Circulating HPV DNA as a Biomarker for Pre-Invasive and Early Invasive Cervical Cancer: A Feasibility Study.

BACKGROUND: High-risk HPV infection is responsible for >99% of cervix cancers (CC). In persistent infections that lead to cancer, the tumour breaches the basement membrane, releasing HPV-DNA into the bloodstream (cHPV-DNA). A next-generation sequencing assay (NGS) for detection of plasma HPV circulating DNA (cHPV-DNA) has demonstrated high sensitivity and specificity in patients with locally advanced cervix cancers. We hypothesised that cHPV-DNA is detectable in early invasive cervical cancers but not in pre-invasive lesions (CIN). METHODS: Blood samples were collected from patients with CIN (n = 52) and FIGO stage 1A-1B CC (n = 12) prior to treatment and at follow-up. DNA extraction from plasma, followed by NGS, was used for the detection of cHPV-DNA. RESULTS: None of the patients with pre-invasive lesions were positive for CHPV-DNA. In invasive tumours, plasma from one patient (10%) reached the threshold of positivity for cHPV-DNA in plasma. CONCLUSION: Low detection of cHPV-DNA in early CC may be explained by small tumour size, poorer access to lymphatics and circulation, and therefore little shedding of cHPV-DNA in plasma at detectable levels. The detection rate of cHPV-DNA in patients with early invasive cervix cancer using even the most sensitive of currently available technologies lacks adequate sensitivity for clinical utility.

Biomarkers of Response and Resistance to Palbociclib Plus Letrozole in Patients With ER+/HER2- Breast Cancer.

PURPOSE: To determine (i) the relationship between candidate biomarkers of the antiproliferative (Ki67) response to letrozole and palbociclib alone and combined in ER+/HER2- breast cancer; and (ii) the pharmacodynamic effect of the agents on the biomarkers. EXPERIMENTAL DESIGN: 307 postmenopausal women with ER+/HER2- primary breast cancer were randomly assigned to neoadjuvant treatment with letrozole for 14 weeks; letrozole for 2 weeks, then letrozole+palbociclib to 14 weeks; palbociclib for 2 weeks, then letrozole+palbociclib to 14 weeks; or letrozole+palbociclib for 14 weeks. Biopsies were taken at baseline, 2 and 14 weeks and surgery at varying times after stopping palbociclib. Immunohistochemical analyses were conducted for Ki67, c-PARP, ER, PgR, RB1, CCNE1, and CCND1. RESULTS: Higher baselines ER and PgR were significantly associated with a greater chance of complete cell-cycle arrest (CCCA: Ki67 <2.7%) at 14 weeks and higher baseline Ki67, c-PARP, and CCNE1 with a lower chance. The interaction with treatment was significant only for c-PARP. CCND1 levels were decreased c.20% by letrozole at 2 and 14 weeks but showed a tendency to increase with palbociclib. CCNE1 levels fell 82% (median) in tumors showing CCCA but were unchanged in those with no CCCA. Only 2/9 tumors showed CCCA 3-9 days after stopping palbociclib. ESR1 mutations were found in 2/4 tumors for which surgery took place ≥6 months after starting treatment. CONCLUSIONS: High CCNE1 levels were confirmed as a biomarker of resistance to letrozole+palbociclib. Ki67 recovery within 3-9 days of discontinuing palbociclib indicates incomplete suppression of proliferation during the "off" week of its schedule.

Molecular characterisation of aromatase inhibitor-resistant advanced breast cancer: the phenotypic effect of ESR1 mutations.

