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&#8217; Gift to the Mayo Clinic Center for Individualized Medicine (CIM).

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 ...

We describe the approach and protocols we used to conduct a &#8220;clinical trial&#8221; 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...

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 former, considered more accurate but technically difficult, involves transplantation directly into the organ of tumor origin. These types of models are believed to precisely mimic histology of the original tumor due to the &#8220;natural&#8217; microenvironment for the tumor6,7. For example, orthotopic transplantation into the bursa of the mouse ovary resulted in tumor dissemination into the peritoneal cavity and the production of ascites, typical of ovarian cancer8. Similarly, injection of breast tumors into the thoracic instead of the abdominal mammary gland affected the PDX success rate and behavior9. However, orthotopic models require sophisticated imaging systems to monitor tumor growth. Heterotopic implantation of solid tumor is typically performed by implanting tissue into the subcutaneous flank of a mouse which allows for easier monitoring of tumor growth and is less expensive and time consuming7. However, tumors grown subcutaneously rarely metastasize unlike as observed in the case of orthotopic implantation10.
The success rate of engraftment has been shown to vary and greatly depend on the tumor type. More aggressive tumors and tissue specimens containing a higher percent of tumor cells were reported to have better success rates12,13. Consistent with this, tumors derived from metastatic sites were shown to engraft at frequencies of 50&#8211;80%, while those from primary sites engraft at frequencies as low as 14%12. In contrast, tissue containing necrotic cells and fewer viable tumor cells engraft poorly. Tumor growth can also be promoted by the addition of basement membrane matrix proteins into the tissue mix at the time of the injection into mice14 without compromising properties of the original tumor. The size and number of tissue pieces intended for implantation were also found to affect the success rate of engraftment. Greater tumor take-rates were reported for implantation in the sub-renal capsule compared to subcutaneous implantation due to the ability of the sub-renal capsule to maintain the original tumor stroma and provide the host stromal cells as well15.
Most studies use NOD/SCID immunodeficient mice, which lack natural killer cells16 and have been shown to increase the tumor engraftment, growth and metastasis compared to other strains14. However, additional monitoring is required as they may develop thymic lymphomas as early as 3-4 month of age13. In ovarian tumor transplants grown in SCID mice, the outgrowth of B cells was successfully inhibited by rituximab, preventing the development of lymphomas but without impacting the engraftment of ovarian tumors17.
More recently, NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice, carrying a null mutation in the gene encoding the interleukin 2 receptor gamma chain18, became a frequently used strain for the generation of PDX models. Tumors from established PDX models passaged to future generations of mice are reported to retain histological and molecular properties for 3 to 6 generations19,20. Numerous studies have shown that the treatment outcomes in PDX models mimic those of their corresponding patients2,3,4,21,22,23. The response rate to chemotherapy in PDX models for non-small lung cancer and colorectal carcinomas was similar to that in clinical trials for the same drugs24,25. Studies conducted in PDX models, developed for patients enrolled in clinical trials, demonstrated responses to tested drugs similar to those observed clinically in corresponding patients2,3,4.
High&#8211;throughput genomic analyses of a patient tumor in conjunction with PDX models provide a powerful tool to study correlations between specific genomic alterations and a therapeutic response. These have been described in a few publications26,27. For example, therapeutic responses to the EGFR inhibitor cetuximab in a set of colorectal PDX models carrying EGFR amplification, paralleled clinical responses to cetuximab in patients28.
There are a few challenges associated with the development and application of PDX models. Among those is tumor heterogeneity29,30 that may compromise the accuracy of treatment response interpretation as a single cell clone with higher proliferative capacity within a PDX can outgrow the other ones31, thus resulting in a loss of heterogeneity. Additionally, when single tumor biopsies are used to develop PDX, some of the cell populations may be missed and will not be represented in the final graft. Multiple samples from the same tumor are recommended for implantation to resolve this issue. Although PDX tumors tend to contain all the cell types of the original donor tumor, these cells are gradually substituted by those of murine origin3. The interplay between murine stroma and human tumor cells in PDX models is not well understood. Nevertheless, stromal cells were shown to recapitulate tumor microenvironment33.
Despite these limitations, PDX models remain among the most valuable tools for translational research as well as personalized medicine for selecting patient therapies. Major applications of PDXs include biomarker discovery and drug testing. PDX models are also successfully used to study drug resistance mechanisms and identify strategies to overcome drug resistance34,35. The approach described in the present manuscript allows the researcher to identify potential therapeutic targets in human tumors and to assess the efficacy of corresponding drugs in vivo, in mice harboring engrafted tumors which were initially genomically characterized. The protocol uses ovarian tumors engrafted intraperitoneally but is applicable to any type of tumor sufficiently aggressive to grow in mice2,3,12.

