Synthesis of the Reported Structures for Kealiinines B and C

Syntheses of the reported structures of kealiinines B and C have been executed. An intermolecular electrophile-induced cyclization of a pendant arene on an ene–guanidine affords the tetracyclic, oxidized naphthimidazole cores

Automated size selection for short cell-free DNA fragments enriches for circulating tumor DNA and improves error correction during next generation sequencing

<div><p>Circulating tumor-derived cell-free DNA (ctDNA) enables non-invasive diagnosis, monitoring, and treatment susceptibility testing in human cancers. However, accurate detection of variant alleles, particularly during untargeted searches, remains a principal obstacle to widespread application of cell-free DNA in clinical oncology. In this study, isolation of short cell-free DNA fragments is shown to enrich for tumor variants and improve correction of PCR- and sequencing-associated errors. Subfractions of the mononucleosome of circulating cell-free DNA (ccfDNA) were isolated from patients with melanoma, pancreatic ductal adenocarcinoma, and colorectal adenocarcinoma using a high-throughput-capable automated gel-extraction platform. Using a 128-gene (128 kb) custom next-generation sequencing panel, variant alleles were on average 2-fold enriched in the short fraction (median insert size: ~142 bp) compared to the original ccfDNA sample, while 0.7-fold reduced in the fraction corresponding to the principal peak of the mononucleosome (median insert size: ~167 bp). Size-selected short fractions compared to the original ccfDNA yielded significantly larger family sizes (i.e., PCR duplicates) during <i>in silico</i> consensus sequence interpretation via unique molecular identifiers. Increments in family size were associated with a progressive reduction of PCR and sequencing errors. Although consensus read depth also decreased at larger family sizes, the variant allele frequency in the short ccfDNA fraction remained consistent, while variant detection in the original ccfDNA was commonly lost at family sizes necessary to minimize errors. These collective findings support the automated extraction of short ccfDNA fragments to enrich for ctDNA while concomitantly reducing false positives through <i>in silico</i> error correction.</p></div

Fragment Length of Circulating Tumor DNA

<div><p>Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134–144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the <i>BRAF</i> V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132–145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the <i>EGFR</i> T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA.</p></div

Periodicity and shorter fragment length of ctDNA derived from GBM8.

<p>In <b>A</b>, coronal <i>f</i> maps and pre- and post-contrast <i>R</i>₁ maps with matched histology (<b>B</b>) and percent of human ctDNA detected in rat plasma (<b>C</b>, colored arrows identify results that correspond to images in <b>A</b>). GBM4₂ is a small tumor (<b>A</b>, white arrow) confirmed on histology (<b>B</b>, black box) with no evidence of a disrupted blood-brain barrier (i.e., post-contrast enhancement on <i>R</i>₁ maps; <b>A</b>). In GBM8₁, a large tumor (white arrow on <i>f</i> map) is associated with disruption of the blood-brain barrier above the corpus callosum, but not below (asterisk on <i>f</i> map). GBM8₃ is an infiltrating tumor (white arrow) with no evidence of blood-brain barrier disruption, but possible invasion into the ventricle as identified on histology (<b>B</b>, black box). GBM8₄ is a large bulky tumor with disruption of the blood-brain barrier (<b>A</b>). Human ctDNA was detected at a level above the control animals for all GBM8 tumors (<b>C</b>). Fragment length distribution for rat cell-free DNA (green line) and human ctDNA (blue line) inferred from paired-end sequencing are plotted in <b>D</b>. All detected ctDNA demonstrated the same strong periodicity and shorter fragment length compared to rat cell-free DNA (<b>E</b>). Distribution of normal rat cell-free DNA was largely consistent between animals (<b>F</b>).</p

Generation of large family sizes in short ccfDNA.

<p>Total reads were similar between sheared buffy coat DNA, unselected ccfDNA, and short ccfDNA (A). Consensus read depth (family size ≥1) was greatest in buffy coat DNA, followed by unselected ccfDNA, and then short ccfDNA (B). Average family size was greatest in the short ccfDNA (C). At the specific variant locations for each patient, consensus read depth in buffy coat DNA rapidly decayed, reaching zero by family size ≥20 (D, gray). In contrast, both the unselected ccfDNA (D, black) and the short ccfDNA (D, purple) showed fewer consensus reads at family size ≥1, but maintained a greater read depth at larger family sizes (D, inset). Consensus read depth at family size ≥20 was greatest in short ccfDNA (E). In (A-C) and (E), solid bars represent the mean value. In (A-E), whiskers correspond to the standard deviation. *** <i>P</i> ≤ 0.001; NS = not significant.</p

Detection of variant alleles in ccfDNA.

<p>Flowchart (A) depicting steps prior to determination of variant allele frequency (VAF). With known variants, VAF can be determined directly from ccfDNA with ddPCR (a), while sequencing requires a multi-step process (b). The addition of truncated adapters followed by extension to full-length in separate steps (b-e) is done to improve resolution during size selection (c, d) of desired subfractions of ccfDNA. There was a strong association (B) between direct measurement of VAF in ccfDNA by ddPCR (A-a) and by the multi-step sequencing process (A-b). This association was present even at VAFs < 1.5% (B-inset). The equations for each colored regression line are shown in a corresponding color. In (C), boxplots of wild type alleles (dark blue) and variant alleles (light blue) by NGS are shown for each cancer patient (C = colorectal adenocarcinoma; M = melanoma; P = pancreatic ductal adenocarcinoma). In (C), data are only shown for insert sizes ≤250 to focus results on the mononucleosome as that length approximates the midpoint between the mononucleosome and dinucleosome lengths associated with ccfDNA. The light gray line identifies the median insert size (167 bp) from all patients. In the majority of patients, the median insert size of the tumor-associated variant allele was shorter than the corresponding wild type allele.</p

In lung cancer patients, mutant alleles occurred more commonly in shorter fragments of cell-free DNA.

