Suppressed immune microenvironment and repertoire in brain metastases from patients with resected non-small-cell lung cancer.

BACKGROUND: The tumor immune microenvironment (TIME) of lung cancer brain metastasis is largely unexplored. We carried out immune profiling and sequencing analysis of paired resected primary tumors and brain metastases of non-small-cell lung carcinoma (NSCLC). PATIENTS AND METHODS: TIME profiling of archival formalin-fixed and paraffin-embedded specimens of paired primary tumors and brain metastases from 39 patients with surgically resected NSCLCs was carried out using a 770 immune gene expression panel and by T-cell receptor beta repertoire (TCRß) sequencing. Immunohistochemistry was carried out for validation. Targeted sequencing was carried out to catalog hot spot mutations in cancer genes. RESULTS: Somatic hot spot mutations were mostly shared between both tumor sites (28/39 patients; 71%). We identified 161 differentially expressed genes, indicating inhibition of dendritic cell maturation, Th1, and leukocyte extravasation signaling pathways, in brain metastases compared with primary tumors (P < 0.01). The proinflammatory cell adhesion molecule vascular cell adhesion protein 1 was significantly suppressed in brain metastases compared with primary tumors. Brain metastases exhibited lower T cell and elevated macrophage infiltration compared with primary tumors (P < 0.001). T-cell clones were expanded in 64% of brain metastases compared with their corresponding primary tumors. Furthermore, while TCR repertoires were largely shared between paired brain metastases and primary tumors, T-cell densities were sparse in the metastases. CONCLUSION: We present findings that suggest that the TIME in brain metastases from NSCLC is immunosuppressed and comprises immune phenotypes (e.g. immunosuppressive tumor-associated macrophages) that may help guide immunotherapeutic strategies for NSCLC brain metastases.

Liquid biopsy assay for lung carcinoma using centrifuged supernatants from fine-needle aspiration specimens.

INTRODUCTION: Tumor mutation profiling is standard-of-care in lung carcinoma patients. However, comprehensive molecular profiling of small specimens, including core needle biopsy (CNB) and fine-needle aspiration (FNA) specimens, may often be inadequate due to limited tissue. Centrifuged FNA supernatants, which are typically discarded, have emerged recently as a novel liquid-based biopsy for molecular testing. In this study, we evaluate the use of lung carcinoma FNA supernatants for detecting clinically relevant mutations. METHODS: Supernatants from lung carcinoma FNA samples (n = 150) were evaluated. Samples were further analyzed using next-generation sequencing (NGS) and ultrasensitive droplet digital PCR (ddPCR). Mutation profiles in a subset of samples were compared with results derived from paired tissue samples from the same patient (n = 67) and available plasma liquid biopsy assay (n = 45). RESULTS: All 150 samples yielded adequate DNA and NGS were carried out successfully on 104 (90%) of 116 selected samples. Somatic mutations were detected in 82% of the samples and in 50% of these patients a clinically relevant mutation was identified that would qualify them for targeted therapy or a clinical trial. There was high overall concordance between the mutation profiles of supernatants and the corresponding tissue samples, with 100% concordance with concurrent FNA and 96% with concurrent CNB samples. Comparison of actionable driver mutations detected in supernatant versus plasma samples showed 84% concordance. CONCLUSIONS: FNA supernatants can provide a valuable specimen source for genotyping lung carcinoma especially in patients with insufficient tumor tissue, thereby reducing multigene mutation profiling failure rates, improving turnaround times, and avoiding repeat biopsies.

Multiplex KRASG12/G13 mutation testing of unamplified cell-free DNA from the plasma of patients with advanced cancers using droplet digital polymerase chain reaction.