<table>
	<tbody>
		<tr>
			<td>3M Vetbond</td>
			<td>3M, Co.</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-AKT antibody</td>
			<td>Cell Signaling Technologies, Inc.</td>
			<td>9272</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GAPDH antibody(G-9)</td>
			<td>Santa Cruz Biotech. Inc.</td>
			<td>sc-365062</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MAPK antibody</td>
			<td>Cell Signaling Technologies, Inc.</td>
			<td>9926</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-AKT antibody</td>
			<td>Cell Signaling Technologies, Inc.</td>
			<td>9271</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mTOR antibody</td>
			<td>Cell Signaling Technologies, Inc.</td>
			<td>2972</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Phospho-mTOR antibody</td>
			<td>Cell Signaling Technologies, Inc.</td>
			<td>2971</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Phospho-S6 antibody</td>
			<td>Cell Signaling Technologies, Inc.</td>
			<td>4858</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Rictor antibody</td>
			<td>Cell Signaling Technologies, Inc.</td>
			<td>2114</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-S6 antibody</td>
			<td>Cell Signaling Technologies, Inc.</td>
			<td>2217</td>
			<td></td>
		</tr>
		<tr>
			<td>Captisol</td>
			<td>ChemScene, Inc.</td>
			<td>cs-0731</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboplatin</td>
			<td>NOVAPLUS, Inc.</td>
			<td>61703-360-18</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Mediatech, Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy-A Hi-Fi PCR Cloning Enzyme</td>
			<td>Agilent, Inc.</td>
			<td>600404-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Lubricant</td>
			<td>Cardinal Healthcare</td>
			<td>82-280</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning, Inc.</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>McCoy&#39;s media</td>
			<td>Mediatech, Inc.</td>
			<td>10-050-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>MK-2206</td>
			<td>ApexBio, Inc.</td>
			<td>A3010</td>
			<td></td>
		</tr>
		<tr>
			<td>MK-8669</td>
			<td>ARIAD Pharmaceuticals, Inc.</td>
			<td>AP23573</td>
			<td></td>
		</tr>
		<tr>
			<td>Nair Sensitive Skin</td>
			<td>Church &#38; Dwight Co.</td>
			<td>Nair Hair Remover Shower Power Sensitive</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD/SCID mice</td>
			<td>Charles River, Inc.</td>
			<td>NOD.CB17-Prkdcscid/NCrCrl</td>
			<td></td>
		</tr>
		<tr>
			<td>Paclitaxel</td>
			<td>NOVAPLUS, Inc.</td>
			<td>55390-304-05</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG400</td>
			<td>Millipore Sigma, Inc.</td>
			<td>88440-250ML-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Perjeta</td>
			<td>Genetech, Co.</td>
			<td>Pertuzumab</td>
			<td></td>
		</tr>
		<tr>
			<td>Rituximab</td>
			<td>Genetech, Co.</td>
			<td>Rituxan</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Mediatech, Inc.</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>SCID mice</td>
			<td>Harlan Laboratories, Inc.</td>
			<td>C.B.-17/IcrHsd-PrkdcscidLystbg</td>
			<td></td>
		</tr>
		<tr>
			<td>SLAx 13-6MHz linear transducer</td>
			<td>FUJIFILM SonoSite, Inc</td>
			<td>HFL38xp</td>
			<td></td>
		</tr>
		<tr>
			<td>SonoSite S-series Ultrasound machine</td>
			<td>FUJIFILM SonoSite, Inc</td>
			<td>SonoSite SII</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Millipore Sigma, Inc.</td>
			<td>P4780-100ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

testing targeted therapies in cancer using structural dna alteration analysis and patient-derived xenografts

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.

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...

