Christine Parkinson

Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA

Cancers acquire resistance to systemic treatment as a result of clonal evolution and selection. Repeat biopsies to study genomic evolution as a result of therapy are difficult, invasive and may be confounded by intra-tumour heterogeneity. Recent studies have shown that genomic alterations in solid cancers can be characterized by massively parallel sequencing of circulating cell-free tumour DNA released from cancer cells into plasma, representing a non-invasive liquid biopsy. Here we report sequencing of cancer exomes in serial plasma samples to track genomic evolution of metastatic cancers in response to therapy. Six patients with advanced breast, ovarian and lung cancers were followed over 1–2 years. For each case, exome sequencing was performed on 2–5 plasma samples (19 in total) spanning multiple courses of treatment, at selected time points when the allele fraction of tumour mutations in plasma was high, allowing improved sensitivity. For two cases, synchronous biopsies were also analysed, confirming genome-wide representation of the tumour genome in plasma. Quantification of allele fractions in plasma identified increased representation of mutant alleles in association with emergence of therapy resistance. These included an activating mutation in PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) following treatment with paclitaxel; a truncating mutation in RB1 (retinoblastoma 1) following treatment with cisplatin; a truncating mutation in MED1 (mediator complex subunit 1) following treatment with tamoxifen and trastuzumab, and following subsequent treatment with lapatinib, a splicing mutation in GAS6 (growth arrest-specific 6) in the same patient; and a resistance-conferring mutation in EGFR (epidermal growth factor receptor; T790M) following treatment with gefitinib. These results establish proof of principle that exome-wide analysis of circulating tumour DNA could complement current invasive biopsy approaches to identify mutations associated with acquired drug resistance in advanced cancers. Serial analysis of cancer genomes in plasma constitutes a new paradigm for the study of clonal evolution in human cancers.

Noninvasive Identification and Monitoring of Cancer Mutations by Targeted Deep Sequencing of Plasma DNA

Sizable genomic regions were screened and low-frequency mutations were identified in circulating DNA of cancer patients using tagged-amplicon deep sequencing (TAm-Seq). Deep Sequencing Tumor DNA in Plasma Five liters of circulating blood contain millions of copies of the genome, broken into short fragments; in cancer patients, a small fraction is circulating tumor DNA (ctDNA). An even smaller number harbor mutations that affect cancer outcome. Looking for diagnostic answers in circulating DNA is a challenge, but Forshew, Murtaza, and colleagues have risen to the occasion by developing a tagged-amplicon deep sequencing (TAm-Seq) method that can amplify and sequence large genomic regions from even single copies of ctDNA. By sequencing such large regions, the authors were able to identify low-level mutations in the plasma of patients with high-grade serous ovarian carcinomas. Forshew et al. designed primers to amplify 5995 bases that covered select regions of cancer-related genes, including TP53, EGFR, BRAF, and KRAS. In plasma obtained from 38 patients with high levels of ctDNA, the authors were able to identify mutations in TP53 at allelic frequencies of 2% to 65%. In plasma samples from one patient, they also identified a de novo mutation in EGFR that had not been detected 15 months prior in the tumor mass itself. Finally, the TAm-Seq approach was used to sequence ctDNA in plasma samples collected from two women with ovarian cancer and one woman with breast cancer at different time points, tracking as many as 10 mutations in parallel. Forshew and coauthors showed that levels of mutant alleles reflected the clinical course of the disease and its treatment—for example, stabilized disease was associated with low allelic frequency, whereas patients at relapse exhibited a rise in frequency. Through several experiments, the authors were able to show that TAm-Seq is a viable method for sequencing large regions of ctDNA. Although this provides a new way to noninvasively identify gene mutations in our blood, TAm-Seq will need to achieve a more sensitive detection limit (<2% allele frequency) to identify mutations in the plasma of patients with less advanced cancers. Nevertheless, once optimized, this “liquid biopsy” approach will be amenable to personalized genomics, where the level and type of mutations in ctDNA would inform clinical decision-making on an individual basis. Plasma of cancer patients contains cell-free tumor DNA that carries information on tumor mutations and tumor burden. Individual mutations have been probed using allele-specific assays, but sequencing of entire genes to detect cancer mutations in circulating DNA has not been demonstrated. We developed a method for tagged-amplicon deep sequencing (TAm-Seq) and screened 5995 genomic bases for low-frequency mutations. Using this method, we identified cancer mutations present in circulating DNA at allele frequencies as low as 2%, with sensitivity and specificity of >97%. We identified mutations throughout the tumor suppressor gene TP53 in circulating DNA from 46 plasma samples of advanced ovarian cancer patients. We demonstrated use of TAm-Seq to noninvasively identify the origin of metastatic relapse in a patient with multiple primary tumors. In another case, we identified in plasma an EGFR mutation not found in an initial ovarian biopsy. We further used TAm-Seq to monitor tumor dynamics, and tracked 10 concomitant mutations in plasma of a metastatic breast cancer patient over 16 months. This low-cost, high-throughput method could facilitate analysis of circulating DNA as a noninvasive “liquid biopsy” for personalized cancer genomics.