Background: Cell-free DNA (cfDNA) from plasma offers easily obtainable material for KRAS mutation analysis. Novel, multiplex, and accurate diagnostic systems using small amounts of DNA are needed to further the use of plasma cfDNA testing in personalized therapy. Patients and methods: Samples of 16 ng of unamplified plasma cfDNA from 121 patients with diverse progressing advanced cancers were tested with a KRASG12/G13 multiplex assay to detect the seven most common mutations in the hotspot of exon 2 using droplet digital polymerase chain reaction (ddPCR). The results were retrospectively compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care. Results: Eighty-eight patients (73%) had KRASG12/G13 mutations in archival tumor specimens collected on average 18.5 months before plasma analysis, and 78 patients (64%) had KRASG12/G13 mutations in plasma cfDNA samples. The two methods had initial overall agreement in 103 (85%) patients (kappa, 0.66; ddPCR sensitivity, 84%; ddPCR specificity, 88%). Of the 18 discordant cases, 12 (67%) were resolved by increasing the amount of cfDNA, using mutation-specific probes, or re-testing the tumor tissue, yielding overall agreement in 115 patients (95%; kappa 0.87; ddPCR sensitivity, 96%; ddPCR specificity, 94%). The presence of ≥ 6.2% of KRASG12/G13 cfDNA in the wild-type background was associated with shorter survival (P = 0.001). Conclusion(s): Multiplex detection of KRASG12/G13 mutations in a small amount of unamplified plasma cfDNA using ddPCR has good sensitivity and specificity and good concordance with conventional clinical mutation testing of archival specimens. A higher percentage of mutant KRASG12/G13 in cfDNA corresponded with shorter survival.

Study on development of accessory sex glands in prepubertal kids using two-dimensional ultrasonography.

AIM: The present study was undertaken to study growth pattern of accessory sex glands in prepubertal kids from 2 weeks to 6 months of age using two-dimensional ultrasonography. MATERIALS AND METHODS: The study was conducted on six Beetal kids. The scanning of accessory sex glands was done in standing position using rectal probe and measurements were recorded. Data collected were statistically analyzed using one-way ANOVA followed by Duncan multiple range test was performed using the SPSS (16.0) system for windows. RESULTS: With the advancement of age all the dimensions of glands increased. Both the lobes of prostate gland showed an increase in width with advancement of age. Width of prostate above the urethra (W1) showed a significant increase at 2, 10, and 20 weeks of age, whereas non-significant increase from 2 to 8, 10 to 19, and 20 to 24 weeks of age was recorded. Width of prostate below the urethra (W2) showed a significant increase at 20 weeks of age, whereas non-significant increase was recorded during rest of period of growth. Left and right bulbourethral gland showed a similar pattern of growth with the advancement of age. The circumference dimensions increased significantly at 2, 16, 20, and 21 weeks of age for both glands. The increase was non-significant from 4 to 14, 16 to 19, and 20 to 23 weeks of age. The same pattern was observed for left and right seminal vesicular gland. CONCLUSION: Significant growth in three accessory sex glands in prepubertal kids was not observed at the same age. The trend observed was that the prostate was the first gland to show significant growth at 10 weeks of age followed by a significant increase in seminal vesicles and bulbourethral gland at 14 and 16 weeks of age, respectively.

An insight into current concepts and techniques in resin bonding to high strength ceramics.

BACKGROUND: Reliable bonding between high strength ceramics and resin composite cement is difficult to achieve because of their chemical inertness and lack of silica content. The aim of this review was to assess the current literature describing methods for resin bonding to ceramics with high flexural strength such as glass-infiltrated alumina and zirconia, densely sintered alumina and yttria-partially stabilized tetragonal zirconia polycrystalline ceramic (Y-TZP) with respect to bond strength and bond durability. METHODS: Suitable peer reviewed publications in the English language were identified through searches performed in PubMed, Google Search and handsearches. The keywords or phrases used were 'resin-ceramic bond', 'silane coupling agents', 'air particle abrasion', 'zirconia ceramic' and 'resin composite cements'. Studies from January 1989 to June 2015 were included. RESULTS: The literature demonstrated that there are multiple techniques available for surface treatments but bond strength testing under different investigations have produced conflicting results. CONCLUSIONS: Within the scope of this review, there is no evidence to support a universal technique of ceramic surface treatment for adhesive cementation. A combination of chemical and mechanical treatments might be the recommended solution. The hydrolytic stability of the resin ceramic bond should be enhanced.

