Merkel cell carcinoma (MCC) is an uncommon, but highly malignant, cutaneous tumor. Merkel cell polyoma virus (MCV) has been implicated in a majority of MCC tumors; however, viralnegative tumors have been reported to be more prevalent in some geographic regions subject to high sun exposure. While the impact of MCV and viral T-antigens on MCC development has been extensively investigated, little is known about the etiology of viralnegative tumors. We performed targeted capture and massively parallel DNA sequencing of 619 cancer genes to compare the gene mutations and copy number alterations in MCV-positive (n ¼ 13) and -negative (n ¼ 21) MCC tumors and cell lines. We found that MCV-positive tumors displayed very low mutation rates, but MCV-negative tumors exhibited a high mutation burden associated with a UV-induced DNA damage signature. All viral-negative tumors harbored mutations in RB1, TP53, and a high frequency of mutations in NOTCH1 and FAT1. Additional mutated or amplified cancer genes of potential clinical importance included PI3K (PIK3CA, AKT1, PIK3CG) and MAPK (HRAS, NF1) pathway members and the receptor tyrosine kinase FGFR2. Furthermore, looking ahead to potential therapeutic strategies encompassing immune checkpoint inhibitors such as anti-PD-L1, we also assessed the status of T-cell-infiltrating lymphocytes (TIL) and PD-L1 in MCC tumors. A subset of viral-negative tumors exhibited high TILs and PD-L1 expression, corresponding with the higher mutation load within these cancers. Taken together, this study provides new insights into the underlying biology of viral-negative MCC and paves the road for further investigation into new treatment opportunities. Cancer Res; 75(24); 5228-34. Ó2015 AACR.

BackgroundClinical specimens undergoing diagnostic molecular pathology testing are fixed in formalin due to the necessity for detailed morphological assessment. However, formalin fixation can cause major issues with molecular testing, as it causes DNA damage such as fragmentation and non-reproducible sequencing artefacts after PCR amplification. In the context of massively parallel sequencing (MPS), distinguishing true low frequency variants from sequencing artefacts remains challenging. The prevalence of formalin-induced DNA damage and its impact on molecular testing and clinical genomics remains poorly understood.MethodsThe Cancer 2015 study is a population-based cancer cohort used to assess the feasibility of mutational screening using MPS in cancer patients from Victoria, Australia. While blocks were formalin-fixed and paraffin-embedded in different anatomical pathology laboratories, they were centrally extracted for DNA utilising the same protocol, and run through the same MPS platform (Illumina TruSeq Amplicon Cancer Panel). The sequencing artefacts in the 1-10% and the 10-25% allele frequency ranges were assessed in 488 formalin-fixed tumours from the pilot phase of the Cancer 2015 cohort. All blocks were less than 2.5 years of age (mean 93 days).ResultsConsistent with the signature of DNA damage due to formalin fixation, many formalin-fixed samples displayed disproportionate levels of C>T/G>A changes in the 1-10% allele frequency range. Artefacts were less apparent in the 10-25% allele frequency range. Significantly, changes were inversely correlated with coverage indicating high levels of sequencing artefacts were associated with samples with low amounts of available amplifiable template due to fragmentation. The degree of fragmentation and sequencing artefacts differed between blocks sourced from different anatomical pathology laboratories. In a limited validation of potentially actionable low frequency mutations, a NRAS G12D mutation in a melanoma was shown to be a false positive.ConclusionsThese findings indicate that DNA damage following formalin fixation remains a major challenge in laboratories working with MPS. Methodologies that assess, minimise or remove formalin-induced DNA damaged templates as part of MPS protocols will aid in the interpretation of genomic results leading to better patient outcomes.