<p>In <b>A-C</b>, histograms of overall cell-free DNA fragment length from the entire 16-gene capture panel determined by sequencing compared between five healthy controls (blue lines) and individual tumor patients (red line). Plasma concentration of cell-free DNA and presence (+) /absence (-) of <i>EGFR</i> and <i>KRAS</i> amplifications are also described for each tumor patient. There was a strong left shift (i.e., shorter fragment lengths) and periodicity in LC5 compared to controls (<b>A</b>). In <b>B</b>, there was a subtle shift towards shorter fragment length that was most apparent at longer lengths (black arrow) where fewer inserts from the tumor patient (LC1) were present compared to the healthy controls. In <b>C</b>, no difference between the tumor patient (LC10) and the healthy controls was observed. In <b>D</b>, the length of fragments containing the WT or mutant <i>EGFR</i> allele is shown for healthy controls (blue dots) and tumor patients with the mutant L858R allele (orange dots). The solid bars indicate the mean fragment length for each sample. In <b>E</b>, a histogram of the fragment lengths of the mutant L858R allele from LC5 (orange line) vs. the WT allele in healthy controls (blue lines) demonstrates a higher prevalence of mutant allele at shorter fragment lengths. The black dashed-line identifies the fragment length that corresponds to the most inserts in the tumor patient. Note that the mutant allele more commonly occurs at shorter fragment lengths while the WT allele in healthy controls occurs more commonly at longer fragment lengths. In <b>F</b>, the fragment length associated with <i>EGFR</i> for the WT allele in the healthy controls (blue dots) and tumor patients with the mutant T790M allele (red dots) is displayed. The solid bars correspond to mean fragment length for each sample. In <b>G</b>, a histogram of the fragment length of the mutant allele (L858R) from LC9 (red line) vs. the WT allele in healthy controls (blue lines) is shown. The black dashed-line identifies the fragment length that corresponds to the most inserts in the tumor patient. Note that the WT allele in healthy controls more commonly occurs at longer fragment lengths. In <b>H</b>, the <i>EGFR</i> fragment length associated with the WT allele (pink dots) and the mutant T790M allele (MA; red dots) in each of the tumor patients are depicted. The mutant allele more commonly occurred at a shorter fragment length compared to the length of the WT allele within the same patient.</p

Demographics and variant allele frequency (VAF).

<p>Demographics and variant allele frequency (VAF).</p

Extraction of cell-free DNA fractions for evaluating mutant allele frequency within specific fragment lengths.

<p>In <b>A</b>, an image of an 8% polyacrylamide gel loaded with a truncated library prepared from the cell-free DNA of a lung cancer patient (LC10, middle column). On either side is a custom-designed ladder made from phage lambda containing double-stranded DNA of 229, 240, and 262 bp in length. Six adjacent samples were excised from the gel corresponding to the colored boxes. In <b>B</b>, densitometry of the full-length libraries made from each fraction and the original library are shown. Colors of each curve and peak correspond to the colors in <b>A</b> (the library is shown in black). In <b>C</b>, the mutant allele frequency as determined by digital droplet PCR is shown for the library and each fraction. Colors for mutant allele frequency (%) correspond to the colors in <b>A</b> and <b>B</b>. Note that the purple fraction (peak fragment length of 320 bp) represented the largest increase (9.1-fold) in mutant allele frequency compared to the library (peak fragment length of 348 bp). Fractions containing longer fragment lengths than the library (e.g., blue fraction: peak fragment lengths of 361 bp) demonstrated a reduction in mutant allele frequency.</p

Transcription Factor Oct1 Is a Somatic and Cancer Stem Cell Determinant

<div><p>Defining master transcription factors governing somatic and cancer stem cell identity is an important goal. Here we show that the Oct4 paralog Oct1, a transcription factor implicated in stress responses, metabolic control, and poised transcription states, regulates normal and pathologic stem cell function. Oct1^HI cells in the colon and small intestine co-express known stem cell markers. In primary malignant tissue, high Oct1 protein but not mRNA levels strongly correlate with the frequency of CD24^LOCD44^HI cancer-initiating cells. Reducing Oct1 expression via RNAi reduces the proportion of ALDH^HI and dye efflux^HI cells, and increasing Oct1 increases the proportion of ALDH^HI cells. Normal ALDH^HI cells harbor elevated Oct1 protein but not mRNA levels. Functionally, we show that Oct1 promotes tumor engraftment frequency and promotes hematopoietic stem cell engraftment potential in competitive and serial transplants. In addition to previously described Oct1 transcriptional targets, we identify four Oct1 targets associated with the stem cell phenotype. Cumulatively, the data indicate that Oct1 regulates normal and cancer stem cell function.</p> </div