The Impact of RNA-DNA Hybrids on Genome Integrity in Bacteria.

During the essential processes of DNA replication and transcription, RNA-DNA hybrid intermediates are formed that pose significant risks to genome integrity when left unresolved. To manage RNA-DNA hybrids, all cells rely on RNase H family enzymes that specifically cleave the RNA portion of the many different types of hybrids that form in vivo. Recent experimental advances have provided new insight into how RNA-DNA hybrids form and the consequences to genome integrity that ensue when persistent hybrids remain unresolved. Here we review the types of RNA-DNA hybrids, including R-loops, RNA primers, and ribonucleotide misincorporations, that form during DNA replication and transcription and discuss how each type of hybrid can contribute to genome instability in bacteria. Further, we discuss how bacterial RNase HI, HII, and HIII and bacterial FEN enzymes contribute to genome maintenance through the resolution of hybrids.

Clinicopathological predictors of survival in resected primary lung adenocarcinoma.

AIMS: Primary lung adenocarcinoma consists of a spectrum of clinical and pathological subtypes that may impact on overall survival (OS). Our study aims to evaluate the impact of adenocarcinoma subtype and intra-alveolar spread on survival after anatomical lung resection and identify different prognostic factors based on stage and histological subtype. METHODS: Newly diagnosed patients undergoing anatomical lung resections without induction therapy, for pT1-3, N0-2 lung adenocarcinoma from April 2011 to March 2013, were included. The effect of clinical-pathological factors on survival was retrospectively assessed. RESULTS: Two hundred and sixty-two patients were enrolled. The 1-year, 3-year and 5-year OS were 88.8%, 64.3% and 51.1%, respectively. Univariate analysis showed lymphovascular, parietal pleural and chest wall invasion to confer a worse 1-year and 5-year prognosis (all p<0.0001). Solid predominant adenocarcinomas exhibited a significantly worse OS (p=0.014). Multivariate analysis did not identify solid subtype as an independent prognostic factor; however, identified stage >IIa, lymphovascular invasion (p=0.002) and intra-alveolar spread (p=0.009) as significant independent predictors of worse OS. Co-presence of intra-alveolar spread and solid predominance significantly reduced OS. Disease-free survival (DFS) was reduced with parietal pleural (p=0.0007) and chest wall invasion (p<0.0001), however, adenocarcinoma subtype had no significant impact on DFS. CONCLUSIONS: Our study demonstrates that solid predominant adenocarcinoma, intra-alveolar spread and lymphovascular invasion confer a worse prognosis and should be used as a prognostic tool to determine appropriate adjuvant treatment.

RecA Is Required for the Assembly of RecN into DNA Repair Complexes on the Nucleoid.

Homologous recombination requires the coordinated effort of several proteins to complete break resection, homologous pairing, and resolution of DNA crossover structures. RecN is a conserved bacterial protein important for double-strand break repair and is a member of the structural maintenance of chromosomes (SMC) protein family. Current models in Bacillus subtilis propose that RecN responds to double-stranded breaks prior to RecA and end processing, suggesting that RecN is among the very first proteins responsible for break detection. Here, we investigate the contribution of RecA and end processing by AddAB to RecN recruitment into repair foci in vivo. Using this approach, we found that recA is required for RecN-green fluorescent protein (GFP) focus formation on the nucleoid during normal growth and in response to DNA damage. In the absence of recA function, RecN foci form in a low percentage of cells, RecN localizes away from the nucleoid, and RecN fails to assemble in response to DNA damage. In contrast, we show that the response of RecA-GFP foci to DNA damage is unchanged in the presence or absence of recN. In further support of RecA activity preceding RecN, we show that ablation of the double-strand break end-processing enzyme addAB results in a failure of RecN to form foci in response to DNA damage. With these results, we conclude that RecA and end processing function prior to RecN, establishing a critical step for the recruitment and participation of RecN during DNA break repair in Bacillus subtilis. IMPORTANCE Homologous recombination is important for the repair of DNA double-strand breaks. RecN is a highly conserved protein that has been shown to be important for sister chromatid cohesion and for surviving break-inducing clastogens. Here, we show that the assembly of RecN into repair foci on the bacterial nucleoid requires the end-processing enzyme AddAB and the recombinase RecA. In the absence of either recA or end-processing RecN-GFP, foci are no longer DNA damage inducible, and foci form in a subset of cells as large complexes in regions away from the nucleoid. Our results establish the stepwise order of action, where double-strand break end processing and RecA association precede the participation of RecN in break repair in Bacillus subtilis.

