Residual ctDNA after treatment predicts early relapse in patients with early-stage non-small cell lung cancer.

BACKGROUND: Identification of residual disease in patients with localized non-small cell lung cancer (NSCLC) following treatment with curative intent holds promise to identify patients at risk of relapse. New methods can detect circulating tumour DNA (ctDNA) in plasma to fractional concentrations as low as a few parts per million, and clinical evidence is required to inform their use. PATIENTS AND METHODS: We analyzed 363 serial plasma samples from 88 patients with early-stage NSCLC (48.9%/28.4%/22.7% at stage I/II/III), predominantly adenocarcinomas (62.5%), treated with curative intent by surgery (n = 61), surgery and adjuvant chemotherapy/radiotherapy (n = 8), or chemoradiotherapy (n = 19). Tumour exome sequencing identified somatic mutations and plasma was analyzed using patient-specific RaDaR™ assays with up to 48 amplicons targeting tumour-specific variants unique to each patient. RESULTS: ctDNA was detected before treatment in 24%, 77% and 87% of patients with stage I, II and III disease, respectively, and in 26% of all longitudinal samples. The median tumour fraction detected was 0.042%, with 63% of samples <0.1% and 36% of samples <0.01%. ctDNA detection had clinical specificity >98.5% and preceded clinical detection of recurrence of the primary tumour by a median of 212.5 days. ctDNA was detected after treatment in 18/28 (64.3%) of patients who had clinical recurrence of their primary tumour. Detection within the landmark timepoint 2 weeks to 4 months after treatment end occurred in 17% of patients, and was associated with shorter recurrence-free survival [hazard ratio (HR): 14.8, P <0.00001] and overall survival (HR: 5.48, P <0.0003). ctDNA was detected 1-3 days after surgery in 25% of patients yet was not associated with disease recurrence. Detection before treatment was associated with shorter overall survival and recurrence-free survival (HR: 2.97 and 3.14, P values 0.01 and 0.003, respectively). CONCLUSIONS: ctDNA detection after initial treatment of patients with early-stage NSCLC using sensitive patient-specific assays has potential to identify patients who may benefit from further therapeutic intervention.

Amplicon-based next-generation sequencing of plasma cell-free DNA for detection of driver and resistance mutations in advanced non-small cell lung cancer.

Background: Genomic analysis of plasma cell-free DNA is transforming lung cancer care; however, available assays are limited by cost, turnaround time, and imperfect accuracy. Here, we study amplicon-based plasma next-generation sequencing (NGS), rather than hybrid-capture-based plasma NGS, hypothesizing this would allow sensitive detection and monitoring of driver and resistance mutations in advanced non-small cell lung cancer (NSCLC). Patients and methods: Plasma samples from patients with NSCLC and a known targetable genotype (EGFR, ALK/ROS1, and other rare genotypes) were collected while on therapy and analyzed blinded to tumor genotype. Plasma NGS was carried out using enhanced tagged amplicon sequencing of hotspots and coding regions from 36 genes, as well as intronic coverage for detection of ALK/ROS1 fusions. Diagnostic accuracy was compared with plasma droplet digital PCR (ddPCR) and tumor genotype. Results: A total of 168 specimens from 46 patients were studied. Matched plasma NGS and ddPCR across 120 variants from 80 samples revealed high concordance of allelic fraction (R2 = 0.95). Pretreatment, sensitivity of plasma NGS for the detection of EGFR driver mutations was 100% (30/30), compared with 87% for ddPCR (26/30). A full spectrum of rare driver oncogenic mutations could be detected including sensitive detection of ALK/ROS1 fusions (8/9 detected, 89%). Studying 25 patients positive for EGFR T790M that developed resistance to osimertinib, 15 resistance mechanisms could be detected including tertiary EGFR mutations (C797S, Q791P) and mutations or amplifications of non-EGFR genes, some of which could be detected pretreatment or months before progression. Conclusions: This blinded analysis demonstrates the ability of amplicon-based plasma NGS to detect a full range of targetable genotypes in NSCLC, including fusion genes, with high accuracy. The ability of plasma NGS to detect a range of preexisting and acquired resistance mechanisms highlights its potential value as an alternative to single mutation digital PCR-based plasma assays for personalizing treatment of TKI resistance in lung cancer.