Clinical massively parallel next-generation sequencing analysis of 409 cancer-related genes for mutations and copy number variations in solid tumours.

BACKGROUND: In a clinical diagnostic laboratory, we evaluated the applicability of the Ion Proton sequencer for screening 409 cancer-related genes in solid tumours. METHODS: DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue biopsy specimens of 55 solid tumours (20 with matched normal tissue) and four cell lines and screened for mutations in 409 genes using the Ion Proton system. The mutation profiles of these samples were known based on prior testing using the Ion Torrent Personal Genome Machine (46-gene hotspot panel), Sanger sequencing, or fluorescence in situ hybridisation (FISH). Concordance with retrospective findings and additional mutations were evaluated. Assay sensitivity and reproducibility were established. Gene copy number variations (CNVs) detected were confirmed by molecular inversion probe (MIP) array. RESULTS: The average Ion Proton (409-gene panel) sequencing output per run was 8 gigabases with 128 million sequencing reads. Of the 15,992 amplicons in the 409-gene panel, 90% achieved a minimum average sequencing depth of 100X. In 59 samples, the Ion Proton detected 100 of 105 expected single-nucleotide variants (SNVs) and all expected deletions (n=8), insertions (n=5), and CNVs (n=7). Five SNVs were not detected due to failed amplification of targeted regions. In 20 tumours with paired normal tissue, Ion Proton detected 37 additional somatic mutations, several in genes of high prognostic or therapeutic significance, such as MET, ALK, TP53, APC, and PTEN. MIP array analysis confirmed all CNVs detected by Ion Proton. CONCLUSIONS: The Ion Proton (409-gene panel) system was found to be well suited for use in a clinical molecular diagnostic laboratory. It can simultaneously screen 409 genes for a variety of sequence variants in multiple samples using a low input of FFPE DNA with high reproducibility and sensitivity.

Clinicopathologic characteristics and gene expression analyses of non-KRAS 12/13, RAS-mutated metastatic colorectal cancer.

BACKGROUND: KRAS mutations in codons 12 and 13 are present in ∼40% of all colorectal cancers (CRC). Activating mutations in codons 61 and 146 of KRAS and in codons 12, 13, and 61 of NRAS also occur but are less frequent. The clinicopathologic features and gene expression profiles of this latter subpopulation of RAS-mutant colorectal tumors have not yet been clearly defined but in general are treated similarly to those with KRAS 12 or 13 mutations. PATIENTS AND METHODS: Records of patients with metastatic CRC (mCRC) treated at MD Anderson Cancer Center between December 2000 and August 2012 were reviewed for RAS (KRAS or NRAS) and BRAF mutation status, clinical characteristics, and survival outcomes. To study further with an independent cohort, data from The Cancer Genome Atlas were analyzed to define a gene expression signature for patients whose tumors feature these atypical RAS mutations and explore differences with KRAS 12/13-mutated colorectal tumors. RESULTS: Among the 484 patients reviewed, KRAS 12/13, KRAS 61/146, NRAS, and BRAF mutations were detected in 47.7%, 3.0%, 4.1%, and 7.4%, respectively, of patients who were tested for each of these aberrations. Lung metastases were more common in both the KRAS 12/13-mutated and atypical RAS-mutated cohorts relative to patients with RAS/BRAF wild-type tumors. Gene expression analyses revealed similar patterns regardless of the site of RAS mutation, and in silico functional algorithms predicted that KRAS and NRAS mutations in codons 12, 13, 61, and 146 alter the protein function and drive tumorgenesis. CONCLUSIONS: Clinicopathologic characteristics, survival outcomes, functional impact, and gene expression profiling were similar between patients with KRAS 12/13 and those with NRAS or KRAS 61/146-mutated mCRC. These clinical and bioinformatic findings support the notion that colorectal tumors driven by these RAS mutations are phenotypically similar.

