The objective of the proposed study is to identify and quantitatively assess the frameshift mutation (FSM) variants in microsatellite instability high (MSI-H) colorectal cancer (CRC), with the long-term goal of targeting frequently expressed FSMs, which generate FS peptides (FSPs), with a preventive vaccine and monitoring FSM or FSP expression to verify vaccine response. We will complete this objective through the following aims:

Aim 1: To assess FSM status in MSI-H tumor tissues and plasma samples, using microsatellite stable (MSS) CRC samples as control. The microsatellite status of the requested samples has already been determined.

Hypothesis: We hypothesize that the FSMs included in our target panel will be detected in MSI-H CRCs, but not in MSS CRC.

Expected results: Plasma samples from MSI-H cases will be requested along with control MSS plasma. Due to defects in the DNA mismatch-repair process in cancers with MSI and the increased replicative potential of cancer, we expect that MSI CRCs will generate an elevated number of FSMs. Furthermore, cancer cells shed DNA fragments into the circulation, called cell-free DNA (cfDNA) or circulating tumor DNA2. Thus, FSMs can be assessed in these MSI-H tumor DNA and cfDNA via next generation sequencing (NGS). Based on our preliminary data in the Msh2LoxP/LoxP;Villin-Cre (VCMsh2) mouse model, 45 FSMs were detected in tumors from VCMsh2 mice (85-93% of positive rate) and 12 out of 18 FSMs were detected in plasma samples (Table 1). Thus, we expect positive detection of FSMs from a large target panel generated based on literature search and the human CRC cell lines. However, due to the low cfDNA yield and low abundance of the mutations in plasma compared to tumors, we expect lower FSM detection rate and variant allele frequency (VAF) in plasma than tumor samples.

Aim 2: To correlate plasma- and tumor-derived FSMs in MSI-H and MSS CRCs.

Hypothesis: We hypothesize that the FSM variants detected in MSI-H CRC plasma will be positively correlated with those detected in the matched tumor tissue.

Expected results: We expect that the plasma-derived FSM profile will be correlated with the matched tumor sample. However, because the abundance of FSMs will be greater in tumor tissue than in plasma, we expect that the detection rate and VAF will be lower in plasma than that in CRC tumor tissues. We also expect that plasma samples collected from late-stage individuals will be better correlated with tumors than those samples collected from early-stage individuals.

The results of the current proposal will be critical to further development and monitor efficacy of vaccines to prevent Lynch syndrome-associated CRCs, which account for approximately 3% of MSI CRC3. Lynch syndrome is a heritable condition which increases one’s risk of developing cancers such as CRC. Due to the significant risk of cancer that Lynch syndrome poses, developing effective prevention modalities is a public health priority. This study will provide necessary data to initiate a prospective cancer prevention study of Lynch syndrome carriers.

Robert Shoemaker (National Cancer Institute)
Holli Loomans-Kropp (The Ohio State University)
Yurong Song (National Cancer Institute)