Developmental transcription factor NFIB is a putative target of oncofetal miRNAs and is associated with tumour aggressiveness in lung adenocarcinoma.

Genes involved in fetal lung development are thought to play crucial roles in the malignant transformation of adult lung cells. Consequently, the study of lung tumour biology in the context of lung development has the potential to reveal key developmentally relevant genes that play critical roles in lung cancer initiation/progression. Here, we describe for the first time a comprehensive characterization of miRNA expression in human fetal lung tissue, with subsequent identification of 37 miRNAs in non-small cell lung cancer (NSCLC) that recapitulate their fetal expression patterns. Nuclear factor I/B (NFIB), a transcription factor essential for lung development, was identified as a potential frequent target for these 'oncofetal' miRNAs. Concordantly, analysis of NFIB expression in multiple NSCLC independent cohorts revealed its recurrent underexpression (in ∼40-70% of tumours). Interrogation of NFIB copy number, methylation, and mutation status revealed that DNA level disruption of this gene is rare, and further supports the notion that oncofetal miRNAs are likely the primary mechanism responsible for NFIB underexpression in NSCLC. Reflecting its functional role in regulating lung differentiation, low expression of NFIB was significantly associated with biologically more aggressive subtypes and, ultimately, poorer survival in lung adenocarcinoma patients. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Distinct evolutionary trajectories of primary high-grade serous ovarian cancers revealed through spatial mutational profiling.

High-grade serous ovarian cancer (HGSC) is characterized by poor outcome, often attributed to the emergence of treatment-resistant subclones. We sought to measure the degree of genomic diversity within primary, untreated HGSCs to examine the natural state of tumour evolution prior to therapy. We performed exome sequencing, copy number analysis, targeted amplicon deep sequencing and gene expression profiling on 31 spatially and temporally separated HGSC tumour specimens (six patients), including ovarian masses, distant metastases and fallopian tube lesions. We found widespread intratumoural variation in mutation, copy number and gene expression profiles, with key driver alterations in genes present in only a subset of samples (eg PIK3CA, CTNNB1, NF1). On average, only 51.5% of mutations were present in every sample of a given case (range 10.2-91.4%), with TP53 as the only somatic mutation consistently present in all samples. Complex segmental aneuploidies, such as whole-genome doubling, were present in a subset of samples from the same individual, with divergent copy number changes segregating independently of point mutation acquisition. Reconstruction of evolutionary histories showed one patient with mixed HGSC and endometrioid histology, with common aetiologic origin in the fallopian tube and subsequent selection of different driver mutations in the histologically distinct samples. In this patient, we observed mixed cell populations in the early fallopian tube lesion, indicating that diversity arises at early stages of tumourigenesis. Our results revealed that HGSCs exhibit highly individual evolutionary trajectories and diverse genomic tapestries prior to therapy, exposing an essential biological characteristic to inform future design of personalized therapeutic solutions and investigation of drug-resistance mechanisms.

Nucleic acid quantity and quality from paraffin blocks: defining optimal fixation, processing and DNA/RNA extraction techniques.

Although the extraction and analysis of nucleic acids from formalin-fixed paraffin-embedded tissues is a routine and growing part of pathology practice, no generally accepted recommendations exist to guide laboratories in their selection of tissue fixation, processing and DNA/RNA extraction techniques. The aim of this study was to determine how fixation method and length, paraffin embedding, processing conditions and nucleic acid extraction methods affect quality and quantity of DNA and RNA, and their performance in downstream applications. Nine tissue samples were subjected to freezing, fixation in formalin for <24 h and 7 days followed by conventional processing, and fixation in molecular fixative for <24 h and 7 days followed by rapid processing. DNA and RNA were isolated using in-house extraction and commercial kits, and assessed by PCR reactions for amplicons with varying sizes ranging from 268 to 1327 bp and one-step RT-PCR for 621 bp and 816 bp amplicons of housekeeping genes. Molecular fixative (MF) appeared to perform well under nearly all circumstances (extraction methods, fixation lengths and longer amplicons), often performing as well as frozen samples. Formalin fixation generally performed well only for shorter length amplicons and short fixation (<24 h). WaxFree kit showed consistently higher success rates for DNA and poorer rates for RNA. RecoverAll kit generally performed suboptimally in combination with prolonged formalin fixation. In conclusion, the Molecular Fixative regardless of fixation length, and the rapid tissue processing system were able to preserve large DNA and RNA fragments in paraffin blocks, making these techniques preferable for use in downstream molecular diagnostic assays.

Transitional cell carcinoma of the ovary is related to high-grade serous carcinoma and is distinct from malignant brenner tumor.

Transitional cell tumors of the ovary include benign, borderline (atypically proliferating), and malignant Brenner tumors (BT), as well as transitional cell carcinoma (TCC). Some TCCs could conceivably be examples of malignant BT where the benign component has been overgrown. Our objectives were: (A) compare the immunophenotypes of BT and TCC and (B) examine a large cohort of ovarian carcinomas for cases with the immunophenotype of BT and transitional features but lacking a benign BT component. Seven BTs (3 benign, 3 borderline/atypically proliferating, 1 malignant) and 7 TCCs were stained for WT1, ER, p53, and p16(INK4a). The BTs were negative for WT1, p53 overexpression, ER (except for weak positivity in 1), and negative or weakly positive for p16(INK4a). In contrast, the TCCs stained as follows: 4/6 positive for WT1, 5/7 positive for ER, 2/7 strongly positive for p16(INK4a), and 6/7 showed abnormal p53, an immunophenotype resembling that of high-grade serous carcinoma. A database of 500 cases of ovarian carcinoma was searched and 116 showed an immunoprofile characteristic of BT: WT1 negative, ER negative, p16(INK4a) or weak positive, p53 negative (77 clear cell carcinoma, 14 endometrioid carcinoma, 12 mucinous carcinoma, 8 high-grade serous carcinoma). None of these tumors showed transitional features on review, indicating that if examples of malignant BT where there has been overgrowth of benign BT components exist, they are rare. Our results suggest that BT and TCC are unrelated, and should not be combined for classification purposes.

Disulfiram, a drug widely used to control alcoholism, suppresses the self-renewal of glioblastoma and over-rides resistance to temozolomide.

Glioblastomas (GBM) are associated with high rates of relapse. These brain tumors are often resistant to chemotherapies like temozolomide (TMZ) and there are very few treatment options available to patients. We recently reported that polo-like kinase-1 (PLK1) is associated with the proliferative subtype of GBM; which has the worst prognosis. In this study, we addressed the potential of repurposing disulfiram (DSF), a drug widely used to control alcoholism for the past six decades. DSF has good safety profiles and penetrates the blood-brain barrier. Here we report that DSF inhibited the growth of TMZ resistant GBM cells, (IC90=100 nM), but did not affect normal human astrocytes. At similar DSF concentrations, self-renewal was blocked by ~100% using neurosphere growth assays. Likewise the drug completely inhibited the self-renewal of the BT74 and GBM4 primary cell lines. Additionally, DSF suppressed growth and self-renewal of primary cells from two GBM tumors.These cells were resistant to TMZ, had unmethylated MGMT, and expressed high levels of PLK1. Consistent with its role in suppressing GBM growth, DSF inhibited the expression of PLK1 in GBM cells. Likewise, PLK1 inhibition with siRNA, or small molecules (BI-2536 or BI-6727) blocked growth of TMZ resistant cells. Our studies suggest that DSF has the potential to be repurposed for treatment of refractory GBM.