Related Experiment Video
Updated: May 5, 2026
Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards
Published on: February 25, 2017
Discovery of Gene Fusions in Driver-Negative Cancer Samples From the National Cancer Institute-Molecular Analysis for
Stefan T Kaluziak1, Elizabeth M Codd1, Rashi Purohit1
1Pathology Department, Massachusetts General Hospital, Boston, MA.
Purpose:
The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) trial was implemented to identify actionable genetic alterations across cancer types and enroll patients accordingly onto treatment arms, irrespective of tumor histology. Using multiplex polymerase chain reaction (PCR) next-generation sequencing, NCI-MATCH genotyped 5,540 patients, discovering gene fusions in 202/5,540 tumors (3.65%). This result, substantially lower than the fusion detection prevalence of 8.5% across all patients with cancer screened at Massachusetts General Hospital's (MGH) clinical laboratories, supported reanalysis of NCI-MATCH samples identified as mutations-of-interest (MOI)-negative. The assay used by NCI-MATCH requires previous knowledge of both fusion genes, cannot detect novel fusions, and may underestimate fusion-positive patients. Anchored multiplex PCR (AMP) technology permits fusion detection with knowledge of just one gene of the fusion partners.
Methods:
Using AMP-based kits, we reprocessed 663 MOI-negative samples. 200 ng of RNA per sample were shipped from the Eastern Cooperative Oncology Group-American College of Radiology Imaging Network biorepository to MGH (n = 319) and Yale University (n = 344), processed, and sequenced on the NextSeq550. Reported fusions were manually reviewed, and novel fusions orthogonally verified via reverse-transcription PCR and Sanger sequencing.
Results:
AMP identified 148 fusions in 142/663 MOI-negative patients (21% [95% CI, 18 to 25]), of which 28 were covered by the Oncomine Comprehensive Assay (OCA) panel but missed, while 120 were not covered by OCA. Among AMP-identified positive patients, 32 had actionable fusions, 24 contained novel fusions, and six had two fusion events. We identified fusions in 12/34 (35% [95% CI, 20 to 54]) cholangiocarcinomas and 43/109 (39% [95% CI, 30 to 49]) sarcomas.
Conclusion:
Technology and awareness of actionable fusions have improved since the NCI-MATCH trial. With AMP-based technology, we identified 142 patients with fusions not detected during NCI-MATCH screening, many potentially actionable. These striking data underscore the need to optimize the fusion-detection capabilities of genotyping assays used in precision medicine.
Insights
Reanalyzing cancer samples with anchored multiplex PCR (AMP) identified significantly more gene fusions than initially detected by the National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) trial, revealing potentially actionable targets for precision medicine.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) trial aimed to match patients to targeted therapies based on genetic alterations, irrespective of tumor type.
- The initial NCI-MATCH screening using multiplex PCR identified gene fusions in 3.65% of 5,540 patients, a rate lower than expected.
- Limitations of the NCI-MATCH assay included the inability to detect novel fusions and a requirement for prior knowledge of both fusion partners.
Purpose of the Study:
- To re-evaluate NCI-MATCH samples negative for mutations-of-interest (MOI) using a more sensitive fusion detection technology.
- To assess the prevalence of gene fusions missed by the original NCI-MATCH assay.
- To identify potentially actionable fusions for precision oncology.
Main Methods:
- 663 MOI-negative samples from the NCI-MATCH trial were reprocessed using anchored multiplex PCR (AMP) kits.
- RNA samples were sequenced, and identified fusions were manually reviewed and orthogonally verified.
- AMP technology allows fusion detection with knowledge of only one fusion partner.
Main Results:
- Anchored multiplex PCR (AMP) identified 148 fusions in 142 out of 663 reanalyzed patients (21%), significantly increasing the detection rate.
- Of the newly identified fusions, 120 were not covered by the original Oncomine Comprehensive Assay (OCA) panel used in NCI-MATCH.
- Actionable fusions were found in 32 patients, including novel fusions in 24 patients. High fusion rates were observed in cholangiocarcinomas (35%) and sarcomas (39%).
Conclusions:
- Advanced fusion detection technology like AMP significantly enhances the identification of gene fusions missed by earlier assays.
- A substantial number of patients in the NCI-MATCH cohort harbored potentially actionable fusions that were previously undetected.
- Optimizing fusion detection capabilities in genotyping assays is crucial for advancing precision medicine and improving patient outcomes.
Related Concept Videos
02:21Cancers Originate from Somatic Mutations in a Single Cell
02:57Targeted Cancer Therapies
There are several types of targeted therapies against...
02:50Combination Therapies and Personalized Medicine
The combination of the drug acetazolamide and sulforaphane is a good example of combination therapy to treat cancer. The cells in the interior of a large tumor often die due to the hypoxic and...
01:05Cancer-Critical Genes II: Tumor Suppressor Genes
When the function of certain critical genes, especially those involved in cell cycle regulation and cell growth signaling cascades, gets disrupted, it upsets the cell cycle progression. Such cells with unchecked cell cycles start proliferating uncontrollably and eventually develop into tumors.
Such genes that act...