RNA polymerase I (Pol I) transcription of ribosomal RNA genes (rDNA) is tightly regulated downstream of oncogenic pathways, and its dysregulation is a common feature in cancer. We evaluated CX-5461, the fi rst-in-class selective rDNA transcription inhibitor, in a fi rst-in-human, phase I dose-escalation study in advanced hematologic cancers. Administration of CX-5461 intravenously once every 3 weeks to 5 cohorts determined an MTD of 170 mg/m 2 , with a predictable pharmacokinetic profi le. The dose-limiting toxicity was palmar-plantar erythrodysesthesia; photosensitivity was a dose-independent adverse event (AE), manageable by preventive measures. CX-5461 induced rapid on-target inhibition of rDNA transcription, with p53 activation detected in tumor cells from one patient achieving a clinical response. One patient with anaplastic large cell lymphoma attained a prolonged partial response and 5 patients with myeloma and diffuse large B-cell lymphoma achieved stable disease as best response. CX-5461 is safe at doses associated with clinical benefi t and dermatologic AEs are manageable. SIGNIFICANCE: CX-5461 is a fi rst-in-class selective inhibitor of rDNA transcription. This fi rst-inhuman study establishes the feasibility of targeting this process, demonstrating single-agent antitumor activity against advanced hematologic cancers with predictable pharmacokinetics and a safety profi le allowing prolonged dosing. Consistent with preclinical data, antitumor activity was observed in TP53 wild-type and mutant malignancies.

ObjectivesTo identify key health outcomes, beyond morbidity and mortality, regarded as important in children and young people with neurodisability, and their parents.DesignQualitative research incorporating a thematic analysis of the data supported by the Framework Approach; the International Classification of Functioning, Disability and Health (ICF) provided a theoretical foundation.SettingThe study was conducted in community settings.ParticipantsParticipants were 54 children and young people with neurodisability: 50 participated in focus groups, and 4 in interviews; 53 parents participated: 47 in focus groups and 6 in interviews. Children/young people and parents were recruited through different networks, and were not related.ResultsChildren/young people and parents viewed health outcomes as inter-related. Achievement in some outcomes appeared valued to the extent that it enabled or supported more valued domains of health. Health outcomes prioritised by both young people and parents were: communication, mobility, pain, self-care, temperament, interpersonal relationships and interactions, community and social life, emotional well-being and gaining independence/future aspirations. Parents also highlighted their child's sleep, behaviour and/or safety.ConclusionsThose responsible for health services for children/young people with neurodisability should take account of the aspects of health identified by families. The aspects of health identified in this study provide a basis for selecting appropriate health indicators and outcome measures.

The clinical management of patients with cancer of unknown primary (CUP) is hampered by the absence of a definitive site of origin. We explored the utility of massively-parallel (next-generation) sequencing for the diagnosis of a primary site of origin and for the identification of novel treatment options. DNA enrichment by hybridization capture of 701 genes of clinical and/or biological importance, followed by massively-parallel sequencing, was performed on 16 CUP patients who had defied attempts to identify a likely site of origin. We obtained high quality data from both fresh-frozen and formalin-fixed, paraffin-embedded samples, demonstrating accessibility to routine diagnostic material. DNA copy-number obtained by massively-parallel sequencing was comparable to that obtained using oligonucleotide microarrays or quantitatively hybridized fluorescently tagged oligonucleotides. Sequencing to an average depth of 458-fold enabled detection of somatically acquired single nucleotide mutations, insertions, deletions and copy-number changes, and measurement of allelic frequency. Common cancer-causing mutations were found in all cancers. Mutation profiling revealed therapeutic gene targets and pathways in 12/16 cases, providing novel treatment options. The presence of driver mutations that are enriched in certain known tumour types, together with mutational signatures indicative of exposure to sunlight or smoking, added to clinical, pathological, and molecular indicators of likely tissue of origin. Massively-parallel DNA sequencing can therefore provide comprehensive mutation, DNA copy-number, and mutational signature data that are of significant clinical value for a majority of CUP patients, providing both cumulative evidence for the diagnosis of primary site and options for future treatment.