Miswak: A periodontist's perspective.

Meticulous plaque control on a daily routine basis is the single most important step to achieve good oral health. Herbal chewing sticks, commonly known as Miswak, are among the ancient and traditional oral hygiene aids popular in India, Pakistan, most of the Arabian countries, and several African countries. But nowadays, because of low cost, free availability, unique chemical composition, and spiritual beliefs, miswak is being used worldwide. A large number of studies have proved that miswak is as effective as, or even superior to the present day's most common oral hygiene aid, i.e., toothbrush. The aim of this review article is to discuss various pharmacological and therapeutic aspects of miswak and also to compare the effectiveness of miswak with modern toothbrushes in terms of oral hygiene practice.

Hypoxia-regulated microRNA-210 modulates mitochondrial function and decreases ISCU and COX10 expression.

The mechanisms of compromised mitochondrial function under various pathological conditions, including hypoxia, remain largely unknown. Recent studies have shown that microRNA-210 (miR-210) is induced by hypoxia under the regulation of hypoxia-inducible factor-1alpha and has an important role in cell survival under hypoxic microenvironment. Hence, we hypothesized that miR-210 has a role in regulating mitochondrial metabolism and investigated miR-210 effects on mitochondrial function in cancer cell lines under normal and hypoxic conditions. Our results demonstrate that miR-210 decreases mitochondrial function and upregulates the glycolysis, thus make cancer cells more sensitive to glycolysis inhibitor. miR-210 can also activate the generation of reactive oxygen species (ROS). ISCU (iron-sulfur cluster scaffold homolog) and COX10 (cytochrome c oxidase assembly protein), two important factors of the mitochondria electron transport chain and the tricarboxylic acid cycle have been identified as potential targets of miR-210. The unique means by which miR-210 regulates mitochondrial function reveals an miRNA-mediated link between microenvironmental stress, oxidative phosphorylation, ROS and iron homeostasis.

MicroRNA-196a targets annexin A1: a microRNA-mediated mechanism of annexin A1 downregulation in cancers.

Suppression of annexin A1 (ANXA1), a mediator of apoptosis and inhibitor of cell proliferation, is well documented in various cancers but the underlying mechanism remains unknown. We investigated whether decreased ANXA1 expression was mediated by microRNAs (miRNAs), which are small, non-coding RNAs that negatively regulate gene expression. Using Sanger miRBase, we identified miR-584, miR-196a and miR-196b as potential miRNAs targeting ANXA1. Only miRNA-196a showed significant inverse correlation with ANXA1 mRNA levels in 12 cancer cell lines of esophageal, breast and endometrial origin (Pearson's correlation -0.66, P=0.019), identifying this as the candidate miRNA targeting ANXA1. Inverse correlation was also observed in 10 esophageal adenocarcinomas (Pearson's correlation -0.64, P=0.047). Analysis of paired normal/tumor tissues from additional 10 patients revealed an increase in miR-196a in the cancers (P=0.003), accompanied by a decrease in ANXA1 mRNA (P=0.004). Increasing miR-196a levels in cells by miR-196a mimics resulted in decreased ANXA1 mRNA and protein. In addition, miR-196a mimics inhibited luciferase expression in luciferase plasmid reporter that included predicted miR-196a recognition sequence from ANXA1 3'-untranslated region confirming that miR-196a directly targets ANXA1. miR-196a promoted cell proliferation, anchorage-independent growth and suppressed apoptosis, suggesting its oncogenic potential. This study demonstrated a novel mechanism of post-transcriptional regulation of ANXA1 expression and identified miR-196a as a marker of esophageal cancer.

