Focus on enhanced assays to detect cancer cells

1st April 2013

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Demand continues to grow for new and improved assays to detect cancer cells. The latest technologies, as Sean Ottewell reports, focus on rare cancer mutations, colorectal cancer recurrence and breast cancer.

RainDance Technologies, the leading provider of microdroplet-based single molecule and single cell analysis solutions, has launched the DeepSeq formalin-fixed paraffin-embedded (FFPE) solution, an end-to-end workflow for the ultra-deep targeted sequencing of FFPE and fresh frozen tissue samples.

DeepSeq FFPE enables researchers to discover common and rare cancer mutations that represent as little as one per cent of a heterogeneous tumour.

For the first time, researchers can analyse as many as 500 genomic targets at up to 50000-fold coverage per loci to discover rare cancers and other disease-specific mutations from heterogeneous samples.

Based on the proven RainStorm microdroplet-based single molecule PCR technology, the DeepSeq Solution delivers high specificity, accurate quantitation and unbiased allelic representation. The company says that the ultra-deep resolution of genomic targets represents a significant enhancement over the 10-30 per cent allelic sensitivity achievable with current next-generation sequencing, target capture/hybridisation, or microarray methods.

It is optimised for both fresh frozen and FFPE tissue, which is the standard archival method for nearly all solid tumours.

Degraded DNA

Historically, researchers have been limited in applying next-generation sequencing to FFPE samples due to challenges with low yield, degraded DNA, and sample extraction. With an estimated 400million FFPE samples stored in tissue banks worldwide, cancer researchers and oncologists can now detect specific novel and causative mutations that drive drug resistance, metastasis and progression of different forms of the disease.

"In order to achieve the promise of targeted therapy cocktails optimised for the individual patient, we need cost-effective tools that address the heterogeneous nature of cancers," said Kelly A Frazer, founding chief of the division of genome information sciences for the department of paediatrics at the UCSD school of medicine. "Technologies, such as those offered by RainDance, will help us identify rare mutations present in as little as onepercent of a tumour biopsy."

The DeepSeq FFPE Solution runs on the RDT 1000, an automated instrument that produces two million single molecule microdroplet-PCR reactions per sample in a single tube in less than one hour (Fig.1).

The solution also introduces a number of innovative enhancements to simplify customer workflow and lower sequencing costs, including custom primer libraries, tailed primers to eliminate library generation, and multi-sample indexing.

Genome amplification

To further enhance the DeepSeq FFPE Solution, RainDance is collaborating with NuGEN Technologies to develop a complementary fast and simple whole genome amplification (WGA) FFPE sample preparation kit.

NuGEN's Ovation WGA FFPE product delivers accurate and unbiased genomic representation and generates microgram amounts of DNA from 100 nanograms of starting FFPE DNA samples.

"FFPE samples represent a huge repository of clinical information that has remained virtually untapped by next-generation sequencing due to the lack of available high-performance sample preparation technologies," said Elizabeth Hutt, ceo of NuGEN. "In this collaboration with RainDance, we will provide cancer researchers with a new, powerful and cost-effective solution to generate high quality next-generation sequencing data."

Agendia, a leader in molecular cancer> diagnostics has received its fifth US Food and Drug Administration (FDA) clearance for MammaPrint, the company's widely used breast cancer ( recurrence assay (Fig.2).

Microarray scanners

The new clearance is comprised of two additional Agilent microarray scanners and two Agilent bioanalysers, expanding laboratory capacity to handle the increasing number of MammaPrint, TargetPrint and BluePrint test requests.

The company said that the presence of FDA cleared equipment in both of its locations will further mitigate risk posed by a potential interruption to its business in the event of an equipment breakdown at either location.

MammaPrint previously received several FDA clearances for clinical use in the US and remains the first FDA cleared in vitro diagnostic multivariate index assay (IVDMIA) on the market and the only FDA cleared breast cancer recurrence test.

"Agendia continues to lead by example in the genomic testing industry by ensuring that our products and equipment are in full compliance with the most recent FDA standards, despite the fact that such oversight is not mandatory," said company founder and ceo Bernhard Sixt.

MammaPrint was previously defined by the FDA as a qualitative in vitro diagnostic test service, performed only in Agendia's Irvine labs. The latest FDA clearance defines a new 'intended use' which allows for the test to be performed in a central laboratory.

