May 1, 2014 (Vol. 34, No. 9)

Padma Sundar Director Affymetrix

Whole Genome Copy Number Analyses Reveal Novel Druggable Targets

The first-line treatment for cancer treatment is usually surgery, with many patients also receiving radiation and chemotherapy. However, chemotherapy has significant side effects, and benefits may be short lived with many patients becoming therapy-resistant. As a result, pharmaceutical researchers and oncologists are increasingly using a personalized medicine approach to cancer, which involves identifying and targeting multiple patient-specific genomic alterations that drive cancer.

In recent years, we have seen dramatic advances in cancer-targeted treatment options as a result of personalized medicine. One of the leading groups to address cancer genomics challenges is The Cancer Genome Atlas (TCGA), launched by the National Institutes of Health (NIH) in 2006. Researchers on this project are creating an atlas of genomic changes involved in a number of specific cancer types. Among the reported findings of the pilot project is the revelation that cancer-related genetic alterations are shared between tumors, independent of tumor tissue type.

Cancer Classes

Tumors are now broadly characterized into two groups—those characterized by widespread copy number changes and those marked by a preponderance of somatic mutations. These findings led to the commitment of major additional resources to TGCA. By the end of 2015, the TCGA Research Network expects to analyze more than 10,000 specimens from more than 25 different tumor types.

Cancers characterized by multiple recurrent chromosomal gains and losses are called “C” class. This class includes almost all serous ovarian and breast carcinoma samples, as well as a large percentage of lung and head and neck squamous cell carcinomas and endometrioid tumors of the serous subtype.

These findings were published in a Nature Genetics Paper in October 2013. The paper lists 14 therapeutically actionable genomic subclasses within the broader “C” class driven by high-level amplifications and homozygous deletions in 23 genes that can be targeted by currently available drugs. When testing is limited to somatic mutations, or low-plex copy number testing, these druggable markers are missed. In fact, researchers on this project have suggested that patients with cancers in specific subclasses be matched with combination therapies targeting the multiple genomic alterations characterizing these subclasses.

While copy number aberrations are now recognized as important in the study of solid tumors, there are several stumbling blocks in assessing this aberration in solid tumor samples. Typically, solid tumor samples are formalin-fixed and paraffin embedded (FFPE). This process results in poor DNA quality as well as limited DNA quantity. Solid tumor samples often have low purity, i.e., tumor tissue is often contaminated with normal tissue, and the tumor may be heterogeneous, i.e., not all tumor cells contain the aberration. This means that copy number changes may be found only in a small fraction of cells in the sample, and technologies must be sensitive enough to detect these changes. The gold standard for copy number information is FISH but it is low-plex technology and the challenges with obtaining reliable copy number information in FFPE samples using FISH are well documented.

No array-based technology is optimized for FFPE sample types, so high quantities of DNA are required for input, and assay performance is unpredictable.

Next-generation sequencing technologies need high depth of coverage to generate reliable data from heterogeneous, low purity, FFPE samples. This challenge has been addressed for mutations by performing targeted sequencing of hotspots at high depth (typically 500x–1000x). However, copy number changes affect large genomic regions, and sequencing is not performed at a high depth of coverage across the entire affected regions; therefore, sequencing misses copy number changes in these samples, specifically lower-level but clinically actionable amplifications, and copy-neutral loss of heterozygosity (LOH).

New Copy Number Assay for FFPE

Affymetrix has developed the Molecular Inversion Probe (MIP)-based OnsoScan assay that has been able to address these challenges (Figure 1).

The MIP assay technology features padlock probes that interrogate only 40 base pairs, and therefore the technology is especially optimized for short fragments from degraded FFPE. There is no amplification of genomic DNA. Only the correct padlock probes that have hybridized to the DNA target of interest are amplified, leading to high assay specificity and quantitative amplification detection of >10+ copies. This is important because several high-level amplifications are druggable. The OnsoScan assay has enabled a deeper understanding of the molecular basis of cancer and its various disease subtypes.

One of many published papers using the MIP technology describes the discovery of a novel breast cancer predictive marker—a PIK3CA high-level amplification that is a marker of therapy resistance for PIK3CA/mTOR inhibitors—using the Affymetrix OncoScan Assay for whole-genome copy number research. This is an important finding given the large number of PIK3CA/mTOR inhibitors that are currently in clinical trials for multiple tissue types.

Figure 1. Molecular inversion probe assay: 40 base pair genomic DNA interrogation; 48 hours from DNA to results.

Affymetrix has developed a novel algorithm, tailored to the OncoScan assay technology that can be used to determine if a consistent percentage of aberrant cells (%AC) and ploidy are present at each copy number change. The algorithm reports the linear integer copy number in the cancer portion only, effectively subtracting the normal component, to enable a comparison between tumor samples with different contributions of normal cell contamination.

The OncoScan Assay Kit, based on the MIP technology, delivers genome-wide copy number in just 48 hours from as little as 80 ng of DNA. The assay performs well with highly degraded DNA that is 20+ years old and has proven compatibility with all major solid tumor tissue types. For example, one beta test customer ran 38 FFPE samples from multiple solid tumor tissues, including glioma, breast carcinoma, and liposarcoma that contained FISH-confirmed aberrations. The ages of the samples ranged from six months to three years. 

Figure 2A. OncoScan Nexus Express Software Chromosome View: Chromosome 7 view of sample showing gain of the EGFR gene at high resolution.

In a single OncoScan assay, 98% of samples passed QC, and gains, losses, and LOH were clearly detected, including copy-neutral mosaic LOH in all of the passing samples. (Figures 2A and 2B) In addition to the FISH-confirmed aberrations, this assay obtained incremental copy number aberrations in the samples. The OncoScan assay can also detect aberrations in subclones, and this makes it compatible with low purity, heterogeneous samples.

Copy number is now established as an important biomarker in the study of solid tumor diseases. OncoScan is the only platform for the reliable detection of all the known actionable copy number markers in FFPE in a single assay. Because it is a whole genome platform it also enables the discovery of new druggable biomarker targets, for the development of novel therapeutics.

Figure 2B. FISH confirmation of the EGFR gain shown in Figure 2A: Vysis LSI EGFR (spectrum orange) and Vysis CEP 7 (spectrum green) probes.

Padma Sundar ([email protected]) is director, solid tumor applications at Affymetrix.

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