RnhP is a plasmid-borne RNase HI that contributes to genome maintenance in the ancestral strain Bacillus subtilis NCIB 3610.

RNA-DNA hybrids form throughout the chromosome during normal growth and under stress conditions. When left unresolved, RNA-DNA hybrids can slow replication fork progression, cause DNA breaks, and increase mutagenesis. To remove hybrids, all organisms use ribonuclease H (RNase H) to specifically degrade the RNA portion. Here we show that, in addition to chromosomally encoded RNase HII and RNase HIII, Bacillus subtilis NCIB 3610 encodes a previously uncharacterized RNase HI protein, RnhP, on the endogenous plasmid pBS32. Like other RNase HI enzymes, RnhP incises Okazaki fragments, ribopatches, and a complementary RNA-DNA hybrid. We show that while chromosomally encoded RNase HIII is required for pBS32 hyper-replication, RnhP compensates for the loss of RNase HIII activity on the chromosome. Consequently, loss of RnhP and RNase HIII impairs bacterial growth. We show that the decreased growth rate can be explained by laggard replication fork progression near the terminus region of the right replichore, resulting in SOS induction and inhibition of cell division. We conclude that all three functional RNase H enzymes are present in B. subtilis NCIB 3610 and that the plasmid-encoded RNase HI contributes to chromosome stability, while the chromosomally encoded RNase HIII is important for chromosome stability and plasmid hyper-replication.

An immunotherapy survivor population: health-related quality of life and toxicity in patients with metastatic melanoma treated with immune checkpoint inhibitors.

PURPOSE: The immune checkpoint inhibitors (ICIs) have resulted in subgroups of patients with metastatic melanoma achieving high-quality durable responses. Metastatic melanoma survivors are a new population in the era of cancer survivorship. The aim of this study was to evaluate metastatic melanoma survivors in terms of health-related quality of life (HRQoL), immune-related adverse events (irAEs) and exposure to immunosuppressive agents in a large single centre in the UK. METHODS: We defined the survivor population as patients with a diagnosis of metastatic melanoma who achieved a durable response to an ICI and had been followed-up for a minimum of 12 months from initiation of ICI without disease progression. HRQoL was assessed using SF-36. Electronic health records were accessed to collect data on demographics, treatments, irAEs and survival. HRQoL data was compared with two norm-based datasets. RESULTS: Eighty-four metastatic melanoma survivors were eligible and 87% (N = 73) completed the SF-36. ICI-related toxicity of any grade occurred in 92% of patients and 43% had experienced a grade 3 or 4 toxicity. Almost half (49%) of the patients required steroids for the treatment of ICI-related toxicity, whilst 14% required treatment with an immunosuppressive agent beyond steroids. Melanoma survivors had statistically significant lower HRQoL scores with regard to physical, social and physical role functioning and general health compared with the normative population. There was a trend towards inferior scores in patients with previous exposure to ipilimumab compared with those never exposed to ipilimumab. CONCLUSIONS: Our results show that metastatic melanoma survivors have potentially experienced significant ICI-related toxicity and experience significant impairments in specific HRQoL domains. Future service planning is required to meet this population's unique survivorship needs.

Long-term benefit from immune modulation and anti-inflammatory treatment in metastatic mesothelioma.

A 64 year old male heating engineer was investigated for a persistent cough and found to have epithelioid mesothelioma with pleural effusion, lung nodules and increased thoracic lymph nodes. He declined standard of care treatment following his own research and he was enrolled in a named patient programme of IMM-101. He was advised to correct his low vitamin D3 level and to start using anti-inflammatories such as aspirin, bromelain and low dose Naltrexone. At review one year later a CT scan showed no change and he continued on the regimen. Four years after the diagnosis a CT scan showed that there was a modest but definite progression of the left malignant pleural thickening, and a new right-sided effusion, enlargement of several intrathoracic nodes which had been noted on the early scans. The chest wall lump eventually broke down and required local radiotherapy. He then developed abdominal pain and found to have peritoneal disease. Last year he obtained the cannabinoids CBD and THC which slowed down the disease and a CT scan after he had been on this for six months, showed that his disease was fairly stable with marginal progression.