Tracking evolution of aromatase inhibitor resistance with circulating tumour DNA analysis in metastatic breast cancer.

Background: Selection of resistance mutations may play a major role in the development of endocrine resistance. ESR1 mutations are rare in primary breast cancer but have high prevalence in patients treated with aromatase inhibitors (AI) for advanced breast cancer. We investigated the evolution of genetic resistance to the first-line AI therapy using sequential ctDNA sampling in patients with advanced breast cancer. Patients and methods: Eighty-three patients on the first-line AI therapy for metastatic breast cancer were enrolled in a prospective study. Plasma samples were collected every 3 months to disease progression and ctDNA analysed by digital droplet PCR and enhanced tagged-amplicon sequencing (eTAm-Seq). Mutations identified in progression samples by sequencing were tracked back through samples before progression to study the evolution of mutations on therapy. The frequency of novel mutations was validated in an independent cohort of available baseline plasma samples in the Study of Faslodex versus Exemestane with or without Arimidex (SoFEA) trial, which enrolled patients with prior sensitivity to AI. Results: Of the 39 patients who progressed on the first-line AI, 56.4% (22/39) had ESR1 mutations detectable at progression, which were polyclonal in 40.9% (9/22) patients. In serial tracking, ESR1 mutations were detectable median 6.7 months (95% confidence interval 3.7-NA) before clinical progression. Utilising eTAm-Seq ctDNA sequencing of progression plasma, ESR1 mutations were demonstrated to be sub-clonal in 72.2% (13/18) patients. Mutations in RAS genes were identified in 15.4% (6/39) of progressing patients (4 KRAS, 1 HRAS, 1 NRAS). In SoFEA, KRAS mutations were detected in 21.2% (24/113) patients although there was no evidence that KRAS mutation status was prognostic for progression free or overall survival. Conclusions: Cancers progressing on the first-line AI show high levels of genetic heterogeneity, with frequent sub-clonal mutations. Sub-clonal KRAS mutations are found at high frequency. The genetic diversity of AI resistant cancers may limit subsequent targeted therapy approaches.

Osimertinib benefit in EGFR-mutant NSCLC patients with T790M-mutation detected by circulating tumour DNA.

Background: Approximately 50% of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) patients treated with EGFR tyrosine kinase inhibitors (TKIs) will acquire resistance by the T790M mutation. Osimertinib is the standard of care in this situation. The present study assesses the efficacy of osimertinib when T790M status is determined in circulating cell-free tumour DNA (ctDNA) from blood samples in progressing advanced EGFR-mutant NSCLC patients. Material and methods: ctDNA T790M mutational status was assessed by Inivata InVision™ (eTAm-Seq™) assay in 48 EGFR-mutant advanced NSCLC patients with acquired resistance to EGFR TKIs without a tissue biopsy between April 2015 and April 2016. Progressing T790M-positive NSCLC patients received osimertinib (80 mg daily). The objectives were to assess the response rate to osimertinib according to Response Evaluation Criteria in Solid Tumours (RECIST) 1.1, the progression-free survival (PFS) on osimertinib, and the percentage of T790M positive in ctDNA. Results: The ctDNA T790M mutation was detected in 50% of NSCLC patients. Among assessable patients, osimertinib gave a partial response rate of 62.5% and a stable disease rate of 37.5%. All responses were confirmed responses. After median follow up of 8 months, median PFS by RECIST criteria was not achieved (95% CI: 4-NA), with 6- and 12-months PFS of 66.7% and 52%, respectively. Conclusion(s): ctDNA from liquid biopsy can be used as a surrogate marker for T790M in tumour tissue.

TERC polymorphisms are associated both with susceptibility to colorectal cancer and with longer telomeres.