MYC translocation in chronic lymphocytic leukaemia is associated with increased prolymphocytes and a poor prognosis.

Chromosomal translocations that involve MYC, characteristic of Burkitt lymphoma, are rare in chronic lymphocytic leukaemia (CLL). We report the clinical, morphological, immunophenotypic, cytogenetic and molecular genetic features of eight CLL cases with MYC rearrangement. The patients, five men and three women (median age, 71 years) had bone marrow involvement and an absolute peripheral blood lymphocytosis; five had lymphadenopathy; seven had splenomegaly. Prolymphocytes were increased (>/=10%) in all cases. Six cases were classified as CLL with increased prolymphocytes (CLL/PL; prolymphocytes 10-55%), and two were classified as CLL in prolymphocytic transformation (CLL/PT; prolymphocytes >55%). All cases co-expressed CD5, CD19, and CD23; five of eight expressed ZAP-70. Of seven cases tested, four had mutated and three had unmutated IGHV genes. Conventional cytogenetic studies demonstrated t(8;14)(q24.1;q32) in five cases, t(8;22)(q24.1;q11) in two cases, and t(2;8)(p12;q24.1) in one case. Seven cases contained additional chromosomal abnormalities. All patients received combination chemotherapy. Two developed Epstein-Barr virus (EBV)-associated diffuse large B-cell lymphomas (DLBCL) that were clonally unrelated to the CLL. At follow-up, two patients are alive, four died of underlying disease, one died of EBV-associated DLBCL, and one died of an unrelated cancer. In summary, MYC rearrangement, which occurs rarely in CLL patients, is associated with increased prolymphocytes, complex cytogenetic abnormalities, and a poor prognosis.

Frequency and clinical significance of BCR-ABL mutations in patients with chronic myeloid leukemia treated with imatinib mesylate.

Mutations of the BCR-ABL kinase domain are a common mechanism of resistance to imatinib in chronic myeloid leukemia. We screened for mutations 171 patients failing imatinib therapy. Sixty-six mutations in 23 amino acids were identified in 62 (36%) patients not responding to imatinib. Phosphate-binding loop (P-loop) mutations were the most frequent (n=24; 36%). By multivariate analysis, factors associated with development of mutations were older age (P=0.026) prior interferon therapy (P=0.026), and accelerated phase or blast phase at time of imatinib failure (P=0.001). After a median follow-up of 38 months (range, 4-68 months) from the start of imatinib therapy, seven patients with non-P-loop and two with P-loop mutation died. By multivariate analysis, development of clonal evolution and higher percentage of peripheral blood basophils were associated with worse survival from the time of imatinib failure. Mutation status had no impact on survival. When survival was measured from the time therapy started, non-P-loop mutations together with duration of response and transformation at the time of failure to imatinib were associated with shorter survival. In conclusion, P-loop mutations were not associated with poor outcome, suggesting that the prognosis of patients who fail imatinib is multifactorial.

Discordant genetic changes in ovarian and endometrial endometrioid carcinomas: a potential pitfall in molecular diagnosis.

Endometrioid carcinoma simultaneously involving ovaries as well as the uterine corpus may present a diagnostic dilemma because of the difficulty in determining whether the lesions are separate primary tumors or metastases. It has been reported that the detection of clonality using microsatellite markers may be useful in solving this dilemma. To determine the usefulness of this technique, we compared the genetic alterations in microsatellite markers present in matched pairs of ovarian tumors from 12 patients. The study includes four ovarian cancer FIGO stage I and eight stage III/IV patients, and four patients also with independent endometrial carcinoma of the uterus. DNA from paraffin-embedded tissue was extracted and amplified using a multiplex polymerase chain reaction, after which the status of microsatellite instability and loss of heterozygosity in four microsatellite loci (BAT25, BAT26, D17S250, and D5S346) were determined. In the four patients with stage I ovarian cancer, four microsatellite markers were identical in one patient and three were identical in the remaining three patients. In high-stage patients, three markers were identical in at least 4/8 cases. In three of four patients with uterine involvement, three of the four markers were identical in the uterine tumor and one of the corresponding ovarian tumors. These results suggest that genetic discordance does not indicate independent origin or metastasis of the tumor but instead a progression of genetic changes at separate sites probably due to the marked genetic instability existing in these tumors. Because of these discordant genetic changes, great caution should be taken when distinguishing between primary and metastatic tumors on the basis of microsatellite markers.

