Association of quantitative MRI-based radiomic features with prognostic factors and recurrence rate in oropharyngeal squamous cell carcinoma.

Radiomics focuses on extracting a large number of quantitative imaging features and testing both their correlation with clinical characteristics and their prognostic and predictive values. We propose a radiomic approach using magnetic resonance imaging (MRI) to decode the tumor phenotype and local recurrence in oropharyngeal squamous cell carcinoma (OPSCC). The contrast-enhanced T1-weighted sequences from baseline MRI examinations of OPSCC patients treated between 2008 and 2016 were retrospectively selected. Radiomic features were extracted using the IBEX software, and hiegrarchical clustering was applied to reduce features redundancy. The association of each radiomic feature with tumor grading and stage, HPV status, loco-regional recurrence within 2 years, considered as main endpoints, was assessed by univariate analysis and then corrected for multiple testing. Statistical analysis was performed with SAS/STAT® software. Thirty-two eligible cases were identified. For each patient, 1286 radiomic features were extracted, subsequently grouped into 16 clusters. Higher grading (G3 vs. G1/G2) was associated with lower values of GOH/65Percentile and GOH/85Percentile features (p=0.04 and 0.01, respectively). Positive HPV status was associated with higher values of GOH/10Percentile (p=0.03) and lower values of GOH/90Percentile (p=0.03). Loco-regional recurrence within 2 years was associated with higher values of GLCM3/4-7Correlation (p=0.04) and lower values of GLCM3/2-1InformationMeasureCorr1 (p=0.04). Results lost the statistical significance after correction for multiple testing. T stage was significantly correlated with 9 features, 4 of which (GLCM25/180-4InformationMeasureCorr2, Shape/MeanBreadth, GLCM25/90-1InverseDiffMomentNorm, and GLCM3/6-1InformationMeasureCorr1) retained statistical significance after False Discovery Rate correction. MRI-based radiomics is a feasible and promising approach for the prediction of tumor phenotype and local recurrence in OPSCC. Some radiomic features seem to be correlated with tumor characteristics and oncologic outcome however, larger collaborative studies are warranted in order to increase the statistical power and to obtain robust and validated results.

HPV in oropharyngeal cancer: the basics to know in clinical practice.

The incidence of oropharyngeal squamous cell carcinoma (OPSCC) is rising in contrast to the decreasing incidence of carcinomas in other subsites of the head and neck, in spite of the reduced prevalence of smoking. Human papilloma virus (HPV) infection, and in particular type 16 (HPV-16), is now recognized as a significant player in the onset of HPV positive OPSCC, with different epidemiological, clinical, anatomical, radiological, behavioural, biological and prognostic characteristics from HPV negative OPSCC. Indeed, the only subsite in the head and neck with a demonstrated aetiological viral link is, at present, the oropharynx. These observations lead to questions regarding management choices for patients based on tumour HPV status with important consequences on treatment, and on the role of vaccines and targeted therapy over the upcoming years.

Viral control of the SUMO pathway: Gam1, a model system.

SUMO (small ubiquitin-related modifier) is a ubiquitin-like family member that is conjugated to its substrates through discrete enzymatic steps: activation, involving the E1 enzyme [SAE (SUMO-activating enzyme) 1-SAE2], conjugation, involving the E2 enzyme [Ubc9 (ubiquitin-conjugating enzyme 9)], and substrate modification, through the co-operation of Ubc9 and E3 protein ligases. Work from our laboratory has shown the first example of a viral protein, Gam1, that binds to the E1 heterodimer, inhibiting its function and causing a complete block of the SUMOylation pathway both in vivo and in vitro, followed by SAE1-SAE2 degradation. The mechanism by which a viral protein inactivates and subsequently degrades an essential cellular enzyme, arresting a key regulatory pathway, will be discussed. Although four distinct SUMO isoforms have been described, I will use SUMO to describe the entire system.

Activation of heat-shock response by an adenovirus is essential for virus replication.

Successful viral infection requires viruses to redirect host biochemistry to replicate the viral genome, and produce and assemble progeny virions. Cellular heat-shock responses, which are characterized as elevation and relocalization of heat-shock proteins, occur during replication of many viruses. Such responses might be host reactions to the synthesis of foreign protein, or might be irrelevant consequences of the viral need to activate transcription. Alternatively, as heat-shock proteins can facilitate protein folding, activating a heat-shock response might be a specific virus function ensuring proper synthesis of viral proteins and virions. It is not possible to determine whether heat-shock response is essential for virus replication, because the implicated viral genes (such as Ad5 EIA, ref. 10) also control other essential replication steps. Here we report that expression of Gam1, a protein encoded by the avian virus CELO (ref. 11), elevates and relocalizes hsp70 and hsp40. Gam1-negative CELO is replication-defective; however, Gam1 function can be partially replaced by either heat shock or forced hsp40 expression. Thus, an essential function of Gam1 during virus replication is to activate host heat-shock responses with hsp40 as a primary target.