BACKGROUND AND AIMS: Shorter telomeres have been associated with increased risk of malignancy, including colorectal cancer (CRC). Telomere length is heritable and may be an intermediate phenotype linked to genetic susceptibility to CRC. METHODS: In a large sample, the study investigated whether candidate single nucleotide polymorphisms (SNP) in 'telomere biology' genes were associated with telomere length in leucocytes. SNP associated with an increased risk of CRC were searched for separately. RESULTS: Carriers of the common allele at SNP rs10936599, near the telomerase RNA component (TERC) locus, had significantly longer telomeres. It was independently found that the same rs10936599 allele was associated with increased risk of both CRC and colorectal adenomas. Neither telomere length nor CRC risk was associated with variation near telomerase reverse transcriptase or other telomere biology genes. In silico analysis showed that SNP rs2293607 was strongly correlated with rs10936599, mapped within TERC transcripts, had a predicted effect on messenger RNA folding and lay at a reported transcription factor binding site. TERC mRNA were expressed, differing only at the alleles of rs2293607, in CRC cell line HCT116. The long-telomere/CRC-risk allele was associated with higher levels of TERC mRNA and the formation of longer telomeres. CONCLUSIONS: Common genetic variation at TERC is associated with both longer telomeres and an increased risk of CRC, a potential mechanism being reduced levels of cell senescence or death. This finding is somewhat paradoxical, given retrospective studies reporting that CRC cases have shorter telomeres than controls. One possibility is that that association actually results from poorer survival in patients with longer telomeres.

APC and the three-hit hypothesis.

The seminal 'two-hit hypothesis' implicitly assumes that bi-allelic tumour suppressor gene (TSG) mutations cause loss of protein function. All subsequent events in that tumour therefore take place on an essentially null background for that TSG protein. We have shown that the two-hit model requires modification for the APC TSG, because mutant APC proteins probably retain some function and the two hits are co-selected to produce an optimal level of Wnt activation. We wondered whether the optimal Wnt level might change during tumour progression, leading to selection for more than two hits at the APC locus. Comprehensive screening of a panel of colorectal cancer (CRC) cell lines and primary CRCs showed that some had indeed acquired third hits at APC. These third hits were mostly copy number gains or deletions, but could be protein-truncating mutations. Third hits were significantly less common when the second hit at APC had arisen by copy-neutral loss of heterozygosity. Both polyploid and near-diploid CRCs had third hits, and the third hits did not simply arise as a result of acquiring a polyploid karyotype. The third hits affected mRNA and protein levels, with potential functional consequences for Wnt signalling and tumour growth. Although some third hits were probably secondary to genomic instability, others did appear specifically to target APC. Whilst it is generally believed that tumours develop and progress through stepwise accumulation of mutations in different functional pathways, it also seems that repeated targeting of the same pathway and/or gene is selected in some cancers.

Array painting reveals a high frequency of balanced translocations in breast cancer cell lines that break in cancer-relevant genes.

Chromosome translocations in the common epithelial cancers are abundant, yet little is known about them. They have been thought to be almost all unbalanced and therefore dismissed as mostly mediating tumour suppressor loss. We present a comprehensive analysis by array painting of the chromosome translocations of breast cancer cell lines HCC1806, HCC1187 and ZR-75-30. In array painting, chromosomes are isolated by flow cytometry, amplified and hybridized to DNA microarrays. A total of 200 breakpoints were identified and all were mapped to 1 Mb resolution on bacterial artificial chromosome (BAC) arrays, then 40 selected breakpoints, including all balanced breakpoints, were further mapped on tiling-path BAC arrays or to around 2 kb resolution using oligonucleotide arrays. Many more of the translocations were balanced at 1 Mb resolution than expected, either reciprocal (eight in total) or balanced for at least one participating chromosome (19 paired breakpoints). Second, many of the breakpoints were at genes that are plausible targets of oncogenic translocation, including balanced breaks at CTCF, EP300/p300 and FOXP4. Two gene fusions were demonstrated, TAX1BP1-AHCY and RIF1-PKD1L1. Our results support the idea that chromosome rearrangements may play an important role in common epithelial cancers such as breast cancer.

Analysis of copy number changes suggests chromosomal instability in a minority of large colorectal adenomas.