Reattachment of fractured fragments of maxillary central incisors--report of a case.

Reattachment of the original tooth fragment to the fractured tooth helps in maintaining the tooth's colour, wear resistance, morphology and translucency in the restoration. This paper describes the reattachment of fractured fragments using metallic post and core in case of a 12 year old patients who suffered a complicated facture of maxillary central incisors.

Salt-tolerant mutants in glycophytic salinity response (GSR) genes in Catharanthus roseus.

The periwinkle Catharanthus roseus shares glycophytic properties of crop plants. To contribute towards an understanding of the glycophytic response to salinity, large populations of M(2) seeds having an origin in nitroso-methyl urea and ethyl methane sulphonate treatments were screened for germination with 250 mM of NaCl. Out of the nine mutant lines so recovered, which tolerated salt stress due to loss of the normal glycophytic salinity response ( GSR), the characteristics of six gsr mutants are reported here. All six, gsr-1 to gsr-6, differed from the wild-type in both seedling and adult-plant morphological characters beside being salt tolerant. The mutations in them were inherited as monogenic recessive alleles at the corresponding wild-type loci. The trans-complementation tests revealed that the gsr-1 to gsr-6 mutants specified six complementation groups. The mutant seedlings generally accumulated more proline and glycine betaine, constitutively, than the wild-type. The mutant plants transpired lower amounts of water and accumulated higher amounts of proline under drought stress. It was inferred that the products of the six GSR genes defined here are involved in the regulation of salt stress, as well as cell division, developmental and/or morphogenetic pathway(s), in C. roseus.

Frequency and risk factors for pterygium in the Barbados Eye Study.

OBJECTIVE: To describe the distribution and risk factors for pterygium in the predominantly black population of the Barbados Eye Study, which was based on a random sample of Barbadian-born citizens between the ages of 40 and 84 years. METHODS: The standardized protocol included ophthalmic and other measurements, automated perimetry, lens gradings, fundus photography, and a detailed interview. A 10% systematic sample of participants and those meeting specific criteria also received a comprehensive ophthalmologic evaluation. RESULTS: The Barbados Eye Study included 4709 participants, of whom 2978 were referred for an ophthalmologic evaluation and 2781 (93%) completed the examination. Cases of pterygium were found among 23.4% of 2617 black, 23.7% of 97 mixed (black and white), and 10.2% of 59 white participants examined. In addition to African ancestry, logistic regression analyses indicated a positive association between pterygium and age (odds ratio [OR], 1.01; 95% confidence interval [CI], 1.00-1.02), fewer years of education (OR, 1.43; 95% CI, 1.01-2.03), and an outdoor job location (OR, 1.87; 95% CI, 1.52-2.29). Having a darker skin complexion (OR, 0.66; 95% CI, 0.52-0.83), always using sunglasses outdoors (OR, 0.18; 95% CI, 0.06-0.59), and the use of prescription glasses (OR, 0.75; 95% CI, 0.60-0.93) were protective factors. CONCLUSIONS: Approximately one quarter of the black participants examined had pterygia, a frequency that was 2.5 to 3 times higher than among whites in the Barbados Eye Study and elsewhere. Pterygium was almost twice as frequent among persons who worked outdoors but was only one fifth as likely among those who always used sunglasses outdoors. Educational interventions to modify these potential exposures may assist in preventing pterygium.