Invasive cervical cancer after treatment of CIN.

INTRODUCTION: A historical audit of 30 post-treatment cervical cancers (10% of 289 cancers, 1999-2016) compared with a one-year-equivalent control group treated for cervical intraepithelial neoplasia (CIN) grade 3 (n = 164). MATERIALS AND METHODS: We compared history and follow up of cancer patients and controls and reviewed initial excision biopsies preceding cancer and, in 41% of controls, high-grade recurrence (n = 17) or consistently negative follow-up (n = 51). RESULTS: Either abnormal post-excision cytology without high-risk human papillomavirus (hrHPV) tests or immediate re-excision was recorded in 70% (19 of 27) of patients with squamous cell carcinoma (SCC). Negative investigations including cytology, colposcopy, re-excision, hysteroscopy, hrHPV, and/or treatment default were recorded in 83% (25 of 30) of all cancers. The mean interval between initial excision and cancer diagnosis was 79.8 ± 30.1 months versus 11.2 ± 30.1 months for CIN3 recurrence. Eight, 13, and 9 patients with cancer had initial excision at age 20-34, 35-49, and 50+ years, respectively, compared with 71%, 23%, and 5% of controls. CIN3 more often preceded SCC than CIN2 (22:1); 5 of 30 initial excisions were originally reported as negative after severe dyskaryosis. No SCC or CIN3 recurrence followed complete excision. Depth of CIN3 2+ mm (20 of 82 reviewed) was strongly associated with cancer/high-grade recurrence or early stromal invasion on review (18 of 20; 90%). Discrepancies were found on review in 10% of biopsies and as occasional abnormal cells in 9 of 34 cytology slides. CONCLUSIONS: Residual disease may be inconspicuous or absent on cytology, colposcopy, and/or histology. Management taking account of risk of recurrence (age, CIN3 depth, incomplete initial excision) could avoid some post-treatment cancers.

Non-invasive classification of non-small cell lung cancer: a comparison between random forest models utilising radiomic and semantic features.

OBJECTIVE: Non-invasive distinction between squamous cell carcinoma and adenocarcinoma subtypes of non-small-cell lung cancer (NSCLC) may be beneficial to patients unfit for invasive diagnostic procedures or when tissue is insufficient for diagnosis. The purpose of our study was to compare the performance of random forest algorithms utilizing CT radiomics and/or semantic features in classifying NSCLC. METHODS: Two thoracic radiologists scored 11 semantic features on CT scans of 106 patients with NSCLC. A set of 115 radiomics features was extracted from the CT scans. Random forest models were developed from semantic (RM-sem), radiomics (RM-rad), and all features combined (RM-all). External validation of models was performed using an independent test data set (n = 100) of CT scans. Model performance was measured with out-of-bag error and area under curve (AUC), and compared using receiver-operating characteristics curve analysis on the test data set. RESULTS: The median (interquartile-range) error rates of the models were: RF-sem 24.5 % (22.6 - 37.5 %), RF-rad 35.8 % (34.9 - 38.7 %), and RM-all 37.7 % (37.7 - 37.7). On training data, both RF-rad and RF-all gave perfect discrimination (AUC = 1), which was significantly higher than that achieved by RF-sem (AUC = 0.78; p < 0.0001). On test data, however, RM-sem model (AUC = 0.82) out-performed RM-rad and RM-all (AUC = 0.5 and AUC = 0.56; p < 0.0001), neither of which was significantly different from random guess ( p = 0.9 and 0.6 respectively). CONCLUSION: Non-invasive classification of NSCLC can be done accurately using random forest classification models based on well-known CT-derived descriptive features. However, radiomics-based classification models performed poorly in this scenario when tested on independent data and should be used with caution, due to their possible lack of generalizability to new data. ADVANCES IN KNOWLEDGE: Our study describes novel CT-derived random forest models based on radiologist-interpretation of CT scans (semantic features) that can assist NSCLC classification when histopathology is equivocal or when histopathological sampling is not possible. It also shows that random forest models based on semantic features may be more useful than those built from computational radiomic features.