BACKGROUND: Several thousand breast cancer patients develop resistance to aromatase inhibitors (AIs) each year in the UK. Rational treatment requires an improved molecular characterisation of resistant disease. MATERIALS AND METHODS: The mutational landscape of 198 regions in 16 key breast cancer genes and RNA expression of 209 genes covering key pathways was evaluated in paired biopsies before AI treatment and at progression on AI from 48 patients. Validity of findings was assessed in another five ESR1-mutated tumours progressing on AI. RESULTS: Eighty-nine mutations were identified in 41 matched pairs (PIK3CA in 27%; CDH1 in 20%). ESR1 (n = 5), ERBB2 (n = 1) and MAP2K4 (n = 1) had mutations in the secondary sample only. There was very high heterogeneity in gene expression between AI-resistant tumours with few patterns apparent. However, in the ESR1-mutated AI-resistant tumours, expression of four classical oestrogen-regulated genes (ERGs) was sevenfold higher than in ESR1 wild-type tumours, a finding confirmed in the second set of ESR1-mutated tumours. In ESR1 wild-type AI-resistant tumours ERG expression remained suppressed and was uncoupled from the recovery seen in proliferation. CONCLUSIONS: Major genotypic and phenotypic heterogeneity exists between AI-resistant disease. ESR1 mutations appear to drive oestrogen-regulated processes in resistant tumours.

Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing.

BACKGROUND: Circulating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with often low variant frequencies. METHODS: We developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes and a novel duplex DNA molecule identification tool for enhanced error correction. RESULTS: Modeling based on cfDNA yields from 58 patients showed that this technology, requiring 25 ng of cfDNA, could be applied to >95% of patients with metastatic colorectal cancer (mCRC). cfDNA-Seq of a 32-gene, 163.3-kbp target region detected 100% of single-nucleotide variants, with 0.15% variant frequency in spike-in experiments. Molecular barcode error correction reduced false-positive mutation calls by 97.5%. In 28 consecutively analyzed patients with mCRC, 80 out of 91 mutations previously detected by tumor tissue sequencing were called in the cfDNA. Call rates were similar for point mutations and indels. cfDNA-Seq identified typical mCRC driver mutations in patients in whom biopsy sequencing had failed or did not include key mCRC driver genes. Mutations only called in cfDNA but undetectable in matched biopsies included a subclonal resistance driver mutation to anti-EGFR antibodies in KRAS, parallel evolution of multiple PIK3CA mutations in 2 cases, and TP53 mutations originating from clonal hematopoiesis. Furthermore, cfDNA-Seq off-target read analysis allowed simultaneous genome-wide copy number profile reconstruction in 20 of 28 cases. Copy number profiles were validated by low-coverage whole-genome sequencing. CONCLUSIONS: This error-corrected, ultradeep cfDNA-Seq technology with a customizable target region and publicly available bioinformatics tools enables broad insights into cancer genomes and evolution. CLINICALTRIALSGOV IDENTIFIER: NCT02112357.

Discordance between oncotype DX recurrence score and RSPC for predicting residual risk of recurrence in ER-positive breast cancer.

PURPOSE: Oncotype DX, a gene expression assay widely employed to aid decision making on adjuvant chemotherapy use in patients with primary oestrogen receptor-positive (ER+) breast cancer, produces a recurrence score (RS) related to distant disease recurrence (DR) risk (RS%). In node-negative patients, RS can be integrated with clinicopathological parameters to derive RS-pathology-clinical (RSPC) that improves prognostic accuracy. METHODS: Data were collected on patients having clinically indicated tests with an intermediate clinical risk of distant recurrence, and for whom the decision to prescribe chemotherapy remained unclear. Correlation between RS% and RSPC scores was examined. An agreement table was constructed using risk-categorised data. Association between RS%-derived categorical risk assignments and treatment recommendation was evaluated. RESULTS: Data on 171 tests (168 patients) were available. Median DR risk by RS% was 11% (range 3-34%), by RSPC it was 15% (range 4-63%). Correlation between RS% and RSPC was 0.702 (p < 0.001). RS% classified 57.3% of cases as low-, 32.2% intermediate- and 10.5% high-risk for DR; by RSPC proportions were 33.9, 35.7, and 30.4%, respectively. The number of patients receiving chemotherapy recommendations was: 14/87 (16.1%) categorised as low-risk by RS%, 27/49 (55.1%) as intermediate-risk and 12/13 (92.3%) as high-risk. Of 149 patients recommended for endocrine treatment alone, 28 (18.8%) were categorised by RS% as low-risk but by RSPC as intermediate- or high-risk. CONCLUSIONS: In this group of patients, RSPC assessed fewer patients as low-risk and more as high-risk than did RS%. The discordances between the scores indicate that RSPC estimates of risk should be considered when selecting patients for endocrine therapy alone.

