Ellen Heitzer

Complex Tumor Genomes Inferred from Single Circulating Tumor Cells by Array-CGH and Next-Generation Sequencing

Circulating tumor cells (CTC) released into blood from primary cancers and metastases reflect the current status of tumor genotypes, which are prone to changes. Here, we conducted the first comprehensive genomic profiling of CTCs using array-comparative genomic hybridization (CGH) and next-generation sequencing. We used the U.S. Food and Drug Administration-cleared CellSearch system, which detected CTCs in 21 of 37 patients (range, 1-202/7.5 mL sample) with stage IV colorectal carcinoma. In total, we were able to isolate 37 intact CTCs from six patients and identified in those multiple colorectal cancer-associated copy number changes, many of which were also present in the respective primary tumor. We then used massive parallel sequencing of a panel of 68 colorectal cancer-associated genes to compare the mutation spectrum in the primary tumors, metastases, and the corresponding CTCs from two of these patients. Mutations in known driver genes [e.g., adenomatous polyposis coli (APC), KRAS, or PIK3CA] found in the primary tumor and metastasis were also detected in corresponding CTCs. However, we also observed mutations exclusively in CTCs. To address whether these mutations were derived from a small subclone in the primary tumor or represented new variants of metastatic cells, we conducted additional deep sequencing of the primary tumor and metastasis and applied a customized statistical algorithm for analysis. We found that most mutations initially found only in CTCs were also present at subclonal level in the primary tumors and metastases from the same patient. This study paves the way to use CTCs as a liquid biopsy in patients with cancer, providing more effective options to monitor tumor genomes that are prone to change during progression, treatment, and relapse.

Circulating Tumor DNA as a Liquid Biopsy for Cancer

BACKGROUND Targeted therapies have markedly changed the treatment of cancer over the past 10 years. However, almost all tumors acquire resistance to systemic treatment as a result of tumor heterogeneity, clonal evolution, and selection. Although genotyping is the most currently used method for categorizing tumors for clinical decisions, tumor tissues provide only a snapshot, or are often difficult to obtain. To overcome these issues, methods are needed for a rapid, cost-effective, and noninvasive identification of biomarkers at various time points during the course of disease. Because cell-free circulating tumor DNA (ctDNA) is a potential surrogate for the entire tumor genome, the use of ctDNA as a liquid biopsy may help to obtain the genetic follow-up data that are urgently needed. CONTENT This review includes recent studies exploring the diagnostic, prognostic, and predictive potential of ctDNA as a liquid biopsy in cancer. In addition, it covers biological and technical aspects, including recent advances in the analytical sensitivity and accuracy of DNA analysis as well as hurdles that have to be overcome before implementation into clinical routine. SUMMARY Although the analysis of ctDNA is a promising area, and despite all efforts to develop suitable tools for a comprehensive analysis of tumor genomes from plasma DNA, the liquid biopsy is not yet routinely used as a clinical application. Harmonization of preanalytical and analytical procedures is needed to provide clinical standards to validate the liquid biopsy as a clinical biomarker in well-designed and sufficiently powered multicenter studies.

Establishment of tumor‐specific copy number alterations from plasma DNA of patients with cancer

With the increasing number of available predictive biomarkers, clinical management of cancer is becoming increasingly reliant on the accurate serial monitoring of tumor genotypes. We tested whether tumor‐specific copy number changes can be inferred from the peripheral blood of patients with cancer. To this end, we determined the plasma DNA size distribution and the fraction of mutated plasma DNA fragments with deep sequencing and an ultrasensitive mutation‐detection method, i.e., the Beads, Emulsion, Amplification, and Magnetics (BEAMing) assay. When analyzing the plasma DNA of 32 patients with Stage IV colorectal carcinoma, we found that a subset of the patients (34.4%) had a biphasic size distribution of plasma DNA fragments that was associated with increased circulating tumor cell numbers and elevated concentration of mutated plasma DNA fragments. In these cases, we were able to establish genome‐wide tumor‐specific copy number alterations directly from plasma DNA. Thus, we could analyze the current copy number status of the tumor genome, which was in some cases many years after diagnosis of the primary tumor. An unexpected finding was that not all patients with progressive metastatic disease appear to release tumor DNA into the circulation in measurable quantities. When we analyzed plasma DNA from 35 patients with metastatic breast cancer, we made similar observations suggesting that our approach may be applicable to a variety of tumor entities. This is the first description of such a biphasic distribution in a surprisingly high proportion of cancer patients which may have important implications for tumor diagnosis and monitoring.

