HistoMosaic: Cancer Tissue Diagnostics by Multiplexed PCR

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Cancer is the second biggest killer in the world after cardiovascular disease, and is projected to overtake it in the near future. Just in the US, every year there are 140k new cases and 49k deaths of colorectal cancer, and 221k new cases and 157k deaths of lung cancer.

Cancer mutations are very important in the treatment of cancer, as some of them are drug-resistant. That is why the American Society for Clinical Oncology recommended in 2009 that colorectal cancer patients are first tested for KRAS before prescribing Cetuximab, which costs $30k per patient per 8 weeks regimen. Similarly, T790M and MET mutations in lung cancer bestow resistance to Tarceva, which costs $90k per patient. ASCO has calculated that testing before giving Cetuximab could save $753M per year.

HistoMosaic is a novel technique for analysis of tissue samples in diagnostics of cancer. State-of-the-art techniques suffer from low sensitivity (immuno-histo-chemistry, in-situ hybridization) or low throughput (laser capture microdissection). Low sensitivity means a significant occurrence of false negatives, leading to expensive drugs being used to no benefit to the patient. Low throughput means inapplicability in the clinical setting. By contrast, our technique is best of both worlds, because it combines the high sensitivity of PCR-based methods with the high throughput of slide-wide clinical techniques.

Briefly, HistoMosaic starts with a conventional tissue slide sample, builds a photoresist layer on top of the tissue, exposes the photoresist to UV through a mask with a honeycomb matrix, and develops the unexposed photoresist (see Slide 1). This results in a photoresist matrix of millions of microwells that conforms to the uneven profile of the tissue on the bottom while leaving a flat surface on the top. Then PCR reagents are added to this construct, and the top is sealed by a microscope slide coated with a cured polydimethylsiloxane layer to serve as a gasket. The assembly is sealed along the edges with epoxy for air-tightness and processed in a standard PCR machine. The PCR assays produce signal in the wells in which the particular mutation is present. Scanning the slide on a fluorescence scanner produces a false-color map of the present cancer mutations. The compartmentalization of the tissue areas allows independent PCR reactions to be performed at the same time with massive parallelism, while the small size of the wells decreases the noise from healthy cells and wild-type cancer cells in the same well.

We have demonstrated that the tissue survives the photolithographic process, that PCR is compatible with photoresist, and that the smallest wells defined on tissue so far are only 50 microns across (see Slide 2), and thus contain less than 10 cells each. We have also demonstrated that the technique amplifies the DNA from the tissue without contamination (see Slide 3). These results prove principle for the technique.

We are currently working on slide-wide scans of the million-well PCR results for colorectal and lung cancer samples.

We have a converted patent application filed Jan 2011.

Please address inquiries to Emil Kartalov, This email address is being protected from spambots. You need JavaScript enabled to view it.

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    Emil Kartalov
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