-Rictor antibody	Cell Signaling Technologies, Inc.	2114
Anti-S6 antibody	Cell Signaling Technologies, Inc.	2217
Captisol	ChemScene, Inc.	cs-0731
Carboplatin	NOVAPLUS, Inc.	61703-360-18
DMEM	Mediatech, Inc.	10-013-CV
Easy-A Hi-Fi PCR Cloning Enzyme	Agilent, Inc.	600404-51
Lubricant	Cardinal Healthcare	82-280
Matrigel	Corning, Inc.	356234
McCoy's media	Mediatech, Inc.	10-050-CV
MK-2206	ApexBio, Inc.	A3010
MK-8669	ARIAD Pharmaceuticals, Inc.	AP23573
Nair Sensitive Skin	Church & Dwight Co.	Nair Hair Remover Shower Power Sensitive
NOD/SCID mice	Charles River, Inc.	NOD.CB17-Prkdcscid/NCrCrl
Paclitaxel	NOVAPLUS, Inc.	55390-304-05
PEG400	Millipore Sigma, Inc.	88440-250ML-F
Perjeta	Genetech, Co.	Pertuzumab
Rituximab	Genetech, Co.	Rituxan
RPMI1640	Mediatech, Inc.	10-040-CV
SCID mice	Harlan Laboratories, Inc.	C.B.-17/IcrHsd-PrkdcscidLystbg
SLAx 13-6MHz linear transducer	FUJIFILM SonoSite, Inc	HFL38xp
SonoSite S-series Ultrasound machine	FUJIFILM SonoSite, Inc	SonoSite SII
Tween 80	Millipore Sigma, Inc.	P4780-100ML