We have examined chromosomal-scale mutations in 34 large colorectal adenomas (CRAs). A small number of changes (median = 2, IQR = 0-4) were found by array-comparative genomic hybridization (aCGH) in most tumours. The most common changes were deletions of chromosomes 1p, 9q, 17, 19, and 22, and gains of chromosomes 13 and 21. SNP-LOH analysis and pseudo-digital SNP-PCR analysis detected occasional copy-neutral LOH. Some aCGH changes found frequently in colorectal carcinomas, such as deletions of chromosomes 4q and 18q, were very infrequent in the adenomas. Almost all copy number changes were of small magnitude, far below the predicted levels even for single copy gain/loss; investigation suggested that these changes were either artefactual or occurred in sub-clones within the tumours. In some cases, these sub-clones may have represented progression towards carcinoma, but comparison with aCGH data from carcinomas showed this to be unlikely in most cases. In two adenomas, there was evidence of a large, outlying number of copy number changes, mostly resulting from part-chromosome deletions. Overall, moreover, there was evidence of a tendency towards part-chromosome deletions-consistent with chromosomal instability (CIN)--in about one-sixth of all tumours. However, there was no evidence of CIN in the form of whole-chromosome copy number changes. Our data did not support previous contentions that CRAs tend to show chromosome breakage at fragile sites owing to CIN associated with an elevated DNA damage response. Chromosomal-scale mutations occur in some CRAs; although CIN is not the norm in these lesions, it probably affects a minority of cases.

Molecular classification and genetic pathways in hyperplastic polyposis syndrome.

Hyperplastic Polyposis (HPPS) is a poorly characterized syndrome that increases colorectal cancer (CRC) risk. We aimed to provide a molecular classification of HPPS. We obtained 282 tumours from 32 putative HPPS patients with >or= 10 hyperplastic polyps (HPs); some patients also had adenomas and CRCs. We found no good evidence of microsatellite instability (MSI) in our samples. The epithelium of HPs was monoclonal. Somatic BRAF mutations occurred in two-thirds of our patients' HPs, and KRAS2 mutations in 10%; both mutations were more common in younger cases. The respective mutation frequencies in a set of 'sporadic' HPs were 18% and 10%. Importantly, the putative HPPS patients generally fell into two readily defined groups, one set whose polyps had BRAF mutations, and another set whose polyps had KRAS2 mutations. The most plausible explanation for this observation is that there exist different forms of inherited predisposition to HPPS, and that these determine whether polyps follow a BRAF or KRAS2 pathway. Most adenomas and CRCs from our putative HPPS patients had 'classical' morphology and few of these lesions had BRAF or KRAS2 mutations. These findings suggest that tumourigenesis in HPPS does not necessarily follow the 'serrated' pathway. Although current definitions of HPPS are sub-optimal, we suggest that diagnosis could benefit from molecular analysis. Specifically, testing BRAF and KRAS2 mutations, and perhaps MSI, in multiple polyps could help to distinguish HPPS from sporadic HPs. We propose a specific model which would have diagnosed five more of our cases as HPPS compared with the WHO clinical criteria.

Investigation of pathogenic mechanisms in multiple colorectal adenoma patients without germline APC or MYH/MUTYH mutations.

Patients with multiple (5-100) colorectal adenomas (MCRAs) often have no germline mutation in known predisposition genes, but probably have a genetic origin. We collected a set of 25 MCRA patients with no detectable germline mutation in APC, MYH/MUTYH or the mismatch repair genes. Extracolonic tumours were absent in these cases. No vertical transmission of the MCRA phenotype was found. Based on the precedent of MYH-associated polyposis (MAP), we searched for a mutational signature in 241 adenomatous polyps from our MCRA cases. Somatic mutation frequencies and spectra at APC, K-ras and BRAF were, however, similar to those in sporadic colorectal adenomas. Our data suggest that the genetic pathway of tumorigenesis in the MCRA patients' tumours is very similar to the classical pathway in sporadic adenomas. In sharp contrast to MAP tumours, we did not find evidence of a specific mutational signature in any individual patient or in the overall set of MCRA cases. These results suggest that hypermutation of APC does not cause our patients' disease and strongly suggests that MAP is not a paradigm for the remaining MCRA patients. Our MCRA patients' colons showed no evidence of microadenomas, unlike in MAP and familial adenomatous polyposis (FAP). However, nuclear beta-catenin expression was significantly greater in MCRA patients' tumours than in sporadic adenomas. We suggest that, at least in some cases, the MCRA phenotype results from germline variation that acts subsequent to tumour initiation, perhaps by causing more rapid or more likely progression from microadenoma to macroadenoma.