Identification of a novel antiapoptotic protein, GAM-1, encoded by the CELO adenovirus.

We have developed a simple screening method to identify genes that mimic bcl-2 or adenovirus E1B 19K in enhancing cell survival after transfection and have used this method to identify such a gene in the avian adenovirus CELO. The gene encodes a novel 30-kDa nuclear protein, which we have named GAM-1, that functions comparably to Bcl-2 and adenovirus E1B 19K in blocking apoptosis. However, GAM-1 has no sequence homology to Bcl-2, E1B 19K, or any other known antiapoptotic proteins and thus defines a novel antiapoptotic function.

The complete DNA sequence and genomic organization of the avian adenovirus CELO.

The complete DNA sequence of the avian adenovirus chicken embryo lethal orphan (CELO) virus (FAV-1) is reported here. The genome was found to be 43,804 bp in length, approximately 8 kb longer than those of the human subgenus C adenoviruses (Ad2 and Ad5). This length is supported by pulsed-field gel electrophoresis analysis of genomes isolated from several related FAV-1 isolates (Indiana C and OTE). The genes for major viral structural proteins (Illa, penton base, hexon, pVI, and pVIII), as well as the 52,000-molecular-weight (52K) and 100K proteins and the early-region 2 genes and IVa2, are present in the expected locations in the genome. CELO virus encodes two fiber proteins and a different set of the DNA-packaging core proteins, which may be important in condensing the longer CELO virus genome. No pV or pIX genes are present. Most surprisingly, CELO virus possesses no identifiable E1, E3, and E4 regions. There is 5 kb at the left end of the CELO virus genome and 15 kb at the right end with no homology to Ad2. The sequences are rich in open reading frames, and it is likely that these encode functions that replace the missing El, E3, and E4 functions.

Moloney murine sarcoma virus MuSVts110 DNA: cloning, nucleotide sequence, and gene expression.

We have cloned Moloney murine sarcoma virus (MuSV) MuSVts110 DNA by assembly of polymerase chain reaction (PCR)-amplified segments of integrated viral DNA from infected NRK cells (6m2 cells) and determined its complete sequence. Previously, by direct sequencing of MuSVts110 RNA transcribed in 6m2 cells, we established that the thermosensitive RNA splicing phenotype uniquely characteristic of MuSVts110 results from a deletion of 1,487 nucleotides of progenitor MuSV-124 sequences. As anticipated, the sequence obtained in this study contained precisely this same deletion. In addition, several other unexpected sequence differences were found between MuSVts110 and MuSV-124. For example, in the noncoding region upstream of the gag gene, MuSVts110 DNA contained a 52-nucleotide tract typical of murine leukemia virus rather than MuSV-124, suggesting that MuSVts110 originated as a MuSV-helper murine leukemia virus recombinant during reverse transcription rather than from a straightforward deletion within MuSV-124. In addition, both MuSVts110 long terminal repeats contained head-to-tail duplications of eight nucleotides in the U3 region. Finally, seven single-nucleotide substitutions were found scattered throughout MuSVts110 DNA. Three of the nucleotide substitutions were in the gag gene, resulting in one coding change in p15 and one in p30. All of the remaining nucleotide changes were found in the noncoding region between the 5' long terminal repeat and the gag gene. In NIH 3T3 cells transfected with the cloned MuSVts110 DNA, the pattern of viral RNA expression conformed with that observed in cells infected with authentic MuSVts110 virus in that viral RNA splicing was 30 to 40% efficient at growth temperatures between 28 and 33 degrees C but reduced to trace levels above 37 degrees C.

Genotypic analysis of N-ethyl-N-nitrosourea-induced mutations by Taq I restriction fragment length polymorphism/polymerase chain reaction in the c-H-ras1 gene.

In genotypic mutation analysis DNA sequence changes are determined without the in vivo or in vitro selection of phenotypically altered cells. We have studied the induction of base-pair changes by N-ethyl-N-nitrosourea in Taq I endonuclease recognition site 2508-2511 (TCGA) of the c-H-ras1 gene in human fibroblasts by the restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) method. This site contains the four bases, and all 12 possible single base-pair changes can be monitored. The transition of guanine to adenine at position 2510 was the major mutation detected by lambda plaque oligonucleotide hybridization and quantitative sequence analysis of the RFLP/PCR products. It involves the G residue of the CpG sequence of the coding strand. Data calibration with an internal mutant standard indicates that absolute frequencies for this transition lie in the range of 4-12 x 10(-7). The present study documents the capacity of the RFLP/PCR approach to measure mutagen-induced base-pair changes in a specific gene sequence without the selection of a phenotypically altered cell.

