Monitoring non-small cell lung cancer progression and treatment response through hyaluronic acid in sputum

We evaluated whether hyaluronan (HA) levels in the sputum could be used as a noninvasive tool to predict progressive disease and treatment response, as detected in a computed tomography scan in non-small cell lung cancer (NSCLC) patients. Sputum samples were collected from 84 patients with histological confirmation of NSCLC, 33 of which were in early-stage and 51 in advanced-stage disease. Patients received systemic chemotherapy (CT) after surgery (n=36), combined CT and immunotherapy (IO) (n=15), or targeted therapy for driver mutation and disease relapse (N=4). The primary end-point was to compare sputum HA levels in two different concentrations of hypertonic saline solution with overall survival (OS) and the secondary and exploratory end-points were radiologic responses to treatment and patient outcome. Higher concentrations of HA in the sputum were significantly associated to factors related to tumor stage, phenotype, response to treatment, and outcome. In the early stage, patients with lower sputum HA levels before treatment achieved a complete tumor response after systemic CT with better progression-free survival (PFS) than those with high HA levels. We also examined the importance of the sputum HA concentration and tumor response in the 51 patients who developed metastatic disease and received CT+IO. Patients with low levels of sputum HA showed a complete tumor response in the computed tomography scan and stable disease after CT+IO treatment, as well as a better PFS than those receiving CT alone. HA levels in sputum of NSCLC patients may serve as a candidate biomarker to detect progressive disease and monitor treatment response in computed tomography scans.

Use of uracil-DNA glycosylase enzyme to reduce DNA-related artifacts from formalin-fixed and paraffin-embedded tissues in diagnostic routine

Background: Detection of somatic mutations is a mandatory practice for therapeutic definition in precision oncology. However, somatic mutation detection protocols use DNA from formalin-fixed and paraffin-embedded (FFPE) tumor tissues, which can result in detection of nonreproducible sequence artifacts, especially C:G > T:A transitions, in DNA. In recent studies, DNA pretreatment with uracil DNA glycosylase (UDG), an enzyme involved in base excision repair, significantly reduced the number of DNA artifacts after mutation detection by next-generation sequencing (NGS) and other methods, without affecting the capacity to detect real mutations. This study aimed to evaluate the effects of UDG enzymatic pretreatment in reducing the number of DNA sequencing artifacts from FFPE tumor samples, to improve the accuracy of genetic testing in the molecular diagnostic routine. Methods: We selected 12 FFPE tumor samples (10 melanoma, 1 lung, and 1 colorectal tumor sample) with different storage times. We compared sequencing results of a 16-hotspot gene panel of NGS libraries prepared with UDG-treated and untreated samples. Results: All UDG-treated samples showed large reductions in the total number of transitions (medium reduction of 80%) and the transition/transversion ratio (medium reduction of 75%). In addition, most sequence artifacts presented a low variant allele frequency (VAF < 10%) which are eliminated with UDG treatment. Conclusion: Including UDG enzymatic treatment before multiplex amplification in the NGS workflow significantly decreased the number of artifactual variants detected in FFPE samples. Thus, including this additional step in the current methodology should improve the rate of true mutation detection in the molecular diagnostic routine.

Tissue hyaluronan expression, as reflected in the sputum of lung cancer patients, is an indicator of malignancy

Hyaluronan (HA) shows promise for detecting cancerous change in pleural effusion and urine. However, there is uncertainty about the localization of HA in tumor tissue and its relationship with different histological types and other components of the extracellular matrix, such as angiogenesis. We evaluated the association between HA and degree of malignancy through expression in lung tumor tissue and sputum. Tumoral tissue had significantly increased HA compared to normal tissue. Strong HA staining intensity associated with cancer cells was significant in squamous cell carcinoma compared to adenocarcinoma and large cell carcinoma. A significant direct association was found between tumors with a high percentage of HA and MVD (microvessel density) in tumoral stroma. Similarly significant was the direct association between N1 tumors and high levels of HA in cancer cells. Cox multivariate analysis showed significant association between better survival and low HA. HA increased in sputum from lung cancer patients compared to cancer-free and healthy volunteers and a significant correlation was found between HA in sputum and HA in cancer tissue. Localization of HA in tumor tissue was related to malignancy and reflected in sputum, making this an emerging factor for an important diagnostic procedure in patients suspected to have lung cancer. Further study in additional patients in a randomized prospective trial is required to finalize these results and to validate our quantitative assessment of HA, as well as to couple it to gold standard sputum cytology.