Investigating the histopathologic correlates of 18F-FDG PET heterogeneity in non-small-cell lung cancer.

PURPOSE: Despite the growing use of fluorine-18-fluorodeoxyglucose (F-FDG) PET texture analysis to measure intratumoural heterogeneity in cancer research, the biologic basis of F-FDG PET-derived texture variables is poorly understood. We aimed to assess correlations between F-FDG PET-derived texture variables and whole-slide image (WSI)-derived metrics of tumour cellularity and spatial heterogeneity. PATIENTS AND METHODS: Twenty-two patients with non-small-cell lung cancer prospectively underwent F-FDG PET imaging before tumour resection. We tested nine F-FDG PET parameters: metabolically active tumour volume, total lesion glycolysis, mean standardized uptake value (SUVmean), first-order entropy, energy, skewness, kurtosis, grey-level co-occurrence matrix entropy and lacunarity (SUV-lacunarity). From the haematoxylin and eosin-stained WSIs, we derived mean tumour-cell density (MCD) and lacunarity (path-lacunarity). Spearman's correlation analysis and agglomerative hierarchical clustering were performed to assess variable associations. RESULTS: Tumour volumes ranged from 2.2 to 74 cm (median: 17.9 cm). MCD correlated positively with total lesion glycolysis (rs: 0.46, P: 0.007) and SUVmean (rs : 0.55; P: 0.008) and negatively with skewness and kurtosis (rs: -0.47 for both; P: 0.028 and 0.026, respectively). SUV-lacunarity and path-lacunarity were positively correlated (rs: 0.5; P: 0.018). On cluster analysis, larger tumours trended towards higher SUVmean and entropy with a predominance of tightly concentrated high SUV-voxels (negative skewness and low kurtosis on the histogram); on WSI analysis such larger tumours also displayed generally higher MCD and low SUV-lacunarity and path-lacunarity. CONCLUSION: Our data suggest that histopathological MCD and lacunarity are associated with several commonly used F-FDG PET-derived indices including SUV-lacunarity, metabolically active tumour volume, SUVmean, entropy, skewness, and kurtosis, and thus may explain the biological basis of F-FDG PET-uptake heterogeneity in non-small-cell lung cancer.

Imaging Heterogeneity in Lung Cancer: Techniques, Applications, and Challenges.

OBJECTIVE: Texture analysis involves the mathematic processing of medical images to derive sets of numeric quantities that measure heterogeneity. Studies on lung cancer have shown that texture analysis may have a role in characterizing tumors and predicting patient outcome. This article outlines the mathematic basis of and the most recent literature on texture analysis in lung cancer imaging. We also describe the challenges facing the clinical implementation of texture analysis. CONCLUSION: Texture analysis of lung cancer images has been applied successfully to FDG PET and CT scans. Different texture parameters have been shown to be predictive of the nature of disease and of patient outcome. In general, it appears that more heterogeneous tumors on imaging tend to be more aggressive and to be associated with poorer outcomes and that tumor heterogeneity on imaging decreases with treatment. Despite these promising results, there is a large variation in the reported data and strengths of association.

Pleurectomy/decortication is superior to extrapleural pneumonectomy in the multimodality management of patients with malignant pleural mesothelioma.