Mutation of FOXL2 in granulosa-cell tumors of the ovary

BACKGROUND Granulosa-cell tumors (GCTs) are the most common type of malignant ovarian sex cord-stromal tumor (SCST). The pathogenesis of these tumors is unknown. Moreover, their histopathological diagnosis can be challenging, and there is no curative treatment beyond surgery. METHODS We analyzed four adult-type GCTs using whole-transcriptome paired-end RNA sequencing. We identified putative GCT-specific mutations that were present in at least three of these samples but were absent from the transcriptomes of 11 epithelial ovarian tumors, published human genomes, and databases of single-nucleotide polymorphisms. We confirmed these variants by direct sequencing of complementary DNA and genomic DNA. We then analyzed additional tumors and matched normal genomic DNA, using a combination of direct sequencing, analyses of restriction-fragment-length polymorphisms, and TaqMan assays. RESULTS All four index GCTs had a missense point mutation, 402C-->G (C134W), in FOXL2, a gene encoding a transcription factor known to be critical for granulosa-cell development. The FOXL2 mutation was present in 86 of 89 additional adult-type GCTs (97%), in 3 of 14 thecomas (21%), and in 1 of 10 juvenile-type GCTs (10%). The mutation was absent in 49 SCSTs of other types and in 329 unrelated ovarian or breast tumors. CONCLUSIONS Whole-transcriptome sequencing of four GCTs identified a single, recurrent somatic mutation (402C-->G) in FOXL2 that was present in almost all morphologically identified adult-type GCTs. Mutant FOXL2 is a potential driver in the pathogenesis of adult-type GCTs.

Spatial and Temporal Heterogeneity in High-Grade Serous Ovarian Cancer: A Phylogenetic Analysis

Background The major clinical challenge in the treatment of high-grade serous ovarian cancer (HGSOC) is the development of progressive resistance to platinum-based chemotherapy. The objective of this study was to determine whether intra-tumour genetic heterogeneity resulting from clonal evolution and the emergence of subclonal tumour populations in HGSOC was associated with the development of resistant disease. Methods and Findings Evolutionary inference and phylogenetic quantification of heterogeneity was performed using the MEDICC algorithm on high-resolution whole genome copy number profiles and selected genome-wide sequencing of 135 spatially and temporally separated samples from 14 patients with HGSOC who received platinum-based chemotherapy. Samples were obtained from the clinical CTCR-OV03/04 studies, and patients were enrolled between 20 July 2007 and 22 October 2009. Median follow-up of the cohort was 31 mo (interquartile range 22–46 mo), censored after 26 October 2013. Outcome measures were overall survival (OS) and progression-free survival (PFS). There were marked differences in the degree of clonal expansion (CE) between patients (median 0.74, interquartile range 0.66–1.15), and dichotimization by median CE showed worse survival in CE-high cases (PFS 12.7 versus 10.1 mo, p = 0.009; OS 42.6 versus 23.5 mo, p = 0.003). Bootstrap analysis with resampling showed that the 95% confidence intervals for the hazard ratios for PFS and OS in the CE-high group were greater than 1.0. These data support a relationship between heterogeneity and survival but do not precisely determine its effect size. Relapsed tissue was available for two patients in the CE-high group, and phylogenetic analysis showed that the prevalent clonal population at clinical recurrence arose from early divergence events. A subclonal population marked by a NF1 deletion showed a progressive increase in tumour allele fraction during chemotherapy. Conclusions This study demonstrates that quantitative measures of intra-tumour heterogeneity may have predictive value for survival after chemotherapy treatment in HGSOC. Subclonal tumour populations are present in pre-treatment biopsies in HGSOC and can undergo expansion during chemotherapy, causing clinical relapse.

Management of solid tumours in organ-transplant recipients

Malignancy is a well-recognised complication of transplantation and can occur de novo, as a recurrence of a pre-existing malignancy, or from transmission of malignancy from the donor. Common de-novo malignancies are those of the skin and the lymphoreticular system. Various solid-organ cancers have also been reported in transplant recipients and each poses a unique management challenge in view of the unusual setting. We review solid-organ cancers in transplant recipients and their management, including surveillance and prevention.