Changes in Colorectal Carcinoma Genomes under Anti-EGFR Therapy Identified by Whole-Genome Plasma DNA Sequencing

Monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR), such as cetuximab and panitumumab, have evolved to important therapeutic options in metastatic colorectal cancer (CRC). However, almost all patients with clinical response to anti-EGFR therapies show disease progression within a few months and little is known about mechanism and timing of resistance evolution. Here we analyzed plasma DNA from ten patients treated with anti-EGFR therapy by whole genome sequencing (plasma-Seq) and ultra-sensitive deep sequencing of genes associated with resistance to anti-EGFR treatment such as KRAS, BRAF, PIK3CA, and EGFR. Surprisingly, we observed that the development of resistance to anti-EGFR therapies was associated with acquired gains of KRAS in four patients (40%), which occurred either as novel focal amplifications (n = 3) or as high level polysomy of 12p (n = 1). In addition, we observed focal amplifications of other genes recently shown to be involved in acquired resistance to anti-EGFR therapies, such as MET (n = 2) and ERBB2 (n = 1). Overrepresentation of the EGFR gene was associated with a good initial anti-EGFR efficacy. Overall, we identified predictive biomarkers associated with anti-EGFR efficacy in seven patients (70%), which correlated well with treatment response. In contrast, ultra-sensitive deep sequencing of KRAS, BRAF, PIK3CA, and EGFR did not reveal the occurrence of novel, acquired mutations. Thus, plasma-Seq enables the identification of novel mutant clones and may therefore facilitate early adjustments of therapies that may delay or prevent disease progression.

Hematogenous dissemination of glioblastoma multiforme

Hematogenous spread of glioblastoma multiforme (GBM) might be responsible for reported extracranial metastases and transmission of GBM by organ transplantation. Circulating Brain Tumor Cells Glioblastoma multiforme is an aggressive brain tumor that is most common in adults. It was generally thought that glioblastoma could not metastasize outside the central nervous system, and patients were even allowed to serve as organ donors. However, some reports of glioblastoma transmission through transplanted organs prompted researchers to reconsider this idea. Now, Müller et al. report that about 20% of glioblastoma patients have circulating tumor cells in their blood, suggesting that these patients should not serve as organ donors and offering new insights into the biology of this generally incurable disease. Glioblastoma multiforme (GBM) is the most frequent and aggressive brain tumor in adults. The dogma that GBM spread is restricted to the brain was challenged by reports on extracranial metastases after organ transplantation from GBM donors. We identified circulating tumor cells (CTCs) in peripheral blood (PB) from 29 of 141 (20.6%) GBM patients by immunostaining of enriched mononuclear cells with antibodies directed against glial fibrillary acidic protein (GFAP). Tumor cell spread was not significantly enhanced by surgical intervention. The tumor nature of GFAP-positive cells was supported by the absence of those cells in healthy volunteers and the presence of tumor-specific aberrations such as EGFR gene amplification and gains and losses in genomic regions of chromosomes 7 and 10. Release of CTCs was associated with EGFR gene amplification, suggesting a growth potential of these cells. We demonstrate that hematogenous GBM spread is an intrinsic feature of GBM biology.

The dynamic range of circulating tumor DNA in metastatic breast cancer

IntroductionThe management of metastatic breast cancer needs improvement. As clinical evaluation is not very accurate in determining the progression of disease, the analysis of circulating tumor DNA (ctDNA) has evolved to a promising noninvasive marker of disease evolution. Indeed, ctDNA was reported to represent a highly sensitive biomarker of metastatic cancer disease directly reflecting tumor burden and dynamics. However, at present little is known about the dynamic range of ctDNA in patients with metastatic breast cancer.MethodsIn this study, 74 plasma DNA samples from 58 patients with metastasized breast cancer were analyzed with a microfluidic device to determine the plasma DNA size distribution and copy number changes in the plasma were identified by whole-genome sequencing (plasma-Seq). Furthermore, in an index patient we conducted whole-genome, exome, or targeted deep sequencing of the primary tumor, metastases, and circulating tumor cells (CTCs). Deep sequencing was done to accurately determine the allele fraction (AFs) of mutated DNA fragments.ResultsAlthough all patients had metastatic disease, plasma analyses demonstrated highly variable AFs of mutant fragments. We analyzed an index patient with more than 100,000 CTCs in detail. We first conducted whole-genome, exome, or targeted deep sequencing of four different regions from the primary tumor and three metastatic lymph node regions, which enabled us to establish the phylogenetic relationships of these lesions, which were consistent with a genetically homogeneous cancer. Subsequent analyses of 551 CTCs confirmed the genetically homogeneous cancer in three serial blood analyses. However, the AFs of ctDNA were only 2% to 3% in each analysis, neither reflecting the tumor burden nor the dynamics of this progressive disease. These results together with high-resolution plasma DNA fragment sizing suggested that differences in phagocytosis and DNA degradation mechanisms likely explain the variable occurrence of mutated DNA fragments in the blood of patients with cancer.ConclusionsThe dynamic range of ctDNA varies substantially in patients with metastatic breast cancer. This has important implications for the use of ctDNA as a predictive and prognostic biomarker.

