Rational Cancer Diagnostics Using Rare Mutation Detection Technology With Massively Parallel Sequencing

Worldwide, cancers remain a leading cause of death. The judicious use of cancer diagnostics -- broadly defined as tests for cancer -- has great potential to reduce disease morbidity and mortality. Impeding this potential is the difficulty of creating effective new tests, as the techniques successful for one type of cancer frequently cannot be generalized to another. Although the ability to detect cancer-specific DNA mutations at the low levels commonly encountered in clinical specimens would yield a promising, broadly applicable diagnostic strategy, existing technologies have been unacceptably limited in throughput or accuracy. Here we describe the development and application of a scalable, generalizable DNA sequence-based technology for the reliable detection of mutations. By drastically reducing artifacts introduced through sample preparation and massively parallel sequencing, rare mutations arising from cancer cells – when present – can be confidently discriminated from a large excess of non-mutant DNA. The technology can be directed to virtually any genomic region, affording rational test design. When applied to routinely collected Pap specimens, our approach detected cancer-specific mutations in 41% (9 of 22) and 100% (24 of 24) of women harboring various stages of ovarian and endometrial cancers, respectively. Our approach was highly specific, as no false positives were detected in a cohort of Pap specimens collected from women without gynecologic cancer. We also demonstrate how the urine of patients with urothelial carcinoma can be utilized to predict disease recurrences. Eighty-eight percent (7 of 8) of patients with a detectable mutation had recurrences while none were detected in the six patients without recurrent disease (P <0.001). Finally we present data suggesting that a wide range of cancers shed mutant DNA into blood and that these mutations are sensitive and specific markers for disease. Taken together, our results demonstrate the potential and feasibility of improved diagnostics for several cancers using a variety of clinical specimens obtainable in a minimally invasive fashion. Larger studies are underway as a prelude to implementing these tests in the clinic -- a critical step in addressing the many unmet clinical needs of patients with cancer

FAST-SeqS: A Simple and Efficient Method for the Detection of Aneuploidy by Massively Parallel Sequencing

<div><p>Massively parallel sequencing of cell-free, maternal plasma DNA was recently demonstrated to be a safe and effective screening method for fetal chromosomal aneuploidies. Here, we report an improved sequencing method achieving significantly increased throughput and decreased cost by replacing laborious sequencing library preparation steps with PCR employing a single primer pair designed to amplify a discrete subset of repeated regions. Using this approach, samples containing as little as 4% trisomy 21 DNA could be readily distinguished from euploid samples.</p> </div

Demonstration of FAST-SeqS reproducibility among different samples, sequencing instruments, and sequencing depth.

<p>FAST-SeqS was performed on eight normal plasma DNA samples, their corresponding matched peripheral blood white blood cell (WBC) DNA, and on the splenic or WBC DNA of an additional eight unrelated individuals. The eight samples within each experiment constituted the reference group (see ‘Materials and Methods’ section) from which the plotted z-scores were calculated. No autosome in any sample had a z-score outside the range of −3.0 and 3.0 (dotted lines). Despite 3-fold less sequencing of the splenic or WBC samples, the z-scores (range: −2.2 to 2.1) were similar to those obtained from the plasma (range: −2.1 to 1.9) and matched WBC DNA samples (range: −2.2 to 1.9).</p

Accurate discrimination of euploid DNA samples from those containing trisomic DNA.

<p>(A) Comparison of z-scores from patients with trisomy 21 (n = 4), trisomy 18 (n = 2), and trisomy 13 (n = 1) with eight normal spleen or peripheral blood white blood cell (WBC) DNAs. The z-scores displayed represent the relevant chromosome for the comparison. The maximum z-score observed for any of the compared normal chromosomes was 1.9 (chr13). (B) Control WBC DNA was analyzed alone (n = 2) or when mixed with DNA from a patient with trisomy 21 at 5% (n = 2), 10% (n = 1), or 25% (n = 1) levels. A tight correlation existed between the expected and observed fractions of extra chromosome 21 (r = 0.997 by Pearson correlation test, n = 6). (C) Control WBC DNA was analyzed alone (z-score range: −0.8 to 1.3) or when mixed with DNA from a patient with trisomy 21 at 4% (z-score range: 4.5 to 7.2) or 8% (z-score range: 8.9 to 10.) levels. Each experiment in (C) was performed in quadruplicate.</p

Comparison of observed and predicted distributions of FAST-SeqS amplification products.