Exploratory Analysis of TP53 Mutations in Circulating Tumour DNA as Biomarkers of Treatment Response for Patients with Relapsed High-Grade Serous Ovarian Carcinoma: A Retrospective Study

Background Circulating tumour DNA (ctDNA) carrying tumour-specific sequence alterations may provide a minimally invasive means to dynamically assess tumour burden and response to treatment in cancer patients. Somatic TP53 mutations are a defining feature of high-grade serous ovarian carcinoma (HGSOC). We tested whether these mutations could be used as personalised markers to monitor tumour burden and early changes as a predictor of response and time to progression (TTP). Methods and Findings We performed a retrospective analysis of serial plasma samples collected during routine clinical visits from 40 patients with HGSOC undergoing heterogeneous standard of care treatment. Patient-specific TP53 assays were developed for 31 unique mutations identified in formalin-fixed paraffin-embedded tumour DNA from these patients. These assays were used to quantify ctDNA in 318 plasma samples using microfluidic digital PCR. The TP53 mutant allele fraction (TP53MAF) was compared to serum CA-125, the current gold-standard response marker for HGSOC in blood, as well as to disease volume on computed tomography scans by volumetric analysis. Changes after one cycle of treatment were compared with TTP. The median TP53MAF prior to treatment in 51 relapsed treatment courses was 8% (interquartile range [IQR] 1.2%–22%) compared to 0.7% (IQR 0.3%–2.0%) for seven untreated newly diagnosed stage IIIC/IV patients. TP53MAF correlated with volumetric measurements (Pearson r = 0.59, p < 0.001), and this correlation improved when patients with ascites were excluded (r = 0.82). The ratio of TP53MAF to volume of disease was higher in relapsed patients (0.04% per cm3) than in untreated patients (0.0008% per cm3, p = 0.004). In nearly all relapsed patients with disease volume > 32 cm3, ctDNA was detected at ≥20 amplifiable copies per millilitre of plasma. In 49 treatment courses for relapsed disease, pre-treatment TP53MAF concentration, but not CA-125, was associated with TTP. Response to chemotherapy was seen earlier with ctDNA, with a median time to nadir of 37 d (IQR 28–54) compared with a median time to nadir of 84 d (IQR 42–116) for CA-125. In 32 relapsed treatment courses evaluable for response after one cycle of chemotherapy, a decrease in TP53MAF of >60% was an independent predictor of TTP in multivariable analysis (hazard ratio 0.22, 95% CI 0.07–0.67, p = 0.008). Conversely, a decrease in TP53MAF of ≤60% was associated with poor response and identified cases with TTP < 6 mo with 71% sensitivity (95% CI 42%–92%) and 88% specificity (95% CI 64%–99%). Specificity was improved when patients with recent drainage of ascites were excluded. Ascites drainage led to a reduction of TP53MAF concentration. The limitations of this study include retrospective design, small sample size, and heterogeneity of treatment within the cohort. Conclusions In this retrospective study, we demonstrated that ctDNA is correlated with volume of disease at the start of treatment in women with HGSOC and that a decrease of ≤60% in TP53MAF after one cycle of chemotherapy was associated with shorter TTP. These results provide evidence that ctDNA has the potential to be a highly specific early molecular response marker in HGSOC and warrants further investigation in larger cohorts receiving uniform treatment.

Repeatability of quantitative FDG-PET/CT and contrast enhanced CT in recurrent ovarian carcinoma: test retest measurements for tumor FDG uptake, diameter and volume