Watch this Scientific Journal Video about Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts at JoVE.com

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

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&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

The protocol described here is aimed to demonstrate an algorithm for testing treatment efficacy using in vivo cancer models. The protocol consists of a combination of several techniques. Whole genome bio-sequencing of human tumor specimens is used to identify genomic alterations.These include both gene rearrangements and gene copy number changes. So the analysis of identified alterations is performed in order to select potentially druggable changes. The drugs selected based on genomic analysis are then used for in vivo treatment of corresponding tumors grown in immunocompromised mice.The developed algorithm represents a promising approach to aid treatment decisions for the care of cancer patients. Use the Panda tool or an analogous software to identify targetable alterations. We can list the genes which were identified by microsequencing resulted as a simple tab delineated file using standard accepted gene symbols.Add the pound sign to the headerline of the list to ensure that the table header is transferred to the pathway level U of the software. Upload the file by clicking on the corresponding navigation tab. Assign a single icon to represent the underlying data by clicking on the icon of choice, and then clicking on the finalize tab.Once then a patient files are uploaded, preview the page to identify a column that displays the number of annotated genes per pathway. This is the last column on the right. Use a pathway filter in the upper left of the main window to restrict the number of pathways displayed to the ones that contain the genes of interest.To identify pathways that have more genes annotated than would be expected by chance, use a function located under the enrichment tab. A column then is added to the main table that shows corresponding p-value from a Fisher's exact test. Select the database to display potential druggable genes from preset annotation by checking an appropriate icon on the left of the main window.To select a pathway for visualization, click on its name displayed in the pathway viewer page. Icons representing each annotation set are displayed next to the associated gene. Clicking on any gene in the pathway will open the corresponding gene cards page.Select the pathways that showed annotated genes of interest and hits for potential drugs for further analysis. Perform the tissue work in a laminar flow hood to maintain sterile conditions. Lay the tumor tissue in a dish containing cold PBS or tissue culture media, such as RPMI, DMEM, containing antibiotics.Identify and isolate viable tumor material from adjacent normal and necrotic tissue with the help from a pathologist. Use sterile forceps and scalpel to remove necrotic material pointed out by a pathologist. To perform subcutaneous engraftment, cut the tumor tissue with sterile forceps, scalpel or surgical scissors into small fragments, roughly 2 x 2 x 2 millimeters in size.Transfer the fragmented tissue to a pre-chilled petri dish on ice. Let cold matrigel into the dish with the fragmented tissue, approximately 200 microliters per 10 pieces of tissue. Mix well and let the tissue fragments soak in the matrigel for 10 minutes.Use sterile surgical scissors and forceps to make a 5-10 millimeters vertical skin incision on both flanks of a mouse. Insert straight forceps gently into the subcutaneous space to create a pocket large enough for a tumor fragment to be placed under the fat pad. Use sterile straight forceps to insert tumor fragments in the previously prepared pocket in each of five mice.Close the skin incision using tissue glue. After implantation, to inhibit lymphocyte proliferation, inject each mouse with 100 microliters of rituximab. Prepare the mouse for oral gavage by pinching the skin of the back and bending it backward so that the head and lips of the mouse are immobilized.Insert the gavage probe down the back of the throat of the mouse until the probe reaches the esophagus. Make sure that the probe is not inserted too far as the lungs of the mouse may perforate causing death. The representative genome float illustrates the landscape of genomic changes in one tumor.Typical for high-grade serosubtype tumors, multiple gains blue lines and losses red lines, were identified indicating high levels of genomic instability. The top trend alteration for therapeutic intervention in the OC101 tumor was an amplification at chromosome 17, involving ERBB2, a gene which codes for HER2 receptor. To validate the results on the DNA level, several sets of specific primers were designed for the edges of the amplified region.And PCR was carried out. No amplification product was absorbed when control human DNA was used. Specific bands were identified for OC101 tumor DNA.In another variant tumor, T14, numerous DNA gains were observed. Those included AKT2 in RICTOR genes. Validation carried out on the protein level, using immunoblotting showed high levels of AKT in RICTOR.A significant reduction in the tumor burden was observed in the chemotherapy treated group by the end of week six. There was an extra benefit over chemotherapy alone in the groups which received a combination treatment. Tumor tissue was collected for molecular analysis of treatment response at the end of the treatment trial.Total and phosphorylated levels of S6, AKT, and mTOR were determined using immunoblotting. Comparison of the levels of these proteins in untreated, chemotherapy-treated, and treated with AKT or mTOR inhibitor showed a marked decrease for the latter two. The presented approach is very useful to conduct the clinical trial in PDX models.It takes advantage of molecular characteristics of the tumor as obtained by genomic profiling to determine the best choice of drugs for testing.

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

Abstract