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Multiplex Super-Selective PCR Assays for the Detection and Quantitation of Rare Somatic Mutations

Eppendorfs microcentrifuge tubes

Professor Fred Kramer spoke at the recent Research & Technology Series exploring Flow Cytometry / qPCR & Digital PCR / Liquid Biopsies. During his presentation, he explained how Super Selective primers enable the simultaneous identification and quantitation of rare somatic mutations in routine multiplex PCR assays, while virtually eliminating signals from abundant closely related wild-type sequences.

“These assays are sensitive, specific, rapid, and low-cost, and can be carried out in widely available spectrofluorometric thermal cyclers. The results of these assays will potentially enable the choice, and subsequent modification if necessary, of effective targeted therapies for the treatment of an individual’s cancer, utilizing frequent non-invasive liquid biopsies.”

Cancer cells are often characterized by the occurrence of many somatic mutations. This is a consequence of cancer, not a cause of cancer. Some of these somatic mutations, which rarely occur in normal cells enable a targeted therapy or an immunotherapy to selectively destroy the cancer cells without significantly harming the normal cells. However, even in the same type of cancer, different somatic mutations occur. To select an effective targeted therapy, it is necessary to identify the somatic mutations that are present. Moreover, during treatment new somatic mutations may arise in the cancer cells making it necessary to change the therapy. Consequently, effective personalized therapy requires frequent analysis of which somatic mutations are present in each patient’s cancer.

Frequent invasive tissue biopsies are, however, inappropriate. They are potentially harmful and often miss the occurrence of new somatic mutations that render therapies ineffective.

Today, there is a great deal of interest in utilizing liquid biopsies to identify DNA fragments in the blood plasma that contain somatic mutations that are transiently present in the blood plasma because of the death of the cancer cells. However, DNA fragments possessing somatic mutations, associated with cancer, diagnosis, prognosis and therapy, are often very rare. Also, closely related wild-type DNA fragments from the normal cells are abundant. To manage this, current techniques employ next-generation sequencing which can analyse many different mutations simultaneously. Unfortunately, next-generation sequencing lacks sensitivity because the required DNA amplification step introduces mutations that mask the presence of the actual mutations. Also, it takes time, it is labour intensive, and it is expensive.

Real-time PCR assays on the other hand are rapid low cost and extremely sensitive. Potentially they can detect even a single template molecule in the sample.

Fred Kramer and his team have been developing multiplex PCR assays that enable a routine blood sample to be analysed on a widely available spectral floor metric. These thermal cyclers can identify and quantitate many different rare somatic mutations that are relevant to cancer diagnosis, prognosis, and therapy. The fundamental problem is the difficulty posed by the need to selectively observe the extremely rare mute DNA fragments without interference from the abundant closely related wild-type DNA fragments.

“The extraordinary sensitivity, of this approach allows for personalized care of a patient using multiplex real-time super selective PCR assays to identify the somatic mutations that are present, if any, and determine the therapeutic action that is needed.”

Professor Fred Kramer

Instruments are currently available that can detect as many as 10 different, fluorescent signals and others are being developed. Super selective PCR assays have great possibilities for the future. Clinicians will be able to follow the patient. Subsequent liquid biopsies will document the success of the treatment and, if a new mutation arises resisting the therapy, enable early identification of mutation and alternate therapies.

Fred Kramer is Professor of Microbiology, Biochemistry and Molecular Genetics at Rutgers University, USA

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