Papel da proteína Mx1 na resposta a interferons e no processo neoplásico / Role of Mx1 protein in the response to interferons and neoplastic process

A proteína Mx1 é codificada por um gene induzido por interferon e compartilha a organização de seus domínios, a capacidade de homo-oligomerização e associação com membranas com as grandes dinaminas GTPases. A proteína Mx1 está envolvida na resposta contra um grande número de vírus de RNA, como aqueles pertencentes à família Buniavírus e o vírus influenza. Curiosamente, o gene MX1 foi encontrado como silenciado por metilação em diversos processos neoplásicos, incluindo carcinomas de cabeça e pescoço de células escamosas. Neste cenário, o silenciamento gênico de MX1 está associado à imortalização de uma série de linhagens celulares neoplásicas. Assim, Mx1 se destaca como uma das principais proteínas envolvidas nas respostas imunes induzidas por interferon e também desempenha um importante papel no controle do ciclo celular. Aqui discutimos os aspectos funcionais da proteína Mx1 abordando sua atividade antiviral, organização estrutural, envolvimento com neoplasias e, principalmente, os aspectos funcionais obtidos pela determinação de seus parceiros celulares.

The Mx1 protein is encoded by an interferon-induced gene and shares domain organization, homo-oligomerization capacity and membrane association with the large dynamin-like GTPases. The Mx1 protein is involved in the response to a large number of RNA viruses, such as the bunyavirus family and the influenza virus. Interestingly, it has also been found as a methylation-silenced gene in several types of neoplasm, including head and neck squamous cell carcinoma. In this scenario, MX1 gene silencing is associated with immortalization in several neoplastic cell lines. Thus, Mx1 stands out as one of the key proteins involved in interferon-induced immune response and also plays an important role in cell cycle control. Here we discuss some of the functions of the Mx1 protein, including its antiviral activity, protein folding and involvement in neoplasia, as well as those revealed by investigating its cellular partners.

Gene expression profiling of clinical stages II and III breast cancer

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.

Mining ORESTES no-match database: can we still contribute to cancer transcriptome?

The Human Cancer Genome Project generated about 1 million expressed sequence tags by the ORESTES method, principally with the aim of obtaining data from cancer. Of this total, 341,680 showed no similarity with sequences in the public transcript databases, referred to as [quot ]no-match[quot ]. Some of them represent low abundance or difficult to detect human transcripts, but part of these sequences represent genomic contamination or immature mRNA. We performed a bioinformatics pipeline to determine the novelty of ORESTES [quot ]no-match[quot ] datasets from prostate or breast tissues. We started with 14,908 clusters mapped on the human genome. A total of 2226 clusters originating from more than two libraries or singletons with gaps upon genome alignment were selected. Ninety-four clusters with canonical splice sites representing the most stringent criteria to be considered a gene were subjected to manual inspection regarding genomic hits. Of the manually inspected clusters, 49.6% contained new sequences where 42.2% were probable low-expression alternative forms of the characterized genes and 7.4% unpredicted genes. RT-PCR followed by sequencing was performed to validate the largest spliced sequence from 8 clusters, resulting in the confirmation of five sequences as true human transcript fragments. Some of them were differentially expressed between tumor and normal tissue by an in silico analysis. We can conclude that after clean up of the no-match dataset, we still have about 939 new exons and 165 unpredicted genes that could complete the prostate or breast transcriptome.

Closure of rRNA related gaps in the Chromobacterium violaceum genome with the PCR-assisted contig extension (PACE) protocol

In the finishing phase of the Chromobacterium violaceum genome project, the shotgun sequences were assembled into 57 contigs that were then organized into 19 scaffolds, using the information from shotgun and cosmid clones. Among the 38 ends resulting from the 19 scaffolds, 10 ended with sequences corresponding to rRNA genes (seven ended with the 5S rRNA gene and three ended with the 16S rRNA gene). The 28 non-ribosomal ends were extended using the PCR-assisted contig extension (PACE) methodology, which immediately closed 15 real gaps. We then applied PACE to the 16S rRNA gene containing ends, resulting in eight different sequences that were correctly assembled within the C. violaceum genome by combinatory PCR strategy, with primers derived from the non-repetitive genomic region flanking the 16S and 5S rRNA gene. An oriented combinatory PCR was used to correctly position the two versions (copy A and copy B, which differ by the presence or absence of a 100-bp insert); it revealed six copies corresponding to copy A, and two to copy B. We estimate that the use of PACE, followed by combinatory PCR, accelerated the finishing phase of the C. violaceum genome project by at least 40 per cent