This empowers Agendia to legally perform tests for the US market in both the Irvine and Amsterdam clinical laboratory improvement amendments (CLIA) and College of American Pathologists (CAP) accredited facilities, and also additional future central labs under Agendia control.

Everist Genomics, a prognostics company focused on developing gene-based molecular assays for the prediction of cancer ( recurrence, has announced the positive results of the first external validation study of its OncoDefender-CRC colorectal cancer ( recurrence test.

Recurrence risk

The study is being conducted by Mayo Validation Support Services and coordinated with investigators and resources from all three Mayo Clinic sites. The study demonstrates that the OncoDefender-CRC assay can accurately predict individual recurrence risk in stage I and II colorectal cancer patients.

"One of the most pressing challenges for physicians who treat colorectal cancer is the early identification of patients who are at risk of recurrence and could potentially benefit from prophylactic post-surgical chemotherapy," said Lisa A Boardman, associate professor of medicine, Mayo Clinic College of Medicine and principal study investigator. "The study's objective is to develop an effective tool to guide treatment decisions and for identifying those patients most likely to benefit from adjuvant therapy following surgical resection of tumor tissue."

Among the key performance indicators of the study, patients who were identified as high risk by the OncoDefender-CRC assay had a significantly higher probability of recurrence within 36 months than patients identified as low risk.

"The OncoDefender-CRC test equips physicians with critical data about the genetic properties of a tumour, which when used in conjunction with standard pathological tests, enables clinicians to make better individualised treatment decisions for high-risk patients, while minimising the exposure of low-risk patients to unnecessary, costly and potentially toxic chemotherapy and radiotherapy," said Peter F Lenehan, chief medical officer of Everist Genomics and principal study investigator.

Meanwhile Compendia Bioscience has entered a new strategic partnership with Althea Diagnostics, the first outcome of which is the joint breast cancer segregation panel assay and analysis service.

The panel measures 96 genes that represent key molecular variability of breast cancer ( and have been derived from a proprietary meta-analysis of cancer ( genomic profiles from more than 5000 clinical samples. The assay has been optimised by Althea Diagnostics to measure the genes using quantitative RT-PCR from FFPE samples to enable rapid translation to real-world clinical settings.

Compendia Bioscience will analyse the data generated by the assay and will perform correlation studies to end-points of interest such as drug response. This will allow for development of multiple drug-specific companion diagnostics from a standardised platform.

"This addresses a critical concern," said Scott Tomlins, a scientific advisor from the University of Michigan. "Clinicians could soon become overwhelmed by the number of molecular diagnostics entering the market, each requiring precious tissue samples. A standard assay that could be used to address a number of different clinical questions will be highly valuable to the field. We are hopeful that future studies will prove the utility of Compendia's approach."

Assay development

The companies have also announced that they plan to develop similar assays for other cancer disease types. They have begun development of a colon segregation panel assay, which will be available later in 2011.

Detecting asbestos-related pulmonary cancers

Experts from the Cancer Institute at NYU Langone Medical Centre presented new research findings at the American Association for Cancer Research 102nd Annual Meeting in Florida from 2-6 April. NYU Cancer Institute researchers discussed various breakthroughs such as a novel test for early-stage asbestos-related pulmonary cancer, a promising treatment strategy for glioblastomas, genome-wide mapping of nickel-related cancer and greater understanding of melanoma and bladder cancer.

For example, malignant pleural mesothelioma is an aggressive, asbestos-related pulmonary cancer that develops in the lining of the lungs. Researchers investigated a novel protein test to detect early-stage, asbestos-related pulmonary cancer.

The new test can accurately identify proteins secreted from cancerous tumors caused by asbestos exposure.

In a blinded test researchers detected 15 of 19 cases of stage one or stage two malignant pleural mesothelioma. The goal of the new diagnostic test is to find the cancer early enough to effectively treat it. The test, called Multiplex SOMAmer Assay by SomaLogic, uses SOMAmers, chemically modified single-stranded DNA molecules to bind specifically to target proteins and quantify biomarkers.

The test successfully measures 19 protein biomarkers for malignant pleural mesothelioma and quantifies the small amount of proteins secreted by tumour cells

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