Genotypic analysis of mutations in Taq I restriction recognition sites by restriction fragment length polymorphism/polymerase chain reaction.

Point mutations in somatic cells play a role in the etiology of several classes of human pathologies. Experimental procedures are required that allow the detection and quantitation of such mutations in disease-related genes in tissue biopsy samples without the need for the selection of mutated cells. We describe the genotypic analysis of single base pair mutations in the Taq I endonuclease recognition sequence TCGA, residues 2508-2511 of exon 2 of the human c-H-ras1 gene, by the restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) approach. The high thermostability of Taq I endonuclease allows the continuous removal of eventual residual wild-type sequences during the thermocycling of the PCR and reduces polymerase errors in the final RFLP/PCR product to a minimum. As few as five copies of a mutant standard containing two base pair changes in the chosen Taq I site could be rescued from 10(8) copies of wild-type DNA. Taq I RFLP/PCR holds promise for the monitoring of mutations in biochemical epidemiology.

Nickel mutagenesis: alteration of the MuSVts110 thermosensitive splicing phenotype by a nickel-induced duplication of the 3' splice site.

We investigated DNA damage caused by carcinogenic metals in a murine sarcoma virus (MuSV)-based mutagenicity assay in which mutations targeted to v-mos expression can be selected. Nickel chloride treatment of NRK cells (termed 6m2 cells) infected with MuSVts110, a retrovirus conditionally defective in viral RNA splicing and cell transformation, caused the outgrowth of transformed "revertants" with changes in the MuSVts110 RNA splicing phenotype. Cadmium and chromium treatment of 6m2 cells resulted in the selection of a second class of revertants with what appeared to be frameshift mutations allowing the translation of a readthrough gag-mos protein. In both classes of metal-induced revertants, viral gene expression was distinct from that observed in revertants arising in untreated 6m2 cultures, arguing that metal treatment did not simply enhance the rate of spontaneous reversion. In one representative nickel revertant line the operative nickel-induced mutation affecting MuSVts110 RNA splicing was a duplication of 70 bases surrounding the 3' splice site. The effect of this mutation was to direct splicing to the most downstream of the duplicated 3' sites and concomitantly relax its characteristic thermosensitivity. These data establish the mutagenic potential of nickel and provide the first example of a defined nickel-induced mutation in a mammalian gene.

Regulation of RNA splicing in gag-deficient mutants of Moloney murine sarcoma virus MuSVts110.

We investigated whether the MuSVts110 gag gene product (P58gag) can regulate the novel growth temperature dependence of MuSVts110 RNA splicing. MuSVts110 mutants with either frameshifts or deletions in the gag gene were tested for their ability to maintain the MuSVts110 splicing phenotype. Only small decreases in splicing efficiency and no changes in the thermosensitivity of viral RNA splicing were observed in MuSVts110 gag gene frameshift mutants. Deletions within the gag gene, however, variably decreased MuSVts110 splicing efficiency but had no effect on its thermosensitivity. Another class of MuSVts110 splicing mutants generated by treatment of MuSVts110-infected cells with NiCl2 was also examined. In these "nickel revertants," P58gag is made, but splicing of the viral transcript is nearly complete at all growth temperatures. The splicing of "tagged" viral RNA transcribed from a modified MuSVts110 DNA introduced into nickel revertant cells remained thermosensitive, arguing against trans effects of viral gene products on splicing efficiency. These experiments indicated that neither the MuSVts110 P58gag protein nor any other viral gene product acts in trans to regulate MuSVts110 splicing.

Differential expression of the c-erbB-2 gene in human small cell and non-small cell lung cancer.

We studied non-small cell lung cancers (NSCLC) from 60 patients for abnormalities in the c-erbB-2 gene. Eleven human lung cancer cell lines, including four derived from small cell lung cancer (SCLC) and seven derived from NSCLC were also examined for altered c-erbB-2 gene expression. Southern blot analysis of paired tumor and normal lung samples demonstrated that amplification of the c-erbB-2 gene is rare in NSCLC (2/60) and not restricted to adenocarcinomas. One patient showed an EcoRI restriction fragment length polymorphism for the c-erbB-2 locus. Four of four SCLC cell lines demonstrated minimal or nondetectable expression of c-erbB-2 mRNA compared to high levels of expression by seven of seven NSCLC lines. The highest expression levels were seen in four of four adenocarcinomas. We conclude that c-erbB-2 expression is different in SCLC compared to NSCLC and high expression of c-erbB-2 is consistently present in lung adenocarcinomas.