<p>(A) A density plot of the expected distribution of fragment lengths, with peaks at 124 and 142 bp. (B) A density plot of the actual tag counts obtained in eight normal plasma DNAs. The 124 bp fragments are preferentially amplified compared to the 142 bp fragments, likely due to an amplification bias towards smaller fragments. Inset: polyacrylamide gel of a representative FAST-SeqS sequencing library. Note: the amplification products contain an additional ∼120 bp of flanking sequence to facilitate sequencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041162#pone.0041162.s002" target="_blank">Table S2</a>). (C) The average representation of the most frequently observed L1 retrotransposon subfamilies in eight normal plasma samples. Roughly 97% of uniquely aligning tags arise from positions representing only seven L1 retrotransposon subfamilies. (D) A detailed examination of the average number of observed positions per chromosome from eight normal plasma DNAs compared with the number predicted by RepeatMasker for each of the seven L1 retrotransposon subfamilies noted in (C). Error bars in each panel depict the range.</p

The potential of circulating tumor DNA (ctDNA) to reshape the design of clinical trials testing adjuvant therapy in patients with early stage cancers

BACKGROUND: The conventional approach to testing the benefit of adjuvant therapies in patients (pts) with relatively favorable prognoses is to follow a large number of pts for long periods of time, hoping that mature outcome data will document an improved outcome compared to control pts. We reasoned that the design of such trials could be improved if pts with minimal residual disease could be identified a priori through the presence of ctDNA, and the effects of adjuvant therapy then assessed through serial ctDNA assays. METHODS: We carried out a prospective trial in 231 pts with Stage II colon cancer. Serial plasma samples were collected every 3 months starting 4-10 weeks after surgery. Somatic mutations in pts' tumors were identified via sequencing of 15 genes commonly mutated in colon cancer. We then designed personalized assays to quantify ctDNA in plasma samples. Adjuvant chemotherapy was administered at clinician discretion, blinded to ctDNA analysis. RESULTS: Somatic mutations were identified in 230 (99.6%) of tumors. Matching ctDNA was detected in the immediate post-operative period in 14 of 178 (8%) pts not treated with chemotherapy, 11 of whom had recurred (79%) at a median follow-up of 27 months. In contrast, recurrence occurred in only 16 (10%) of the 164 pts with negative ctDNA not treated with chemotherapy (HR 15.66, log-rank P<0.0001). ctDNA was detected in the immediate post-operative period in 6 of 52 pts who went on to receive chemotherapy. The ctDNA status turned from positive to negative during adjuvant treatment phase in all 6 pts (100%) but became positive again following completion of chemotherapy in 2 pts, both of whom have recurred. In patients with serial samples available, the median lead-time between ctDNA detection and radiologic-recurrence was 167 days. CONCLUSIONS: Detection of ctDNA in pts with resected stage II colon cancer provides direct evidence of residual disease. As well as defining pts at very high risk of later radiologic-recurrence, serial ctDNA analysis may provide an early readout of adjuvant treatment effect. Including ctDNA analyses would increase the efficiency of clinical trials testing the benefit of adjuvant treatment

The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers

Colorectal tumours that are wild type for KRAS are often sensitive to EGFR blockade, but almost always develop resistance within several months of initiating therapy. The mechanisms underlying this acquired resistance to anti-EGFR antibodies are largely unknown. This situation is in marked contrast to that of small-molecule targeted agents, such as inhibitors of ABL, EGFR, BRAF and MEK, in which mutations in the genes encoding the protein targets render the tumours resistant to the effects of the drugs. The simplest hypothesis to account for the development of resistance to EGFR blockade is that rare cells with KRAS mutations pre-exist at low levels in tumours with ostensibly wild-type KRAS genes. Although this hypothesis would seem readily testable, there is no evidence in pre-clinical models to support it, nor is there data from patients. To test this hypothesis, we determined whether mutant KRAS DNA could be detected in the circulation of 28 patients receiving monotherapy with panitumumab, a therapeutic anti-EGFR antibody. We found that 9 out of 24 (38%) patients whose tumours were initially KRAS wild type developed detectable mutations in KRAS in their sera, three of which developed multiple different KRAS mutations. The appearance of these mutations was very consistent, generally occurring between 5 and 6months following treatment. Mathematical modelling indicated that the mutations were present in expanded subclones before the initiation of panitumumab treatment. These results suggest that the emergence of KRAS mutations is a mediator of acquired resistance to EGFR blockade and that these mutations can be detected in a non-invasive manner. They explain why solid tumours develop resistance to targeted therapies in a highly reproducible fashion