Mechanisms of Therapy Resistance in Patient-Derived Xenograft Models of BRCA1-Deficient Breast Cancer.

BACKGROUND: Although BRCA1-deficient tumors are extremely sensitive to DNA-damaging drugs and poly(ADP-ribose) polymerase (PARP) inhibitors, recurrences do occur and, consequently, resistance to therapy remains a serious clinical problem. To study the underlying mechanisms, we induced therapy resistance in patient-derived xenograft (PDX) models of BRCA1-mutated and BRCA1-methylated triple-negative breast cancer. METHODS: A cohort of 75 mice carrying BRCA1-deficient breast PDX tumors was treated with cisplatin, melphalan, nimustine, or olaparib, and treatment sensitivity was determined. In tumors that acquired therapy resistance, BRCA1 expression was investigated using quantitative real-time polymerase chain reaction and immunoblotting. Next-generation sequencing, methylation-specific multiplex ligation-dependent probe amplification (MLPA) and Target Locus Amplification (TLA)-based sequencing were used to determine mechanisms of BRCA1 re-expression in therapy-resistant tumors. RESULTS: BRCA1 protein was not detected in therapy-sensitive tumors but was found in 31 out of 42 resistant cases. Apart from previously described mechanisms involving BRCA1-intragenic deletions and loss of BRCA1 promoter hypermethylation, a novel resistance mechanism was identified in four out of seven BRCA1-methylated PDX tumors that re-expressed BRCA1 but retained BRCA1 promoter hypermethylation. In these tumors, we found de novo gene fusions that placed BRCA1 under the transcriptional control of a heterologous promoter, resulting in re-expression of BRCA1 and acquisition of therapy resistance. CONCLUSIONS: In addition to previously described clinically relevant resistance mechanisms in BRCA1-deficient tumors, we describe a novel resistance mechanism in BRCA1-methylated PDX tumors involving de novo rearrangements at the BRCA1 locus, demonstrating that BRCA1-methylated breast cancers may acquire therapy resistance via both epigenetic and genetic mechanisms.

Serial Next-Generation Sequencing of Circulating Cell-Free DNA Evaluating Tumor Clone Response To Molecularly Targeted Drug Administration.

PURPOSE: We evaluated whether next-generation sequencing (NGS) of circulating cell-free DNA (cfDNA) could be used for patient selection and as a tumor clone response biomarker in patients with advanced cancers participating in early-phase clinical trials of targeted drugs. EXPERIMENTAL DESIGN: Plasma samples from patients with known tumor mutations who completed at least two courses of investigational targeted therapy were collected monthly, until disease progression. NGS was performed sequentially on the Ion Torrent PGM platform. RESULTS: cfDNA was extracted from 39 patients with various tumor types. Treatments administered targeted mainly the PI3K-AKT-mTOR pathway (n = 28) or MEK (n = 7). Overall, 159 plasma samples were sequenced with a mean sequencing coverage achieved of 1,685X across experiments. At trial initiation (C1D1), 23 of 39 (59%) patients had at least one mutation identified in cfDNA (mean 2, range 1-5). Out of the 44 mutations identified at C1D1, TP53, PIK3CA and KRAS were the top 3 mutated genes identified, with 18 (41%), 9 (20%), 8 (18%) different mutations, respectively. Out of these 23 patients, 13 received a targeted drug matching their tumor profile. For the 23 patients with cfDNA mutation at C1D1, the monitoring of mutation allele frequency (AF) in consecutive plasma samples during treatment with targeted drugs demonstrated potential treatment associated clonal responses. Longitudinal monitoring of cfDNA samples with multiple mutations indicated the presence of separate clones behaving discordantly. Molecular changes at cfDNA mutation level were associated with time to disease progression by RECIST criteria. CONCLUSIONS: Targeted NGS of cfDNA has potential clinical utility to monitor the delivery of targeted therapies.