Disease severity and genetic pathways in attenuated familial adenomatous polyposis vary greatly but depend on the site of the germline mutation.

BACKGROUND: Attenuated familial adenomatous polyposis (AFAP) is associated with germline mutations in the 5', 3', and exon 9 of the adenomatous polyposis coli (APC) gene. These mutations probably encode a limited amount of functional APC protein. METHODS AND RESULTS: We found that colonic polyp number varied greatly among AFAP patients but members of the same family tended to have more similar disease severity. 5' Mutants generally had more polyps than other patients. We analysed somatic APC mutations/loss of heterozygosity (LOH) in 235 tumours from 35 patients (16 families) with a variety of AFAP associated germline mutations. In common with two previous studies of individual kindreds, we found biallelic changes ("third hits") in some polyps. We found that the "third hit" probably initiated tumorigenesis. Somatic mutation spectra were similar in 5' and 3' mutant patients, often resembling classical FAP. In exon 9 mutants, in contrast, "third hits" were more common. Most "third hits" left three 20 amino acid repeats (20AARs) on the germline mutant APC allele, with LOH (or proximal somatic mutation) of the wild-type allele; but some polyps had loss of the germline mutant with mutation leaving one 20AAR on the wild-type allele. CONCLUSIONS: We propose that mutations, such as nt4661insA, that leave three 20AARs are preferentially selected in cis with some AFAP mutations because the residual protein function is near optimal for tumorigenesis. Not all AFAP polyps appear to need "three hits" however. AFAP is phenotypically and genetically heterogeneous. In addition to effects of different germline mutations, modifier genes may be acting on the AFAP phenotype, perhaps influencing the quantity of functional protein produced by the germline mutant allele.

A sinonasal primary Ewing's sarcoma.

Nasal fractures are a common complaint familiar to all otolaryngologists. Sinonasal primary Ewing's sarcomas are extremely rare. The case of a 9-year-old boy is presented whose nasal fracture and subsequent lateral nasal wall hematoma revealed an underlying Ewing's sarcoma. There are several unusual features in the history and clinical course of this patient. Following biopsies, immunohistochemistry proved essential in distinguishing a Ewing's sarcoma from other small cell tumours. It is important that a seemingly common condition can be the first presentation of a less common, more sinister pathology.

Lobular capillary haemangioma of the nasal vestibule: a rare entity.

The authors report a case of an 8-year-old boy with lobular capillary haemangioma (LCH) of the nasal vestibule presenting with nasal blockage, nasal discharge and epistaxis. LCH of the nasal vestibule is rarely reported in the English literature, and the authors feel that it should be considered in the differential diagnosis of lesion of the nasal vestibule. Unnecessary investigations could be avoided if its diagnosis is considered, especially in children.

Prognostic value of electrocochleography in patients with unilateral Menière's disease undergoing saccus surgery.

Although a controversial operation, saccus surgery has continued to be used in patients with severe Menière's disease in whom hearing preservation is the objective. The aim of this study was to determine if electrocochleography (ECoG) could predict the patients who would be free of vertigo, 2 and 5 years after surgery. Seventy patients with severe Menière's syndrome underwent ECoG prior to endolymphatic sac surgery. Eighty-four per cent of the patients had an abnormal electrocochleogram. The patients were interviewed 2 and 5 years after surgery. At 2 years, 4/10 (40%) patients with normal ECoG were relieved of dizziness compared to 38/51 (75%) with abnormal ECoG (P < 0.05). At 5 years, 5/8 (63%) patients with normal ECoG were relieved of dizziness compared to 30/30 (100%) with abnormal ECoG (P < 0.001). The patients with a normal ECoG do have less severe measurable hearing impairment (P < 0.05) although they are more likely (P < 0.05) to complain of constant tinnitus. Their history of dizziness is longer and their dizziness more disabling before surgery. In conclusion, patients with a normal ECoG will be less likely to benefit from saccus surgery in the first 5 years. These patients may represent a different nosological entity.