Neuropathic cancer pain: Prevalence, severity, analgesics and impact from the European Palliative Care Research Collaborative–Computerised Symptom Assessment study

Background: Neuropathic pain causes greater pain intensity and worse quality of life than nociceptive pain. There are no published data that confirm this in the cancer population. Aim: We hypothesised that patients with neuropathic cancer pain had more intense pain, experienced greater suffering and were treated with more analgesics than those with nociceptive cancer pain, and a neuropathic pain screening tool, painDETECT, would perform as well in those with cancer pain as is reported in those with non-cancer pain. Design: The data were obtained from an international cross-sectional observational study. Setting/Participants: A total of 1051 patients from inpatients and outpatients, with incurable cancer completed a computerised assessment on symptoms, function and quality of life. In all, 17 centres within eight countries participated. Medical data were recorded by physicians. Pain type was a clinical diagnosis recorded on the Edmonton Classification System for Cancer Pain. Results: Of the patients, 670 had pain: 534 with nociceptive pain, 113 with neuropathic pain and 23 were unclassified. Patients with neuropathic cancer pain were significantly more likely to be receiving oncological treatment, strong opioids and adjuvant analgesia and have a reduced performance status. They reported worse physical, cognitive and social function. Sensitivity and specificity of painDETECT for identifying neuropathic cancer pain was less accurate than when used in non-cancer populations. Conclusions: Neuropathic cancer pain is associated with a negative impact on daily living and greater analgesic requirements than nociceptive cancer pain. Validated assessment methods are needed to enable early identification of neuropathic cancer pain, leading to more appropriate treatment and reduced burden on patients.

Replicative DNA polymerase mutations in cancer.

Three DNA polymerases — Pol α, Pol δ and Pol ɛ — are essential for DNA replication. After initiation of DNA synthesis by Pol α, Pol δ or Pol ɛ take over on the lagging and leading strand respectively. Pol δ and Pol ɛ perform the bulk of replication with very high fidelity, which is ensured by Watson–Crick base pairing and 3′exonuclease (proofreading) activity. Yeast models have shown that mutations in the exonuclease domain of Pol δ and Pol ɛ homologues can cause a mutator phenotype. Recently, we identified germline exonuclease domain mutations (EDMs) in human POLD1 and POLE that predispose to ‘polymerase proofreading associated polyposis’ (PPAP), a disease characterised by multiple colorectal adenomas and carcinoma, with high penetrance and dominant inheritance. Moreover, somatic EDMs in POLE have also been found in sporadic colorectal and endometrial cancers. Tumors with EDMs are microsatellite stable and show an ‘ultramutator’ phenotype, with a dramatic increase in base substitutions.

p14ARF Hypermethylation Is Common but INK4a-ARF Locus or p53 Mutations Are Rare in Merkel Cell Carcinoma

Although the exact molecular mechanisms of Merkel cell carcinoma (MCC) tumorigenesis are unknown, they likely involve complex genetic alterations and mutations similar to those seen in many other cancers. In this study, we obtained MCCs from 21 elderly patients (19 women, 2 men) and analyzed their DNA for mutation of exons of interest in several tumor-suppressor genes or oncogenes known to be frequently mutated in skin cancer: p53 (exons 4-8), Ras (exons 1 and 2), c-Kit (exon 11), and the INK4a-ARF locus (encoding p14 and p16) (exons 1 and 2). Direct sequence analysis revealed p53 mutations (that is, at codons 224, 234, and 294) in three tumors (14%) and p16INK4a mutations (that is, at codon 6) in one (5%). No mutations were detected in Ha-Ras, Ki-Ras, N-Ras, c-Kit, or p14ARF. On the other hand, methylation-specific PCR revealed methylation of p14ARF promoter DNA in eight of 19 analyzable tumor samples (42%) and p16INK4a promoter DNA in one of 19 analyzable tumor samples (5%). Together, these findings suggest that p14ARF silencing may be an important mechanism in MCC tumorigenesis, and thus a potential target for therapeutic intervention in this highly aggressive tumor type.

High Quality Assessment of DNA Methylation in Archival Tissues from Colorectal Cancer Patients Using Quantitative High-Resolution Melting Analysis

High-resolution melting (HRM) analysis is a novel tool for analysis of promoter methylation. The aim of the present study was to establish and validate HRM analysis for detection of promoter methylation on archival formalin-fixed paraffin-embedded tissues from colorectal cancer patients. We first evaluated HRM assays for O(6)-methylguanine-DNA methyltransferase (MGMT) and adenomatous polyposis coli (APC) promoter methylation on a methylated DNA dilution matrix and DNA extracted from eight fresh or formalin-fixed paraffin-embedded human cancer cell lines. Then we used these assays for the analysis of MGMT and APC promoter methylation in a subset of archival formalin-fixed paraffin-embedded colorectal tumor specimens. All samples with promoter methylation of MGMT or APC and randomly selected samples without promoter methylation were analyzed twice. All results generated by HRM were validated with MGMT and APC MethyLight assays. APC and MGMT promoter methylation data were consistent and reproducible throughout the dilutions and all three replicates in the methylated DNA dilution matrix and between two experiments in clinical samples. There was high concordance between HRM and MethyLight results. HRM for APC promoter methylation revealed consistent results between fresh and formalin-fixed paraffin-embedded human cancer cell line DNA. The methylation status in archival tumor specimens from patients with colorectal cancer can therefore be determined with high quality by HRM. The ability to analyze archival tissues greatly facilitates further research and its clinical implementation.