INTRODUCTION: To compare the outcomes of two different multimodality regimens involving neoadjuvant chemotherapy, extrapleural pneumonectomy (EPP) and adjuvant radiotherapy versus pleurectomy/decortication (P/D), hyperthermic pleural lavage with povidone-iodine, and adjuvant chemotherapy in patients with malignant pleural mesothelioma. METHODS: Nonrandomized prospective study of patients treated by multimodality therapy and operated on between January 2004 and June 2011. Second-line treatments were administered when appropriate. Survival and prognostic factors were analyzed by the Kaplan Meier method, log rank test, and Cox regression analysis. RESULTS: Twenty-five consecutive patients received neoadjuvant chemotherapy, 22 underwent EPP, and 17 received adjuvant radiotherapy. Over the same period, 54 consecutive patients underwent P/D and hyperthermic pleural lavage and received prophylactic radiotherapy and adjuvant chemotherapy. The 30-day mortality rate was 4.5%in the EPP group and nil in the P/D group. Fifteen patients (68%) in the EPP group and 15 (27.7%) in the P/D group experienced complications. There were no differences between the EPP and P/D groups for age, sex, histology, pathologic stage, and nodal status. Trimodality therapy was completed by 68%of the patients in the EPP group and 100%in the P/D group. Survival was significantly better in the P/D group: median survival was 23 months versus 12.8 months, 2-year survival was 49%versus 18.2 %, and 5-year survival was 30.1%versus 9%, respectively (p = 0.004). At multivariate analysis, epithelioid histology, P/D, and completeness of resection were independent prognostic factors. CONCLUSIONS: In our experience, P/D, hyperthermic pleural lavage with povidone-iodine, and adjuvant chemotherapy were superior to neoadjuvant chemotherapy, EPP, and adjuvant radiotherapy.

Screening for EGFR and KRAS mutations in endobronchial ultrasound derived transbronchial needle aspirates in non-small cell lung cancer using COLD-PCR.

EGFR mutations correlate with improved clinical outcome whereas KRAS mutations are associated with lack of response to tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Endobronchial ultrasound (EBUS)-transbronchial needle aspiration (TBNA) is being increasingly used in the management of NSCLC. Co-amplification at lower denaturation temperature (COLD)-polymerase chain reaction (PCR) (COLD-PCR) is a sensitive assay for the detection of genetic mutations in solid tumours. This study assessed the feasibility of using COLD-PCR to screen for EGFR and KRAS mutations in cytology samples obtained by EBUS-TBNA in routine clinical practice. Samples obtained from NSCLC patients undergoing EBUS-TBNA were evaluated according to our standard clinical protocols. DNA extracted from these samples was subjected to COLD-PCR to amplify exons 18-21 of EGFR and exons two and three of KRAS followed by direct sequencing. Mutation analysis was performed in 131 of 132 (99.3%) NSCLC patients (70F/62M) with confirmed lymph node metastases (94/132 (71.2%) adenocarcinoma; 17/132 (12.8%) squamous cell; 2/132 (0.15%) large cell neuroendocrine; 1/132 (0.07%) large cell carcinoma; 18/132 (13.6%) NSCL-not otherwise specified (NOS)). Molecular analysis of all EGFR and KRAS target sequences was achieved in 126 of 132 (95.5%) and 130 of 132 (98.4%) of cases respectively. EGFR mutations were identified in 13 (10.5%) of fully evaluated cases (11 in adenocarcinoma and two in NSCLC-NOS) including two novel mutations. KRAS mutations were identified in 23 (17.5%) of fully analysed patient samples (18 adenocarcinoma and five NSCLC-NOS). We conclude that EBUS-TBNA of lymph nodes infiltrated by NSCLC can provide sufficient tumour material for EGFR and KRAS mutation analysis in most patients, and that COLD-PCR and sequencing is a robust screening assay for EGFR and KRAS mutation analysis in this clinical context.

Extended right pneumonectomy in an adult with a double aortic arch: a therapeutic dilemma.

We report the case of a 69-year-old man presenting with a primary right lung cancer and a complete double aortic arch. An extended right pneumonectomy was successfully performed and the patient remained well at the one-year follow-up. We discuss the surgical approach and the technical considerations imposed by this rare vascular abnormality.

Lung mass appearing as a dilated left ventricle on Tc-99m tetrofosmin myocardial perfusion scintigraphy.

Tc-99m tetrofosmin is a common tracer used in myocardial perfusion scintigraphy. Several benign and malignant tumors also take up tetrofosmin. We present a case of a 60-year-old woman with a history of a left lung mass awaiting resection. The patient was referred for a myocardial perfusion scan for preoperative risk assessment. The myocardial perfusion scan revealed a large cavitated lesion mimicking a dilated left ventricle and the CT scan revealed a large mass in the left lung with central necrosis displacing the heart and mediastinum. The patient underwent thoracotomy with resection of the mass and the histology confirmed atypical carcinoid. This case highlights noncardiac uptake of Tc-99m tetrofosmin in an atypical carcinoid.