Purpose: Repeatability of baseline FDG-PET/CT measurements has not been tested in ovarian cancer. This dual-center, prospective study assessed variation in tumor 2[18F]fluoro-2-deoxy-D-glucose (FDG) uptake, tumor diameter, and tumor volume from sequential FDG-PET/CT and contrast-enhanced computed tomography (CECT) in patients with recurrent platinum-sensitive ovarian cancer. Experimental Design: Patients underwent two pretreatment baseline FDG-PET/CT (n = 21) and CECT (n = 20) at two clinical sites with different PET/CT instruments. Patients were included if they had at least one target lesion in the abdomen with a standardized uptake value (SUV) maximum (SUVmax) of ≥2.5 and a long axis diameter of ≥15 mm. Two independent reading methods were used to evaluate repeatability of tumor diameter and SUV uptake: on site and at an imaging clinical research organization (CRO). Tumor volume reads were only performed by CRO. In each reading set, target lesions were independently measured on sequential imaging. Results: Median time between FDG-PET/CT was two days (range 1–7). For site reads, concordance correlation coefficients (CCC) for SUVmean, SUVmax, and tumor diameter were 0.95, 0.94, and 0.99, respectively. Repeatability coefficients were 16.3%, 17.3%, and 8.8% for SUVmean, SUVmax, and tumor diameter, respectively. Similar results were observed for CRO reads. Tumor volume CCC was 0.99 with a repeatability coefficient of 28.1%. Conclusions: There was excellent test–retest repeatability for FDG-PET/CT quantitative measurements across two sites and two independent reading methods. Cutoff values for determining change in SUVmean, SUVmax, and tumor volume establish limits to determine metabolic and/or volumetric response to treatment in platinum-sensitive relapsed ovarian cancer. Clin Cancer Res; 20(10); 2751–60. ©2014 AACR.

Management of malignant ovarian germ cell tumors.

Malignant ovarian germ cell tumors are rare, highly curable cancers of young women. The majority of patients can be cured with either fertility-preserving surgery alone or a combination of surgery and chemotherapy. Relapses occur in 10% to 20% of patients, and the significant proportion of them can be salvaged with chemotherapy. There is no evidence that treatment for malignant ovarian germ cell tumors will adversely affect menstrual or reproductive functions, increase future pregnancy loss, or increase the risk of congenital malformations of the fetus. Late effects, such as secondary leukemia, from chemotherapy are reported but rare. Target Audience: Obstetricians & Gynecologists and Family Physicians. Learning Objective: After completing this CME activity, physicians should be better able to diagnose ovarian germ cell tumors, outline management of malignant ovarian germ cell tumors, and understand the impact of treatment on fertility and late effects.

New paradigms for BRCA1/BRCA2 testing in women with ovarian cancer: results of the Genetic Testing in Epithelial Ovarian Cancer (GTEOC) study

Background Over recent years genetic testing for germline mutations in BRCA1/BRCA2 has become more readily available because of technological advances and reducing costs. Objective To explore the feasibility and acceptability of offering genetic testing to all women recently diagnosed with epithelial ovarian cancer (EOC). Methods Between 1 July 2013 and 30 June 2015 women newly diagnosed with EOC were recruited through six sites in East Anglia, UK into the Genetic Testing in Epithelial Ovarian Cancer (GTEOC) study. Eligibility was irrespective of patient age and family history of cancer. The psychosocial arm of the study used self-report, psychometrically validated questionnaires (Depression Anxiety and Stress Scale (DASS-21); Impact of Event Scale (IES)) and cost analysis was performed. Results 232 women were recruited and 18 mutations were detected (12 in BRCA1, 6 in BRCA2), giving a mutation yield of 8%, which increased to 12% in unselected women aged <70 years (17/146) but was only 1% in unselected women aged ≥70 years (1/86). IES and DASS-21 scores in response to genetic testing were significantly lower than equivalent scores in response to cancer diagnosis (p<0.001). Correlation tests indicated that although older age is a protective factor against any traumatic impacts of genetic testing, no significant correlation exists between age and distress outcomes. Conclusions The mutation yield in unselected women diagnosed with EOC from a heterogeneous population with no founder mutations was 8% in all ages and 12% in women under 70. Unselected genetic testing in women with EOC was acceptable to patients and is potentially less resource-intensive than current standard practice.

Multidisciplinary management of malignant ovarian germ cell tumours

OBJECTIVES Malignant ovarian germ cell tumours (MOGCT) are rare cancers of young women. Limited prospective trials exist from which evidence-based management can be developed. This review summarizes the available literature concerning MOGT in order to provide the clinician with information relevant to their multidisciplinary management. METHODS MEDLINE was searched between 1966 and 2010 for all publications in English where the studied population included women diagnosed with malignant ovarian germ cell tumours. RESULTS The majority of patients can be cured with fertility-preserving surgery with or without combination chemotherapy. Long term survival approaches 100% in early stage disease and is approximately 75% in advanced stage disease. Most studies suggest that the treatment has little, if any, effect on future fertility and limited data suggest that there is no adverse effect on the future quality of life. CONCLUSION MOGCTs are rare tumours of young women the majority of which can be successfully treated with fertility-preserving surgery with or without chemotherapy with preservation of reproductive function. Minimisation of chemotherapy in good prognostic groups and improved treatment in resistant and relapsed MOGCT are important goals for the future. Further studies are needed to quantify the late adverse effects of treatment in long term survivors.